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Sayan Chatterjee,Lakshmi Vineela Nalla,Monika Sharma,Nishant Sharma,Aditya A. Singh,Fehmina Mushtaque Malim,Manasi Ghatage,Mohd Mukarram,Abhijeet Pawar,Nidhi Parihar,Neha Arya,Amit Khairnar

Association of COVID-19 with Comorbidities: An Update

27-02-2023

COVID-19,comorbidity,diabetes,cancer,Parkinson’s disease,cardiovascular disease

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which was identified in Wuhan, China in December 2019 and jeopardized human lives. It spreads at an unprecedented rate worldwide, with serious and still-unfolding health conditions and economic ramifications. Based on the clinical investigations, the severity of COVID-19 appears to be highly variable, ranging from mild to severe infections including the death of an infected individual. To add to this, patients with comorbid conditions such as age or concomitant illnesses are significant predictors of the disease’s severity and progression. SARS-CoV-2 enters inside the host cells through ACE2 (angiotensin converting enzyme2) receptor expression; therefore, comorbidities associated with higher ACE2 expression may enhance the virus entry and the severity of COVID-19 infection. It has already been recognized that age-related comorbidities such as Parkinson’s disease, cancer, diabetes, and cardiovascular diseases may lead to life-threatening illnesses in COVID-19-infected patients. COVID-19 infection results in the excessive release of cytokines, called “cytokine storm”, which causes the worsening of comorbid disease conditions. Different mechanisms of COVID-19 infections leading to intensive care unit (ICU) admissions or deaths have been hypothesized. This review provides insights into the relationship between various comorbidities and COVID-19 infection. We further discuss the potential pathophysiological correlation between COVID-19 disease and comorbidities with the medical interventions for comorbid patients. Toward the end, different therapeutic options have been discussed for COVID-19-infected comorbid patients.

Association of COVID-19 with Comorbidities: An Update Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which was identified in Wuhan, China in December 2019 and jeopardized human lives. It spreads at an unprecedented rate worldwide, with serious and still-unfolding health conditions and economic ramifications. Based on the clinical investigations, the severity of COVID-19 appears to be highly variable, ranging from mild to severe infections including the death of an infected individual. To add to this, patients with comorbid conditions such as age or concomitant illnesses are significant predictors of the disease’s severity and progression. SARS-CoV-2 enters inside the host cells through ACE2 (angiotensin converting enzyme2) receptor expression; therefore, comorbidities associated with higher ACE2 expression may enhance the virus entry and the severity of COVID-19 infection. It has already been recognized that age-related comorbidities such as Parkinson’s disease, cancer, diabetes, and cardiovascular diseases may lead to life-threatening illnesses in COVID-19-infected patients. COVID-19 infection results in the excessive release of cytokines, called “cytokine storm”, which causes the worsening of comorbid disease conditions. Different mechanisms of COVID-19 infections leading to intensive care unit (ICU) admissions or deaths have been hypothesized. This review provides insights into the relationship between various comorbidities and COVID-19 infection. We further discuss the potential pathophysiological correlation between COVID-19 disease and comorbidities with the medical interventions for comorbid patients. Toward the end, different therapeutic options have been discussed for COVID-19-infected comorbid patients. Coronavirus disease (COVID-19) is a communicable disease associated with the dysfunction of the upper respiratory tract caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The city of Wuhan, China was the first to document pneumonia cases of unknown etiology at the end of December 2019. It was spread to around 188 nations, resulting in many confirmed cases and severe health and socioeconomic consequences. The total number of confirmed cases worldwide reached 574,818,625 with 6,395,451 fatalities by the fourth week of July 2022; this data was presented by the Coronavirus Resource Center at Johns Hopkins University. Ejaz and colleagues reported that SARS-CoV-2 infections could cause various symptoms, from mild diseases that go away independently to dangerous ones that affect many organs. There are mainly four types of coronavirus (CoV), classified as α-CoV, β-CoV, γ-CoV, and δ-CoV. Of these, only α-CoV and β-CoV have been shown to cause animal sickness. Further, β-CoV was responsible for SARS in 2003 and the Middle East respiratory syndrome (MERS) in 2012. According to several genomic studies, SARS-CoV-2 is an encapsulated virus with a positive sense-RNA genome. The genome of SARS-CoV-2 is approximately 96% similar to that of bat CoV RaTG13. Furthermore, the genomic sequence and evolution of the analysis of SARS-CoV-2 have a 79.5% genomic similarity to the severe acute respiratory syndrome-coronavirus (SARS-CoV). SARS-CoV-2 enters human cells by attaching to the angiotensin convertase enzyme2 (ACE2) receptor of the upper respiratory tract, which acts as an entry point for this virus. Although the virus travels by intranasal and oral pathways, it affects olfactory sensory neurons. Eventually, it infects the central nervous system (CNS), causing hyposmia (loss of sensation of smell) and hypogeusia (loss of taste) as well as other sensory symptoms. Although SARS-CoV-2 infects people of all ages and genders, research shows that individuals with comorbidities are more susceptible to COVID-19 infection. Further evidence suggests that male patients 50 years of age with or without comorbidities show a significantly increased risk of death. According to the Centers for Disease Control and Prevention (CDC), USA, individuals aged 65 and above accounted for around 30% of COVID-19 infections, 45% of hospitalizations, 53% of intensive care unit (ICU) admissions, and 80% of deaths. In addition, after COVID-19 infection, those with a compromised immune system due to cancer treatments or steroids requiring hospitalization are prone to mortality. Given those the events of ICU admissions or mortality following COVID-19 infection increase. It is vital to comprehend the mechanisms and treatment alternatives that are most suited for marginalized populations. Based on clinical data on COVID-19, comorbidities like cardiovascular disease (CVD) including hypertension and diabetes have been the most prevalent. In this review, we focused on the link between COVID-19 and comorbidities such as Parkinson’s disease (PD), cancer, diabetes, and CVD. We also looked at epidemiological data, pathological relationships, and potential treatment options for COVID-19-infected people with comorbidities. As was highlighted, this review illustrates the relationship between several comorbidities and COVID-19. A literature search was conducted online using several databases and search engines like PubMed to exploit this. The keywords such as COVID-19, SARS-CoV-2, and comorbidity in COVID-19 were used to get the most relevant articles that support this study. On the other hand, the relationship between PD, cancer, diabetes mellitus, CVD, and hypertension with COVID-19 articles were also used to accomplish this study. Besides, the rest of the articles with mismatched or irrelevant keywords were not considered for this study. All the publications were examined and referenced based on their relevancy and compatibility with the current topic of discussion. We used PubMed’s “Boolean Operators” (AND, NOT, and OR) search criterion to acquire relevant search results for this. Figure 1 depicts the strategy for obtaining the data and subsequently filtering the articles, and Table 1 provides the keywords used for searching through Pubmed. The significant comorbidities of COVID-19, such as PD, cancer, diabetes, and CVD, have been included after a literature search and screening of published research articles, meta-analyses, and systemic review studies. In this study, 198 articles were discussed in depth to show how these significant comorbidities were to blame for hospitalization, ICU admission, and mortality in most cases. In addition, we looked at the molecular and cellular mechanisms of COVID-19 and how they relate to its pathophysiology, linked with substantial comorbidities. For instance, cytokine storm is typical of all the main comorbidities described above. We looked for several inflammatory cytokines connected to COVID-19-related comorbidities in this context. Then, we checked the further information on clinical trials web site to learn more about the clinical studies that used the repurposed drugs for the appropriate comorbidities. Furthermore, we have also included future projections as well as several treatment strategies for each comorbidity. Patients with COVID-19 have received treatment using a wide range of therapeutic modalities globally. In the absence of a vaccine or a SARS-CoV neutralizing antibody, convalescent plasma therapy and pharmaceutical repurposing lead the charge. During genomic replication, a virus’s genetic code changes (gene mutation), a phenomenon also prevalent with the SARS-CoV-2 virus, wherein constant gene mutations have led to many variants (lineage) of the same virus over time. A lineage is a set of genetically related viral variants that share a common ancestor. SARS-CoV-2 has several lineages, all of which produce COVID-19 infection. Some lineage changes propagate more rapidly and easily than others, perhaps contributing to make COVID-19 cases more common. A rise in the number of cases have imposed a higher burden on healthcare resources, resulting in additional hospitalizations and, perhaps, fatalities. In the USA, epidemiological investigations into viral genetic sequence-based monitoring and laboratory research are routinely conducted to track SARS-CoV-2 genetic lineages. The SARS-CoV-2 Interagency Group (SIG) of the US government categorized Omicron as a Variant of Concern on November 30, 2021 (Control and Prevention, 2021). According to SIG, there are four types of SARS-CoV-2 variants: 1. Variant Being Monitored (VBM): Alpha (B.1.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P.1 and descendent lineages), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1), Zeta (P.2); 2. Variant of Interest (VOI): None of the variant(s) yet identified; 3. A variant of Concern (VOC): Delta (B.1.617.2 and AY lineages), Omicron (B.1.1.529 and BA lineages); 4. A variants of High Consequence (VOHC): This sort of variation is yet to be detected internationally. Omicron is constantly evolving mutations even after 3 years of the pandemic and still giving rise to several new subvariants, such as BA.2.75 and BA.4.6. Importantly, several of these new variations, including BA.2.3.20, BA.2.75.2, CA.1, BR.2, BN.1, BM.1.1.1, BU.1, BQ.1.1, and XBB, exhibit notable growth advantages over BA.5. Recently reported in the second week of October 2022 is the name of the latest lineages variant of Omicron is XBB and sublineages XBB.1 (S:252 V) found in major regions such as China, United Kindom (UK), Europe, and North America, resulting in all these countries announcing that they are now going on nationwide lockdown-like restriction once again due to the sudden surge of the new COVID variant. XBB and XBB.1 (S:252 V) are mainly found in Bangladesh, Singapore, and India. The prevalence of Delta and Omicron (BA.1) coinfections and Omicron lineages BA.1 and BA.2 coinfections were estimated at 0.18% and 0.26%, respectively. Among 6,242 hospitalized patients, ICU admission rates were 1.64%, 4.81, and 15.38% in Omicron, Delta, and Delta/Omicron patients, respectively. Among patients admitted to the ICU, there were no reports of BA.1/BA.2 coinfections. A total of 21 patients (39.6%) of the 53 coinfected patients missed vaccinations. Even though SARS-CoV-2 coinfections were rare in their clinical study, it is still essential to accurately identify them so that they can figure out how they affect patients and how likely they will make recombinants. One clinical study has been conducted in France to detect the prevalence of SARS-CoV-2 coinfection during spread of the Delta, Omicron, Delta/Omicron variant. This study was held from December 2021 to February 2022. They tested the effectiveness of four sets of whole-genome sequencing primers using 11 blends of Delta/Omicron isolates at multiple ratios, and they developed a bioinformatics technique that is impartial for identifying coinfections involving various genetic SARS-CoV-2 lineages. Applied to 21,387 samples collected from 6 December 2021 to 27 February 2022, random genomic surveillance in France, they detected 53 coinfections between different lineages. The Delta variation was the most susceptible and transmissible of all of these variants, that resulted in an increase in the percentage of fatalities as well as comorbidities such as hospitalization of older persons. The Omicron variant may spread more quickly than other variants, such as Delta; however, Omicron was less lethal compared to the Delta variant. These differences in response could be attributed to many factors such as the less efficient cleaving of the S protein fraction of omicron and more α helix stabilization than the delta variant. COVID-19 infection is associated with aging, which is a major risk factor for severe illness and mortality, especially for those who are in long-term care facilities. In addition, people at any age with serious underlying medical conditions are more at risk of getting COVID-19 infection. The elderly, SARS-CoV-2 infected persons with comorbidities, including PD, diabetes, cancer, and hypertension (HTN) and CVD, are at higher risk of death. Early evidence from several epidemiological data sets shows that the COVID-19 case fatality ratio (CFR) increases with age. Table 2 reflects the number of CFRs in various countries; overall, the CFR of China and USA is 2.3% and 2.7%, respectively, while the global CFR was at 2.8%. Italy was the first nation to be affected by the pandemic after China. The total CFR of Italy was higher (7.2%) compared to China (2.3%). This is attributed to a more significant proportion of older adults (22.8% and 11.9%, respectively) in Italy. In addition, an 82-year-old man in Brooklyn was the first COVID-19 fatality reported in New York City. A significant case series of 5,700 COVID-19 patients admitted to hospitals in New York City revealed a similar pattern of COVID-19 fatalities with age. However, the mechanism of SARS-CoV-2 infection is still unknown. However, the primary mechanism of SARS-CoV-2 underlies the ACE2 enzyme’s expression and utilization of the ACE2 receptor, which helps to enter the SARS-CoV-2 inside the cell. Lymphopenia, abnormal respiration, and a high level of pro-inflammatory cytokines in plasma are the main manifestations ascribed to individuals with COVID-19 infection along with very high body temperature and respiratory issues. COVID-19 is caused by several metabolic and viral disorders, all of which have a part in the developing of the more complicated symptoms. The CFR for India seems to be lower than in several European nations. This might attributed to low percentage of population (6.38%) to be above the age of 65 as per 2019 statistics. According to reports, COVID-19 puts elderly persons (those over 60) at a greater risk of mortality. The relationship between CFR and several other health and socioeconomic factors, variations in the virulence of SARS-CoV-2 across geographical areas, and COVID-19 response indicators unique to certain countries has to be further studied. The projected CFRs (July 2020) based on the random- and fixed-effect models were 1.42% (95% Cl 1.19–1.70%) and 2.97% (95% CI 2.94–3.00%), respectively. Estimates made using the random-effects model were more likely to accurately reflect the real CFR for India because of the high level of variability. In earlier research, the COVID-19 CFR was estimated using random-effect models, or the CFR was provided using both random- and fixed-effect models. We made sure that states with a lot of cases and fatalities got more weightage than those with fewer cases and deaths by using a random effects model.Table 3 shows that COVID-19-infected patients with comorbidities had a higher death risk. As a consequence of aging, the body experiences progressive biological alterations in immune function, concurrently causing an increased susceptibility to age-related inflammation (inflammaging) and other associated inflammatory conditions, which makes the elderly population vulnerable to enhanced risk of infection following exposure to the virus. Inflammaging is a chronic low-stage inflammation mediated by dysfunction in the basal responses of the pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). A progressive impairment in autophagic signals affect the PRR signals in the aging population and consequently cause the exorbitant release of reactive oxygen species (ROS). This condition may further be worsened with the binding of the virus to immune cells as they also work in synchronization with PAMPs and PRRs, thereby causing oxidative damage to cells in older individuals. Another hallmark of inflammaging is increased production of interleukins (ILs), especially IL-1β and IL-18, due to activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, in COVID-19 infections. NLRP3 activation is strongly correlated to the aging population. This consequently causes increased pyroptosis, which is central to cell death post-infection; increased release of IL-1β, IL-18 as well as damage-associated molecular patterns (DAMPs) further cumulates inflammatory responses in the elderly. This mechanism has also been reported to be the root cause of inflammatory conditions such as cancer, diabetes, PD, and acute myocardial injury. Furthermore, with age, there is a progressive hardening of the endothelial cells, causing the increased formation of plaque, and further leading to a hypercoagulative state. Following COVID-19 infection, reports have demonstrated that a hypercoagulative state is associated with an increased risk of comorbid conditions such as ischemic stroke and myocardial infarction. Therefore, it can be concluded that aging and comorbid conditions are crucial factors in eliciting the response mediated by COVID-19 infection alone. So, in the subsequent sections, we discussed the latest reports of age-related comorbidities, such as PD, cancer, diabetes, and CVD, and how they relate to the severity and pathology of COVID-19. The population of patients needing hospital admission is disproportionately composed of older adults, men, and people with comorbid conditions, including diabetes and CVDs. After COVID-19 expanded to multiethnic communities in Western Europe and North America, multiple studies claimed that Black, Asian, or other minority ethnic groups were more likely to be affected by the illness. According to the statistical report from USA, in several cities or cumulative analyses across large states, Black and Hispanic people had higher per capita mortality rates than White people, but the underlying reasons were unknown. A large cohort study has been conducted in the United Kingdom (UK), wherein they reported higher overall death rates for Black and South Asian people compared to White people. However, the study ignores the wide variances in the ethnic makeup of local populations across various geographic locations. In this regard, a case-control and cohort study was conducted at King’s College Hospital Foundation Trust, UK to investigate if ethnic origin influences the probability of hospital admission with severe COVID-19 and/or in-hospital mortality. Inner city adult patients with confirmed COVID-19 admitted to the hospital (n = 872 cases) were compared with 3,488 matched controls randomly drawn from a primary healthcare database consisting of 344,083 people dwelling in the same area. For the cohort study, the authors examined 1,827 people continuously hospitalized with COVID-19. Self-defined ethnicity served as the primary exposure factor and analyses were adjusted for socio-demographic and clinical characteristics. Based on the results obtained by conditional logistic regression analysis, it was demonstrated that the Black and Mixed/Other ethnicity were linked with greater admission risk than the White. Further, the Black and Mixed ethinicity could be linked to disease severity but not to in-hospital mortality. This was majorly attributed to ethinicity and partly to comorbities and socioeconomic factors. In addition, the study elucidated the association of increased in-hospital mortality with ICU admission for Asian ethinicity. Therefore,it could be concluded that COVID-19 disease outcome is influenced by the ethnic background. Although aging is a prominent factor for comorbidities, it may not be as labeled as the only confounding factor for the disease severity. Reports have suggested that hospitalized males had the highest mortality rate as compared to females and this association was more prominent with patients with predisposing conditions such as hypertension, diabetes and obesity in an age-dependent manner. One study has shown male patients’ as a predictor of ICU admissions. Contrastingly, in context of the long term COVID-19 manifestations, women were more likely to report uneasiness, breathlessness, and fatigue following recovery. Outcomes in severity also resulted from biochemical differences in males and females; compared to male patients, females had higher lymphocyte counts, higher levels of high-density lipoprotein, as well as lower levels of highly sensitive C-reactive protein. Another hypothesis for gender disparity in protection is that the females have biallelic Toll-like receptor (TLR)7 expression, thereby leading to a better interferon (IFN)-mediated response after early infection. Interestingly, studies have reported that estrogen confers some protection against the severity of COVID-19. This has been validated in a preclinical setting, where mice infected with the SARS-CoV virus had a higher mortality rate following ovariectomy or estrogen receptor antagonist administration. Further, pregnant women with mild infection demonstrate same outcome as uninfected pregnant women. However, those with severe infection demonstrate a higher risk of perinatal infection as well as mortality, usually having a tendency to feel unwell and this further exacerbates during COVID-19 infection and may result in worsening of conditions of the patients. Children are disportionately infected with COVID-19 compared to older population but with low infection severity and could be attributed to lower concentration of ACE2 receptors in children as well as trained/acquired immunity as a result of vaccination, indigenous virus competition, as well as maternal immunity. Thus, it can be concluded that sex differences play a major role in the outcomes and severity of COVID-19 infection. Due to the pivotal role of female hormones, they may be less susceptible to long-term manifestations which are prevalent in male patients. SARS-CoV-2 is a neurotropic virus, that can enter the CNS either by hematogenous or neuronal retrograde dissemination. In an autopsy study, viral RNA in the brains of several COVID-19 patients was detected. Patients having neurological diseases are generally more vulnerable to the respiratory system infections.and could be attributed to the involvement of central respiratory centers in the case of COVID-19 infections. There are shreds of evidence that suggest the potential of SARS-CoV-2 to enter the brain through the olfactory epithelium and cause neuronal death in mice. Furthermore, there have been cases of COVID-19 individuals developing symptomatic parkinsonism after 2–5 weeks of viral infection. SARS-CoV was discovered in the cerebral fluid of individuals suffering from acute SARS-CoV disease, as was also reported in the COVID-19 cases. Recent data from a study performed in three designated COVID-19 care hospitals of the Union Hospital of Huazhong University of Science and Technology in Wuhan, China suggests that 78 patients out of 214 COVID-19 patients demonstrated neurologic manifestations. This involved the CNS, peripheral nervous system, and skeletal muscles, subsequently indicating the neurotropic potential of this virus. Hitherto, there is not enough evidence regarding the susceptibility of PD patients to COVID-19. According to a study from the Parkinson’s and Movement Disorders Unit in Padua, Italy and the Parkinson’s Foundation Centre of Excellence at King’s College Hospital in London, UK, PD patients with an average age of more than 78.3 years and with a disease period of greater than 12.7 years are more prone to COVID-19. They also have a significantly high mortality rate of 40%. The patients in extreme conditions of PD with respiratory muscle rigidity, dyspnoea, and on deep brain stimulation or levodopa infusion therapy showed high vulnerability and 50% mortality. Moreover, SARS-CoV and H1N1 viruses (structurally and functionally similar to COVID-19) can aggravate the mechanisms involved in PD pathophysiology as supported by previous studies. A few studies also suggest the role of the CNS in COVID-19 infection indicating that PD patients might be more prone to COVID-19 infection. There are various theories about how SARS-CoV-2 enters the CNS. Peripheral SARS-CoV-2 infection can lead to a cytokine storm, which might disrupt the blood–brain barrier integrity and might be a mechanism for SARS-CoV-2 to infiltrate into the CNS. Besides this, ACE2 receptors are highly expressed in the substantia nigra and striatum (the regions which are potentially affected in PD) making dopaminergic neurons present in these brain regions more susceptible to SARS-CoV-2 infection. Furthermore, the accumulation of alpha-synuclein (α-Syn) is the major hallmark of PD. According to previous findings, the entrance of SARS-CoV-2 into the CNS may upregulate this protein, causing aggregation. On the contrary, few studies have indicated α-Syn’s protective effect in blocking viral entry and propagation into the CNS. Moreover, its expression in the neurons can act as a barrier to viral RNA replication. In a retrospective cohort study conducted in Japan, PD patients suffering from pneumonia showed a lower mortality rate. Furthermore, the main pathophysiological pathway involved in PD development, includes autophagy disruption, ER stress, and mitochondrial dysfunction. As a result, COVID-19 may trigger frequent modulations in these pathways, as seen in SARS-CoV and influenza A virus. Proteostasis plays a vital role in protein translation, folding, and subsequent clearance with the help of heat shock proteins (HSPs). Viral infection hijacks the host cellular machinery for its replication and disrupts the proteostasis pathways by interacting with Hsp40. The viral-Hsp40 interaction results in the binding of Hsp40 with two subunits of viral RNA polymerase, which further assists the viral genome to get translocated into the nucleus via interaction with the viral nucleoprotein and inhibition of protein kinase R (PKR) activation, thereby restricting the host from producing an antiviral response. Hsp90 also modulates the activity of viral RNA polymerase after it enters the nucleus. Under normal conditions, Hsp90 and Hsp70 restrict apoptosis initiation pathways thereby reducing apoptosis. However, infection with SARS-CoV-2 suppresses Hsp90 and Hsp70 function, leading to activation of caspase cascade followed by apoptosis and subsequent propogation of infection. Autophagy lysosomal pathways and ubiquitin-proteasome pathways are two important components of proteostasis and are responsible for the degradation of impaired proteins. It has been reported that H1N1 infection obstructs autophagic flux at the initial stages resulting in the reduced number of autophagosomes and hindering autophagosome-lysosome fusion at later stages of autophagy. Both these activities result in autophagy disruption in human dopaminergic neurons and mouse brain and subsequently lead to α-Syn aggregation. Furthermore, when H1N1 was instilled intranasally in Rag knockout mice, α-Syn aggregates were found in the cells near olfactory bulbs, which may further spread in a prion-like manner to other regions of the brain and originate downstream events of PD pathogenesis. Furthermore, the ubiquitin-proteasome system can destroy viral proteins by ubiquitination; however, the H1N1 virus hijacks this mechanism and inhibits the host cell opponents of viral reproduction. This disrupts proteostasis and toxic protein aggregation. Further, SARS-CoV-2 has also been shown to act similarly to H1N1 virus and might be involved in α-Syn accumulation. Besides this, ER has also been reported as a target of various viruses. SARS-CoV-2 utilizes ER for the synthesis and processing of viral proteins. It has been shown previously that the Spike (S) protein gets collected in the ER and induces unfolded protein response (UPR) by transcriptional activation of several UPR effectors, including glucose-regulated protein 78 (GRP78), GRP94, and CCAAT/Enhancer-binding protein (C/EBP) homologous protein to aid viral replication. UPR may result in ER stress, which further activates cellular signals triggering neuronal death, are implicated in PD. Another study discovered that SARS-CoV open reading frames (ORF) 6 and 7a produce ER stress via GRP94 activation. Mitochondrial dysfunction is another pathway that connects PD with COVID-19. ORF-9b of SARS-CoV-2 degrades dynamin-like protein 1 (Drp1), involved in mitochondrial fission, thus causing mitochondrial elongation. Moreover, it suppresses antiviral cellular signaling by targeting the mitochondrial-associated adaptor molecule signalosome. ORF3b is located partially in mitochondria and is involved in apoptosis along with other accessory proteins (ORF3a, ORF6, and ORF 7a of SARS-CoV). The virus utilizes the mitochondria for caspase activation for apoptosis, thereby causing viral dissemination to other cells. The aforementioned cellular malfunctions result in increased ROS, redox imbalance, and mitochondrial and lysosomal dysfunction making the cells more susceptible to infection. According to current research, neuroinflammation is a defining factor in COVID-19 infection. Proinflammatory cytokine levels are higher in the periphery and cerebrospinal fluid in PD patients. Strikingly, studies have reported that viral infection can also induce neuroinflammation. Due to the compromised anti-inflammatory mechanisms in old age, the older population is more susceptible to develop neurodegenerative diseases as well as severe COVID-19 infection. TLRs may play a role in the immunological response to coronavirus infections indicated by the presence of PAMPs (lipopolysaccharides, dsDNA/RNA, ssRNA) in the host cells recognized by specific TLRs derived from viruses following infection. TLR 3 is known to be activated in the case of HSV-I and influenza A infection. Whenever TLRs are triggered, pro-inflammatory cytokines are released (IL-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha)) and type I IFN-α/β via MyD88-dependent and MyD88-independent pathways, which further translocate nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), interferon regulatory factor (IRF), IRF-3 and IRF-7, inside the nucleus. Moreover, NF-κB has been reported to contribute to the pathogenesis of PD by triggering the release of pro-inflammatory mediators and subsequent neuroinflammation. Therefore, it can be concluded that NF-κB plays a common role in inflammation in both PD and COVID-19 pathogenesis. Neuroinflammation can also trigger misfolding and aggregation of α-Syn. Aggregated α-Syn leads to the activation of microglia which further favors the production of pro-inflammatory cytokines, ultimately causing neurodegeneration (Figure 2). None of the anti-Parkinsonian drugs render PD patients at risk for COVID-19; therefore, PD patients should not alter or stop any medicine without a clinician’s consultation. However, in order to avoid possible interactions, PD patients with COVID-19 infection should not use cough suppressants containing dextromethorphan and pseudoephedrine with Selegiline. Previous data suggest that the therapies used for COVID-19 such as angiotensin converting enzyme inhibitors (ACEi), might be safe for PD patients as well. Neuroprotective effects of ACEi like captopril and perindopril have been observed in PD animal models, which act by preventing dopaminergic cell loss and increasing striatal dopamine content, respectively. ACEi also work as antioxidants by reducing oxidative stress, which has been linked to PD and has been found to decrease the number of falls in a cross-sectional study involving 91 PD patients. Furthermore, hydroxychloroquine exhibits anti-Parkinsonian effects by raising Nurr1 expression, inhibiting glycogen synthase kinase-3 beta (GSK-3β) and functioning as an anti-inflammatory drug, making it a viable treatment option for PD patients infected with COVID-19. In another study, a COVID-19 patient with PD was cured with remdesivir in a clinical trial. In addition, the antiviral drug amantadine used in PD treatment has also been used for years in the treatment of influenza. Amantadine inhibits viral replication by blocking the influenza M2 ion channel, thereby preventing the delivery of viral ribonucleoprotein into the cytoplasm of the host and might have a disruptive effect on the lysosomal pathway. As a result, amantadine might be advantageous for treatment in COVID-19-positive PD patients as a potential treatment approach to lower viral load in these individuals. Oseltamivir phosphate, an anti-influenza drug, was found to be useful in PD as it inhibits H1N1-induced α-Syn aggregation. Many studies have highlighted that older people are typically deficient in vitamin D; vitamin D might have antiviral properties. As a result, vitamin D3 supplementation (2000–5000 IU/day) has been recommended in older PD patients, which might protect them against COVID-19. Moreover, it has been suggested that vitamin D3 can slow down the progression of PD. Above all, Fenoldopam, a dopamine D1 receptor agonist, was shown to be protective against inflammation as well as lung permeability and pulmonary edema in an endotoxin-induced acute lung injury mouse model. Taken together, it can be concluded that therapies used in PD and COVID-19 infection do not have any detrimental drug interaction. However, diagnosis presents a challenge in PD patients especially in the older population as they might neglect the COVID-19 symptoms because of other chronic diseases. Furthermore, early symptoms of COVID-19, for example dyssomnia, might be neglected by PD patients as they commonly suffer from olfactory dysfunction. Still, people with COVID-19 pneumonia who take medications for PD should have their doses changed because motor symptoms can impair breathing thereby worsening the condition. There is a scarcity of data linking PD and COVID-19 outcomes. However, evidence from molecular processes of SARS coronaviruses changing proteostasis, mitochondrial and ER malfunction, and α-Syn aggregation suggests that it is necessary to identify medications acting on these pathways as a treatment for PD patients suffering from COVID-19. Detailed investigations on these mechanisms are required to determine PD patients’ sensitivity to COVID-19 as well as the safety of antiviral medicines, vaccinations, ACEi, and other antiviral drugs used for COVID-19. The indirect effect of this pandemic on PD patients including no direct patient–doctor visits, depression due to social distancing, reduced physical activity, and battery failure in patients on deep brain stimulation therapy also need attention and should be taken care of with the help of video conferencing and the availability of sufficient stock of medications. Reports suggest patients with lung cancer, hematological cancer, or any metastatic cancer are potentially at high risk of COVID-19 infection either due to treatment or disease susceptibility. Toward this, COVID-19-infected cancer patients were studied retrospectively; analysis showed a higher incidence of severe events following infection of COVID-19 infection, especially in the patients who received the anticancer treatment for 14 days. In one study, a total of 15 (53.6%) patients developed severe clinical event, and 28.6% were found to be morbid. Similar studies suggested being vigilant toward cancer patients who are on anticancer treatments as they are prone to COVID-19 infection as a result of reduced immunity. As per a nationwide study in China, roughly 39% (7 out of 18) of cancer patients infected with COVID-19 experienced severe symptoms, compared to only 8% (124 out of 1572) of patients not suffering with cancer. Another collective report has stated that there were 52 different studies worldwide involving 18,650 cancer patients infected with COVID-19 with 4,243 deaths. The data further implied that the risk of mortality of cancer patients is about 25.6 % COVID-19 as compared to 2.3% in the normal population. COVID-19 puts lung cancer patients at significant risk of developing severe episodes. In this regard, a study reported that out of 102 lung cancer patients infected with COVID-19 infection, 62% of lung cancer patients were hospitalized, and the mortality rate was approximately 25%. In another study, 55% (6 out of 11) mortality was observed in lung cancer patients infected with COVID-19 which was very high compared to other cancers. SARS-CoV-2 infects the host cell via the ACE2, which is a cell surface receptor. It is highly expressed on the lung epithelial cells and subsequently gets cleaved with the help of the host transmembrane serine protease2 (TMPRSS2). The viral internalization evokes the host immune response through the activation of alveolar macrophages and the complement cascade. This activation leads to a massive release of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and IFN-γ. This phenomenon, termed the cytokine storm, further causes alveolar endothelial tissue damage. Among these cytokines, IL-6 is involved in the pathophysiology of cancer, especially, lung cancer and other chronic diseases. It has been reported that IL-6 promotes tumorigenesis and anti-apoptotic signaling and is an important biomarker for cancer diagnosis and prognosis. The involvement of IL-6 in abnormally immune-activated conditions like cancer inflammation and immunosuppression have also been reported. Apart from IL-6, the activation of serine/threonine p21 activated kinase1 (PAK1), an essential component of malaria and some viral infections, is also a critical mediator of cytokine storm and gets overexpressed in SARS-CoV-2 infected lungs, resulting in mortality of COVID-19 patients. One study has reported that the human PAK, is an important component of host–pathogen interactions. PAK paralogues (Group I PAKs, include PAK1, PAK2, and PAK3; Group II PAKs, including PAK4, PAK5, and PAK6) are found in nearly all mammalian tissues, wherein they play important roles in a variety of processes including cell survival and proliferation, cell cycle progression, and cytoskeletal organization and are involved in different types of cancer. In drug development, the role of PAKs in cell survival along with proliferation, as well as participation in a variety of malignancies, is of significant interest. PAK1 activation can lead to the development of lung fibrosis by stimulation of the chemokine (C–C motif) ligand 2 (CCL2) production, thereby aggravating the patient’s condition. PAK1 blockers can help in restoring the immune response thereby combating virus-induced lung fibrosis. In this regard, the PAK1 inhibitor (propolis) was tested as a therapeutic approach for treating COVID-19 patients. Its extract components were shown to have inhibitory effects against other targets like ACE2 and TMPRSS2 (Figure 3). Besides, underlying conditions and altered immune responses increase the risk of developing venous thromboembolism, microvascular COVID-19 lung and vessels obstructive thrombo-inflammatory syndrome in cancer patients. The progressive endothelial thrombo-inflammatory syndrome may also cover the brain’s microvascular bed and other vital organs, resulting in multiple organ failures and death. Additionally, Bhotla et al. and colleagues hypothesized that platelets are getting infected due to COVID-19 infection which exacerbate the SARS-CoV-2 infection and ultimately lead to the bronchopneumonia or death. In conclusion, the biochemical and immunological characteristics outlined above demonstrate that cancer patients, are more susceptible to COVID-19 infections than the general population. However, non-biological variables such as increased contact with the healthcare system for cancer treatment may potentially contribute to the increase in COVID-19 prevalence in cancer patients. Because of the current pandemic situation, as well as the associated risk factors, cancer patients must take additional precautions. As a result, in order to minimize the adverse consequences of the COVID-19 pandemic on highly susceptible cancer patients, hospitals should have, more robust management procedures in place. To this end, chemotherapy or surgery should be postponed, intense treatment should be provided, greater personal protection should be provided, telemedicine should be used, and a separate treatment approach for COVID-19 cancer patients should be implemented. As discussed in the previous section, IL-6 has been recognized as a crucial component of the immune response to SARS-CoV-2. Many clinical trials are currently underway to investigate treatment strategies targeting IL-6 by repurposing anti-IL-6 therapeutics for COVID-19 in cancer patients. Tocilizumab, a monoclonal antibody against the IL-6 receptor, has shown promising results in a double-blind, placebo-controlled phase-III study called EMPACTA (NCT04372186). Similarly, siltuximab, an IL-6 receptor chimeric mouse–human monoclonal antibody, has already exhibited its antitumor efficacy and is under diverse randomized control trials for further assessing its efficacy for COVID-19 patients (NCT04486521, NCT04330638, and NCT04329650). In addition, tumor reversion therapy might be the near future therapy for the treatment of cancer. The molecular biology behind the tumor reversal process is not only fascinating but alluring. Some chemical compounds used for tumor reversion include LY294002, metformin, sertraline, and ellipticine. Apart from this, T cell therapy such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T cell (CAR-T) therapy may increase the risk of cytokine release resulting in increased severity of COVID-19 infection. Cytokine storm is directly linked to COVID-19-associated diseases such as acute respiratory distress syndrome (ARDS) and multiorgan failure. Moreover, an immunocompromised cancer patient with immunocompromised therapy and also those at risk for immune-related side effects in response to immuno-oncology treatments should be monitored closely. Another treatment approach is mesenchymal stem cell therapy; this is approved and is now used for cancer treatment alone or in combination with other drugs. It was even applied on 7 patients with COVID-19 infection, resulting in a negative effect on the expression of ACE2 along with TMPRSS2. On the sixth day, cytokine-secreting cells (CXCR3+ CD4+ T, CXCR3+ CD8+ T, and NK CXCR3+ cells) disappeared as peripheral lymphocyte counts increased. While the TNF-alpha levels were reduced, dendritic cell populations and IL10 levels were increased. Along with IL-6, other cytokines associated with the pathogenesis of COVID-19 infection in cancer patients include type I IFN, IL-1, IL-7, IL-17, and TNF-alpha. Even a clinical study named Bee-COVID was conducted with the Brazilian Green Propolis Extract for the treatment of the COVID-19 condition (NCT04480593). In conclusion, targeting these inflammatory markers may serve as a good approach to treating COVID-19 infection in cancer patients. The COVID-19 outbreak is a global threat to the health system. Despite the substantial study, there is currently no recognized treatment for COVID-19. Still, it is unclear why some people respond abruptly to SARS-CoV-2 infection and others are asymptomatic. Further, why people with coexisting comorbidities are more susceptible to severe clinical events of COVID-19 is unclear. Being at high risk of infection and deteriorating outcomes, cancer patients are suggested to be more cautious and follow the guidelines issued by World Health Organization and the European Society for Medical Oncology. Diabetes mellitus (DM) is a common metabolic disorder with multiple etiologies primarily associated with a deficiency of insulin secretion and/or its action. Besides the clinical complication of the disease, an individual with diabetes is more susceptible to a broad range of infections (such as foot infection, rhinocerebral mucormycosis, malignant external otitis, and gangrenous cholecystitis) as well as predisposed to certain conditions that primarily affect lungs like influenza, tuberculosis, and legionella pneumonia. Moreover, diabetes and its complications such as disrupted glycemic control and ketoacidosis were found to be a potential risk factor for mortality in the influenza A (H1N1) pandemic in 2009, SARS-COV, and MERS coronavirus infection. Several studies have reported high mortality in COVID-19 patients with diabetes. More specifically, diabetes was the most common underlying comorbidity in approximately 22% of the 32 nonsurvivors from a cohort of 52 COVID-19 patients in intensive care. Detailed clinical research on 140 hospitalized COVID-19 patients in Wuhan indicated the second highest prevalence was diabetes (12.1%) after hypertension. Another study on a subset of 1099 patients discovered that out of 177 severe cases, 16.2% of patients with seriously infected conditions had diabetes. Eighteen of these patients had composite outcomes, including death, use of mechanical ventilation, and admission to an ICU. Recent epidemiological research of 72,314 COVID-19 patients at the Chinese Center for Disease Control and Prevention found that diabetics mortality in diabetic patients was three times greater than non-diabetic patients (7.3% mortality rate as compared to the overall 2.3% mortality rate). Besides, a recent retrospective multicenter cohort study on 191 laboratory-confirmed COVID-19 cases observed a statistically significant association between diabetes and increased mortality. In yet another study, Fadini and his colleagues at the University Hospital of Padova discovered that the mortality rate of diabetic SARS-CoV-2 patients were 1.75 times greater than the general population. This research, coupled with earlier research, suggested that diabetes may exacerbate the outcome of a new coronavirus illness. The CDC data further suggested that COVID-19-infected diabetic patients have a higher risk of the developing severe symptoms. Toward this, in a retrospective research conducted in Wuhan, China, 32% of cases patients had comorbidities, 20% of which were diabetics. In addition, hyperglycemia or dysregulated glycemic management is associated with a high risk of complications. Diabetes as a major risk factor for the course and prognosis of COVID-19 was further mentioned according to a study of 174 COVID-19 patients admitted to Wuhan Union Hospital. COVID-19 infection in diabetic patients might increase stress hormone levels such as glucocorticoids and catecholamines, leading to high glucose levels. Hyperglycemia in diabetes induces glucose allowing non-enzymatic glycosylation of lung collagen and elastin by advanced glycation end products thereby resulting in reduced elasticity of the lungs COVID-19 infection. This causes thickening of the alveolar epithelial basal lamina and microvascular alterations in the pulmonary capillary beds, further leading to a reduction in pulmonary capillary blood volume and diffusing capacity, which influence the patient’s overall survival. Even a brief period of hyperglycemia has the potential to alter immune cell function. Diabetes increases pro-inflammatory cytokines, including IL-1, IL-6, and TNF-alpha. IL-6, C-reactive protein, serum ferritin, and coagulation index, D-dimer are significantly higher in diabetic individuals than in those without diabetes. Current research suggests that diabetic patients are more prone to cytokine storms. This may be further exaggerated in response to a stimulus as seen in patients with COVID-19 infection. In a study, patients infected with COVID-19 have developed a fatal hyperinflammatory syndrome characterized by a fulminant and fatal hypertyrosinemia, and was thought to be associated with disease severity as demonstrated increased IL-2, IL-7, IL-12, TNF-alpha, and IFN-γ inducible protein 10, macrophage inflammatory protein-1α (MIP-1α/CCL3). A prolonged hyperglycemic state causes an elevated immune response, which further leads to inflammation. Interestingly, increased glucose levels were discovered to directly promote SARS-CoV-2 replication in human monocytes and to sustain SARS-CoV-2 replication through the formation of ROS and activation of hypoxia-inducible factor-1α. This massive influx of inflammatory cells has the potential to disrupt the activities of the primary insulin-responsive organs, i.e., the skeletal muscles and liver, which are primarily responsible for insulin-mediated glucose absorption. High glucose concentration in the plasma leads to cytokine production, glucotoxicity, and viral-induced oxidative stress; these factors promoted a greater risk of thromboembolic problems as well as damage to important organs in diabetic patients (Figure 4). Moreover, one enzyme, dipeptidyl peptidase-4 (CD26 or adenosine deaminase complexing protein 2), tends to bind with the virus and promote the ACE2 expression which is involved to initiate the infectious disease. Furthermore, studies have shown severe lung pathology with dysregulated immune response in mice (with existing Type2 DM (T2DM)) infected with MERS-CoV. In addition, COVID-19 patients with diabetes have an easier progression to acute respiratory distress syndrome and septic shock resulting in multiple organ failures. Another direct metabolic link that exists between coronavirus infection and diabetes is based on the expression of ACE2 present in specific tissue. ACE2 is a transmembrane glycoprotein that converts angiotensin II (Ang II) to angiotensin (Ang). ACE2 is also expressed in blood vessels, macrophages, and monocytes. The expression of ACE2 is of prime importance in the etiology of SARS-CoV-2. It has been observed that inflammatory signals produced by macrophages, such as type I IFN, increase ACE2 receptor expression. The infection of pancreatic macrophages may have triggered these inflammatory signals. As a result, more immune cells particularly pro-inflammatory monocyte/macrophages are recruited, causing further damage to islets of Langerhans and β-cells in the pancreas. Subsequently, it reduces insulin release, subsequently resulting in acute hyperglycemia and transitory diabetes in healthy people. Additionally, macrophages and monocytes are mobile, and once infected with the SARS-CoV-2 virus, they can infiltrate the cells of the pancreatic islets and spread the virus throughout the pancreas. Furthermore, SARS-CoV-2 infection of the β-cells can directly damage them, causing apoptosis, and further worsening the glycemic control of diabetic patients. Furthermore, anti-diabetic medicines, such as glucagon-like peptide 1 agonists, antihypertensive drugs, and statins, also increase ACE2 expression. COVID-19-infected patients were treated with different medications, such as systemic corticosteroids and antiviral medicines, which may potentially cause hyperglycemia. Glucocorticoid-induced DM (GIDM) is a frequent and potentially critical issue to address in clinical practice, although it may contribute to worsening hyperglycemia and ultimately aggravate the diabetic condition associated with COVID-19 infection. The detailed mechanism causing glucocorticoid-induced hyperglycemia is the promotion of weight gain, decrease in peripheral insulin sensitivity, increase in the production of glucose with the promotion of gluconeogenesis, β cell injury due to the destruction of pancreatic cells, increase in the levels of fatty acids, and impairment of insulin release. However, in the case of SARS-CoV-2 infection, the effect of diabetes on ACE2 expression still needs to be studied in detail. In a study, an increased expression of ACE2 was observed in the kidney of diabetic patients in the early stage followed by a decreased expression in later stages that relatively overlaps with the occurrence of diabetic nephropathy. Each of these pathways working synergistically may worsen the situation for diabetic patients making them frailer and further increasing the severity of COVID-19 disease. The glucose level in diabetic patients is mainly maintained with the administration of insulin and is mainly recommended for critically ill patients infected with SARS-CoV-2. It has also been observed that insulin infusion significantly reduces the inflammatory cytokines and helps in lowering the severity of COVID-19. Metformin has been proven to have anti-inflammatory properties in preclinical investigations, and it has also been demonstrated to lower circulating levels of inflammatory biomarkers in persons with T2DM. Besides the anti-inflammatory action of metformin, it is potentially used against the SARS-CoV-2 virus. Metformin acts through inhibiting the virus–host-cell association as well as prevents the expression of ACE2 through the activation of adenosine monophosphate-activated protein kinase. Another promising molecule, DPP-4 antagonist (Linagliptin), an anti-diabetic agent with potential anti-aging properties, was repurposed to combat COVID-19 infection also demonstrates antiaging properties. Further, plant-based natural compounds such as resveratrol, catechin, curcumin, procyanidin, and theaflavin have been tested for the treatment of COVID-19 disease through in silico, in vitro, and in vivo studies. More recently, convalescent plasma therapy has also been applied for COVID-19 associated comorbidities wherein it was used to downregulate the inflammatory cytokines and the viral load in COVID-19 patients. Sulfonylurea must be avoided in COVID-19 patients comorbid with T2DM, as they can cause hypoglycemia. Thiazolidinediones can be used in mild diseases with caution as they have protective effects on the cardiovascular system. However, due to weight gain, edema, and heart failure, thiazolidinediones were not used in moderate and severe conditions. COVID-19 pathology majorly involves inflammation that eventually leads to multiple organ failure and even death. Comorbid diabetic patients are highly susceptible to hospitalization-based COVID-19 infection. Diabetes is characterized by a persistent state of hyperglycemia that can aggravate SARS-CoV-2-induced inflammation. Therefore, diabetic patients’ medication should be monitored as some drugs can increase the expression of viral entry receptors leading to a poor prognosis of COVID-19 in these patients. Further, insulin resistance has also been reported in comorbid, especially, T2DM patients. Hence, management of diabetes is of utmost importance in infected patients. Drugs that do not interact with ACE2 receptors could be given, though insulin treatment is unanimously given for COVID-19 patients with comorbid status of diabetes. CVD is a broad classification for a range of conditions that involve dysregulation of the heart and vascular systems. Since COVID-19 infection is a multiplexed pathophysiological condition, it is obvious that the cardiovascular systems are at the crux of exposure. Toward this, in a multicenter study conducted in Wuhan involving 191 patients, 24% of the patients that died had coronary heart disease. In addition, in a systemic review of 199 patients with COVID-19, 40% of them were diagnosed with myocarditis. Further, a meta-analysis study reported that 25% of the patients developed acute cardiac injury and the mortality rate was 20 times higher than those pre-existing comorbidities in comparison to those with no pre-existing comorbidities. As discussed in the previous section, systemic inflammation due to viral infection is prevalent and is a consequence of the cytokine storm. This inflammation is of particular concern for cardiac tissues and has been shown to cause myocarditis. The mechanism proposed for this pathology demonstrates the involvement of hepatocyte growth factor release, followed by priming of the immune cells such as T cells and subsequent release of IL-6 As a result, COVID-19 patients have been found to have pericardial effusion, which can lead to inflammation of the membranes around the heart and pericarditis. Conversely, the safety of mRNA vaccines is of particular concern as they have been reported to cause myocarditis. In the earlier section we discussed PD, its association with COVID-19 and the importance of olfactory nerves and the brain. Although nerves express ACE2 receptors, which are of utmost importance for infectivity, one cannot rule out the importance of the possible involvement of the heart–brain axis. This bidirectional communication is important as it is mediated by the control of endothelial cells and the regulation of cerebral blood flow by somatosensory signaling mechanisms. Thus, infection with COVID-19 disrupts this normal physiological function and, as a consequence, vascular–neuronal communication is disturbed thereby causing headaches and increased incidences of stroke in infected patients. Along with this, serious implications such as the neural spread of viruses and consequent, central disturbances such as anxiety are predisposed due to neuroinflammation and lower brain-derived neurotropic factor levels. Elevated levels of inflammatory biomarkers, CRP and D-dimer are the most common markers for COVID-19-associated coagulopathy. Particularly, elevated levels of D-dimer in hypertensive patients indicate the severity of the disease since COVID-19 patients’ death rates are more likely to rise with D-dimers upon admission or throughout time. Alternatively, in patients with diabetic complications, depletion of the ACE2 receptor causes activation of the renin-angiotensin-aldosterone system (RAAS), leading to β-cell destruction and an increased risk of cardiomyopathy in an age-dependent manner. However, these concepts are still premature and need a discussion on hypertension, which has been studied to a great extent with COVID-19 and is a predisposing factor for other cardiovascular complications as well. HTN is the most common disorder in people aged 50 years or more. Epidemiological studies have reported a high risk of hospitalization for patients with CVD such as HTN after COVID-19 infection. One study has shown hypertension (30%) and coronary heart disease (20%) as the most prevalent comorbidities (8%) in COVID-19-infected patients. Another investigation has shown that HTN (27%) and cardiovascular complications were the most prevalent comorbidities among COVID-19 patients with acute respiratory distress syndrome (6%). The high prevalence of hypertension in COVID-19 patients is not surprising, nor does it necessarily imply a causal relationship between hypertension and COVID-19 or its severity. Further, hypertension is extremely common in the elderly and older people appear to be at a higher risk of contracting SARS-CoV-2 and developing severe forms and complications of COVID-19. Hypertensive patients are frequently treated with ACEi or angiotensin receptor blockers (ARBs) to lower the volume of blood and ultimately blood pressure. However, the application of ACEi or ARBs in hypertensive patients is questionable as ACEi or ARBs are reported to increase the levels of ACE2 in hypertensive patients, and importantly, SARS-CoV-2, is reported to enter inside the lung cells through ACE2 receptors. Therefore, it is crucial to understand how RAAS reacts to COVID-19 infection and whether it is feasible to use ACEi or ARBs. ACE2 is a monocarboxypeptidase that is homologous to ACE and has an an extracellular active site. Angiotensin I (Ang I) is cleaved by ACE to produce Ang II, which constricts blood vessels and increases salt and fluid retention by binding to and activating the angiotensin receptor 1 (AT1 receptor), causing HTN. However, the membrane-bound ACE2 inactivates Ang II to Angiotensin I–VII (Ang I–VII) which then binds to the Mas1 oncogene (Mas receptor), which has further shown to possess a vasodilator effect. Furthermore, ACE2 converts Ang II into Angiotensin I–IX (Ang I–IX) which is then transformed into Ang I–VII by ACE. In addition, ACE2 converts Ang I with less binding affinity as compared to Ang II. ACE2 acts as a negative regulator of the RAAS, modulating vasoconstriction, fibrosis, and hypertrophy. Hypertensive individuals have lower gene expression and/or ACE2 activity than normotensive patients. Ang II, on the other hand, inhibits ACE2. It has been reported in preclinical studies that ACE2 deficiency induces HTN in rats when Ang II exceeds. In several studies, SARS-CoV infection lowered ACE2 expression in cells, causing severe organ damage by disturbing the physiological homeostasis between ACE/ACE2 and Ang II/Ang I–VII. The ACE2 transmembrane domain is internalized along with the virus during SARS-CoV-2 infection, which reduces ACE2 expression. For some transmembrane proteinases and proteins, disintegrin is one of the proteins that may be involved in the binding and membrane fusion processes and ADAM metallopeptidases domain 17 (ADAM17), TMPRSS2, and TNF-converting enzyme. TMPRSS2 cleaves ACE2 to increase viral uptake, and ADAM17 can cleave ACE2 to produce ectodomain shedding. Importantly, lower levels of ACE2 in COVID-19-infected hypertensive patients resulted in a lower degradation rate of Ang II, overexpression of the AT1 receptor with reduced activity of Ang I–IX and I–VII and promotion of hypertension, ARDS, hypertrophy, and myocardial injury. The ACE2/Ang I–VII/Mas axis has been shown to play a beneficial role in the heart. It can enhance post-ischemic heart functions by inducing coronary vessel vasorelaxation, inhibiting oxidative stress, attenuating abnormal cardiac remodeling, and inhibiting oxidative stress. ACE2 expression rises early in the course of a heart attack but declines as the disease progresses. ACE2 knockout mice develop myocardial hypertrophy and interstitial fibrosis, which accelerates heart failure. Furthermore, ACE2 deletion in mice exacerbates diabetes-related heart failure. ACE2 expression in cardiac cells has been shown to be significantly reduced in both SARS-CoV-infected humans and mice. Due to the significant downregulation of ACE2 and overexpression of Ang II in COVID-19 infection, the lack of protective actions of Ang I–VII may exacerbate and perpetuate cardiac damage. According to current research and several clinical studies, HTN is a comorbidity in a significant proportion of individuals with severe illness. RAAS overactivation may have already occurred in these people before infection. The absence of the protective effects of Ang I–VII may accelerate and perpetuate cardiac damage due to considerable downregulation of ACE2 and overexpression of Ang II in COVID-19 infection. ACE2/Ang I–VII/Mas receptor axis counteracts excessively activated ACE/Ang-II/AT1 receptor axis as seen in HTN (Figure 5). Notably, soluble ACE2 molecules have been demonstrated to limit SARS-CoV infection and suggested that a soluble recombinant form of ACE2 molecules can act as competitive interceptor of SARS-CoV-2 virus and prevent it from latching onto cellular membrane-bound ACE2. In ARDS, recombinant human soluble ACE2 was planned to be tested clinically for its efficacy against COVID-19 infection so that a larger phase IIB trial can be performed which may potentially benefit the hypertensive COVID-19-infected patients (NCT04287686). Cardiac damage induced by SARS-CoV and SARS-CoV-2 is a major cause of mortality and morbidity, affecting up to a third of those who have the disease in its most severe form. SARS-CoV was found in one-third of human autopsy hearts, accompanied by a substantial drop in cellular ACE2. Also, the involvement of ACE2 has been studied in critically ill patients, wherein continued treatment with ACE2i (ACE2 inhibitor) was deemed to demonstrate less load on the heart as the alveolar spaces are critically dismantled in these patients. Even though the powerful immune response seen in these people might affect cardiac dysfunction similar to the lungs, Ang II is expected to contribute to the negative effects of SARS-CoV on the heart and SARS-associated cardiomyopathy. Inflammatory signals are thought to lower ACE2 cell-surface expression and transcription. Some contrasting studies report that ACE2 may also be involved in vasodilation, anti-inflammatory and antioxidant roles due to the generation of fragment Ang (I–VII) from Ang II. However, this is scantly reported and as a result, the classical hypothesis of disease worsening is most prevalent. In conclusion, a decrease in cellular ACE2 may render the cells less sensitive to SARS-CoV-2, whereas overexpression of the AT1 receptor causes more severe tissue damage. On the other hand, as AT1 receptor activity is decreased, the cell membrane becomes more sensitive to viral particles with increase in ACE2 levels. Both ACEi and ARB have been demonstrated to upregulate ACE2, and some researchers have speculated that ARB and ACEi treatments may have a deleterious effect on SARS-CoV-2 infection. Given how commonly these compounds are used to treat HTN and heart failure, this might be a major source of concern. In animal studies, ACEi treatment increased plasma Ang I–VII levels, decreased plasma Ang II levels, and increased ACE2 expression in the heart. In contrast, Ang II receptor blockers (ARBs) increase plasma levels of both Ang II and Ang I–VII, as well as ACE2 expression and activity in the heart. ACEi/ARBs, renin inhibitors, and Ang I–VII analogs may minimize organ damage by blocking the RAAS pathway and/or increasing Ang I–VII levels. In population-based research, the use of ACEi and ARBs significantly lowered the 30-day mortality in pneumonia patients requiring hospitalization. Treatment with ACEi/ARBs has also prompted concerns that increasing the expression of ACE2 in target organs might facilitate the infection-induced development in severe COVID-19 infection. According to two large cohort studies, administration of ACEi/ARBs were connected to hospitalized patients’ having a lower risk of all-cause mortality rather than an increased chance of SARS-CoV-2 infection. However, further research is warranted to examine the protective effects of ACEi/ARBs in COVID-19. Despite the various probable confounders, a decrease in membrane ACE2 expression might explain many of the anomalies seen in SARS-CoV-2 infection. There is not enough clinical evidence to suggest that there is a higher chance of acquiring a severe COVID-19 infection; further, it is unclear if continuation or discontinuation of ARB/ACEi is a wise decision. In addition, we do not know if switching to another treatment approach could worsen the patient’s situation, notably in individuals with heart failure and a poor ejection fraction. Further, whether RAAS inhibitor medication is useful or detrimental for virally induced lesions and switching to another medicine could make the patient’s situation even worse. Clinical trials to detect the effect of losartan as a potential treatment approach for COVID-19, are currently starting (NCT04311177 and NCT04312009). Further, trial to detect if stopping or continuing ACEi/ARB treatment has any consequences are underway (NCT04338009). ACE inhibitors and angiotensin-converting enzyme inhibitors (ARBs) are not only used to treat HTN and heart failure, but they also have a minor impact on ACE2. Although β-blockers are unlikely to interact with ACE or ACE2, they do lower plasma Ang II levels by preventing the conversion of pro-renin to renin. Calcium channel blockers appear to reduce Ang II-induced ACE2 downregulation. However, the data is limited to a single research article that studies nifedipine’s effect on fractionated cell extracts. Thiazides and mineralocorticoid receptor antagonists did not improve the hypertensive rats’ naturally low ACE2 activity, although mineralocorticoid receptor antagonists did reduce ACE expression. In heart failure patients, mineralocorticoid receptor antagonists, on the other hand, increase membrane ACE2 activity. Newer therapies such as DNA aptamers, short oligonucleotide sequences that bind to specific proteins, are being investigated for masking the ACE2 binding domain. In this regard, a group reported the synthesis of novel DNA aptamers that were shown to specifically bind to the ACE2-K353 domain and blocked the entry of the virus through ACE2 receptors. It is still controversial that non-ACEi/BRA drugs (β-blockers, calcium channel blockers, diuretics) are more likely to increase the risk of adverse outcomes than ACEi/BRA drugs that increase ACE2 and provide theoretical protection if the reduction in membranous ACE2 seen in HTN and obesity is important in the pathophysiology of severe COVID-19. It has been confirmed that SARS-CoV-2 enters the lung through the ACE2 receptor followed by other tissues like the liver, bile duct, gastrointestinal system (small intestine, duodenum), esophagus, and kidney. SARS-CoV-2 can damage these organs associated with the heart and transmit it from human to human rapidly, resulting in serious illness and life-threatening diseases such as heart attack or cancer. Although higher levels of ACE2 enzyme may be expressed in hypertensive patients, this can be a marker for the severity of COVID-19. Effective treatment and prevention of coronavirus infection should begin immediately. However, developing vaccines or drugs for humans in a shorter period is difficult. Nevertheless, only Covaxin and Covishield are currently available in India. In the current scenario, if any person is infected with SARS-CoV-2, then he/she should be isolated and should be controlling the origin of the infection. Scientists are trying to develop vaccines or repurposing drugs with the help of different in vivo or in vitro studies resulting in some positive evidence for the treatment of COVID-19, but these pieces of evidence are not sufficient to cure the infection. As COVID-19 treatment options are evaluated, it will be important to understand the possible side effects of CVD, with a focus on drug–drug interactions. Further, in order to learn more about how COVID-19 affects the heart, we need comprehensive molecular tests that may look at how things work and prospective and retrospective studies with good clinical methodology. The COVID-19 pandemic is still globally stressful. SARS-CoV-2 has infected the entire globe and has caused pneumonia-like symptoms in severely infected populations. Conversely, some new variants have been reported which have less serious effects with some patients being asymptomatic post-infection. However, the population afflicted with comorbid conditions is found to be more vulnerable to death or ICU admissions comparable to people without comorbid conditions. Age is found to be the highest risk factor in COVID-19 infection, as aged people show enhanced sensitivity to immune responses. Similarly, SARS-CoV-2 causes the cytokine storm, which makes aged people more sensitive to severe infection. There was a significant increase in mortality observed with COVID-19-infected people having age-related disease conditions such as PD, cancer, diabetes, and CVD. COVID-19 infection in comorbid patients causes mitochondrial and ER malfunction that induces α-Syn aggregation, activates TLRs, and ultimately causes nuclear translocation of NFκB through JAK/STAT3 pathway in cancer patients. SARS-CoV-2 infection in diabetic individuals leads to depletion of beta cells of islets of Langerhans creating imbalance in glucose levels. Hypertensive patients infected with COVID-19 demonstrate enhanced Ang-II levels which are responsible for vasoconstriction ultimately worsening hypertensive condition. Therefore, the population infected with SARS-CoV-2 and with comorbid conditions needs to be monitored carefully at the onset of the infection, as the symptoms may worsen with time which may lead to severe life-threatening conditions. Toward this, more clinical studies are warranted for an improved mechanistic understanding of the susceptibility of comorbid patients to COVID-19 infection. Further, the comorbid population should be vaccinated on a priority basis as compared to people without comorbid conditions. Though various studies indicate the importance of comorbid disorders as the possible determinants for COVID-19 infected individuals, a comprehensive evaluation of an clarification regarding the vulnerability in COVID-19 patients is required. This article has limits on clinical-epidemiological conclusions and lacks critical and statistical analysis of COVID-19 and comorbidities and the reader should be aware of the aforementioned restrictions in our review.

PMC10000025

Qi Min,Lu Yang,Yu Wang,Yili Liu,Mingfeng Jiang

Transcriptome-Based Evaluation of Optimal Reference Genes for Quantitative Real-Time PCR in Yak Stomach throughout the Growth Cycle

03-03-2023

yak,stomach,transcriptome-wide,reference gene,RT-qPCR

Simple Summary The stomach is one of the primary sites for the digestion and absorption of nutrients. Quantifying related gene expression patterns using quantitative real-time PCR (RT-qPCR) is conducive to further understanding the molecular mechanisms underlying nutrition metabolism in the yak stomach. The authenticity of RT-qPCR data is affected by the selection of reference genes. Unfortunately, no studies have demonstrated suitable reference genes for the normalization of RT-qPCR data in the yak stomach. In this study, 15 candidate reference genes (CRGs) were identified according to transcriptome sequencing (RNA-seq) results and the previous literature. Five algorithms were used to evaluate the stability of the CRGs across the entire developmental stage in the yak stomach. RPS15, MRPL39, and RPS23 were found to be the most stable genes in the yak stomach from birth to adulthood. This study indicates the appropriate reference genes for gene expression analysis via RT-qPCR in the yak stomach. Abstract Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that RPS15, MRPL39 and RPS23 are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of HMGCS2 were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining RPS15, MRPL39 and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle.

Transcriptome-Based Evaluation of Optimal Reference Genes for Quantitative Real-Time PCR in Yak Stomach throughout the Growth Cycle The stomach is one of the primary sites for the digestion and absorption of nutrients. Quantifying related gene expression patterns using quantitative real-time PCR (RT-qPCR) is conducive to further understanding the molecular mechanisms underlying nutrition metabolism in the yak stomach. The authenticity of RT-qPCR data is affected by the selection of reference genes. Unfortunately, no studies have demonstrated suitable reference genes for the normalization of RT-qPCR data in the yak stomach. In this study, 15 candidate reference genes (CRGs) were identified according to transcriptome sequencing (RNA-seq) results and the previous literature. Five algorithms were used to evaluate the stability of the CRGs across the entire developmental stage in the yak stomach. RPS15, MRPL39, and RPS23 were found to be the most stable genes in the yak stomach from birth to adulthood. This study indicates the appropriate reference genes for gene expression analysis via RT-qPCR in the yak stomach. Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that RPS15, MRPL39 and RPS23 are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of HMGCS2 were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining RPS15, MRPL39 and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle. The yak (Bos grunniens), a precious domesticated ruminant, also known as the “boat on the plateau”, is mostly found on the Qinghai-Tibetan Plateau and nearby areas at an altitude above 3000 m. As the most significant livestock in this region, yaks are capable of surviving and providing milk, meat, hair and cheese for local herders in a hostile environment [1]. A previous study found that efficient nutritional assimilation and energy metabolism in the yak stomach contributes to their adaption to a harsh environment [2]. In ruminants, the stomach and small intestine are mostly where nutrients are digested and absorbed [3]. Additionally, the development of the yak stomach at different stages plays a vital role in digestive ability and nutrient supply [4]. Thus, accurate analysis of gene expression profiles in the yak stomach are of major priority to further reveal the molecular mechanisms of nutrient and energy metabolism. As a typical ruminant, a remarkable feature of the yak is that it has a complex stomach consisting of four gastric compartments: rumen, reticulum, omasum and abomasum [5]. The first three compartments of the compound stomach (i.e., rumen, reticulum and omasum) are commonly referred to as the “forestomach” and perform cooperative functions [6]. They serve as fermentative chambers where bacteria break down the ingested cellulose, producing enormous amounts of gas [7]. By contrast, only the abomasum can generate digestive juices and gastric enzymes [7]. Hence, the abomasum is also called the true stomach. In newborn ruminants, dietary requirements are fulfilled by the uptake of colostrum, which is digested in the abomasum to provide energy and essential nutrients, as well as immunity molecules [8]. In comparison, the rumen acts as the primary location of digestion and absorption in grown ruminants, and microorganisms decompose ingested feed in the rumen to produce volatile fatty acids (VFA) that serve as the main source of energy [9]. Understanding which genes in the stomach are crucial for nutrient absorption and digestion and how they might be regulated to contribute to growth and maintenance is a major concern in the field of yak research. RT-qPCR is extensively used for the analysis of gene expression patterns due to its sensitivity, accuracy and specificity, as well as practical simplicity [10,11]. However, several drawbacks such as nucleic acid quality, poor choice of primers or probes and inappropriate data and statistical analyses encumber the authenticity of RT-qPCR results [12]. Therefore, various strategies have been applied to normalize RT-qPCR results. The use of reference genes that are not affected by study conditions is a generally accepted strategy for normalizing RT-qPCR data [13]. Despite the fact that several genes such as ACTB and GAPDH are commonly employed as reference genes in a wide range of studies, it is unlikely that any genes have enough overall expression stability to be appropriate for any kind of experiment [14]. Therefore, it is essential to select reliable reference genes for the specific experimental context under study. To date, no studies have shown suitable reference genes for the normalization of RT-qPCR data in the yak stomach. Many studies have concentrated on verifying subsets of frequently used reference genes for specific experimental contexts [14]. However, it is biased to select the CRGs from a minority of genes and assume that at least a few of those genes are appropriate for the certain experimental context. The emergence of high-throughput RNA-seq technology provides a novel strategy for identifying reference genes [15]. Based on the RNA-seq dataset, CRGs with stable expression and high abundance were preliminarily selected. Subsequently, the expression levels of CRGs were quantified using RT-qPCR and their stabilities were evaluated using geNorm [16], NormFinder [17], BestKeeper [18] and the comparative CT method [19]. This strategy has been successful for identifying reference genes for fish [20], Holstein cows [21], goats [22], and so on. The purpose of this study was to select and validate reliable reference genes from across the transcriptome that can serve as internal controls for longitudinal gene expression studies in the yak stomach throughout the growth cycle. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Southwest Minzu University (permit number: 2020-07-02-11). All Maiwa yaks were raised in Hongyuan County of Sichuan Province and fed with natural lactation and pasture. A total of 15 Maiwa yaks (7 males and 8 females) were selected from the same herd at 5 different growth stages: 0 days (lactating stage), 20 days (lactating stage and starting to graze), 60 days (lactating stage and graze stage), 15 months (graze stage but still lactating) and 3 years old (natural graze stage). For sample collection, three separate yaks of each age were slaughtered. The stomach tissues of the yaks, including rumen, reticulum, omasum and abomasum, were rinsed immediately in 0.1% DEPC water after slaughter and frozen in liquid nitrogen until processing for total RNA extraction. The total RNA of the rumen, reticulum, omasum and abomasum tissues were extracted using the mirVana miRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The purity and concentration of total RNA were confirmed using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of total RNA was assessed using 1% agarose gel electrophoresis. The cDNA was generated from 1000 ng total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) in a reaction mixture of 20 µL. The cDNA was stored at −80 °C until required. Based on our previous RNA-seq results of the compound stomach at five stages in fifteen yaks (unpublished data), 7 CRGs, ribosomal protein S15 (RPS15), ribosomal protein S23 (RPS23), 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), ribosomal protein L13a (RPL13A), β-actin (ACTB), ribosomal protein S9 (RPS9) and glyceraldehyde-3-phos-phate dehydrogenase (GAPDH), were selected according to the fragments per kilobase of exon model per million mapped reads (FPKM) and the coefficient of variation (CV, %). The value of FPKM was higher than 100 and the CV value was less than 20%. FPKM = cDNA fragments/[mapped fragments (millions) × transcript length (kb)] and CV = standard deviation (SD) FPKM/MeanFPKM × 100%. Subsequently, based on the previous literature, eight genes were selected as CRGs: ubiquitously expressed prefoldin-like chaperone (UXT), dystrobrevin binding protein (DBNDD2), DEAD box polypeptide 54 (DDX54), hydroxymethylbilane synthase (HMBS), protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11), mitochondrial ribosomal protein S15 (MRPS15), mitochondrial ribosomal protein L39 (MRPL39) and TATA box binding protein (TBP). Primers for RT-qPCR were designed using Primer-BlAST with a length of 20 ± 3 bases and amplicon sizes ranging from 100 to 150 bp. The sequences of the CRGs were obtained from NCBI (https://www.ncbi.nlm.nih.gov/ accessed on 25 June 2022). The primer specificity of each CRG was verified using 2% agarose gel electrophoresis and melting curve analysis. To validate the specificity of each primer pair, the products of PCR were purified and sequenced using a 3730 DNA analyzer (ABI, Carlsbad, CA, USA), and the sequencing results were compared with all potential transcript sequences in NCBI using BLAST. All RT-qPCR assays were carried out in triplicate for each sample using the LightCycler 96 System (Roche Diagnostics, Indianapolis, IN, USA). The total volume of each reaction mixture was 20 µL, including 10 µL of TB Green Premix Ex Taq II (TaKaRa, Dalian, China), 2 µL of diluted cDNA, 0.5 µL of each of 10 μM forward and reverse primers and 7 µL of RNase Free dH2O. The PCR program was conducted as follows: 95 °C for 30 s (pre-denaturation), 40 cycles of 95 °C for 5 s and 60 °C for 30 s (quantitative analysis), 95 °C for 5 s and 60 °C for 1 min (melting curves analysis). To determine the correlation coefficient (R2) and amplification efficiency (E) for each primer pair, a five-point standard curve was generated using a five-fold dilution of cDNA. The correlation coefficient (R2) and amplification efficiency (E) of each primer pair were calculated using the LightCycler 96 System. A modified Pfaffl equation was used to determine the relative quantity (RQ) of each gene [23]: Cq (calibrator) = Cq for the arithmetic mean of all samples at 5 stages, Cq (sample) = Cq for the sample. The formula for calculating the relative expression level of a target gene is as follows: RQGOI: the RQ value of the target gene, Geomean[RQREFS]: the geometric mean of the RQ value of selected reference genes. The normalization factor (NF) was calculated using the geometric mean of the RQ value of the selected reference genes [23]. The expression stability of 15 CRGs was evaluated using 4 algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. In addition, RefFinder (http://blooge.cn/RefFinder/ accessed on 10 October 2022) was used to synthesize the evaluation results of the above four algorithms to give an overall ranking. HMGCS2 is the key rate-limiting enzyme in the ketogenic pathway and plays an important role in the digestion and absorption of nutrients in the stomach. The expression levels of HMGCS2 were quantified using RT-qPCR to validate the selected reference genes. The expression levels of HMGCS2 in the stomach at 5 stages were normalized using the three most stable gene combinations and the three most unstable gene combinations identified from this study. The relative mRNA expression of HMGCS2 was calculated using the 2−∆∆Ct method. In addition, statistical significance was analyzed using one-way analysis of variance via SPSS 25.0 software (IBM, Armonk, NY, USA). A p value below 0.05 was regarded as statistically significant. The 260/280 ratio of total RNA for each sample ranged from 1.8 to 2.2, and the purity and concentration were qualified for subsequent experiments (Table S1). The RNA of all samples clearly displayed two prospective bands at 18 s and 28 s without any signs that the products were degraded (Figure S1). The above results indicate that the RNAs of all samples were equipped for cDNA synthesis. The criteria for preliminary selection of reference genes were relatively high transcriptome abundance and low expression variation [15]. As a result, preliminary selection comprised genes with relatively high transcriptome abundance (FPKM > 100) as identified by the mean FPKM value and low variability as identified by the coefficient of variation (CV < 20%). A total of 80 CRGs were preliminarily selected using our previous RNA-seq results of the stomach at five stages in fifteen yaks (Table S2). Furthermore, 7 genes (RPS15, RPS23, YWHAZ, RPL13A, ACTB, GAPDH, and RPS9) were considered as CRGs due to their lower CV values, higher FPKM values, and easier primer designs. In addition, eight CRGs were selected based on previous studies. Among these CRGs: UXT, HMBS, MRPS15, PPP1R11, MRPL39 and TBP were validated to be appropriate reference genes for RT-qPCR in yak [13,24,25,26]. Additionally, DBNDD2 and DDX54 were verified as suitable reference genes for RT-qPCR in the rumen epithelium of cows [27]. In conclusion, 15 genes were chosen as CRGs for further evaluation. The details of primer pairs of 15 CRGs are displayed in Table 1. Primer pairs of amplification efficiency (%) ranged from 91 to 109%, the amplicon’s size lay in 100–286 bp and the R2 of each primer pair was not less than 0.99. The specificity of primer pairs for each gene was verified via 2% agarose gel electrophoresis (Figure S2) and melting curve analysis (Figure S3). To further validate the specificity of each primer pair, the products of PCR were purified and sequenced. Then, the sequencing results were compared with all potential transcript sequences in NCBI using BLAST (Table S3). The mean Cq values of all tested samples calculated to determine the expression levels of the 15 CRGs are illustrated in Figure 1. Cq value had a negative correlation with gene expression level. In other words, higher gene expression levels are associated with lower Cq values and vice versa. The Cq values of all CRGs ranged from 18.49 to 32.67. For each CRG, the mean and median Cq values were relatively close. Among all the CRGs, RPS23 demonstrated the highest expression level, with Cq = 20.18 ± 0.76, while PPP1R11 had the lowest expression level, with Cq = 30.59 ± 0.89. In this study, four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method were used to evaluate CRGs for stability ranking. The stability rankings obtained from the four algorithms were different. Thus, RefFinder was employed to obtain a total score that was used to rank the stability of the 15 CRGs (Table 2). The M-value was calculated via geNorm analysis to identify gene expression stability. Then, the M-value was used to rank the stability of expression for the 15 CRGs, and the M-value was negatively correlated with the stability of gene expression. According to the geNorm method, the results show that RPS15 and DBNDD2 were the most stable CRGs with the lowest M-value of 0.48, while RPL13A was the least stable gene with the highest M-value of 0.74 in the yak stomach throughout the growth cycle. The NormFinder algorithm was used to calculate the stability value (SV) to identify the ranking of the CRGs, with the most stable gene showing the lowest SV. For the yak stomach throughout the growth cycle, the most stable gene was RPS15 with the lowest SV of 0.35, and RPL13A was the most unstable gene with the highest SV of 0.62. The BestKeeper and the comparative CT method regard standard deviation (SD) as one of the criteria to evaluate the stability of gene expression.. The lower the SD value, the more stable the gene expression. Based on BestKeeper analysis, RPS15 was the most stable gene, whereas YWHAZ was the least stable gene with the highest SD value. By contrast, according to the comparative CT method, MRPL39 had the highest stability, and the YWHAZ was the most unstable gene. Based on the results obtained using these four algorithms, RefFinder was used for comprehensive ranking. As a result, the comprehensive rankings according to stability from the highest to the lowest are RPS15 > MRPL39 > RPS23 > DDX54 > DBNDD2 > GAPDH > TBP > RPL13A > MRPS15 > PPP1R11 > ACTB > HMBS > RPS9 > UXT > YWHAZ. The pairwise variation values (V) were calculated using geNorm software, which is a valid tool to identify the optimal number of reference genes for RT-qPCR. Vandesompele et al. [16] proposed taking 0.15 as a cut-off value below which the inclusion of additional reference genes is not necessary. Thus, according to the cut-off value (V = 0.15), the results indicate that the combination of three genes was the optimal number for normalization of RT-qPCR data in the yak stomach throughout the growth cycle (Figure 2A). Furthermore, low pairwise variation values correspond to a high correlation coefficient [16]. Clearly, there is no need to include an additional gene when using the three most stable reference genes for calculating the NF (Figure 2C). In contrast, it is essential to have more than an additional gene when using the two most stable reference genes for calculation of NF (Figure 2B). Thus, we recommend the combination of the three most stable genes (RPS15, MRPL39, and RPS23) to normalize RT-qPCR data in the yak stomach throughout the growth cycle. To verify the effect of the combination of RPS15, MRPL39 and RPS23 for the normalization of RT-qPCR data, the expression of HMGCS2 was quantified via RT-qPCR in yak stomach at 5 stages (0 d, 20 d, 60 d, 15 m and adult). Moreover, the expression patterns of HMGCS2 in yak stomach at 5 stages were also identified using the FPKM of RNA-seq results. The results show a correspondence between the RT-qPCR and RNA-seq, indicating the RT-qPCR data of HMGCS2 using the RPS15, MRPL39 and RPS23 for normalization were reliable (Figure 3). To further validate the selection of CRGs, the three most stable CRGs (RPS15, MRPL39, and RPS23) and the three least stable CRGs (RPS9, UXT and YWHAZ) were used to normalize the expression of HMGCS2. As shown in Figure 4A,B, the expression patterns of HMGCS2 in the rumen, reticulum, omasum and abomasum at five stages (0 d, 20 d, 60 d, 15 m and adult) were similarly obtained using FPKM based on RNA-seq results and the combination of three most stable CRGs (RPS15, MRPL39, and RPS23) for normalization. Furthermore, the expression of HMGCS2 in the rumen, reticulum and omasum were the lowest at 0 d and the highest at adulthood, while the opposite was true in the abomasum. However, compared with the expression of HMGCS2 based on RNA-seq results (Figure 4A), normalization of HMGCS2 expression using the three least stable CRGs (RPS9, UXT and YWHAZ) demonstrated significant differences (Figure 4C). Hence, it is essential to select suitable reference gene combinations to normalize the expression of target genes. Gene expression analysis via RT-qPCR is a dependable and extensively used method to reveal the molecular mechanism of digestion and absorption of nutrients in the stomach. The use of reference genes is the most credible strategy for taking into account the initial concentration of RNA, sample loss during experimentation, the efficiency of cDNA synthesis, and so on [28]. However, the selection of inappropriate reference genes also affects the authenticity of RT-qPCR data [29]. Therefore, selecting suitable reference genes is essential to obtain meaningful RT-qPCR results. Until now, strategies for identifying reference genes from the transcriptome have been widely used. Reference genes selected from the transcriptome increase the reproducibility and sensitivity of results, give a stronger correlation between protein expression levels, and have better detection and coverage [30]. Although RNA-seq screening has many merits in predicting reference genes, this strategy is not absolutely trustworthy and needs further validation via RT-qPCR [15,31]. In this study, 15 CRGs were determined via RNA-seq and the previous literature, and further verified using RT-qPCR. In this study, geNorm, NormFinder, BestKeeper and the comparative CT method were employed to assess the stability of 15 CRGs. Ribosomal protein S15 (RPS15) is a component of the 40S ribosomal subunit and functions as a nuclear export factor [32]. Although different algorithms were used for the stability ranking of 15CRGs, RPS15 had the best stability in all the algorithms except the comparative CT method (geNorm, NormFinder and BestKeeper) (Table 2). Furthermore, RPS15 was also the most stable gene in the comprehensive ranking of the results of the four algorithms using RefFinder. This is consistent with Bionaz et al. [28] finding that RPS15 is one of the best reference genes used for the normalization of gene expression data in the bovine mammary gland during the lactation cycle. Tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), belonging to the 14-3-3 protein family, participates in various cell activities including cell growth, cell cycle, apoptosis, and so on [33,34]. Some studies have demonstrated the consideration of YWHAZ as an appropriate reference gene due to its high stability in cattle [35], buffaloes [36] and yak [25]. By comparison, it seems that YWHAZ was the least stable gene in our study (Table 2). Even so, the M value of YWHAZ (0.73) derived from the geNorm analysis is well below the threshold (M = 1.5) proposed by Vandesompele et al. [16], suggesting that it is also a relatively stable gene in the yak stomach. These results indicate that the stability of reference genes is highly specific and should be evaluated for a given experimental context. Although RPS15 had the highest stability in our evaluation, we still do not recommend using it alone as the reference gene for the normalization of RT-qPCR data in the yak stomach. Many studies show that using a single gene as the reference gene should be avoided [16,18,28]. It has been reported that using a single reference gene results in significant bias [37]. To date, no specific theory prescribes a certain number of reference genes to be used. Use of geNorm can provide the optimal number of reference genes needed to eliminate the majority of technical variation [11]. Accuracy and practicality are trade-offs when determining the optimal number of reference genes. It is an unnecessary waste of resources to use more reference genes if the inclusion of additional genes has no significant effect on NF [16]. In our study, there was no significant change between the NF calculated with the three most stable CRGs and that calculated with the four most stable CRGs, indicating that it was superfluous to add a gene for normalization (Figure 2C). In addition, the digestive tract of the yak has three developmental stages: pre-rumination (0–20 days), transition from pre-rumination to rumination (20–60 days), and rumination (after 60 days). The diets of yaks are different at different developmental stages. Therefore, we evaluated the stability of these genes in yak stomach tissue over five developmental stages. Our results support the use of these reference genes in the normalization of RT-qPCR data under different dietary conditions. As a result, we recommend using the combination of three most stable genes (RPS15, MRPL39, and RPS23) to calculate the NF for normalization of RT-qPCR data in the yak stomach throughout the growth cycle. The expression profiles of HMGCS2 were quantified via RT-qPCR in the yak stomach at five developmental stages, and its expression levels were normalized by the selected combination of reference genes. HMGCS2 is the key rate-limiting enzyme in the ketogenic pathway and induces the biosynthesis of HMG-CoA, which is the central metabolite of rumen epithelial cells [38,39]. The ketogenic capacity of ruminal epithelium in ruminants increases with age, and newborn ruminants have no ketogenic capacity [40]. Thus, we hypothesized that the expression level of HMGCS2 in the rumen should increase in terms of age, as well as those in the reticulum and omasum because they serve analogous functions to the rumen. In this paper, the expression level of HMGCS2 did increase with age, as determined using RNA-seq and RT-qPCR in the rumen, reticulum and omasum (Figure 3A–C). For newborn ruminants, the rumen was not fully developed and ingested colostrum is instead digested in the abomasum [8]. Consequently, the expression level of HMGCS2 in the abomasum should be highest at birth and lowest in adulthood (Figure 3D). In addition, although target genes with significant expression changes can be identified using less stable reference genes, target genes with imperceptible expression changes can only be detected using the best reference genes [20,37]. Our results confirm that significant changes in the expression of HMGCS2 between birth and adulthood could be identified using either the three most stable CRGs (RPS15, MRPL39, and RPS23) or the three least stable CRGs (RPS9, UXT and YWHAZ). However, when the change in HMGCS2 expression is slight, errors may occur when using RPS9, UXT and YWHAZ for normalization. For example, in the rumen, there were no significant differences in HMGCS2 expression levels between 60 days and 15 months either based on the results of RNA-seq or using RPS15, MRPL39, and RPS23 for normalization, whereas its expression levels normalized using RPS9, UXT and YWHAZ had significant differences (p < 0.05) between 60 days and 15 months (Figure 4). These results imply that using suitable reference genes is essential for accurate normalization of target gene expression. In this study, 15 CRGs were selected using transcriptome sequencing results and the previous literature, and their expression stability was evaluated using five algorithms. Therefore, we recommend the combination of the three most stable genes RPS15, MRPL39, and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle.

PMC10000032

Maria Brockmann,Christoph Leineweber,Tom Hellebuyck,An Martel,Frank Pasmans,Michaela Gentil,Elisabeth Müller,Rachel E. Marschang

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards

28-02-2023

Devriesea agamarum,polymerase chain reaction,Uromastyx sp.,Pogona vitticeps,bearded dragon,lizard,reptile,cheilitis,dermatitis

Simple Summary Bacterial infections can play an important role in dermatitis in lizards. The bacterial species Devriesea (D.) agamarum is a known cause of dermatitis, cheilitis and even fatal disease in lizards. Disease has most often been reported in Uromastyx species, but other lizards may also be affected. However, some are asymptomatic carriers, increasing the risk of spreading D. agamarum. Usually, D. agamarum is detected with culture-based methods. It was the aim of this study to establish a real-time PCR assay to expand diagnostic options in routine diagnostics. The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods. This enables a fast therapeutic approach for affected animals and decreases the risk of spread. Abstract (1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards Bacterial infections can play an important role in dermatitis in lizards. The bacterial species Devriesea (D.) agamarum is a known cause of dermatitis, cheilitis and even fatal disease in lizards. Disease has most often been reported in Uromastyx species, but other lizards may also be affected. However, some are asymptomatic carriers, increasing the risk of spreading D. agamarum. Usually, D. agamarum is detected with culture-based methods. It was the aim of this study to establish a real-time PCR assay to expand diagnostic options in routine diagnostics. The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods. This enables a fast therapeutic approach for affected animals and decreases the risk of spread. (1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods. Devriesea (D.) agamarum is a bacterial species known to cause dermatitis and cheilitis in lizards. Disease has most often been described in Uromastyx spp. [1,2,3,4,5]. However, D. agamarum can also infect other lizards [6,7]. It has been reported in captive [8,9] as well as in free-ranging lizards [7]. Clinical signs of disease generally include dermatitis or cheilitis, often described with a yellow crusty appearance [10]. Disease outbreaks with extensive mortality have also been reported, especially if lizards developed septicaemia. [7,11]. Bearded dragons have been described to asymptomatically carry D. agamarum in their oral cavities [2,3]. Treatment of affected animals usually includes debridement of dermal lesions and systemic use of antibiotics—especially cephalosporines are considered effective [4,12]—and may also require disinfection of the enclosure [13]. Autovaccines have also been discussed as a treatment method [14]. Therefore, a fast and reliable diagnostic approach is an important consideration, both in clinical disease with suspected D. agamarum infection and in entry controls. D. agamarum is relatively easily cultured at 37 °C but also grows at temperatures of 25−42 °C on Columbia agar with 5% sheep blood [11]. Diagnosis can, however, be complicated in laboratories with limited experience with this pathogen. Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS), one of the most frequently used standard techniques for the identification of bacteria in routine laboratory diagnostics, may not (yet) be able to identify D. agamarum when working with standardized databases [15]. However, this issue is likely to be overcome as databases expand. Currently, laboratories can improve D. agamarum identification by implementing and expanding their own MALDI-TOF MS databases or using 16S rRNA gene sequencing to identify cultured but unidentified isolates. Another option would be a specific PCR assay for D. agamarum, which might prove especially valuable if other infectious agents, such as viral or fungal pathogens, are also suspected, allowing concurrent testing from the same sample. The aim of this study was, therefore, to develop a PCR assay for the detection of D. agamarum. In total, 14 D. agamarum isolates were used in this study as positive controls. Three D. agamarum isolates (GenBank accession numbers: MT664091-93) were obtained from routine diagnostic submissions at Laboklin GmbH & Co. KG (Bad Kissingen Germany) in 2019 [15], while 11 were isolated between 2005 and 2009 at the Faculty of Veterinary Medicine, Ghent University (Table 1). Non-D. agamarum isolates (n = 34) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Some were included in order to determine the ability of the assay to exclude a broad spectrum of different bacterial species. Others, like Brachybacterium sp. or Dermabacter sp., were included as their sequences were described to be highly similar to D. agamarum [11] (Table 2). These 34 isolates served to determine the specificity of the PCR. Pure cultures of each strain were incubated in 750 μL lysis buffer (MagNA Pure DNA Tissue Lysis Buffer, Roche, Mannheim, Germany) and 75 μL proteinase K (proteinase K, lyophilisiert, ≥30 U/mg, Carl Roth GmbH & Co KG, Karlsruhe, Germany) for one hour at 65 °C. From this, 200 μL were utilized for automated nucleic acid (NA) extraction using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The resulting NAs were eluted in a volume of 100 μL. The isolated NAs were kept at −18 °C until the PCR tests were performed. The DNA used for the dilution series was extracted manually using the QIAamp® DNA MicroKit (50) (Qiagen, Hilden, Germany), and the DNA concentration was measured with a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Inc., Wilmington, NC, USA). The 16S rRNA gene was selected as the target region based on the availability of sequence data from a variety of isolates. Sequences of D. agamarum (NZ_LN849456.1, LN849456.1, NR_044368.1, EU009865.1, KF647330.1) were retrieved from GenBank, and multiple sequence alignment was performed with other sequences of different bacterial species (e.g., Agromyces species, Arthobacter species, Brachybacterium species, Dermabacter species, Pseudomonas species) using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/ last accessed on 20 February 2023). The primers and probe were designed using primer3 (https://primer3.ut.ee/ last accessed on 20 February 2023). Reactions included 1.0 µL of each primer (10 µM), 1.0 µL of the probe (2 µM), 4.0 µL DNA Process Control Detection Kit qPCR Reaction Mix and 5.0 µL template DNA in a total volume of 20.0 µL. Amplification was performed with a LightCycler 96 (Roche, Mannheim, Germany) in a 96-well format. The following protocol was used: Preincubation at 95 °C for 30 s followed by 40 cycles of two-step-amplification (95 °C for 5 s and 60 °C for 30 s). PCR-grade water (Roche, Mannheim, Germany) served as a negative control. While all other bacterial samples were negative, Dermabacter hominis produced positive PCR results at an annealing temperature of 60 °C (Figure 1). Therefore, DNA of Dermabacter hominis (DSM 30958) from the DSMZ as well as DNA of D. agamarum (GenBank Accession number: MT664092.1/0919Ur) [15], was tested in duplicate with different annealing temperatures (protocol 1: 65.0 °C, protocol 2: 66.0 °C, protocol 3: 67.0 °C) using a LightCycler 96 (Roche, Mannheim, Germany). Various non-D. agamarum bacterial species (Table 2) were tested in duplicate for positive reactions at 66 °C. The following protocol was used: Preincubation at 95 °C for 30 s followed by 40 cycles of two-step-amplification (95 °C for 5 s and 66 °C for 30 s). PCR-grade water (Roche, Mannheim, Germany) served as a negative control. DNA of 14 D. agamarum isolates (Table 1) was also tested. To assess the intraassay repeatability of the PCR, standard deviations were calculated for 10-fold serial dilutions in triplicate on a single plate. For the interassay reproducibility, standard deviations were calculated for a 10-fold serial dilution series which was amplified three times daily for two days. The standard deviations of the CT values were used to calculate the percent of coefficient of variation (CV%). Detection limit for bacterial cell dilutions: In order to determine the sensitivity of the assay, a culture of D. agamarum (isolate MT664091.1/0219Bf) was used, starting with a dilution (D0) of 0.5 McFarland (1.5 × 108 per mL). This suspension (D0) was 10-fold serially diluted (D1–D10) in duplicate, and 1 mL of each dilution was inoculated onto Columbia agar with defibrinated sheep blood (Becton Dickinson GmbH, Heidelberg, Germany/Oxoid GmbH, Wesel, Germany), incubated at 36 °C and checked for growth after 30 h. Colony forming units (CFU) were counted if the result was expected to be between 0 and 300 CFU. These results were then used to determine the limit of detection of the assay. DNA was extracted from 200 µL of each dilution (D1–D10) as described above, and PCR was carried out in duplicate. The detection of Dermabacter hominis (DSM 30958) was quantified in the same way. Detection limit for DNA from pure culture: To evaluate the assay’s sensitivity, PCR was carried out in triplicate using serial 10-fold dilutions of DNA prepared from colonies of D. agamarum (isolate MT664091.1/0219Bf). Spiked-in matrix: The assay was also evaluated using a spiked-in matrix (D. agamarum-negative-tested lizard skin with a known concentration of target DNA). For these assays, 10 µL of the above-described dilutions D0 to D6 were inoculated onto D. agamarum-negative-tested lizard skin. The skin was incubated in 500 µL lysis buffer, and 50 µL proteinase K and NA were extracted from 200 µL of this suspension as described above and eluted in a total volume of 100 µL NA. The PCR was carried out in duplicate as described above. Clinical samples from lizards for which appropriate material (skin, crusts, dry swab) was submitted to a commercial veterinary laboratory between March 2022 and December 2022 and for which the submitting veterinarian indicated an interest in D. agamarum diagnostics were tested using the established protocol to evaluate the PCR for use with clinical samples. Some of the samples were derived from animals showing clinical signs, others from asymptomatic animals tested in the context of a health check—often when D. agamarum had been isolated from animals in the group previously. If suitable material was available (skin or swab in a transport medium), bacteriology was performed (as previously described [15]). Identification of isolates was based on growth characteristics on agar plates (Columbia Agar with defibrinated sheep blood and Endo Agar, Becton Dickinson GmbH, Heidelberg, Germany), biochemical parameters and MALDI-TOF MS. In doubt, colonies were also re-checked via PCR. If the results of the bacteriological culture were available, the results of the PCR and culture would be compared. Samples were considered PCR positive if the cycle threshold (CT) was <35.0 and equivocal if the CT was ≥35.0, but a signal was obtained. If swabs of different origins regarding the localisation (e.g., dermal and oral) of the same animal were received, all samples were tested separately. Amplicons from positive samples were sequenced (ABI PRISM 3130 XL Genetic Analyser, Applied Biosystems, Foster City, CA, USA) and sequences were analysed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on 20 February 2023). The selected primer and probe sequences (Eurofins MWG Operon, Ebersberg, Germany) are shown in Table 3. The product size was expected to be 246 base pairs. Optimisation of annealing temperature was performed using three protocols with different annealing temperatures. In protocol 1 (65 °C), D. agamarum DNA was detected with CT values of 14.58 and 16.49 and Dermabacter hominis with CT values of 29.43 and 29.62. In protocol 2 (66 °C), D. agamarum DNA was detected with CT values of 16.22 and 16.14, and Dermabacter hominis showed values of 34.28 and 35.19. In protocol 3 (67 °C), D. agamarum was detected with CT values of 15.57 and 16.57, while Dermabacter hominis was not detected. Therefore, an annealing temperature of 66 °C was chosen for all further analyses as it was considered sufficient to discriminate between pure cultures of D. agamarum and Dermabacter hominis DNA without losing sensitivity for the detection of D. agamarum. Specificity of the assay using the protocol with 66 °C as an annealing temperature was determined using 14 D. agamarum isolates (Table 1) and 34 non-D. agamarum bacterial isolates (Table 2) in duplicate. No signal was obtained from any bacterial DNA from isolates other than D. agamarum except for Dermabacter hominis. The CT value obtained using the DNA from pure cultures of Dermabacter hominis were high (34.55 and 34.48) in comparison to those reached using DNA from D. agamarum (12.08–18.14) but still below the threshold set for clinical samples. As these are the results for DNA extracted from pure culture, 66 °C was considered sufficient for further testing of samples without prior cultivation as samples without prior cultivation are expected to yield a lower pathogen level. The intraassay CV was calculated to be 1.31%, while the interassay CV was 1.80%. Detection limit for bacterial cell dilutions: A positive PCR signal was detected for D. agamarum dilutions D0–D4. An equivocal signal was detected for D5 and D6. In culture, D4 corresponded to 2 × 104 colonies per mL. Therefore, the assay sensitivity was 2 × 104 colonies per mL with dilutions of bacterial cell culture serving as a template. This corresponds to approximately 200 CFU per PCR. For the serially diluted culture of Dermabacter hominis, no positive PCR signals were observed. Two dilutions (D0 and D1) resulted in equivocal CT values, with D0 being set to 0.5 McFarland (1.5 × 108 per mL). Detection limit for DNA from pure culture: The DNA concentration was determined to be 42.5 ng/µL (A260/A280: 1.94). D. agamarum DNA was detectable in dilutions up to 1:105. Therefore, DNA concentrations of as low as 425 fg/µL were detectable in the PCR. Spiked-in matrix: Spiked-in matrixes produced clearly positive results up to skin spiked with 10 µL of D2 with D2 corresponding to 2 × 106 colonies per mL (approximately 360 CFU per PCR considering dilution during sample preparation). In order to test the use of the developed PCR for clinical samples, a total of 48 samples from 38 lizards were tested for the presence of D. agamarum (Table 4). The samples were derived from several species, mostly agamids (Pogona spp. and Uromastyx spp.), and were of different origins (zoological collection, animal rescue centre, private owner) from Germany and the Netherlands. Some of these animals were asymptomatic and were tested in the context of a health check. Others showed clinical signs such as skin lesions, hyperkeratosis or stomatitis (Table 4). Samples were mostly derived from the oral cavity or skin/crusts. Of the 38 animals tested, D. agamarum was detected by PCR in 16 animals (42.10%). A further five animals (13.16%) were considered to have equivocal results, and 17 animals (44.74%) were negative for D. agamarum. One animal (animal 3) that tested negative proved to be infected with a fungus of the family Onygenaceae. A bacteriological examination was performed for 33 of the 38 animals, but D. agamarum was not cultured. However, in most of these cases, various other bacterial species (Table 4) were cultured when bacteriology was performed. D. agamarum is an important pathogen causing skin lesions and, in some cases, systemic disease in lizards. Depending on the species, some animals can be inapparent carriers, while others may develop severe diseases. Diagnosis of the causative agent is therefore important in order to facilitate treatment as well as to prevent the spread of disease. Since animals may suffer when untreated and the risks of spreading increase with time, a fast diagnostic approach is important. The detection of D. agamarum is commonly achieved via culture, followed in some cases by 16S rRNA gene sequencing [11,16]. The PCR developed in this study provides a time-saving tool compared to culture and bacterial identification. Detection of D. agamarum and concurrent bacteriological examination was performed in 33 of the 38 animals, resulting in 13 of 33 clearly PCR-positive animals but no culture-positives. D. agamarum is expected to be abundantly present in symptomatic animals. Culturing of D. agamarum is not considered difficult and has been successfully performed in this laboratory before [15]. However, a successful culture depends on the quality of the submitted samples. Appropriate samples include affected tissue below hyperkeratotic crusts or inside of the crusts as well as organs in septicaemic lizards and subcutaneous granulomas. In asymptomatic animals as well as in symptomatic animals, isolation from the oral cavity, gastrointestinal tract or healthy skin may be challenging. D. agamarum was cultured in the laboratory in which the study was performed during the study period, but these samples were excluded from the study as no suitable material for concurrent PCR testing (e.g., dry swab, skin) was available. In this study, six animals (1, 2, 21 and 22–24) were known to have been symptomatic and had positive PCR results. Bacteriological culture was performed in three of these animals (22–24). In animal 24, a positive PCR result was only obtained from the skin sample, which was not tested by bacteriological culture. Animal 6 might have been symptomatic (no information was received, but due to the reported previous treatment, it seemed likely). It was treated with antibiotics prior to sampling, which might have influenced the bacteriology results. Possible reasons for the failure of culturing D. agamarum out of positive clinical samples in this study include previous antibiotic treatment, incorrect sampling techniques, contamination with (oral) microbiota, increased transport time, inappropriate transport conditions or overgrowth by other bacteria. The latter is especially important as in the presented cases, no selective media for gram-positive bacteria were used, and various different bacterial species are expected to be present on the skin [17,18]. PCR analysis is useful if the performance of bacterial culture is difficult, e.g., due to previous treatment with antibiotics, inadequate preanalytical conditions (such as increased or decreased temperature, increased transport time, inadequate transport medium), or in cases in which overgrowth by other bacteria make detection challenging or impossible. The detection of D. agamarum via PCR can also simplify concurrent PCR testing for other known pathogens, e.g., viral or fungal pathogens known to cause dermatitis [19,20], since the same extracted nucleic acids can be used. In general, PCR is advantageous when culturable samples are unavailable, for example, when older samples are tested or stored DNA is examined. However, bacterial DNA can persist in the environment [21], and D. agamarum has been shown to survive for several months in the environment, depending on the conditions [13]. A PCR could therefore detect bacteria even in cases in which these were not responsible for clinical signs or in which no replication-competent bacteria were present. The PCR developed here was not 100% specific. Pure cultures of Dermabacter hominis did result in a weak positive signal. However, if diluted, only equivocal results were observed. Dermabacter hominis is genetically closely related to D. agamarum [2,11,22]. Dermabacter hominis is associated with the human microbiome [23]. It is occasionally described in human clinical samples such as abscesses or blood cultures [24,25,26] but is usually found to be of minor clinical significance [27]. So far, its clinical importance in reptiles is very unclear. Contamination during sampling or sample preparation should be considered a possible option leading to false equivocal results. However, clinical samples are expected to yield less bacterial DNA, making false equivocal results less likely. The 16S rRNA gene is known to be highly conserved between bacterial species, which makes it a useful target if the aim is to identify different bacterial species. It is a commonly used target for bacterial detection, and therefore a large amount of sequence data is available for a wide range of bacterial species. However, it may not be ideal for differentiating closely related bacteria. Currently, the availability of sequence data for D. agamarum other than the 16S rRNA gene is limited, but in the future, other targets may prove to be better options. In the meantime, especially equivocal CT values should be evaluated with caution in the face of clinical signs, sampling and sample preparation, and ideally, retesting is recommended. Possibly, skin samples might prove more useful than swabs as they yielded lower CT values in two of the three animals for which both sample types were available, but this might be highly dependent on the sampling method. The PCR protocol developed in this study proved helpful for the detection of D. agamarum in clinical samples. D. agamarum was detected in oral swabs from clinically healthy Pogona species and serrated casquehead iguana (Laemanctus serratus), while the results in which equivocal results were obtained were also from clinically healthy Pogona species as well as from clinically healthy black hardun (Laudakia stellio picea). Pogona species have previously been shown to be possible inapparent carriers of D. agamarum and a possible source of infection for more sensitive species [2,3]. Therefore, this PCR protocol may not only be useful for clinical cases but also as a screening tool. However, the number of tested samples is still small, and testing of larger sample numbers is necessary in order to confirm the usefulness of this method for clinical practice. A real-time PCR was developed that is able to detect D. agamarum in clinical samples. The assay provides a fast method for the detection of this important pathogen of lizards but should be evaluated with further samples in the clinical context.

PMC10000039

Neelam A. Topno,Veerbhan Kesarwani,Sandeep Kumar Kushwaha,Sarwar Azam,Mohammad Kadivella,Ravi Kumar Gandham,Subeer S. Majumdar

Non-Synonymous Variants in Fat QTL Genes among High- and Low-Milk-Yielding Indigenous Breeds

28-02-2023

milk fat,whole-genome sequencing,SNPs,genomic variation,variant calling,indigenous breeds

Highlights What are the main findings? Differentially expressed milk fat QTL genes explored with whole genome se-quencing for variant analysis. Identified non-synonymous SNPs for hub and bottleneck QTL genes associated with milk fat traits. What is the implication of the main finding? Identified differential pattern(s) of SNPs in fat QTLs between high and low milk yield breeds. Impact of the identified SNP pattern(s) on milk fat traits can be further explored. Simple Summary Milk fat is a crucial trait that varies significantly among cattle breeds and determines the milk quality and pricing value. Indigenous breeds have disparity in milk quantity and quality. Our study is one of a kind which helps to decipher the variations at the genetic level correlated with transcriptional level among high and low milk-yielding cattle breeds exploring the fat QTLs. We assessed and unveiled a few key differences between the high and low-milk-yield breeds. Abstract The effect of breed on milk components—fat, protein, lactose, and water—has been observed to be significant. As fat is one of the major price-determining factors for milk, exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. Here, on whole-genome sequencing, 25 differentially expressed hub or bottleneck fat QTLs were explored for variations across indigenous breeds. Out of these, 20 genes were identified as having nonsynonymous substitutions. A fixed SNP pattern in high-milk-yielding breeds in comparison to low-milk-yielding breeds was identified in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E and, vice versa, in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The identified SNPs were ratified by pyrosequencing to prove that key differences exist in fat QTLs between the high- and low-milk-yielding breeds.

Non-Synonymous Variants in Fat QTL Genes among High- and Low-Milk-Yielding Indigenous Breeds What are the main findings? Differentially expressed milk fat QTL genes explored with whole genome se-quencing for variant analysis. Identified non-synonymous SNPs for hub and bottleneck QTL genes associated with milk fat traits. What is the implication of the main finding? Identified differential pattern(s) of SNPs in fat QTLs between high and low milk yield breeds. Impact of the identified SNP pattern(s) on milk fat traits can be further explored. Milk fat is a crucial trait that varies significantly among cattle breeds and determines the milk quality and pricing value. Indigenous breeds have disparity in milk quantity and quality. Our study is one of a kind which helps to decipher the variations at the genetic level correlated with transcriptional level among high and low milk-yielding cattle breeds exploring the fat QTLs. We assessed and unveiled a few key differences between the high and low-milk-yield breeds. The effect of breed on milk components—fat, protein, lactose, and water—has been observed to be significant. As fat is one of the major price-determining factors for milk, exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. Here, on whole-genome sequencing, 25 differentially expressed hub or bottleneck fat QTLs were explored for variations across indigenous breeds. Out of these, 20 genes were identified as having nonsynonymous substitutions. A fixed SNP pattern in high-milk-yielding breeds in comparison to low-milk-yielding breeds was identified in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E and, vice versa, in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The identified SNPs were ratified by pyrosequencing to prove that key differences exist in fat QTLs between the high- and low-milk-yielding breeds. India has become the largest milk producer in the world [1]. Several schemes involving crossbreeding have been implemented to enhance milk production in the country. As a result, the number of crossbred cattle increased and contributed to around 28 percent of total milk production in India (ca. 188 million tons), surpassing the contribution of indigenous cattle [2]. However, indigenous cattle breeds are well known for their heat tolerance and disease resistance [3], and the crossbreds have been found to be susceptible to tropical diseases and harsh climatic conditions and require constant good management practices. To strike a balance between increasing demand for milk and the change in the environment due to global warming, exploring the genomic merit of indigenous cattle/breeds becomes even more important. Milk being a polygenic trait with medium heritability, the majority of animal breeding research has centered on quantitative trait loci (QTLs) with moderate to large effects on milk production traits. The DGAT1 on chromosome 14 [4,5,6], the growth hormone receptor (GHR) on chromosome 20 [7], and the ABCG2 [8] or SPP1 (Osteopontin) on chromosome 6 [9] are well-known QTL genes that have been fully characterized with a strong putative or well-confirmed causal mutation. The two QTLs DGAT1 (K232A) and ABCG2 (Y581S) in Bos taurus have been suggested to be associated with increased fat yield and fat and protein percent in milk with a decrease in milk yield [6,8,10,11,12,13]. The GHR mutation F279Y has been observed to have a significant effect on milk composition (fat and protein percentage) and milk yield [14]. The locus c.8514C > T in the intronic region of SPP1 has also been found to have a significant effect on milk production and milk composition [15]. However, the DGAT1 and ABCG2 genes have been found to be fixed among Indian breeds Sahiwal, Rathi, Deoni, Tharparkar, Red Kandhari, and Punganur [16]. Currently, the animal QTL database (QTLdb) contains 1,93,216 QTLs for different bovine traits, out of which 83,458 QTLs have been reported for milk traits [17]. Milk is the primary source of nutrition for infants, as well as adults. Besides its nutritional value, it has a major role in imparting growth and immunity through intrinsic milk components such as growth factors, chemokines, anti-inflammatory molecules, antioxidants, prebiotics, and probiotics [8,9]. Milk has four major components, fat (3.6%), protein (3.2%), lactose (4.7%), and water (87%), along with other various kinds of minerals, enzymes, vitamins, and dissolved gases. Various research studies have shown that several factors, such as lactation stage, genetics, environmental factors, and diet management, influence milk quality. The variability of milk composition among popular dairy breeds Brown Swiss, Holstein Friesian, Jersey, Simmental, Grey Alpine, and Rendena under the same dairy management practices has been explored, and Holstein Friesian had higher milk yield with lower fat content (27.45 kg/d, 4.04%) [18], whereas Jersey had lower milk yield with relatively higher fat content (17.27 kg/d, 5.65%) [13]. A low fat percentage has also been reported for Ayrshire, Brown Swiss, Guernsey, Holstein Friesian, and Jersey breeds in the United States [19]. Furthermore, the effect of breed has been found to significantly influence the water (p ≤ 0.0001), protein (p ≤ 0.05), total solids (p ≤ 0.05), fat (p ≤ 0.05), milk urea nitrogen (p ≤ 0.001), and ash (p ≤ 0.0001) content of milk [20]. India, with a huge diversity of 50 cattle breeds, forms an ideal ground to study genetic variation at the genomic level vis-à-vis milk traits [21]. The cost of the milk world-over varies with the percentage of fat present in the milk. Exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. No such studies have been reported in the past for indigenous cattle breeds to evaluate the variation across indigenous breeds within the fat QTLs. Therefore, the objective of the present study was to explore genomic variation(s) within the fat QTLs that were identified to be differentially expressed in lactation across indigenous breeds, which were divided into high- (Sahiwal and Gir) and low-milk-yield (Gaolao, Deoni, Pulikulam, Hallikar, Dangi, and Amritmahal) groups. QTL genes associated with milk fat traits (milk fat content and percentage) and metabolism were extracted from the Animal QTLdb database [22], and the duplicates were removed. QTL genes (286 and 256 (total of 542)) were identified for milk fat yield and milk fat percentage, respectively, with 417 unique genes for both traits (Supplementary File 1). Functional annotation of the 125 QTL genes commonly associated with milk fat yield and milk fat percentage was performed in g:Profiler [23] and ShinyGO [24] to identify the enriched biological processes. Protein interaction network analysis of the 417 genes was performed using the Search Tool for the Retrieval of Interacting Genes Search Tool for the Retrieval of Interacting Genes 11.0 (STRING 11.0) database at a confidence score value of 0.5 against model species Bos taurus [25]. The interaction network was imported into the Cytoscape 3.8.0 software (Institute for System Biology, CA, USA) for visualization. The hub and bottleneck genes were identified in the interaction network using the Cytohubba plugin of Cytoscape [26] considering the degree of association between the genes and by taking the bottleneck approach, which takes into account the top 20% of the degree of distribution of the proteins in the network [26]. A total of 74 QTL genes were identified to be hub/ bottleneck genes. The hub genes were the genes that had the highest degree of association, and the bottleneck genes were the key connectors having a high betweenness (measure the centrality of the nodes) among different clusters in protein interactions [27]. The publicly available milk transcriptome bioproject ID (PRJNA419906) was used to analyze the expression of QTL genes associated with milk fat traits. This bioproject was considered in this study as it has data generated from Jersey (a breed with high fat content ranging from 4.10–4.86%) and Kashmiri (a breed with low fat content ranging from 3.20–3.94%) [28]. The data were generated from mammary epithelial cells (MECs) collected on Day 15 (D15), D90, and D250 from six lactating cows (three Jersey and three Kashmiri cattle). These days represent early, mid-, and late lactation, respectively [28]. The data were downloaded from the Sequence Read Archive (SRA) of the NCBI database, and the fastq-dump program of SRAtoolkit [29] was used to extract the fastq reads. Quality assessment and control of RNA-seq data were performed through Fast QC Version 0.11.5 [30], MultiQC Version 1.8 [31], and trimmomatic Version 0.39 [32]. All the high-quality reads were mapped to the Bos indicus genome (GCF_003369695.1) using STAR Version 2.5.4b with the default parameters [33]. Gene expression was estimated using RSEM [34], and differential gene expression was performed through the DESeq2-R package [35]. The differentially expressed genes among the 74 fat QTL hub and bottleneck genes were identified. Indigenous cattle breeds for the proposed study were grouped into high- and low-milk-yield-breed groups. The high-milk-yield group (avg milk yield per day 8 kg) included Sahiwal (n = 4) and Gir (n = 4), and the low-milk-yield group (avg milk yield per day 2.5 kg) included 6 animals representing the breeds Gaolao, Deoni, Hallikar, Dangi, Pulikulam, and Amritmahal [36]. Animals of different breeds were considered in the study to have a true representation of both the high- and low-milk-yield groups. Genomic DNA (gDNA) was extracted from the blood samples of the animals from these breeds using the nucleospin blood L-kit (Macherey-Nagel), and the integrity of the genomic DNA was checked on agarose. After estimating the concentration of gDNA (Nanodrop2000, ThermoFischer Scientific), DNA libraries were prepared as per the manufacturer’s protocol (Illumina sequencing platform) for paired-end sequencing (2150 bp). Sequencing data generated on an Illumina platform were pre-processed for quality assessment and improvement (base quality, nucleotide distribution, GC content, adaptor sequence, duplication, length distribution, etc.) by FastP [37]. All high-quality reads were mapped to the Bos indicus reference genome (Brahman-GCF_003369695.1) using BWA aligner [38]. Variant calling was performed from the aligned data using freebayes [39] and GATK [40]. For the GATK- and freebayes-generated vcf files, only SNPs were selected, leaving aside all indels and insertions. After freebayes variant calling, the low-quality variants were filtered by vcftools Version 1.10 [41] for Q > 20. In the GATK pipeline, the paired-end Illumina Hi-Seq raw reads for each individual were first converted into an unaligned bam; the illumina adapters were marked; the sam was converted back to FASTQ; the reads were mapped to the reference Brahman genome (GCF 003369695.1); the unaligned and mapped bams were combined. Finally, the duplicate marked clean bam was used to generated GVCF for each animal using GATK haplotypecaller. The GVCFs generated for all the animals were combined to call the variants using the genotypeGVCF module. The parameters for GATK VariantRecalibration to generate the VQSLOD score were QD < 2.0, MQRankSum < −8.5, ReadPosRankSum < −8.0, FS > 60.0, MQ < 40.0, SOR > 3.0, and DP 30x (depth or coverage). The final set of SNPs after recalibration by GATK included the selection of SNPs that passed. We further used the GATK-filtered set and the freebayes-filtered set to identify the common SNPs across these variant callers. From this vcf file, the SNPs in the differentially expressed hub and bottleneck genes (i.e., genes that are hub/bottleneck and are differentially expressed as identified in Section 2.2) were extracted using an in-house perl script. The non-synonymous SNPs (nsSNPs) in the coding regions of these were identified through the SnpEff tool [42]. Three nsSNPs that were found to be distinctly different between the high- and low-milk-yield groups were selected for validation and were genotyped in PyroMark Q48 (Qiagen) as per the manufacturer’s protocol. These nsSNPs were found in the differentially expressed hub and bottleneck genes GHR, LPIN1, and TLR4. GHR was one of the genes having the maximum number of interactions in the network. LPIN1 had the maximum SNP count of 10, whereas TLR4 was one of the top 10 highly upregulated genes. PCR and sequencing primers were designed using PyroMark Assay Design Software 2.0 (Qiagen). The PCR amplification was performed in a 20 µL reaction, with the thermal cycling conditions, which included an initial denaturation of 95 °C for 3 min followed by 40 cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. Sequence analysis was performed by PyroMark Q48 Autoprep software Version 2.4.2 in SNP analysis assay mode for 14 animals. The schematic representation of the study is given in Supplementary File 1: Figure S1. Animal QTLdb was used to extract fat-trait-associated QTL genes. In the QTLdb, after the removal of duplicate genes (Supplementary File 1), 286 and 256 genes were found associated with milk fat yield and percentage, respectively, whereas 125 common genes were found between milk fat yield and percentage (Figure 1A). A total of 417 unique genes were found to be associated with milk fat and other milk traits. These genes were also found to be annotated for other milk traits such as milk yield, protein yield, and percentage. Among all genes, 24 genes were found to be associated with both fat yield and fat percentage traits only (Supplementary File 1). The common genes (125) were found enriched in biosynthetic-, catabolic-, regulatory-, transportation-, and cellular-response-associated metabolic processes. Among the metabolic genes associated with milk fat traits, the genes associated with milk fatty acid metabolism were FASN, GPAT4, DGKG, ELOVL6, and LIPIN1 (Supplementary File 5: Table S1). The publicly available RNA-seq bioproject (PRJNA419906) has library sizes ranging from 7764992200–13682843400 bp and 6842583600–12088807400 bp, for Jersey and Kashmiri, respectively (Supplementary File 5: Figure S2A). Further, gene expression counts per million (CPM), principal component analysis (PCA), and multidimensional scaling (MDS) (Supplementary File 5: Figure S2A,C,D) of the samples were assessed. The PCA and MDS plots of sequenced RNA-seq libraries showed a high level of similarity within breeds and relatively low variation between the lactation stages of breeds. A total of 70 genes were found to be upregulated and 52 genes downregulated in the Jersey breed in comparison with the Kashmiri breed. Differentially expressed transcripts (DETs) were also explored for fat QTL genes (Supplementary File 2). The volcano plots of differentially expressed genes (DEGs) and DETs depicting the distribution of upregulated and downregulated genes are shown in Figure 2. In both the Jersey and Kashmiri breeds, Beta-lactoglobulin (LOC113901792), Casein beta (CSN2), and Casein alpha s1 (CSN1S1) were identified to be among the highly expressed top 20 genes (Supplementary File 5: Table S2). The DETs are listed in Supplementary File 2. In the Jersey breed, CXCL-8, TLR4, and OLR1 were among the highly upregulated genes. CXCL-8/IL8, produced by macrophages, epithelial cells, and airway smooth muscle cells, is a neutrophil chemotactic factor that induces chemotaxis in target cells and other granulocytes to initiate movement toward infection sites, whereas OLR1, a receptor on macrophages, epithelial cells, and airway smooth muscle cells, is involved in rapid oxidization of low-density lipoprotein (LDL), which is more readily recognized by the TLR4 receptor. The list of top 10 upregulated and downregulated DEGs is given in Supplementary File 5: Table S3. A protein interaction network analysis was performed among 417 milk fat QTL genes, and the interaction network was generated with 403 nodes and 671 edges. Based on the degree of association, 50 hub and 50 bottleneck genes were selected from the network (Supplementary File 2). A total of 74 QTL genes were found to be either hub or bottleneck genes (Figure 1B). Out of these, 25 genes (which accounted for 18 hubs/17 bottleneck genes) were differentially expressed in Jersey and Kashmiri (Figure 1C, Supplementary File 2). Out of these genes, ten genes possessed both hub and bottleneck gene characteristics. The SRC and DGAT1 genes were among the top differentially expressed hub and bottleneck genes (Table 1). SRC had the highest degree of association (30) with a log2 fold change of 1.480587, followed by DGAT1 with a degree of 25 and a log2FC of 0.921104. Illumina short read (Paired end) data of 14 samples from both groups of high- and-low-milk-yield breeds had 12.77 billion reads. After preprocessing, clean data included 11.02 billion reads, which is ca. 1516 Gb data. Each dataset had a minimum sequencing depth of ≥30x with an average GC content of 45.26%. The processed datasets contained on average 97.91% Q20 bases and 93.97% Q30 bases. The high-quality trimmed data aligned to the Brahman reference genome with an overall alignment rate of >95%. Initial variant calling on the aligned data provided 63,357,363 variants, which were filtered for high quality. After quality filtering on Q20, a total of 33,976,892 SNPs were identified across the genomes (Supplementary File 3). Upon GATK analysis, 39,625,917 variants were found to pass the variant calibration. A total of 25,956,231 SNPs were found to be common among the variant callers. From these, SNPs in the 25 differentially expressed hub and bottleneck milk fat QTL genes were extracted, out of which 20 genes were found to have non-synonymous substitutions in the coding regions (Table 2). The variants identified in these 20 genes were further explored for two kinds of genomic variant patterns, i.e., fixed SNP pattern in the cattle of the high-milk-yield group vs. variable SNP pattern in cattle of the low-milk-yield group, or vice versa. The fixed SNP pattern in high-milk-yield breeds in comparison to low-milk-yield breeds was observed in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E (Table 3), and the opposite was observed in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E (Table 4). SNPs C/G, C/A, and G/A were confirmed in GHR, TLR4, and LPIN1 in the Amritmahal, Pulikulam, and Dangi breeds (low-milk-yield) as against SNPs C/C, C/C, and G/G in the Gir and Sahiwal breed (high-milk-yield), respectively (Figure 3) (Supplementary File 4). In the TLR4 gene, variant g.107083326A>C was found in the low-milk-yield group, but the same variant was fixed in the high-milk-yield group. Similarly, in the LPIN1 gene variant, g.85211528C>G was found in the low-milk-yield group, but this variant was fixed in the high-milk-yield group. In the NUP98 gene and LPIN1 variants, g.32707374G>A and g.85205642T>G, respectively, were found in the high-milk-yield group, but were found to be fixed (g.32707374G>G; g.85205642T>T) in the low-milk-yield group. Another LPIN1 variant, g.85205642T>G, was observed in the high-milk-yield group, but was fixed in the low-milk-yield group. LPIN1 and ITGA1 had the maximum SNP count of 10, and the genes PTK2, IGF1R, DDIT3, CXCL8, and LPL had the least SNP count (Table 2). The Bos indicus genome is an interesting model to study the genomic potential of different indigenous cattle breeds such as Sahiwal, Gir, Amritmahal, Dangi, Gaolao, Deoni, Pulikulam, and Hallikar, which are highly adapted to different tropical conditions with varying milking potential. The availability of bovine QTL resources such as the Animal QTL database and the collection of QTLs for different traits have provided the opportunity to investigate genomic variation among indigenous breeds for milk-associated traits. Milk quality such as fat yield and percentage are highly variable traits among breeds. Jersey is one among the high-milk-producing breeds worldwide, whereas Kashmiri is one of the poorly performing breeds in the Kashmir region of India. Therefore, we aimed at differences in the expression of fat QTL genes between the two contrasting breeds, Jersey and Kashmiri. In this study, significantly expressed hub and bottleneck fat QTL genes were further analyzed to identify the genomic variants from the whole-genome sequence data between high- (Sahiwal and Gir) and low- (Amritmahal, Dangi, Gaolao, Deoni, Pulikulam, and Hallikar) milk-yield indigenous breeds. This understanding of low- and high-milk-yield breeds for milk fat quality may help in enhancing the quality of milk in the long run. To explore the fat QTL genes, MEC RNA-seq data were processed and analyzed. The high level of similarity within breeds and relatively low variation between lactation confirmed the selection of RNA-seq data to explore differences between breeds rather than to explore difference in lactation stages of breeds. Among the highly expressed genes identified, it was observed that the Jersey breed has allocated more resources for the immune system, whereas the Kashmiri breed for regulation of ribosomal proteins. Among the top 10 upregulated genes, CXC motif chemokine ligand 8 was the most-upregulated gene. It is reported to be involved in various biological pathways such as increased insulin resistance, uncoupling of the GH/IGF1 axis, and an increase in mammary cell proliferation to improve metabolic health and milk yield [43]. Diacylglycerol kinase gamma (DGKG), another upregulated gene, is a member of the type I diacylglycerol kinases and is highly upregulated (log2FC = 4.03) in Jersey. It plays a role in lipid metabolism by modulating the balance between diacylglycerol and phosphatidic acid. Phosphatidic acid is a lipid second messenger to activate protein kinase C isoforms, ras guanyl nucleotide-releasing proteins, and some transient receptor potential channels [44]. Most of the top-upregulated genes in Jersey have been found to have a role in adipogenesis (ETS2) [45], adipocyte differentiation (OLR1, PARM1) [46,47], glucose transport (SLC6A9) (log2FC = 4.49) [48], glucose uptake (SLC45A4) [49], thyroid hormone synthesis (TG) [50], and aldosterone secretion (KCNK9) [51]. The upregulation of these genes in Jersey indicates their involvement in lipid biosynthesis in the mammary gland during lactation. Furthermore, UDP-glucose 6-dehydrogenase (UGDH), which is involved in the biosynthesis of glycosaminoglycans, hyaluronan, chondroitin sulfate, and heparan sulfate, has been found (log2FC = 0.75) to be upregulated in the Jersey breed. UGDH’s expression pattern in liver cells has been associated with an indispensable role in the metabolism of carbohydrates, fats, and proteins in dairy cattle [52]. Moreover, UGDH has been found close to two reported QTLs for fat yield, fat percentage, and protein yield [53]. During lactation, various morphological changes happen in the mammary tissue to support cellular differentiation, tissue elasticity, and reduced fat storage capacity in the animal. Upregulated GRH13 is a transcription factor that mediates the proliferation of epithelial cells [54]. Similarly, MTMR3/3-PAP, a catalytically inactive member of the myotubularin gene family that coprecipitates the activity of lipid phosphatidylinositol 3-phosphate-3-phosphatase, is upregulated [55] in Jersey. Matrilin 2 (MATN2) and prolyl 4 hydroxylase (P4HA3), which are important to maintaining the newly synthesized collagen’s stability, are also upregulated [56,57]. P4HA3 catalyzes the formation of 4-hydroxyproline (Hyp), which ensures the proper folding of procollagens during post-translational modification. The upregulation of MATN2 and P4HA3 probably may help in increasing the elasticity of the udder gland in Jersey during lactation. Further, the downregulation of MFGE8 and ELOVL16 may be responsible for the high fat content in milk and the shift to C18 from C16 fatty acids, respectively, in Jersey. MFGE8 regulates the absorption of free fatty acids and increases intracellular triglycerides’ hydrolase activity, thereby restricting the storage of fat [58], whereas ELOVL fatty acid elongase (ELOVL16) elongates C16 saturated and monounsaturated fatty acids to C18 fatty acids [59]. Further, IDH1, which catalyzes the conversion of isocitrate to α-ketoglutarate and generates the primary source of NADPH for de novo fatty acid synthesis [60], has been to be found downregulated in Jersey (log2FC = −1.632). The dysregulation of the genes MFGE8, EVOLVL16, and IDH1 might be responsible for the variation in the fat yield and composition in Jersey and Kashmiri cows. In addition, the decreased expression of these genes may be linked to the increased expression of genes involved in metabolism, glucose transport, and other transport activities, leading to higher milk production performance in Jersey. Among the ten QTLdb milk and milk trait genes that were differentially expressed and had hub and bottleneck gene characteristics, four genes were found enriched in metabolic pathways (Supplementary File 5: Table S1). SRC was identified as the top hub gene interacting with several genes in the protein–protein interaction network. SRC, a non-receptor tyrosine kinase, performs a wide variety of cellular functions in terms of metabolism and is primarily involved in impaired glucose uptake [61]. Genes Diacylglycerol O-acyltransferase 1 (DGAT1) and ecto-5′-nucleotidase (NT5E) possess hub and bottleneck gene features. DGAT1 encodes a protein that catalyzes the conversion of diacylglycerol and fatty acyl CoA to triacylglycerol. DGAT1 is one of the highly studied genes for milk yield and fat quality [62,63]. The NT5E gene encodes a plasma membrane protein that catalyzes the conversion of extracellular nucleotides to membrane-permeable nucleosides. SRC and DGAT1 were found to be highly upregulated in Jersey with log2FC = 1.48 and log2FC = 0.92, respectively. In the network, DGAT1 was found to interact with Glycerol-3-phosphate acyltransferase 4 (GPAT4) (also known as AGPAT6). This gene has been found to be involved in triglyceride biosynthesis and is comprised of acyltransferase motifs, which are essential for binding to substrates and catalyzing acyltransferase reactions. The AGPAT6 gene, which is highly expressed in mammary gland epithelium during lactation [64], was also observed to be upregulated. NT5E was found to interact with BDNF, NTRK2, and ZNRF4. NT5E is involved in various biological process such as adenosine biosynthetic process, AMP catabolic process, leukocyte cell–cell adhesion, and negative regulation of inflammatory response [65]. DGKG, which was upregulated, was found to be a bottleneck gene in the network and had interactions with PRKCG and RNT. Phosphatidate phosphatase (LPIN1), an enzyme involved in lipid metabolism [66], was upregulated and found as a hub gene in the network having interactions with PPARA and PPARGC1A. LPIN1 is involved in various biological processes related to lipid metabolism such as the triglyceride biosynthetic process, the fatty acid catabolic process, and the regulation of transcription by RNA polymerase II [66]. The gene PTK2 is well known for its association with milk production traits [67], and CACNA1A has a role in hormone regulation of lactation [68]. Similarly, ZBTB16 is involved in bovine adipogenesis [69]. NUP98 is associated with protein percentage [70]. TLR4 is a mastitis-associated marker [71]. FGF2 expression is reported to be associated with milk production traits [72]. In this study, breed (high-milk-yield and low-milk-yield groups) differences in nsSNPs within these hub and bottleneck genes (GHR, TLR4, LPIN1, CACNA1C, MFGE8, PTK2, ZBTB16, FGF2, and NUP98) were identified. These fat QTL nsSNPs may have a role in the existing fat and milk yield differences between the breed groups. However, further studies to evaluate the impact of these SNPs on fat yield/percentage or milk yield need to be carried out. In this study, initially, DEGs in Jersey epithelial cells were identified, and these were further explored in the QTL database as being the major hub and bottleneck genes. The transcriptome in Jersey indicated higher expression of genes involved in metabolism, glucose transport, and other transport activities, leading to higher milk production performance. The 20 differentially expressed hub and bottleneck fat QTL genes were explored for non-synonymous genomic variants in the whole-genome sequence data, which were generated from fourteen animals. The fixed SNP pattern in high-milk-yield breeds in comparison to low-milk-yield breeds was observed in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E, and the opposite was observed in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The role of these SNPs needs to be further explored.

PMC10000040

Yue Sun,Yanze Yu,Jinhao Guo,Linqiang Zhong,Minghai Zhang

Alterations in Fecal Microbiota Linked to Environment and Sex in Red Deer (Cervus elaphus)

04-03-2023

cervidae,KEGG,fecal microbiota,16s rRNA

Simple Summary The gut microbiota forms a complex microecosystem in vertebrates and is affected by various factors. Wild and captive red deer currently live in the same region but have vastly different diets. In this study, the 16S rRNA sequencing technology was performed to evaluate variations in the fecal microbiota of wild and captive individuals of both sexes of red deer. It was found that the composition and function of fecal microbiota in wild and captive environments were significantly different. As a key intrinsic factor, sex has a persistent impact on the formation and development of gut microbiota. Overall, this study reveals differences in the in the fecal microbiota of red deer based on environment and sex. These data could guide future applications of population management in red deer conservation. Abstract Gut microbiota play an important role in impacting the host’s metabolism, immunity, speciation, and many other functions. How sex and environment affect the structure and function of fecal microbiota in red deer (Cervus elaphus) is still unclear, particularly with regard to the intake of different diets. In this study, non-invasive molecular sexing techniques were used to determine the sex of fecal samples from both wild and captive red deer during the overwintering period. Fecal microbiota composition and diversity analyses were performed using amplicons from the V4–V5 region of the 16S rRNA gene sequenced on the Illumina HiSeq platform. Based on Picrust2 prediction software, potential function distribution information was evaluated by comparing the Kyoto Encyclopedia of Genes and Genome (KEGG). The results showed that the fecal microbiota of the wild deer (WF, n = 10; WM, n = 12) was significantly enriched in Firmicutes and decreased in Bacteroidetes, while the captive deer (CF, n = 8; CM, n = 3) had a significantly higher number of Bacteroidetes. The dominant species of fecal microbiota in the wild and captive red deer were similar at the genus level. The alpha diversity index shows significant difference in fecal microbiota diversity between the males and females in wild deer (p < 0.05). Beta diversity shows significant inter-group differences between wild and captive deer (p < 0.05) but no significant differences between female and male in wild or captive deer. The metabolism was the most important pathway at the first level of KEGG pathway analysis. In the secondary pathway of metabolism, glycan biosynthesis and metabolism, energy metabolism, and the metabolism of other amino acids were significantly different. In summary, these compositional and functional variations in the fecal microbiota of red deer may be helpful for guiding conservation management and policy decision-making, providing important information for future applications of population management and conservation.

Alterations in Fecal Microbiota Linked to Environment and Sex in Red Deer (Cervus elaphus) The gut microbiota forms a complex microecosystem in vertebrates and is affected by various factors. Wild and captive red deer currently live in the same region but have vastly different diets. In this study, the 16S rRNA sequencing technology was performed to evaluate variations in the fecal microbiota of wild and captive individuals of both sexes of red deer. It was found that the composition and function of fecal microbiota in wild and captive environments were significantly different. As a key intrinsic factor, sex has a persistent impact on the formation and development of gut microbiota. Overall, this study reveals differences in the in the fecal microbiota of red deer based on environment and sex. These data could guide future applications of population management in red deer conservation. Gut microbiota play an important role in impacting the host’s metabolism, immunity, speciation, and many other functions. How sex and environment affect the structure and function of fecal microbiota in red deer (Cervus elaphus) is still unclear, particularly with regard to the intake of different diets. In this study, non-invasive molecular sexing techniques were used to determine the sex of fecal samples from both wild and captive red deer during the overwintering period. Fecal microbiota composition and diversity analyses were performed using amplicons from the V4–V5 region of the 16S rRNA gene sequenced on the Illumina HiSeq platform. Based on Picrust2 prediction software, potential function distribution information was evaluated by comparing the Kyoto Encyclopedia of Genes and Genome (KEGG). The results showed that the fecal microbiota of the wild deer (WF, n = 10; WM, n = 12) was significantly enriched in Firmicutes and decreased in Bacteroidetes, while the captive deer (CF, n = 8; CM, n = 3) had a significantly higher number of Bacteroidetes. The dominant species of fecal microbiota in the wild and captive red deer were similar at the genus level. The alpha diversity index shows significant difference in fecal microbiota diversity between the males and females in wild deer (p < 0.05). Beta diversity shows significant inter-group differences between wild and captive deer (p < 0.05) but no significant differences between female and male in wild or captive deer. The metabolism was the most important pathway at the first level of KEGG pathway analysis. In the secondary pathway of metabolism, glycan biosynthesis and metabolism, energy metabolism, and the metabolism of other amino acids were significantly different. In summary, these compositional and functional variations in the fecal microbiota of red deer may be helpful for guiding conservation management and policy decision-making, providing important information for future applications of population management and conservation. Red deer (Cervus elaphus), which belong to the family Cervidae, order Artiodactyla, distributed in Asia, Europe, North America, and North Africa [1]. The red deer is a typical forest-inhabiting mammal in northeast China and has an important ecological status in the forest ecosystem [2]. Owing to habitat fragmentation, the populations of red deer in the wild are currently in sharp decline [2]. Using captive populations as reintroduction resources is an effective strategy to restore the populations of wild red deer [3]. The complex gut microbiota systems in the mammalian gut are composed of large fractions of microbes [4]. The gut microbiota are a complex product of the long-term evolution of hosts and microbes [4]. Recent studies have shown that not only are gut microbiota a part of the host, but they also have a significant impact on the health of the host, such as promoting immunity, digestion, metabolism, and intestinal endocrine hormones, among others [5,6,7]. Simultaneously, the complex and flexible gut microbiota can be affected by multiple environmental and host genotypes [8]. Many studies have shown that diet is an important factor that affects the structure and function of the fecal microbiota [9,10,11]. For example, changes in diet alter the function and diversity of fecal microbiota as well as the relative abundance of some microorganisms [12]. Moreover, diet-induced loss of microbial function and diversity will increase the risk of diversity loss and extinction through generational amplification [13]. It was necessary to investigate the gut microbiome by comparing differences between wild and captive red deer. However, to date, there has been a lack of studies comparing the gut microbiota between wild and captive red deer [11]. Because of sex differences in behavior and physiology, sex as an important intrinsic factor leads to differences in gut microbiota among individuals within species [14,15,16]. Although the results are inconsistent, animal species with significant sexual dimorphism and human studies have shown sex-related differences in gut microbiota. In mice (Mus musculus), poultry, and forest musk deer (Moschus berezovskii), the composition of the gut or fecal microbiota shows sex differences [17,18,19]. At present, few studies have analyzed the sexual dimorphism of fecal microbiota in red deer. In order to save endangered populations, artificial breeding of wild populations is carried out. The food types and nutrient intake ratios obtained in captivity and wild environments are very different, especially for endangered cervidae [20]. Therefore, monitoring the digestive system of captive animals and identifying standardized levels of nutritional requirements and fiber composition is critical for captive wild animals to determine whether they have acclimated to artificially provided food and new environments—a part of wildlife conservation’s main problem [21]. Using captive populations as reintroduction resources is an effective strategy to restore the populations of wild red deer. The composition of gut microbiota in wild populations can be a good indicator of the breeding direction of the captive population [9]. Therefore, understanding the impact of dietary differences between wild and captive red deer on the fecal microbiota can help to assess and ensure the long-term viability of this species [9]. At present, the research methods for fecal microbiota have also shifted from traditional methods to 16S rRNA gene sequencing technology, from simple microbial composition, community structure, and core microbiota research to microbial function research, which has become a hot frontier in ungulate research today [22]. The main goal of this study was to characterize the composition of the fecal microbiota of red deer of different sex and feeding plus environment. We used high-throughput 16S rRNA sequencing technology to comprehensively analyze. Thus, we hypothesized that: (1) the fecal microbiota composition and function are different between wild and captive deer; and (2) under the wild or captive environment, the microbiota diversity and evenness are different between females and males. This study was conducted at the Gaogestai National Nature Reserve in Chifeng, Inner Mongolia (119°02′30″, 119°39′08″ E; 44°41′03″, 45°08′44″ N). The total area is 106,284 hm2. It is a typical transition zone forest-steppe ecosystem in the southern foothills of Greater Khingan Mountains, including forests, shrubs, grasslands, wetlands, and other diverse ecosystems. In February 2019, 75 line transects were randomly laid in the Gogestai protection area. Positive and reverse footprint chain tracking was carried out after the foodprints of red deer were found through line transect investigation. Disposable PE gloves were worn to collect red deer feces. While tracking the footprint chain, set 2 m × 2 m plant quadrate every 200 m to 250 m along the footprint chain, and collect all kinds of plant branches eaten by deer in the quadrate as far as possible [23]. A total of 162 fecal samples were collected and stored at −20 °C within 2 h. The feces of red deer from different areas of the Reserve were identified as coming from different individuals, and 43 feces were identified individually in the laboratory. In February 2019, the HanShan Forest Farm in Chifeng City, Inner Mongolia, China (adjacent to the Gaogestai Nature Reserve) had a total of 11 healthy adult red deer of similar age and size. Ear tags were used to differentiate each individual red deer. Through continuous observation, feces were collected immediately after excretion by different red deer individuals and stored at −20 °C. We measured crude protein, energy, neutral detergent fiber (NDF), and total non-structural carbohydrates in red deer diets. We used a qiaamp DNA Fecal Mini-kit (QIAGEN, Hilden, Germany) to extract host deoxyribonucleic acid (DNA) from the fecal samples of red deer as previously described [24]. Microsatellite PCR technology was used with nine pairs of microsatellite primers (BM848, BMC1009, BM757, T108, T507, T530, DarAE129, BM1706, and ILST0S058) [25,26] with good polymorphism that were selected based on the research results of previous studies. These nine pairs of primers can amplify fecal DNA stably and efficiently. A fluorescence marker (TAMRA, HEX, or FAM) was added to the 5′ end of upstream primers at each site (Supplementary Table S1). Primer information, PCR amplification, and genotype identification procedures are described in the literature [27]. Multi-tube PCR amplification was used for genotyping [28], and 3–4 positive amplifications were performed for each locus to determine the final genotype [29]. The excel microsatellite toolkit [30] was used to search for matching genotypes from the data. Samples are judged to be from the same individual if all loci have the same genotype or if only one allele differs at a locus. The microsatellite data were analyzed by Cervus 3.0 software, and the genotyping was completed [31]. Male and female individuals were identified by detecting the existence of genes after the individual identification of red deer was completed. Sry gene primers (F:5′-3′ TGAACGCTTTCATTGTGTGGTC; R:5′-3′ GCCAGTAGTCTCTGTGCCTCCT) were designed, and the amplification system was determined. To minimize the occurrence of false positives or false negatives that could affect results, the Sry gene was repeated three times to expand and increase during the experiment, and samples with target bands that appeared on the second and third occasions were determined to be male [32]. The total microbial DNA of fecal samples was extracted using an E.Z.N.A® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA). The DNA integrity of the extracted samples was determined by 1% agarose gel electrophoresis. Targeting a 420 bp fragment encompassing the V4-V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR using primers 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 907R (5′-CCG TCA ATT CMT TTR AGT TT-3′). NEB 154 Q5 DNA high-fidelity polymerase (NEB, Ipswich, MA, USA) was used in PCR amplifications (Supplementary Table S1). A 1:1 mixture containing the same volume of 1XTAE buffer and the PCR products were loaded on a 2% agarose gel for electrophoretic detection. PCR products were mixed in equidensity ratios. Then, the mixture of PCR products was purified using the Quant-iTPicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were generated using the TruSeq Nano DNA LT Library Prep kit (Illumina, San Diego, CA, USA) following the manufacturer’s recommendations, and index codes were added. The library’s quality was assessed on the Agilent 5400 (Agilent Technologies Co. Ltd., Santa Clara, CA, USA). At last, the library was sequenced on an Illumina NovaSeq 6000 platform, and 250 bp paired-end reads were generated. Microbiome bioinformatics were performed with QIIME2 2019.4 [33] with slight modification according to the official tutorials (https://docs.qiime2.org/2019.4/tutorials/ (accessed on 30 September 2022)). Briefly, raw data FASTQ files were imported into the format that could be operated by the QIIME2 system using the qiime tools import program. The DADA2 [34] process is to obtain amplified variant sequences through de-duplication. In the process, clustering is not carried out based on similarity, but only de-duplication is carried out. Demultiplexed sequences from each sample were quality filtered and trimmed, de-noised, merged, and then the chimeric sequences were identified and removed using the QIIME2 DADA2 plugin to obtain the feature table of amplicon sequence variants (ASV) [34]. The QIIME2 feature-classifier plugin was then used to align ASV sequences to a pre-trained GREENGENES 13_8 99% database (trimmed to the V4V5 around a 420bp region bound by the 515F/907R primer pair) to generate the taxonomy table [35]. In order to unify the sequence effort, samples were rarefied at a depth of 25,318 sequences per sample before alpha and beta diversity analysis. Rarefaction allows one to randomly select a similar number of sequences from each sample to reach a unified depth. Sequence data analyses were mainly performed using QIIME2 and R software (v3.2.0). ASV-level alpha diversity indices, such as the Chao1 richness estimator and Pielou’s evenness, were calculated using the ASV table in QIIME2 [36,37], and visualized as box plots (R software, package “ggplot2”). Beta diversity analysis was performed to investigate the structural variation of microbial communities across samples using weighted or unweighted UniFrac distance metrics [38,39] and visualized via principal coordinate analysis (PCoA) (R software, package “ape”). The significance of differentiation of microbiota structure among groups was assessed by PERMANOVA (permutational multivariate analysis of variance) [40]. Random forest analysis (R software, package “randomForest”) was applied to sort the importance of microbiota with differences in abundance between groups and screen the most critical phyla and genera that lead to microbial structural differences between groups using QIIME2 with default settings [41,42]. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (Picrust2) [43] is software that predicts the functional abundance from the sequencing data of marker genes (typically 16S rRNA). An ASV’s abundance table is used for standardization, and the corresponding relationship of each ASV is compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) library to obtain the functional information and functional abundance spectrum. A total of 22 red deer individuals were identified from 43 fecal samples, including 12 males and 10 females (Supplementary Table S2). The female captive deer were CF1, CF2, CF3, CF4, CF5, CF6, CF7, and CF8. The male captive deer were CM1, CM2, and CM3. We divided all the red deer (22 wild and 11 captive) into four groups: wild females (WF) (n = 10), wild males (WM) (n = 12), captive females (CF) (n = 8), and captive males (CM) (n = 3). The information about identification, location, sex, and diet is summarized in Supplementary Table S2. The wild red deer were fed on 16 species of plants in the winter. The edible plants belonged to 16 species of 16 genera and 9 families. Since the frequency of occurrence of other edible plants in red deer, such as Mongolian oak (Quercus mongolica) and Chinese maple (Acer sinensis), was less than 7%, the nutrient content of these plants was not measured. In addition, we hypothesized that they had little influence on the nutritional strategy of red deer. Therefore, the primary nutrient contents of 14 types of edible plants were determined. The food and nutritional composition of wild red deer are shown in Supplementary Table S3. When the captive red deer were fed, each type of food was fed separately at different times. The nutritional content of the primary food of captive red deer from the farm (adjacent to the Gaogestai Nature Reserve) in winter is shown in Supplementary Table S4. Only one kind of diet were provided to captive deer at each feeding time with all captive deer feeding together. Captive red deer feed on leaves and high protein given by artificial feeding. Compared with captive red deer, wild deer have a wider feeding range and no dietary limitations. Substantial differences exist between these two feeding methods. A total of 1,561,654 high-quality sequences were obtained from the fresh winter feces of 22 wild deer and 11 captive deer. Rarefaction curves based on the Chao1 diversity index reached asymptotes at 22,500. The results showed that with the increase in amount of sequencing, the curve tended to be flat and no longer changed, indicating that the amount of sequencing in this study basically reflected the diversity of red deer fecal microbiota in this study (Supplementary Figure S1). A total of 15,228 ASVs were obtained using a 100% similarity clustering method. The WF, WM, CF, and CM groups included 3056 ASVs, 3924 ASVs, 6661 ASVs, and 1587 ASVs, respectively. We found significant differences in fecal microbial composition between wild and captive red deer based on environment. The fecal microbial communities of four groups (WF, WM, CF, and CM) were dominated by the phyla Firmicutes and Bacteroidetes (Figure 1A). The phylum Firmicutes was the most abundant in WF (81.12 ± 2.87%), followed by WM (79.03 ± 2.19%), CF (58.24 ± 3.17%), and CM (59.66 ± 0.47%). Secondly, Bacteroidetes was abundant in WF (15.19 ± 2.09), WM (16.89 ± 2.08%), CF (33.02 ± 5.48), and CM (31.55 ± 1.61%). At the genus level, the genera from the four groups with abundance > 1% were Oscillospira, a candidate genus 5-7N15 from the family Bacteroidaceae, Ruminococcus, Roseburia, Clostridium, and Prevotella (Figure 1B and Table 1). The chao1 diversity indices demonstrate a significant difference between the WF and WM groups (p < 0.01). There was no statistically significant difference between the CF and CM groups (p > 0.05). Pieluo’s diversity index showed that no significant differences occurred between WF and WM groups (p > 0.05) or CF and CM groups (p > 0.05) (Figure 2). Wild and captive red deer also differed in beta-diversity. An PCoA plot based on the Unweighted Unifrac and Weighted Unifrac distance matrix revealed clear separation of the fecal microbiota between wild and captive red deer (Figure 3A). The results of a PCoA analysis showed that the fecal microbial structures of the CF and CM groups were more similar than those of the WF and WM communities (F = 13.82, p = 0.001; and unweighted: F = 5.983939, p = 0.001; Figure 3A; Supplementary Table S5). A random forest analysis showed that Firmicutes and Bacteroidetes were the primary microorganisms that had differences between the wild and captive populations by (an importance > 0.1) (Figure 3C, D). This analysis indicated that there were significant differences in the abundances of Firmicute and Bacteroidetes between the four groups (an importance > 0.1), which were the primary phyla that caused differences in the microbial communities between groups (Figure 3C). Ruminococcus, Treponema, Akkermansia, a candidate genus 5-7N15 belonging to family Bacteroidaceae, and a candidate genus rc4-4 belonging to family Peptococcaceae were the main genera that caused differences in microbial communities between sex and environment (importance > 0.04; Figure 3D). Metabolism was found to be the most common function prediction performed on fecal microbial communities and included the most important pathways for microbial clustering (76.67%). The second pathway of metabolism included amino acid metabolism (17.26%), carbohydrate metabolism (17.85%), metabolism of cofactors and vitamins (16.57%), and metabolism of terpenoids and polyketides (12.66%) (Figure 4A). A PCoA analysis showed that the WF and WM groups had more similar microbial function clusters (Figure 4B). It was found that there were significant differences in the three metabolic pathways of glycan biosynthesis and metabolism (GBM), energy metabolism (EM), and metabolism of other amino acids (MAA) (p < 0.05) (Figure 5). This is the first study to apply high-throughput sequencing to describe the fecal bacterial microbiota of wild and captive red deer by sex. Analysis of the differences in fecal microbiota is a key step in releasing captive red deer to help expand the wild population. In general, the fecal bacterial microbiota of red deer was similar to that of other cervidae, such as elk (Cervus canadensis), white tailed deer (Odocoileus virginianus) [38], and white-lipped deer (Cervus albirostris) [39], at least at the bacterial phylum level, with high proportions of the phyla Firmicutes and Bacteroidetes. In the digestive tract of herbivores, the role of Firmicutes is mainly to decompose cellulose and convert it into volatile fatty acids, thereby promoting food digestion and host growth and development. The enrichment of Firmicutes plays an important role in promoting the ability of red deer to obtain abundant nutrients from food and, at the same time, affects the metabolic function of the fecal microbiota. Bacteroidetes can improve the metabolism of organisms, promote the development of the gastrointestinal immune system, participate in the body’s bile acid, protein, and fat metabolisms, and also have a certain regulatory effect on carbohydrate metabolism. It can also produce special glycans and polysaccharides, which have a strong inhibitory effect on inflammation [43]. Differences in microbiota may be explained by changes in diet. Data from previous local and overseas studies have shown that diet is the main factor affecting the gut microbiota in mammals [40]. It is likely that wild deer have a more varied diet, more than captive deer. These phyla, Firmicutes and Bacteroidetes, are involved in important processes such as food digestion, nutrient regulation and absorption, energy metabolism, and host intestinal defense against foreign pathogens [40,41,42]. Alpha diversity alterations may be attributed to differential diet or hormonal influences on the gut microbiota. Fecal microbiota richness in wild populations is higher than that in captive animals, such as the Tibetan wild ass (Equus kiang), bharal (Pseudois nayaur), Tibetan sheep (Ovis arise), and yak (Bos mutus) [44,45,46,47,48]. Nevertheless, other studies also found that captivity might increase the alpha diversity of fecal microbiota in most Cervidae compared with other animals, for example, sika deer (Genus Cervus), Père David’s (Elaphurus davidianus), and white-tailed deer (Odocoileus virginianus) [49,50]. It may be that some environmental stresses in the wild or the special structure of the stomach and intestines in these deer lead to decreased alpha diversity of fecal microbiota in wild deer [50]. This phenomenon needs further research to determine its cause. Our results showed that the richness of the fecal microbial community in wild red deer differed by sex (Figure 2). In wild deer, the microbiota diversity was higher for females than males. Microbial community alterations by sex could be attributed to hormonal [51]. The sampling time was during the gestation period of red deer. Levels of female growth hormone during pregnancy may affect the fecal microbiota. Reproductive hormones have also been associated with sex and gut microbial changes in wild animals [17,52,53]. Increased evidence indicates that sex steroid hormone levels are associated with the human gut microbiota [54,55]. Futher, Edwards et al. reported that estrogen and progesterone had an impact on gut function [56]. The captive deer also had the smallest sample size (n = 3 males and 8 females), which limited our ability to detect these differences. In this study, the functional pathway composition of wild red deer is more similar (Figure 5B), which is completely opposite to the microbial structure (Figure 3A). The change in microbial structure does not necessarily lead to the change in function, which may be due to the same function in different microbial communities [57]. In recent years, studies have shown that gut microbiota are involved in various metabolic processes such as amino acids, carbohydrates, and energy, confirming their primary role in assisting host digestion and absorption [58]. It has also been found to be involved in environmental information processing, suggesting that the gut microbiota plays an important role in facilitating acclimation to changing environments [59]. The metabolism of gut microbiota is closely related to the feeding habits of the host. In the long-term evolution process, the gut microbiota will respond to changes in diet types or specific diets by adjusting the content of certain digestive enzymes [4,60]. Studies have shown that the decrease of fecal microbial diversity can lead to a reduction in the functional microbiota, in the efficiency of the microbiota, and in the resistance to pathogen invasion [61]. The decrease in fecal microbial diversity in captive populations resulted in a decrease in functional microbiota [61]. Ruminococcaceae and Lachnospiraceae are two of the most common bacterial families within the Firmicutes phylum [62]. It has been hypothesized that they have an important role as active plant degraders [63,64]. According to our results, the level of Ruminococcaceae in the captive groups is significantly lower than that in the wild group, which could suggest that the fiber-reduced diet in captivity is modifying the ability of the fecal microbiota to degrade recalcitrant substrates such as cellulose, hemicellulose, and lignocellulose, among others, that are commonly found on the main resources of the wild red deer diet. The captive deer’s consequent reduction of diet resources might trigger the decline of important metabolic pathways associated with nutrient use [64]. 16S rRNA analysis constitutes a valuable and cost-efficient approach for surveillance and monitoring wild populations as well as captive individuals. Picrust2 prediction accuracy is dependent on the availability of closely related annotated bacterial genomes in the database and the phylogenetic distance from the reference genome. However, the prediction results are still uncertain, which does not mean that the correlation between the predicted genes and the real metagenome of the microbiota is 100% [65]. At present, due to the difficulty of cultivation, the mechanism by which some functional bacteria exert their effects remain unclear. Therefore, in the follow-up work, it is necessary to repeatedly cultivate the conditions of some intestinal anaerobic bacteria, the most extensive of which are Firmicutes and some Bacteroidetes. The microbiota was cultured in vitro by simulating the gut environment, and its functions were speculated and further verified in combination with multiple groups of studies (metagenomics, meta transcriptome, and proteome, etc.). At the same time, the unknown functional microbiota and its genome sequence information can be explored and studied. These works will help to understand the metabolic activities of the complex microbiota and further explore the host physiological processes involved in gut microbiota. In conclusion, our study provided information on the structure and function of the fecal microbiome of red deer through the 16S rRNA gene of fecal samples. Comparing analyses identified significant variations of fecal microbiota composition and functions between captive and wild populations and also indicated that environment and sex have a great influence on these variations. These findings were of great significance for the reintroduction of captive red deer, given that the differences in fecal microbiota composition and functions between captive and wild red deer would greatly impact the ability of captive red deer to adapt to the wild environment. For further study, incorporating novel methods (e.g., transcriptome) to study the functional annotation of gene content and the functional traits of the host would be essential for better understanding the physiology and immunology of red deer.

PMC10000043

Asmaa Sadat,Alshimaa M. M. Farag,Driss Elhanafi,Amal Awad,Ehab Kotb Elmahallawy,Noorah Alsowayeh,Manal F. El-khadragy,Gehad E. Elshopakey

Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection

01-03-2023

mastitis,E. coli,S. aureus,oxidative/antioxidant molecules,APP,inflammatory cytokines

Simple Summary Establishing reliable biomarkers of udder bacterial infection and its bovine immune response at the early stage of the mastitic infection is considered an urgent matter. Thus, reliable biomarkers were measured in our study for accurate detection of the changes in biochemical and immunological parameters related to both clinical and sub-clinical mastitis. Ultimately, APP and cytokines, along with antioxidant markers can be used as early indicators of subclinical and clinical mastitis. Abstract The study aimed to investigate the mastitis’ emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1β, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis.

Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection Establishing reliable biomarkers of udder bacterial infection and its bovine immune response at the early stage of the mastitic infection is considered an urgent matter. Thus, reliable biomarkers were measured in our study for accurate detection of the changes in biochemical and immunological parameters related to both clinical and sub-clinical mastitis. Ultimately, APP and cytokines, along with antioxidant markers can be used as early indicators of subclinical and clinical mastitis. The study aimed to investigate the mastitis’ emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1β, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis. In veterinary medicine, bovine mastitis is considered one of the most common and economically important diseases affecting dairy herds worldwide. It mostly causes major economic losses [1,2]. Hence, the economic impact of mastitis is usually due to decreased milk production, increased costs of veterinary treatment, and premature culling of infected animals [3]; in addition, it causes alterations in the milk and udder physically, chemically, and pathologically [4,5]. Staphylococcus aureus is one of the major common pathogens causing mastitis in dairy animals [6]. The ability of the organism to cause infections is probably due to the expression of various toxins, virulence factors, and cell wall adhesion proteins. The bacterium can survive phagocytosis in the udder and often causes chronic inflammation [7]. These infections frequently do not respond to routine therapy. However, other microorganisms may be attributed to mastitic infections like Escherichia coli, Salmonella spp., and Streptococci or Listeria spp. [8,9,10]. Bovine mastitis poses a great one-health concern and zoonotic danger to humans due to the capability of this food-borne pathogen to be transmitted through milk or even through workers to consumers [3] The use of antimicrobial agents on cattle farms is often useful but the excessive use of it to control or prevent the mastitic infections may be contributing to the global antimicrobial resistance development [3]. Besides, antimicrobial residue leads to a decrease in the quality of milk [11]. Bovine mastitis is classified into two categories: clinical mastitis and subclinical mastitis; clinical mastitis is characterized by observable clinical signs of the udder and signs of inflammation with general health disturbance [12,13,14,15]; subclinical mastitis is difficult to be detected at once due to the lack of visible signs. It takes days or weeks to be observed [13], producing huge losses in the milk production due to the long-time persistence of infection and alterations of physical and chemical properties of the milk [16]. Usually, subclinical mastitis is more common than clinical mastitis [17,18]. The number of Somatic cells in the milk may be considered a diagnostic marker for subclinical mastitis [13]; these somatic cells consist of macrophages, neutrophils, lymphocytes, and some mammary epithelial cells. In the case of the healthy udder, macrophages are the predominant cell, while during early mastitis neutrophils become the predominant cells [19]. Host-pathogen interactions lead to the activation of the innate immune response to recognize infected microorganisms or rechange immune components by increasing the number of macrophages and releasing cytokines, which recruit leukocytes at the site of infection and trigger the local and systemic acute phase response [19,20]. Furthermore, acute phase proteins such as haptoglobin, cathelicidins, and peptidoglycan recognition protein were recorded at higher rates during infection [21]; similarly, high levels of IL-1β, IL-8, and TNF-α were expressed [22]. The pathogenesis of udder bacterial infection and bovine immune response must be studied to establish reliable biomarkers which can be detected at the early stage of the infection. Thus, these reliable biomarkers were measured in our study for accurate detection of the changes assigned with the bacteriological agent invasion and accompanied changes in biochemical and immunological parameters related to both clinical and sub-clinical mastitis. Meanwhile, the antimicrobial resistance of our strains was assessed to measure the current danger of multidrug resistance strains affecting public health. The present study was conducted in January 2021 on 100 dairy cows: healthy cows (n = 20), clinical mastitis-infected cows (n = 30), and subclinical mastitis-infected cows (n = 50) from two different farms located on the road of Gamasa city, Dakahlia Governorate, Egypt. Their ages ranged between 4 and 7 years. All dairy cattle diagnosed in our study were Holstein Friesian. The study was performed on cows that had not received any medication in the week before sampling. On the farm, modern management techniques and good hygiene standards were applied. Only automatic milking machines were used at both farms. All investigated cows were subjected to a thorough clinical examination. After milking the diseased cows, the size, the conformation of the udder, and the relative size of all quarters were inspected. The udder tissue and supramammary lymph nodes were also thoroughly examined. Cows with clinical mastitis were identified if one or more of the following signs were observed: cardinal signs of inflammation in one or more of an udder’s quarters, signs of systemic reactions (fever, depression, and disturbed appetite), or abnormal physical character of milk (clot formation, discoloration, and alterations in viscosity, aberrant smell, and the presence of blood). Due to the absence of observable clinical signs in cows with subclinical mastitis, the presumptive diagnosis was based on the laboratory diagnostic tests of milk samples, the California mastitis test (CMT), and somatic cell count (SCC). Cows with positive CMT or having SCC > 200,000 cells/mL and lacking clinical signs were considered affected with subclinical mastitis. Briefly, the California mastitis test (CMT), [23] was performed as follows: a plastic vessel with 4 shallow wells was used for collecting approximately 2 mL of milk from each udder quarter. Then, an equal amount of alkali reagent (kerbl® reagent) was added. A gentle circular motion was applied to the mixtures in a horizontal plane for 5 s and the different degrees of gel were recorded, according to the system used in the Nordic countries as the scoring is made from 1–5. Meanwhile, all the milk samples were examined automatically for somatic cell count by using The Nucleo Counter® SCC-100™. The sample was warmed in a water bath at 35 °C for 5 min and then mixed automatically before reading [24]. All procedures were performed following the principles and specific guidelines presented in the Mansoura University Animal care and approved by its Ethical Committee. Milk and blood samples were collected from each individual dairy cow (n = 100), including healthy cows (n = 20), clinical (n = 30), and subclinical mastitis cows (n = 50). The teats were wiped using alcohol for sterilization and all samples were taken in sterilized vials. After milk samples were taken aseptically, they were transported in coolers (4 to 8 °C) for subsequent bacteriological analyses within 6 hrs. All milk samples were sent to the Department of Bacteriology, Mycology and Immunology lab, Faculty of Veterinary Medicine, Mansoura University, Egypt. For blood samples, the halter was used to position the animal’s head in a slightly elevated manner to expose the jugular vein with minimal restraint to get the blood samples without causing injury. One blood sample was collected with an anticoagulant (EDTA) for complete blood counting. The second blood sample was collected in a heparinized tube which was rapidly centrifuged at 3000× g for 10 min for separation of blood plasma. The collected plasma was used for the estimation of fibrinogen. The third blood sample was collected in plain test tubes, left for 15 min to clot, and centrifuged at 3000 rpm (4 °C) for serum separation. The separated serum was stored at −80 °C for further determination of biochemical, inflammatory, and oxidative stress/antioxidant parameters. All collected milk samples were centrifuged for 10 min. A drop from the sediment was cultivated onto Baird-Parker agar, 5% sheep blood agar, MacConkey’s agar, and Mannitol salt agar, (Oxoid, Ltd., Basingstoke, UK) at 37 °C for 24–48 h. Bacterial colonies were classified according to their phenotypic characters on the culture media. All milk samples that give more than two bacterial species on bacterial culture plates were considered contaminated samples and were discarded. Presumptive characterization of the isolated bacteria was carried out based on Gram’s stain and biochemical characteristics according to Quinn and his group [25]. The DNA was extracted from all suspected isolates by the boiling method [26]. Two to three colonies from suspected S. aureus and E. coli isolates were suspended in 200 uL deionized free water and boiled for 10 min, and then centrifugated for 10 min. The supernatants were transferred to a new sterile Eppendorf tube and stored at −20 °C as a DNA sample. All the isolates suspected to be S. aureus by biochemical typing were confirmed by amplification of species-specific nuc gene (encoding for the S. aureus-specific thermonuclease) using the primers: nuc-F:(GCGATTGATGGTGATACGGTT) and nuc-R: (AGCCAAGCCTTGACGAACTAA AGC) by using PCR amplification technique according to Sallam et al. [27]. In brief, A 25 μL PCR reaction mixture contained 5 μL of DNA template, 12.5 μL of 2X PCR master mix (Thermo Scientific, United States), 6.5 μL of deionized nuclease-free water, and 1 μL of each primer. PCR conditions were 94 °C for 2 min for initial denaturation, followed by 35 cycles of 98 °C for 10 s, 58 °C for 30 s, and 68 °C for 60 s, then 68 °C for 7 min as a final extension step; this cyclic reaction was run using a 96 well Applied Biosystem, 2720 thermal cycler. The PCR products were visualized using 1% agarose gel using a UV transilluminator and a Gel Documentation System (cleaver scientific ltd UV gel documentation system, USA). S. aureus strain from a previous study was used as a positive control [28]. Furthermore, all the biochemically characterized E. coli isolates were subjected to PCR amplification for encoding of the Genus-specific primer 16S-rRNA gene using the following primer pair: 16S-rRNA-F-GCGGACGGGTGAGTAATGT and 16S-rRNA-R-TCATCCTCTCAGACCAGCTA (Table 1). PCR protocol was performed as per Teichmann et al. [29]. The reaction was performed in a total volume of 25 μL with 5 μL DNA template, 12.5 μL 2X PCR master mix (Thermo Scientific, United States), 6.5 μL deionized nuclease-free water, and 1 μL of each primer. A 96-well Applied Biosystem and a 2720 thermal cycler were used for PCR amplification. The following PCR amplification reaction was applied: initial denaturation for 5 min at 94 °C; 30 cycles of 94 °C for 15 s, 69 °C for 30 s, and 72 °C for 30 s; final extension for 7 min at 72 °C. PCR products were run on 1% agarose gel and were visualized using a UV transilluminator and a Gel Documentation System (cleaver scientific ltd UV gel documentation system, USA). The strain used as PCR positive control was supplied from a previous study [30]. All the confirmed bacterial isolates were characterized for their antimicrobial sensitivity tests on Muller Hinton agar (Oxoid, Ltd., Basingstoke, UK) by using the disc diffusion technique according to CLSI [31]. Firstly, the twenty-one E. coli isolates were tested for antimicrobial sensitivity against 12 different antimicrobial compounds by disc diffusion test. The following antimicrobial discs (Oxoid, Ltd., Basingstoke, UK) were used: penicillin (10 mg), cefuroxime (30 mg), cefoperazone (30 mg), amikacin (30 mg), streptomycin (15 mg), neomycin (5 mg), azithromycin (15 mg), nalidixic acid (30 mg), trimethoprim-Sulfamethazole (25 mg), gentamycin (10 mg), chloramphenicol (30 mg), and rifamycin (5 mg) (Table 2). In addition, thirty-eight S. aureus isolates were examined against 10 antimicrobial agents for their antimicrobial sensitivity using a disc diffusion test on Muller Hinton agar (Oxoid, Ltd., Basingstoke, UK). These antimicrobial discs (Oxoid, Ltd., Basingstoke, UK) were ampicillin (10 mg), oxacillin (15 mg), ceftazidime (30 mg), kanamycin (30 mg), streptomycin (15 mg), norfloxacin (10 mg), ciprofloxacin (5 mg), chloramphenicol (30 mg), tetracycline (30 mg), and gentamycin (10 mg) (Table 2). The results were interpreted according to CLSI [31]. Resistance to more than two antibiotics from different antimicrobial classes was recorded as MDR [32]. A multiple antibiotic resistance (MAR) index was calculated according to Krumperman [33]. Red blood cell count (RBCs), hemoglobin (Hb), hematocrit value (PCV), and total and differential leukocytic count were analyzed according to Morar et al. [34]. The serum activity of aspartate aminotransferase (AST, catalog No.; AS101) was estimated using kits obtained from the Randox company (Kearneysville, VA, USA). Lactate dehydrogenase (LDH, catalog No.; TK41214), total protein (Catalog No.; MD1001291), and albumin (Catalog No.; MX1001020) were assessed using kits obtained from the Spinreact company (Santa Coloma, Spain) according to the described methods of its manufacturer’s instructions. Serum levels of acute-phase protein (APP) including haptoglobin (Catalog No.; ab137977), amyloid A (Catalog No.; ab274407), fibrinogen (Catalog No.; ab108842), ceruloplasmin (Catalog No.; ab108818), and ferritin (Catalog No.; ab108698) were estimated using kits obtained from the Abcam company (Cambridge, UK) according to the standard protocol of their specific pamphlets. Serum tumor necrosis factor-alpha (TNF-α, Catalog No.; MBS2609886), interleukins, IL-1β, IL-6, (Catalog No.; # ESS0027, and # RBOIL6I) (Invitrogen-Thermo Fisher Co., Waltham, MA, USA) and IL-10, (Catalog No.; ab277386) (Abcam Co., Cambridge, UK) levels were estimated using specific bovine ELISA commercial kits according to the methodology of each enclosed pamphlet. All parameters were measured spectrophotometrically using a 5010 Photometer (ROBERT RIELE GmbH & Co KG, Berlin, Germany). The serum levels of malondialdehyde, total antioxidant capacity (TAC) superoxide dismutase (SOD), and catalase were estimated spectrophotometrically following the illustrated approaches of Ohkawa et al. [35], Benzie and Strain [36], Nishikimi et al. [37], and Aebi [38], respectively. Data analysis was carried out using a statistical software program (SPSS for Windows, Version 21, Cary, NC, USA). The Kolmogorov–Smirnov test was selected to assess the normal distribution of the data. The assessed data were normally distributed; therefore, the means and standard mean of error (SME) for each variable were statistically analyzed and presented. The post hoc test with ANOVA (Tukey’s test) was used to assess statistical differences between the two groups. In all statistical analyses, the results were considered significant at p < 0.05. Out of all investigated mastitic milk samples, a total of 70% (21/30) E. coli isolates and 76% (38/50) S. aureus isolates were identified from clinical mastitis milk and sub-clinical mastitis milk, respectively, using PCR assay. All the other samples were discarded and counted as contaminated samples. A total of twenty milk samples from healthy cows were negative for bacterial culture. Due to the absence of observable clinical signs in animals infected with subclinical mastitis, a presumptive diagnosis was done based on laboratory diagnostic tests of milk samples using the CMT and SCC. Cows with positive CMT or those having SCC > 200,000 cells/mL but lacking clinical signs were considered affected with subclinical mastitis. The results of antimicrobial susceptibility testing of E. coli isolates (n = 21) are listed in Table 2. E. coli isolates were resistant to penicillin, cefuroxime, cefoperazone, azithromycin, nalidixic acid, trimethoprim-sulfamethoxazole, rifamycin, and gentamycin. They displayed intermediate resistance to neomycin (61.9%), amikacin (52.4%), and chloramphenicol (47.6%). E. coli isolates were more sensitive to streptomycin (38.1%). Multiple drug resistance (MDR) was detected in all tested E. coli isolates (resistant to ≥3 antimicrobial class) and the most prevalent antimicrobial pattern was ‘’C, P, DA, STX, RA, N, NA, CXM, CEP, AZM’’ (Table 3). Moreover, the antimicrobial susceptibility testing of S. aureus isolates (n = 38) is listed in Table 2. S. aureus isolates were highly resistant to ampicillin, oxacillin, and gentamycin (76.3%, 76.3%, and 73.7%), respectively, and they displayed intermediate resistance to ceftazidime (52.6%), kanamycin (47.4%), and streptomycin (50%). S. aureus was more sensitive to norfloxacin (34.2%), ciprofloxacin (36.8%), chloramphenicol (26.3%), and tetracycline (23.7%). Multiple drug resistance (MDR) was detected in 94.74% of the total tested isolates (resistant to ≥3 antimicrobial classes). Only two S. aureus strains were found to be sensitive to all antimicrobials tested. The most prevalent antimicrobial pattern was K, NOR DA, TE, OX, CIP, CAZ, AMP, and S (Table 3). As presented in Table 4, the obtained results showed significantly lower RBCs count (p < 0.01), Hb (p < 0.001), and PCV (p < 0.01) values in mastitic cows compared with both subclinical mastitic and control cows. Moreover, WBCs (p < 0.01), and neutrophil (p < 0.001) counts were significantly diminished in clinical and subclinical mastitic cows compared to the controls. Significantly higher levels of AST (p < 0.001, p < 0.01), LDH (p < 0.001, p < 0.01), total protein (p < 0.001, p < 0.05), and globulin (p < 0.001, p < 0.05) in both mastitic and subclinical mastitic cows unlike that of the controls. However, the serum level of albumin (p < 0.05) statistically declined in mastitic cows only compared to other groups. To assess the mechanisms involved in the progression and damage of mammary gland tissue during mastitis, serum acute-phase proteins (APP) and inflammatory cytokines were estimated in our study. The haptoglobin (p < 0.001), fibrinogen (p < 0.01), and Amyloid A (p < 0.001, p < 0.01) levels were found to be highly elevated in the serum of both mastitic and subclinical mastitic cows unlike that of the controls (Figure 1). Moreover, ceruloplasmin (p < 0.05) levels were significantly raised in the serum of mastitic cows compared to the healthy controls (Figure 1D). No significantly valuable differences in serum ferritin levels of all the groups were identified (Figure 1E). TNF-α (p < 0.001), IL-1β (p < 0.001, p < 0.01), and IL-6 (p < 0.001, p < 0.01) were statistically increased in mastitic and subclinical mastitic cows compared to the healthy controls (Figure 2). Meanwhile, the IL-10 (p < 0.01) decreased only in mastitic cows relative to the remaining groups (Figure 2D). During the progression of mastitis, the bacterial infection caused the generation of an accentuated reactive oxygen species (ROS) and impairment of antioxidant molecules confirmed in our results by higher malondialdehyde (MDA) levels (p < 0.001), along with a reduction of total antioxidant capacity (TAC) (p < 0.05, p < 0.001) and catalase (p < 0.01, p < 0.001) in mastitic and subclinical mastitic cows compared to the control non-infected one (Figure 3). Superoxide dismutase (SOD) (p < 0.05) was statistically diminished only in mastitic cows compared to the controls (Figure 3C). Mastitis is a mammary gland inflammatory illness that causes huge, enormous economic losses in the dairy industry [39]. It occurs when a pathogenic microbe enters the mammary gland, usually by disrupting physical barriers like the teat canal [40]. Once the barrier is breached, a prompt and effective defense response is required to prevent the spread of pathogenic organisms and additional injury to mammary gland tissue [41]. Mastitis, in its clinical and subclinical forms, is considered one of the most devastating diseases that affect dairy herds and represents 21% of reported diseases in dairy cattle, with an annual incidence of 37% [42]. To our knowledge, there were no studies applied to clinical or subclinical mastitic cows that directly link the bacterial causative agent, and serum biochemical, antioxidant, and inflammatory markers (APP & cytokines) with mastitis risk. That is why the goal of this study was to investigate the bacterial cause and detect serum biochemical, antioxidant, and inflammatory markers in dairy cows that were linked to clinical and subclinical mastitis susceptibility. Mastitis is a complex disease with varied etiological causes, contagious bacterial, environmental, and opportunist [43]. In our study, the bacterial agent was detected in high proportion 73.75% (59/80) of all mastitic milk samples. This confirms previous studies which mentioned bacterial mastitis as the most common etiology [44,45]. Furthermore, our findings revealed that E. coli and S. aureus were the causative agents responsible for our mastitic cases which are in accordance with previous studies that described these two microorganisms to be the most common and significant bacterial agents responsible for mastitis [46]. E. coli is an environmental agent capable of invading the cow’s teat through ascending infection [47]. E. coli was recovered from 70% (21/30) of our clinical mastitis cases. Higher results were obtained in France and Egypt; investigating more than 80% of the clinical mastitic cases were caused by E. coli [48,49]. In Egypt, a previous report showed a slightly low occurrence (about 31%) of clinical mastitic cases where E. coli was the causative agent [50]. Ahmed et al. [51] recovered a low incidence (9.1%) of E. coli-related mastitic cases. S. aureus is a contagious microorganism that invades the udder and infects all the quarters. In our study, S. aureus recovered at a rate of 76% (38/50) from the subclinical cases which came following other studies in Egypt [52,53]. From Egypt, another study was found to have higher results than ours; about 91.48% of the samples were identified as S. aureus [54]. Antimicrobial resistance is an important hazard that is mentioned as the silent tsunami facing modern medicine. Nowadays, MDR has been a serious challenge facing scientists [55]. Examination of our isolates from farms that did not receive any previous medication the last week relived a dangerous result for MDR. Our E. coli mastitic strains showed extensive resistance against seven antimicrobial classes, β-lactam, cephalosporin, macrolide, quinolone, sulphonamide, lincosamide, and rifamycin. Previous reports discussed E. coli raised antimicrobial resistance against ampicillin, streptomycin, and sulfonamides [56,57,58]; in addition, extended-spectrum β -lactamases resistance was investigated in other reports [59,60]. These results might be attributed to the broad-spectrum antimicrobials, trimethoprim–sulfonamides, oxytetracycline, fluoroquinolones, cefquinome, and ceftiofur which are used in the farms for the systematic treatment of mastitic coliform [61,62]. Thus, E. coli have developed resistance to these extensively used antimicrobial classes. In this study, the isolated Staphylococcus aureus strains showed slightly lower antimicrobial resistance results than the E. coli strain. Our study exhibited higher resistance against ampicillin which came in accordance with previous studies [53,63]. S. aureus produces penicillinase enzyme which makes the treatment by β-lactamase antimicrobial class more problematic. Third-generation cephalosporin was considered for treatment of the mastitis cases caused by S. aureus. It was revealed that about half of the strains were ceftazidime resistant which was the same as other previous studies [53]. We observed that about 73% of the strains developed resistance to gentamicin which was higher than in other studies [64,65]. Reports from China showed that antimicrobial agents such as penicillin, ampicillin, streptomycin, gentamicin, ciprofloxacin, and sulfamethoxazole-trimethoprim were usually applied for mastitis in dairy farms [66]; these antimicrobial agents act by inhibiting protein synthesis [67,68]. Resistance against penicillin, ampicillin, erythromycin, tetracycline, and clindamycin was reported in different countries [69,70,71,72]. In this study, the low resistance against tetracycline and chloramphenicol was reasonable. Since tetracycline resistance is usually acquired by horizontal gene transfer; besides, chloramphenicol is not used anymore in the veterinary field. The proportion of MDR S. aureus in our study was 94.74%. These results are similar to previous studies from Malaysia and Brazil [71,73]. The inappropriate use of antimicrobials in farms may be involved in the emergence or spread of antimicrobial-resistant strains; these strains could be transmitted to humans via direct contact with animals or via ingestion of infected food [74,75]. Thus, misuse led to the environmental release of the antimicrobial resistance genes and resistant bacterium. In this case, the environment and the animals will serve as an end reservoir of the antimicrobial resistance genes [76]. This limited the options used for the therapy against these agents [77]. Periodical surveillance strategies must be taken to control this dramatic development of MDR bacterial strains [78]. Nowadays, the emergence of novel strategies such as the usage of antimicrobial combination, phage therapy, and peptide therapy had been studied [79,80]. Changes in hemato-biochemical parameters and leukocyte counts can be employed as crucial indications of the animal’s physiological or pathological status (mastitis). Following our results, the mastitis-affected cows showed a significant decrease in Hb, PCV, and RBCs counts, with an increase in TLC as compared to healthy cows [81]. These findings were also consistent with a prior study that found a significant fall in RBC, Hb, and PCV values in mastitis-affected animals, resulting in anemia [82]. Earlier studies also found that mastitic cows had a higher leukocyte, granulocyte count, lymphopenia, and anemia [83]. TLC levels were recorded to be increased in affected animals, as well as monocyte, neutrophil, and eosinophil count [82]. Various immunomodulatory actions may be responsible for the considerable increase in WBC and neutrophilic counts in mastitic cows [84]. Furthermore, Abba et al. [85] attributed those neutrophils enhanced the chemotactic molecules generated by infectious pathogens, as well as other immune system components that activate neutrophil recruitment to infection sites. Compared to healthy cows, the activities of AST and LDH exhibited a considerable increase in mastitic cows, which could be attributed to stressful conditions. Our findings were consistent with prior research [81,82,86,87]. In terms of total protein findings, our results revealed a significant increase in serum total protein and globulin with lower albumin levels in mastitic groups when compared to healthy ones. These findings agreed with Ali et al. [88] and Garba et al. [89] who found a significant increase in total protein levels with significantly lower albumin levels in cows with clinical and subclinical mastitis. Similarly, previous reports recorded high levels of globulin and total protein in mastitic cows [88,90]. The immunological response is the main cause of the decrease in albumin levels that are linked to udder infection [89]. As well, hypoalbuminemia may be interpreted as the stress that occurs during mastitis, which increases protein catabolism [91]. Increased serum globulin levels could be related to the development of antibodies in the form of gamma-globulin, which is responsible for neutralizing the invading microorganism’s effect [92]. Alternatively, Sarvesha et al. [93], and Krishnappa et al. [94] recorded the mastitic group had a significantly lower level of total proteins in mastitic in indigenous cows, crossbred cattle, and buffaloes. To identify cows with subclinical mastitis, laboratory diagnostics is crucial. Mastitis control is primarily dependent on determining SCC and the CMT, which aim to detect the number of cells in the milk sample. The other useful diagnostic tool is microbial culture, which complements SCC and CMT [95]. However, all mentioned diagnostic methods have their limitations and therefore novel biomarkers of subclinical mastitis are highly desired. These sensitive indicators include cytokines and acute phase protein measurements, particularly haptoglobin, fibrinogen, and amyloid A which could be determined in cow serum and/or milk, and in the future may become useful in early mastitis diagnostics as well as a preventive tool. This may contribute to increase the detection of mammary gland inflammation in cows, especially in subclinical form, and consequently improve milk quality and quantity. Over the last few decades, research has revealed that acute-phase protein quantitation (APP) e.g., ceruloplasmin, fibrinogen, haptoglobin, and amyloid A, can be found in blood plasma or serum and it can be used for disease diagnosis and prognosis detection, as well as to assess the degree of inflammation [96,97]. In the current investigation, we found that ill cows had significantly higher levels of haptoglobin, fibrinogen, amyloid A, and ceruloplasmin in subclinical and clinical mastitic animals than in the healthy cows. The production of proinflammatory cytokines like TNF-α, which exacerbate the inflammatory process and stimulate neutrophil phagocytic activity, could be to blame for the increased amounts of APPs [98]. The elevated levels of APP are in accordance with those obtained by Winter et al. [99] in cows with Listeria monocytogenes-induced mastitis, as well as in clinical and subclinical mastitic dairy cows [100,101]. Data about subclinical and clinical mastitis demonstrate inflammatory responses to the intramammary infection driven by IL-1β, IL-6, and TNF-α. Moreover, the host defense response in mastitis is characterized by the continuation or resolution of initial inflammation [102]. Additionally, our aim of the study was to determine inflammatory and regulatory cytokines in the serum of dairy cows with subclinical and clinical mastitis and to help in the diagnosis of subclinical or clinical mastitis. Knowledge about the inflammatory and regulatory cytokines in naturally occurring mastitis is lacking as most studies focus on pathogen-induced mastitis. In naturally occurring mastitis, the study of cytokines is a potential tool for early and timely diagnosis and in prospective pathogenesis-based treatment. Our finding recorded statistically increased TNF-α, IL-1β, and IL-6 in mastitic and subclinical mastitic cows compared to the healthy controls, while the IL-10 was decreased only in mastitic cows relative to the other groups. Similarly, it has been shown that pro-inflammatory cytokines are important in fighting the original infection. Increased levels of TNF-α and IL-1β in subclinical mastitis were discovered in this study, suggesting that they play a role in the early stages of mastitis development [103,104,105]. The pro-inflammatory cytokines IL-12, IL-6, TNF-α, and IL-1β were found to be significantly elevated in lactating cows suffering from clinical mastitis [102]. Variations in these proinflammatory concentrations corresponded to the onset of illness signs. As previously reported, increased pH and significant decreases in fat, SNF, protein, and lactose were identified in the mammary gland tissue of these cows [106]. In contrast to our findings, previous research showed a non-significant rise in IL-2 and IL-6 markers in cows with subclinical mastitis [107]. All the living organisms, particularly dairy cows, produce free radicals because of their active metabolism. Oxidative stress occurs when homeostasis is disrupted, which is mostly caused by the generation and accumulation of free radicals, which can lead to mastitis in dairy cows [108]. Antioxidants protect the body from oxidative damage caused by free radicals by scavenging them directly or by inhibiting the action of oxidizing enzymes [109]. In the present study, there was a significant increase in MDA, along with a reduction of TAC, SOD, and catalase in subclinical and clinical mastitic animals compared to the control non-infected ones. The considerable increase in MDA levels, as well as the significant drop in TAC and catalase activity, such findings could imply an increase in free radical activity, which would reflect the state of oxidative stress that happens in such situations [110]. Previously similar findings have been reported by Sharma et al., [111] and Jhambh et al. [112]. Increased consumption to neutralize ROS generated by the inflamed gland could explain the decreased antioxidant enzymatic activity, indicating a weakened antioxidant defense mechanism [90]. Furthermore, these changes could be due to the high demand for high SOD, catalase, and GPx activities for elevated levels of oxidant damage caused by inflammatory reactions in the mammary gland tissue or insufficient nutrition, which has a significant impact on the level of blood lipid peroxidation and lack of energy increases blood plasma levels of MDA in clinical mastitis animals [113]. Nonetheless, this study has limitations to discuss. our baseline scenario assumed that blood parameters were only affected by the mastitis inflammation caused by the bacterial infection, while the bacterial-caused mastitis might result in a decrease in the immune system efficiency which would permit the entrance of secondary invaders or decrease cows’ food intake causing a nutritional deficiency sign. Thus, to solve those limitations a future study on broad aspects should be done where a large scale of mastitic cows in different geographical regions inside Egypt examined for different microbiological, environmental, and nutritional disorders besides, performing a correlation between the revealed data to a better understanding the linkage of mastitis inflammation and microbes. This can be accomplished with the help of new computational biology techniques or through sophisticated technology and artificial intelligence algorithms. Moreover, the study emphasizes the necessity to evaluate additional cytokines to the already studied, including Interleukin-1 alpha (IL-1α) Interleukin-4 (IL-4), Interleukin-17 (IL-17), Interleukin-13 (IL-13), cathelicidin LL37, nuclear factor kappa B (NF-κB), and transforming growth factor-beta 1 (TGF-β1). Together would explain their relationships in detailed interrelations by correlation analysis to substantiate the knowledge of the broad cytokine participating in both host immune defense and mastitis inflammation. Finally, it might be the simplest way to promote the pasteurization of milk so that humans will not get exposed to resistant bacteria. In conclusion, the current findings show that dairy cow mastitis is related to significant immunological, and oxidative changes, including hematological, biochemical indicators, oxidative acute phase protein (APP), and inflammatory cytokine with the predisposing factors for mastitis resistance/susceptibility highlighted by our findings. These findings suggest that variations in these biomarkers could be utilized to diagnose such illnesses. The variable pattern of antioxidant, APP, and inflammatory cytokines in subclinical and clinical mastitic dairy cows could be a biomarker for bovine immune status which not only predicts the most susceptible risk time for disease occurrence but also builds up an effective management protocol to improve health through proper breeding and vaccination regimens.

PMC10000048

Duy Ngoc Do,Prashanth Suravajhala

Editorial: Role of Non-Coding RNAs in Animals

23-02-2023

Editorial: Role of Non-Coding RNAs in Animals The importance of non-coding RNAs (ncRNAs), such as microRNAs (miRNA), long non-coding RNAs (lncRNA), and circular RNAs (circRNA), in gene regulation is increasingly being appreciated in many species. Thanks to the next generation of sequencing methods, thousands of ncRNAs have been identified in different livestock species and their functions are undergoing characterization. Clearly, ncRNAs are involved in many gene regulation processes [1] and pathways related to economically important traits in livestock species [2,3,4,5,6]. In addition, ncRNAs are also considered to be potential biomarkers for infectious diseases in different farm animal species [7,8]. This Special Issue contains 13 articles summarizing the diverse roles of ncRNAs in livestock [9,10,11,12,13,14,15,16,17,18,19,20,21]. In particular, we report on different species of animal that have been used to study the functions of ncRNAs. These include chicken [12,15,18], pigs [11,17,21], goats [9], sheep [14,16], cattle [13,19], mice [20], and insects [10]. Exploring the expression of ncRNAs in different biological conditions is an important step in understanding their function. Bo et al. [9] identified 20,269 lincRNAs, including 16,931 novel lncRNAs, expressed in the testes of Yiling goats. The authors also suggested that ENSCHIT00000000777, ENSCHIT00000002069, and ENSCHIT00000005076 were the key lincRNAs in the process of testis development, a fact derived via co-expression analyses. Gan et al. [11] used RNA sequencing to identify 302 miRNAs expressed in pig umbilical venous blood (UVB) and umbilical arterial blood (UAB). As a result, 106 and 22 miRNAs were specifically expressed in the UVB and UAB, respectively. The authors also suggested that miR-423 and miR-122-5p can be used as characteristic miRNAs of UVB and UAB, respectively. Hao et al. [14] performed RNA-Seq to study the roles of lncRNAs in the mammary glands of lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes. The authors identified 1894 lncRNAs that were differentially expressed, among which 31 and 37 lncRNAs were up and downregulated respectively, when comparing STH ewes with GAM ewes. In addition, the authors also found that the development and proliferation of mammary epithelial cells via the enrichment of the target genes, followed by the morphogenesis of the mammary gland, ErbB signaling pathway, and Wnt signaling pathway could be important for mammary gland and milk yield regulation. To explore the function of lncRNAs in pig spleens at different stages of development, Li et al. [17] profiled the systematic characters of mRNA and lncRNA repertoires in three groups of spleens from nine Yorkshire pigs, with three aged seven days, 90 days, and 180 days, respectively. The authors identified 19,647 genes in addition to 219 known and 3219 putative lncRNA transcripts; 1729 genes and 64 lncRNAs therein were found to express differentially. The authors also found that differentially expressed genes and the potential target genes of differentially expressed lncRNAs both performed the crucial roles of up-regulation in immune activation and hematopoiesis, and down-regulation in cell replication and division, in 90- and 180-day-old pigs compared to seven-day-old pigs. MiRNAs are the most well-studied molecules in the ncRNAs. Several studies contained within this Special Issue attempt to validate their functions. Guo et al. indicated the potential roles of miR-149-5b in the regulation of bovine adipocyte differentiation [13]. Xu et al. show that, by targeting PTEN, MicroRNA-148a can regulate the proliferation and differentiation of ovine preadipocytes. Qiao et al. provided evidence of the role of miR-F4-C12 50 in the regulation of adipose accumulation, finding that it performed this role by targeting PIK3R1 in castrated male pigs [21]. Wang et al. [20] identified 852 known miRNAs and 179 novel miRNAs in female ICR mice mammary glands at the virgin stage, day 16 of pregnancy, day 12 of lactation, day 1 of forced weaning, and day 3 of forced weaning. The authors also discovered a novel miRNA (novel-mmu-miR424-5p) involved in regulating the expression of the CSN2 gene. Other studies also validated the function of circRNAs [18,19] and lncRNAs [15]. Regarding the circRNA function, Wang et al. indicated that circEZH2 can sponge miR-378b to regulate milk fat metabolism [19], while Shen et al. showed that circular PPP1R13B can target miR-9-5p to promote chicken skeletal muscle satellite cell proliferation and differentiation [18]. Lastly, via RNA sequencing and the follow-up validation, Jian et al. [15] confirmed that the roles of lncPGC in the regulation of primordial germ cell formation in chickens were mediated by TCF7L2. This Special Issue focused not only on livestock species but also on the roles of lncRNAs in insects. Choudhary et al. [10] provided an updated review of the functions and molecular mechanisms of the mode of action of different insect lncRNAs. Taken together, these contributions enlighten the research community on contemporary breakthroughs and suggest approaches for performing future functional studies of the regulatory roles of ncRNAs in animals. We anticipate that both expert scientists and readers can benefit from the state-of-the-art studies addressing the roles of ncRNAs contained within this Special Issue.

PMC10000050

Rosa Marchetti,Valerio Faeti,Maurizio Gallo,Massimo Pindo,Davide Bochicchio,Luca Buttazzoni,Giacinto Della Casa

Protein Content in the Diet Influences Growth and Diarrhea in Weaning Piglets

22-02-2023

piglet,post-weaning diarrhea,dietary protein,fecal microbiota,feces composition

Simple Summary Weaning (that is, removal from the sow) and the following two months are the riskiest periods in a pig’s life, especially for pig’s gastrointestinal health. The change in diet due to the suspension of the mother’s milk, accompanied by an acceleration of both morphological and enzymatic maturation of the intestinal mucosa of the piglets, can worsen digestion and absorption. In this context, the protein requirement of piglets, which are in a phase of rapid growth, may be greater than the intestine’s ability to digest proteins. Undigested proteins are the best pabulum for the proliferation of the pathogenic bacterial flora that causes diarrhea. Since these problems can no longer be resolved with prophylactic use of antibiotics, the best balance between intestinal health and growth performance must be found. A diet low in crude protein and supplemented with synthetic amino acids can help achieve this goal. Abstract The aim of this research has been to assess the effect of the dietary protein level on piglet growth and post-weaning diarrhea (PWD) incidence. Piglet fecal microbiota and feces composition were also assessed. The experiment was carried out on 144 weaned piglets (Duroc × Large White; 72 piglets per treatment) and lasted from weaning (at 25 days of age) until the end of the post-weaning phase (at 95 days). Two dietary protein levels were compared: high (HP; 17.5% crude protein on average, during the experiment) and low (LP; 15.5% on average). Lower (p < 0.01) average daily gain and feed conversion ratio were observed in LP piglets in the first growth phase. However, at the end of the post-weaning period, the growth parameters were not significantly different in the two diets. Diarrhea scores were lower in piglets fed LP diets than in piglets fed HP diets (28.6% of the total vs. 71.4% in the HP piglets). Fibrobacteres, Proteobacteria, and Spirochaetes were more abundant in the feces of the piglets fed LP diets. Feces nitrogen content was lower in piglets fed LP diets. In conclusion, low protein levels in the diet can reduce the incidence of PWD while only marginally affecting growth parameters.

Protein Content in the Diet Influences Growth and Diarrhea in Weaning Piglets Weaning (that is, removal from the sow) and the following two months are the riskiest periods in a pig’s life, especially for pig’s gastrointestinal health. The change in diet due to the suspension of the mother’s milk, accompanied by an acceleration of both morphological and enzymatic maturation of the intestinal mucosa of the piglets, can worsen digestion and absorption. In this context, the protein requirement of piglets, which are in a phase of rapid growth, may be greater than the intestine’s ability to digest proteins. Undigested proteins are the best pabulum for the proliferation of the pathogenic bacterial flora that causes diarrhea. Since these problems can no longer be resolved with prophylactic use of antibiotics, the best balance between intestinal health and growth performance must be found. A diet low in crude protein and supplemented with synthetic amino acids can help achieve this goal. The aim of this research has been to assess the effect of the dietary protein level on piglet growth and post-weaning diarrhea (PWD) incidence. Piglet fecal microbiota and feces composition were also assessed. The experiment was carried out on 144 weaned piglets (Duroc × Large White; 72 piglets per treatment) and lasted from weaning (at 25 days of age) until the end of the post-weaning phase (at 95 days). Two dietary protein levels were compared: high (HP; 17.5% crude protein on average, during the experiment) and low (LP; 15.5% on average). Lower (p < 0.01) average daily gain and feed conversion ratio were observed in LP piglets in the first growth phase. However, at the end of the post-weaning period, the growth parameters were not significantly different in the two diets. Diarrhea scores were lower in piglets fed LP diets than in piglets fed HP diets (28.6% of the total vs. 71.4% in the HP piglets). Fibrobacteres, Proteobacteria, and Spirochaetes were more abundant in the feces of the piglets fed LP diets. Feces nitrogen content was lower in piglets fed LP diets. In conclusion, low protein levels in the diet can reduce the incidence of PWD while only marginally affecting growth parameters. The period between weaning (i.e., removal from the mother) and reaching the bodyweight for transfer to the fattening boxes (at about three months of age and 35–40 kg of body weight) is more delicate from the point of view of health and functioning of the piglet digestive system. Among the numerous factors that can intervene to destabilize the delicate balance of the piglet’s intestine, linked to an acceleration phase of both morphological and enzymatic maturation, diet-linked factors undoubtedly play a fundamental role [1]. Among the dietary factors, the quantity of protein fed to piglets plays a leading role. In fact, in this phase, the coverage of nutritional needs requires a protein level higher than the digestive potential of the piglet. According to Kim et al. [2], a protein level of between 21.5% and 24% is required for modern fast-growing lines, a level that, in fact, is higher than the piglet’s digestive capacity; these authors suggest a protein level not higher than 18% in the first days after weaning and with a consistent addition of synthetic amino acids. De Lange et al. [3] pointed out that low protein levels are beneficial for the gut health of piglets because the presence of undigested proteins, as it can occur with high dietary protein levels, allows the proliferation of a bacterial flora producing toxins capable of altering the intestinal barrier. This alteration implies: the colonization of the intestinal epithelium by pathogenic microorganisms; the acceleration of the production of enterocytes that, being immature, have an exudative rather than absorbent attitude; greater ease of crossing the cellular barrier by specific bacterial toxins (edema disease). Zhang et al. [4] highlighted that high-protein diets increase the microbial fermentation of proteins, peptides, and amino acids. According to Gao et al. [1], high protein levels favor the production of ammonia and branched-chain-fatty acids and, therefore, the proliferation of pathogenic bacterial flora, while low protein levels favor the production of short-chain fatty acids (SCFAs), primarily butyric acid, which favors the proliferation of beneficial bacterial flora. The same authors, comparing two protein levels (17% vs. 30%), both obtained exclusively with casein, highlighted that with a high protein level, the bacterial diversity of the microbiota is reduced. The increase in ammonia can negatively affect the formation of intestinal epithelial cells [5]. The reduction of beneficial Lactobacilli that accompanies the maturation of the pig’s intestine and the variations in the buffering effect of pH due to protein fermentation can make the intestinal environment more susceptible to the emergence of opportunistic pathogens, such as Bacteroides and Clostridium species [6]. Opapeju et al. [7] compared four diets administered to piglets with an initial weight of about 6.5 kg: control feed with 21% crude protein (CP); feed with 19% CP and deficient in isoleucine; 19% raw protein feed supplemented with synthetic isoleucine to reach the isoleucine level of the control feed; 17% raw protein feed supplemented with isoleucine and valine to reach the ratio indicated by the ideal protein. The control group showed better production performance in terms of growth and conversion index, although they showed softer feces, a greater amount of ammonia in the feces, and a greater depth of the crypts of the intestinal mucosa, indicating an acceleration of production of enterocytes; essentially, better production performance, but greater susceptibility to the onset of a syndrome affecting the gastrointestinal system. This situation of precarious equilibrium could be easily kept under control only with targeted antibiotic prophylaxis, which, however, is no longer allowed, and even the spaces for metaphylaxis, albeit careful, become increasingly restricted. Therefore, it is necessary to identify feeding strategies that reduce the risk of the appearance of alterations in the gastrointestinal function of the pig [8] and reserve the use of antibiotics for clinically overt pathological situations. Knowledge of the relationship between pig microbiota and diet can be used to orient the intestinal microbial dynamics in the desired direction by diet manipulation [9]. The microbiota of healthy piglets susceptible to post-weaning diarrhea (PWD) has been the subject of numerous investigations [10,11], which highlighted how the state of health of the pig and its susceptibility to diseases, such as PWD, can be related to the change in the composition of the microbiota during the early stages of growth. All factors affecting PWD susceptibility also affect microbiota composition. Among these, pig feeding plays a primary role. Heo et al. [12] observed a reduction in PWD in piglets challenged with an enterotoxigenic strain of Escherichia coli, when fed with lower protein levels. Rist et al. [13] suggested reducing proteins and increasing fermentable carbohydrates in the diet to reduce harmful protein fermentations. Luise et al. [14] showed that lower dietary protein levels could reduce the intestinal fermentation of undigested proteins and the consequent risk of diarrhea. The aim of this research has been to assess the effect of the dietary protein level on piglet growth and PWD incidence. Piglet fecal microbiota and feces composition were also assessed to support the understanding of the results. All animal procedures were performed in strict accordance with the Code of Ethics of the World Medical Association (https://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm. Accessed on 19 February 2023). The experiment was carried out at our experimental pig farm in San Cesario sul Panaro (Modena, Italy), on Duroc Italiana × Large White Italiana crossing lines. A total of 144 weaned piglets was used, half barrows and half females. The experiment lasted from weaning, when the age of the piglets was 25 ± 1.5 days, until the end of the post-weaning phase, corresponding to 70 days of trial and 95 days of age of the animals (Table 1). Two dietary protein levels were compared (Table 2): high (HP) and low (LP), in two growth phases characterized by different diet compositions. The diet composition (Table 3) was changed 25 days after the start of the experiment, at the expected piglet body weight of 15 kg (and actual weight of 17 kg). Synthetic amino acids were also supplemented to ensure a balanced feed formulation. Specifically, total lysine levels in the first and second feeding periods were set at 1.40% and 1.20% of the feed, respectively. The percentages of methionine, cystine, threonine, and tryptophan were balanced according to the proportion of the ideal protein [15] with the addition of synthetic amino acids. After separation from the mother, the piglets were housed in cages of 12 individuals each, distributed as evenly as possible within each cage by body weight, age, and litter of origin. Since it was not possible to accommodate 12 cages (3.3 m2 each) within the same room, the males were housed in one room (6 cages) and the females in another (6 cages). In this way, it was necessary to accept that we were dealing with a confused effect (room and sex), while the factor of interest of the experiment (protein level) was homogeneously represented in both rooms. Each cage was equipped with a hard-plastic floor. The complete feed was administered ad libitum in a hopper feeder with 4 places to ensure sufficient access to feed for all piglets; in each cage, there was a nipple drinker. The temperature of the air in the rooms was 23 °C ± 1 °C, whereas the humidity was not controlled. Forty-six days after weaning, the piglets were moved to larger pens in the fattening area and housed in 12 pens (9 m2 each) of 12 piglets each. The floor was thermally insulated concrete. The pens were arranged in 2 rows of 6 pens (3 adjacent pens for males and 3 adjacent pens for females), one row for each protein level. The animals were given 1 kg of feed per animal and per day in two daily meals. In this phase, the administration of feed was limited to reduce the risk of diarrhea related to the stress of the change of housing. In the first two days, the meal was dry and distributed on the ground, gradually passing to a wet meal in the trough over the next three days. In each pen, the water was still available through a nipple drinker. The assignment of the treatment to the pens followed the criterion of minimizing the possibility of mixing feces from pigs fed different protein levels. In the fattening area, 21 °C was always ensured during the experiment. The effects of dietary protein level on piglet growth, health status, and feces microbiota were considered for the following periods (Table 1): - Period I: From the start of the experiment until the change of feed; - Period II: From the change of feed until the change of housing; - Period III: From the change of housing to the end of the post-weaning period (end of the experiment). The animals were weighed individually at the start of the experiment, at the day of diet change, at the day of change in housing, and at the end of the experiment. For each period, average daily gain (ADG = [body weight at the end of the period—body weight at the beginning of the period]/day), average daily feed intake (ADFI = feed consumption in the period/day), and feed conversion rate (FCR = ADFI/ADG) were calculated. Since feed consumption was known only at the cage/pen level, the values of the ADFI and FCR variables were not known at the individual level and thus were calculated at the cage/pen level. For homogeneity, the ADG values, although individually known, were also processed at the cage/pen level, with six replicates in total for each treatment. The health of the piglets was monitored daily. A score was assigned to each cage/pen based on the number of cases and the extent (mild, medium, severe) of diarrheal phenomena in piglets, visually assessed from the consistency of the feces (Table 4). For each growth period, individual diarrhea scores were summed for each treatment and related to the period’s total diarrhea score. Piglets suffering from diarrhea before the change of diet (Table S1) were treated (Table S2) parenterally with the antibiotics enrofloxacin or marbofloxacin. No antibiotics were administered between the time of the diet change and the date of transfer to the pens because there were no cases of diarrhea. After the transfer, all individuals were treated orally with colistin sulfate, starting 5 days after the transfer and for 8 days. It was decided to resort to oral mass therapy because the appearance of overt diarrhea (score 4 or higher) occurred five days after the moving, in four out of six boxes of the HP treatment and in one out of six boxes of the LP treatment (Table S3). No treatment was applied in the last 12 days before the third and final sampling of feces. Feces samples were collected at the end of each growth period by piglet rectal ampoule stimulation. For each cage/pen, individual samples were pooled into a composite sample. Samples were immediately frozen and stored at −20 °C until analysis. Dry matter, organic matter, total (Kjeldahl) nitrogen and ammonium nitrogen contents, and pH, were determined according to the APHA methods [16]. Crude fiber (CF) and fiber fractions were determined on fecal samples dried at 60 °C. Crude fiber was determined according to [17]. Hemicellulose and cellulose concentrations were estimated by determining neutral-detergent (NDF) and acid-detergent (ADF) fiber fractions and acid-detergent lignin (ADL) according to [18]. The difference between NDF and ADF is an estimate of the hemicellulose content; that between ADF and ADL of the cellulose content. For volatile fatty acid determination, 1 g of the sample was diluted with 3 mL distilled water, then centrifuged at 4000 rpm for 15 min. The supernatant was used for the analyses. Half mL of sample supernatant was added to 0.25 mL 4% H3PO4 and 0.25 mL internal standard to a final volume of 1 mL. A microliter of this mixture was injected in the injection port of the gas-chromatograph (Shimadzu GC 2010 Pro), equipped with a NukolTM capillary column (Supelco, cat. no. 24107), 30 m × 0.25 mm internal diameter, 0.25 μm film thickness. Total volatile fatty acid content (mg L−1) was calculated as the sum of the individual concentrations of acetic, propionic, butyric and iso-butyric, valeric and iso-valeric, and caproic and iso-caproic acids. Total DNA was extracted from the fecal samples after thawing using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, The Netherlands) according to the manufacturer’s instructions. The V3-V4 regions of the 16S rRNA gene were amplified using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805Rmod (5′-GACTACNVGGGTWTCTAATCC-3′) (based on [19], with degenerate bases) primers. Library construction and sequencing were performed at the Sequencing Platform, Fondazione Edmund Mach, Italy. More in detail: each sample was amplified by PCR using a 25 µL reaction mixture with 1 µM of each primer. More in detail, 12.5 µL of 2× KAPA HiFi HotStart ReadyMix and 10 µL forward and reverse primers, were used in combination with 2.5 µL of template DNA (5 ng/µL). The PCR reactions were carried out by GeneAmp PCR System 9700 (Thermo Fisher Scientific) and the following cycling conditions: initial denaturation step at 95 °C for 3 min (one cycle); 25 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; final extension step at 72 °C for 5 min (1 cycle). The amplification products were checked on 1.5% agarose gel and purified using the CleanNGS beads (CleanNA, Waddinxveen, The Netherlands) following the manufacturer’s instructions. Afterward, a second PCR was used to apply dual indices and Illumina sequencing adapters Nextera XT Index Primer (Illumina), by 7 cycles PCR (16S Metagenomic Sequencing Library Preparation, Illumina). The amplicon libraries were purified using the CleanNGS beads (CleanNA, The Netherlands), and the quality control was performed on a Typestation 2200 platform (Agilent Technologies, Santa Clara, CA, USA). Finally, all barcoded libraries were pooled in an equimolar way and sequenced on an Illumina® MiSeq (PE300) platform (MiSeq Control Software 2.5.0.5 and Real-Time Analysis software 1.18.54.0). A total of 3.093.548 raw reads were detected across the samples by the Illumina MiSeq sequencing platform (PE300) (Illumina, Santa Monica, CA, USA). The estimate of the effect of the protein level in the diet on growth performance was carried out by means of a one-factor analysis of variance. The effect of the sex/room factor was included in the block effect. Values of F with p > 0.05 were considered not significant (NS). The analysis of variance (ANOVA) for the effect of diet on diarrhea incidence was applied to the sum of the scores of the period for each treatment replication. Since in the second period the sum of the scores was very low, for the purposes of ANOVA, the first two periods were merged into a single period. Two-way ANOVA (fixed sources of variation: time of sampling, protein level, time of sampling × protein level) was applied to operational taxonomic units (OTU) percentages to estimate the effect of diet and growth period on the relative abundance of phyla, families, and genera in the analyzed samples, using the procedure MIXED, SAS language [20]. A threshold equal to 0.1% of the total reads was adopted to include the phylum, family, or genus in the analysis. Mean multiple comparisons were performed using the statement LSMEANS and the Tukey HSD test. The same two-way ANOVA design was applied to the statistical analysis of feces composition. Data were pre-processed using the MICCA v. 1.7 [21] pipeline and rarefied to an equal depth of 45.225 reads per sample. OTUs were created de novo by clustering sequences with 97% sequence identity and classified using the RDP [22] software version 2.11. The alpha diversity of the populations, the relative abundance (%) of microbial components down to the family and sex level, and their grouping based on the sources of variation were estimated. Alpha diversity is an index of the richness (number) and diversity (relative abundance) of OTUs in a population. The richness of species is indicated by the total number of OTUs (“Observed”) in the microbial community: the higher the number, the more species are present. The CHAO1 index estimates the richness of species, giving more weight to the less abundant ones. The value of CHAO1 is at least equal to “Observed” and increases as the number of rarer species increases. It can be calculated as follows: where Sobs is the number of observed species and F1 and F2 are the count of singletons and doubletons, respectively. The Shannon index is calculated as follows: where: Σ is the summation from 1 to the total number of OTUs, and pi is the proportion of the community represented by the OTU i. It increases with increasing species richness, uniformity, and uncertainty of the estimate. The samples were grouped for compositional similarity (beta diversity) using Principal Coordinate Analysis (PcoA), which is a multivariate method of data analysis used to explore and to visualize similarities or dissimilarities of data [23]. The piglets fed HP level showed a greater ADG (p < 0.01) than those fed LP (Table 5) in the first 25 days of the experiment until the change of diet, while the increases were the same for the two protein levels in the period from the change of diet to the change of housing. The difference remained significant (p < 0.05) during the entire phase in the cages. The FCR value was also significantly better in HP. Conversely, ADFI was not significantly different in piglets fed different protein levels. In the pen housing, no differences were detected between the two treatments as the feed was rationed. The body weight of the piglets at the end of the post-weaning period was not different in the two diets. Diarrhea cases started to occur in the early post-weaning period (Table S1). They were concentrated in the period between weaning and change of feed (Period I; score summation: 107 out of 234, i.e., 45.7% of the total; Table 6) and in the period between change of housing and the conclusion of the post-weaning period (Period III; 121 out of 234: 51.7% of the total). In Period II, overall, feces had normal consistency, as well as in the first and last part of Period III (Tables S1 and S3). The overall incidence of diarrhea in the LP treatment was 28.6% of the total (67 out of 234 scores) and 71.4% in the HP treatment. Specifically, from weaning until feed change (Period I), the diarrhea scores in the LP treatment were 15.0% of the total (16 out of 107); after feed change (Period II), they were 16.7% of the total (1 out of 6), while after the change of housing (Period III) they rose to 41.3% (50 out of 121). The effect of the dietary protein level on diarrhea score summations was highly significant (p < 0.01) until change of housing (Period I + Period II), whereas it was not significant (p = 0.140) from the change of housing until the end of the experiment (Period III). Poor feces consistency was noted in all the boxes in the HP treatment, starting from the twelfth day of the experiment (Table S1). The phenomenon initially extended to the whole pen in three out of six pens and then progressively became reduced, presumably due to therapeutic interventions (Table S2), until it disappeared two days after the change of feed. The alfa diversity of the microbiota (intra-sample diversity) increased after the change of feed and further increased after the change of housing (Figure 1a), while it was not influenced by the protein level in the diet (Figure 1b). The analysis of beta diversity (inter-sample diversity) using the PCoA tool allowed the clustering of the fecal samples into three groups, corresponding to the three times of sampling (Figure 2). The protein level in the diet allowed only partial separation of the treatments. The most represented phyla in the piglet fecal samples (Table 7) were Bacteroidetes and Firmicutes, which together accounted for 90.6% of reads. The relative abundance of phyla changed in relation to the sampling date: at the first sampling, 25 days after weaning, Firmicutes and Actinobacteria were more abundant than in the two subsequent samplings, whereas the other classified phyla: Bacteroidetes, Spirochaetes, Proteobacteria, and Fibrobacteres were more abundant in the two samplings following the first, without significant differences between the second sampling, at the end of a period characterized by a change of feed, and the third sampling, at the end of a period that had begun with the change of housing. Fibrobacteres, Proteobacteria, and Spirocheaetes were, on average, more represented in the feces of piglets on a low-protein diet, whereas Firmicutes were more abundant in the feces of piglets on a high-protein diet. Overall, the most represented bacterial families were: Prevotellaceae, Lachnospiraceae, Ruminococcaceae, and Porphyromonadaceae (65.1% of the total reads). While the influence of the sampling time was evident, the same cannot be said for that of the treatment (Table S4). Low protein levels in the diet were associated with a higher abundance of Fibrobacteraceae, Succinivibrionaceae, Sutterellaceae, and Spirochetaceae, whereas Firmicutes-belonging families prevailed in the HP fecal samples: Lachnospiraceae and Eubacteriaceae were more abundant along all the post-weaning period, whereas Erisipelotrichaceae, Clostridiaceae 1, and Peptostreptococcaceae were more abundant only in the first sampling event. Lactobacillaceae were the only Firmicutes more abundant in the feces of piglets on a low-protein diet and only in the first sampling event (significant interaction effect: Sampling time × Protein level). A few classified families were little or not at all influenced by both the period of growth and the protein level; among these, Enterobacteriaceae. Among the classified genera, the most abundant were Prevotella (27.2%), followed by Clostridium sensu strictu (3.9%), Lactobacillus (3.3%), Alloprevotella (3.2%), Treponema (3.1%) (Table S5). Selected genera were more represented in the families significantly influenced by the dietary protein level, alone or in interaction with the sampling time (Table 8). Lactobacillus and Treponema were more represented in the LP fecal microbiota, whereas Roseburia, Blautia, and some Clostridium genera were more abundant in HP. Almost all the traits analyzed varied significantly over time (Table 9). More specifically, the concentration of dry matter, organic matter, crude fiber, and fiber fractions increased over time, whereas total and ammonium nitrogen concentrations decreased. The pH slightly increased after the feed change. The dietary protein level also affected the feces composition. Piglets fed low protein had feces that were richer in dry and organic matter and lower in total and ammonium nitrogen all over the experiment period. Even though crude fiber concentration was not affected by the dietary protein level, however, the fiber fractions were. In fact, hemicellulose and cellulose concentrations were higher in the LP fecal samples. As for volatile fatty acids, the concentrations of propionic, valeric, and especially isovaleric acid slightly increased after the change of feeding. No effect on the protein level was detected, except for the isovaleric acid concentration, which tended to be lower in low-protein diets. In our experiment, the low protein level negatively affected the growth of the piglets (ADG and FCR) only in the first post-weaning period (up to the change of diet), whereas it had a clear positive impact on diarrhea intensity reduction. Literature on the matter reports contrasting results, as also highlighted by Wang et al. [24]. Heo et al. [12] reported a higher incidence of PWD when feeding weaned piglets at a high (24.3%) compared with a low (17.3%) CP diet. Wen et al. [25] observed a higher incidence of PWD for piglets fed higher protein levels (up to 23%). Rattigan et al. [26] obtained contrasting results by working in sanitary vs. unsanitary conditions. Reducing the dietary CP level did not affect growth performance; however, in sanitary conditions, it increased the Enterobacteriaceae abundance in the colon and the incidence of diarrhea occurrence, whereas the opposite occurred in unsanitary conditions. Limbach [27] tested the effect on growth and PWD incidence in piglets fed soybean-maize diets at three protein levels (22% AA-balanced, 19% AA-balanced, and 16% protein AA-unbalanced) and concluded that low CP diets may be used for the initial post-weaning period to reduce piglet susceptibility to PWD without largely impacting growth performance. Lynegaard et al. [28] overall reduced CP in the first weaning phase (6–9 kg) from 19.1% to 16.6 and 14%, and from 18.4% to 16.2–17.4%, in the second phase (9–15 kg), depending on the treatment, while they left the protein almost constant in the third phase (15–30 kg) of growth. Under these conditions, they observed a decrease in PWD incidence for treatments with reduced dietary protein, as well as lower ADG and FCR values in piglets fed lower protein levels up to 15 kg body weight. The disadvantage, especially in terms of ADG, also remained in the following period. In their experiment, however, the reduction of CP in the early post-weaning growth period was more pronounced than in our experiment. According to [29], CP levels too low (i.e., 3% below average: 17%) are considered harmful in consideration of maladaptive changes to small intestinal morphology and pepsin activity in weaned piglets. The observed increase in the microbiota’s alfa diversity during piglet growth has also been reported by other authors [30], and it can be linked to changes in diet as well as to the progressive maturation of the intestinal system [31]. On the contrary, dietary protein levels have been reported not to influence alpha diversity in pigs [32,33]. Our results confirm these findings. The most represented phyla (Table 7) are Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria (96.9% relative abundance), and the most abundant bacterial families: Prevotellaceae, Lachnospiraceae, Ruminococcaceae, and Porphyromonadaceae in the piglet fecal samples of this experiment are those recurring in the feces of healthy piglets [34,35]. Clostridium sensu stricto, Roseburia, Paraprevotella, Clostridium XIVa, and Blautia have been reported as major representative genera after weaning [36,37]. In general, the results in the literature refer to piglets stressed on purpose or younger than those in this experiment. The considered microbiota is more often that of the intestinal system, different from the fecal one [38]. In our experiment, we considered the microbiota of the feces of piglets raised in protected conditions, which should be the most likely or, in any case, desired in real farms. This protection has been applied both at the environmental level (attention to the absence of causes of stress) and at the health level (preventive interventions with drugs). This may explain the low presence of Enterobacteriaceae, which is normally associated with stressful conditions. The effect of dietary protein level on the composition of gut microbiota has mainly been studied for finishing pigs. Moderate protein levels in the diet have been found to modify the gut microbiota composition and to improve the ileal barrier function [39,40]. On the contrary, information on piglets is scarce, especially when referring to the composition of the fecal microbiota. In our experiment, differences in the composition of the fecal microbiota due to the protein level in the diet were found above all in the first sampling, that is, at the end of the first period after weaning and before the change of feed, just when the effect of protein intake on the incidence of diarrhea phenomena was most evident. Given this coincidence, we can think of associating the higher susceptibility of the piglets to PWD with the greater presence of Firmicutes-belonging families (with the exclusion of Lactobacillaceae), which were more abundant in T1 in the fecal microbiota of piglets fed HP diets. Conversely, a lower incidence of PWD can be associated with the prevailing abundance in LP diets of families belonging to Lactobacillaceae (gen. Lactobacillus), Fibrobacteraceae (gen. Fibrobacter), Succinivibrionaceae, and Spirochetaceae (gen. Treponema). Yang et al. [30] compared the fecal microbiota of healthy piglets and that of diarrheal piglets in three stages of growth: lactation, intermediate stage, and weaning (solid diet for piglets) and noted that, with the transition to solid feeding, the incidence of Lactobacillus and E. coli decreased while that of Prevotella increased. They related reduced numbers of Bacteroides, Ruminococcus, Bulleidia, and Treponema, which are responsible for the digestion of solid foods, to the onset of post-weaning piglet diarrhea. The genera prevalent in the fecal microbiota of piglets fed HP diets, such as Roseburia, Blautia, Eubacterium, and selected Clostridium species, are commonly found in piglet fecal microbiota and are all fermentative. Their greater abundance in the HP feces can be the consequence of the incomplete digestion of complex fermentable substrates because less digestible feed components are more available for microbial activities in the gut terminal tract. The greater presence of Clostridia in the microbiota of piglets fed HP diets can be related to their ability to metabolize amino acids [4]. Conversely, the increased presence of Fibrobacter in low-protein diets can be related to the availability of higher amounts of undigested cellulose in the final intestinal tract. In fact, Fibrobacter is a fibrolytic bacterial species commonly present in the pig intestinal microbiota [41], although it is a typical colonizer of the rumen. Fibrobacter uses glucose, cellulose, and cellobiose as carbon and energy sources with the production of succinate, acetate, and formate [42]. Fibrobacter and Treponema have been reported as more abundant in diets of ruminants fed with higher amounts of lignocellulosic components [43]. Feces composition clearly depends on the type of food ingested and on the use that the animal can make of the food. The less digestible the food, and the worse the digestive abilities of the animal, the higher the quantity of feed that remains undigested in the feces. Higher crude fiber contents accompanying the change of feed can be the reason for worse digestibility: a higher crude fiber content was, in fact, found in the piglet feces at the second and third sampling (i.e., at the change of housing and at the end of the post-weaning period). An important side effect of the reduction in dietary protein was the lower concentration of nitrogen in the feces, possibly because a greater quantity of ingested nitrogen was assimilated instead of wasted. This result has been reported by several authors. Zhao et al. [44] found a significant reduction in N excretion in 90-day-old pigs fed protein diets 3.5% lower than standard. Yang et al. [45] observed a linear decrease in fecal total nitrogen for decreasing dietary protein levels in the diet of growing pigs. Other components in the diet may interfere with nitrogen metabolism. In fact, in maize, the nitrogen-free extractives consist of starch, whereas in soybeans, the starch is less than 1%, and soluble carbohydrates are mainly sucrose, raffinose, and stachyose [46]. High amounts of raffinose and stachyose from soybean in the diet are supposed to reduce the digestibility of nitrogen and amino acids in growing pigs [47]. Therefore, in HP-fed piglets, the greater nitrogen excretion can also derive from a lower digestibility due to the higher percentages of soybean oligosaccharides. Interestingly, Zhang et al. [48] showed a higher incidence of diarrhea cases in piglets fed with soybean flour as is or with added stachyose compared to the control consisting of corn with concentrated soybean protein. Short-chain fatty acids are final metabolites of the intestinal microbiota, produced mainly in the large intestine, where they are used by mono-gastrics as a source of energy. The energy contribution of the SCFA in pig metabolism is important. Weaning has been reported to affect the concentrations of SCFA in the intestine [49]. In our experiment, the dietary protein level did not affect the SCFA content, apart from the case of isovaleric acid, which was less abundant in LP diets. Some confirmations and some interesting insights emerge from this experience. The first of these is whether the body weight differences found in the experiment cancel out during the subsequent stages of growth of the animals and, once this has been established, how far one can go with the reduction of the protein content in particularly critical phases (first 15 days after weaning, sudden changes in the environment) without compromising the subsequent productive career. The second interesting point is to investigate the evolution of the microbiota to identify moments in which an analysis of this can serve as an indicator of the evolution of the state of health of the gastrointestinal system. From our results, it appears that the phase immediately following weaning is the most subject to changes in the fecal microbiota composition and most suitable for earlier identification of possible stress conditions. Everything must be understood in terms of optimizing individual and mass therapeutic interventions in order both to improve the profitability of breeding and to reduce the risk of the appearance of antibiotic-resistance phenomena. This research confirms that a reduction in the protein content of feed can reduce the appearance and severity of gastrointestinal syndromes in piglets in particularly stressful stages of rearing (removal from the mother, change of housing) while only marginally affecting growth performance. Low-protein diets, resulting in excreta with lower quantities of nitrogen than those of standard diets, may allow for potential environmental benefits. The variations in the microbiota are largely determined by the growth phase, which in turn is accompanied by an evolution of the diet. The different protein levels at the same age caused slight but significant variations in some components of the intestinal microbiota. Further research is needed to ascertain whether microorganisms found in low-protein diets can be considered indicators of lower susceptibility to diarrhea in piglets.

PMC10000055

Xin Yang,Xuemei Wu,Shuang Huang,Qian Yao,Xi Chen,Junke Song,Yingying Fan,Guanghui Zhao

C3a/C3aR Affects the Propagation of Cryptosporidium parvum in the Ileum Tissues of Mice by Regulating the Gut Barrier, Cell Proliferation, and CD4+ T Cell Main Effectors

24-02-2023

Cryptosporidium parvum,C3a/C3aR signaling,propagation,intestinal barrier function,cell proliferation,CD4+ T cell-related cytokines

Simple Summary The complement system plays important roles in both innate and adaptive immunity. The present study explored the function of host C3a/C3aR signaling during Cryptosporidium parvum infection, and found that the C3a/C3aR signaling likely affected the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors. Abstract Cryptosporidium parvum is an important zoonotic protozoon that threatens the health of humans and animals, but the interaction mechanisms between C. parvum and hosts are poorly understood. Our previous study indicated that the expression levels of C3a and C3aR were up-regulated in mice during C. parvum infection, but the mechanisms of C3a/C3aR signaling during C. parvum infection have not been elucidated. In the present study, an optimized BALB/c suckling mouse model infected with C. parvum was used to explore the function of C3a/C3aR signaling during C. parvum infection. The expression levels of C3aR in the ileum tissues of mice infected with C. parvum were analyzed using real-time PCR, Western blot and immunohistochemistry. The mRNA levels of the Cryptosporidium 18S rRNA gene, tight junction proteins (zo-1, claudin 3, and occludin), intestinal stem cell marker lgr5, cell proliferation marker ki67, Th1 cell-related cytokine ifn-γ, and Treg cell-related cytokine tgf-β in mouse ileum tissues were analyzed by real-time PCR. The pathological injury of ileal mucosa was examined by histopathology analysis. The mRNA expression levels of Cryptosporidium 18S rRNA gene were significantly up-regulated in the ileum tissues of C3aR-inhibited mice during C. parvum infection. Meanwhile, histopathology analysis of ileal mucosa in mice showed that inhibition of C3aR significantly aggravated the changes in villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth during C. parvum infection. Further studies found inhibition of C3aR aggravated the down-regulation of occludin at most time points during C. parvum infection. The mRNA levels of ki67 and lgr5 in the ileum tissues of mice infected with C. parvum were significantly down-regulated. Inhibition of C3aR significantly down-regulated the mRNA expression levels of lgr5 at most time points, but significantly up-regulated the mRNA expression levels of ki67 at most time points. The mRNA expression levels of ifn-γ and tgf-β were significantly up-regulated and down-regulated in the ileum tissues of mice infected with C. parvum, respectively. However, inhibition of C3aR significantly increased the mRNA expression levels of ifn-γ and tgf-β in the ileum tissues of mice infected with C. parvum. Taken together, C3a/C3aR signaling could possibly affect the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors, which would contribute to our understanding of the interaction between Cryptosporidium and hosts.

C3a/C3aR Affects the Propagation of Cryptosporidium parvum in the Ileum Tissues of Mice by Regulating the Gut Barrier, Cell Proliferation, and CD4+ T Cell Main Effectors The complement system plays important roles in both innate and adaptive immunity. The present study explored the function of host C3a/C3aR signaling during Cryptosporidium parvum infection, and found that the C3a/C3aR signaling likely affected the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors. Cryptosporidium parvum is an important zoonotic protozoon that threatens the health of humans and animals, but the interaction mechanisms between C. parvum and hosts are poorly understood. Our previous study indicated that the expression levels of C3a and C3aR were up-regulated in mice during C. parvum infection, but the mechanisms of C3a/C3aR signaling during C. parvum infection have not been elucidated. In the present study, an optimized BALB/c suckling mouse model infected with C. parvum was used to explore the function of C3a/C3aR signaling during C. parvum infection. The expression levels of C3aR in the ileum tissues of mice infected with C. parvum were analyzed using real-time PCR, Western blot and immunohistochemistry. The mRNA levels of the Cryptosporidium 18S rRNA gene, tight junction proteins (zo-1, claudin 3, and occludin), intestinal stem cell marker lgr5, cell proliferation marker ki67, Th1 cell-related cytokine ifn-γ, and Treg cell-related cytokine tgf-β in mouse ileum tissues were analyzed by real-time PCR. The pathological injury of ileal mucosa was examined by histopathology analysis. The mRNA expression levels of Cryptosporidium 18S rRNA gene were significantly up-regulated in the ileum tissues of C3aR-inhibited mice during C. parvum infection. Meanwhile, histopathology analysis of ileal mucosa in mice showed that inhibition of C3aR significantly aggravated the changes in villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth during C. parvum infection. Further studies found inhibition of C3aR aggravated the down-regulation of occludin at most time points during C. parvum infection. The mRNA levels of ki67 and lgr5 in the ileum tissues of mice infected with C. parvum were significantly down-regulated. Inhibition of C3aR significantly down-regulated the mRNA expression levels of lgr5 at most time points, but significantly up-regulated the mRNA expression levels of ki67 at most time points. The mRNA expression levels of ifn-γ and tgf-β were significantly up-regulated and down-regulated in the ileum tissues of mice infected with C. parvum, respectively. However, inhibition of C3aR significantly increased the mRNA expression levels of ifn-γ and tgf-β in the ileum tissues of mice infected with C. parvum. Taken together, C3a/C3aR signaling could possibly affect the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors, which would contribute to our understanding of the interaction between Cryptosporidium and hosts. Cryptosporidium spp. are important diarrheal pathogens threatening the health of humans and hundreds of animals [1,2]. Cryptosporidium spp. are primary diarrheal pathogens in dairy cattle, with over 20% of young animals infected with the pathogens [3,4]. Meanwhile, children and immunosuppressed populations are highly susceptible to Cryptosporidium [5]. Cryptosporidium spp. have been recognized as one of five major diarrheal pathogens in children under two years of age in developing countries [5], and have led to hundreds of outbreaks of diarrhea in industrialized nations [6]. Nitazoxanide is the only FDA-approved drug in the treatment of human cryptosporidiosis, but it is almost useless in children and immunosuppressed populations [7]. Therefore, hitherto, there are still no effective strategies for the treatment of cryptosporidiosis in humans and animals. Knowledge on the interaction between Cryptosporidium and hosts can shed the light on the discovery of novel targets for the development of vaccines and drugs, providing resources for the prevention and control of cryptosporidiosis in humans and animals. Previous studies have found that both innate and acquired immune responses played important roles in the early defense and late elimination of Cryptosporidium [8]. During the early stages of acute infection, innate immune response participates in the host defense against Cryptosporidium invasion by monitoring immunocytes, cytokines, chemokines and complement molecules [8,9,10,11,12]. More importantly, activated innate immune response can trigger and regulate acquired immune responses [10]. Acquired immune responses are necessary to eliminate Cryptosporidium, especially for the cellular immune response dominated by CD4+ T cells [8,13,14,15]. As the main components of innate immune response, complement molecules can play roles in dissolving pathogens, regulating phagocytosis, and mediating inflammation, immune adhesion and cytotoxicity in infected areas through the classical pathway, alternative pathway and lectin pathway [16]. Moreover, activated complement components (e.g., C3d, complement receptor CD21/35 and C5aR) can be involved in the regulation of inflammation and acquired immune response dominated by CD4+ T cells, and linked with the innate immunity with acquired immunity [17,18,19,20]. Complement C3 hydrolyzes to form anaphylaxis toxin C3a after the activation of the complement system, and C3a can bind to its specific receptor C3aR on the surface of cells; it then functions in inflammation, intestinal injury repairment, and CD4+ T cell differentiation and development [21,22,23]. Our previous studies indicated the up-regulation of C3a and C3aR in mice during C. parvum infection, but the role of C3a/C3aR signaling during Cryptosporidium infection was still unknown [24]. Therefore, the present study applied an optimized BALB/c mouse model infected with C. parvum to explore the function of C3a/C3aR signaling during C. parvum infection, which would contribute to our understanding of the interaction between C. parvum and hosts. The C. parvum IIdA19G1 strain was isolated from one pre-weaned dairy calf with diarrhea in the Shaanxi province of China, and identified based on sequence analysis of the 18S rRNA and the gp60 gene loci [25,26]. The strain was isolated in the lab of parasitology of Northwest A&F University and passaged in pre-weaned dairy calves in specific pathogen-free conditions. Cryptosporidium parvum oocysts in the faeces were purified using the Sheather’s sugar flotation technique and cesium chloride density gradient centrifugation, then stored in PBS with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin B solutions for less than 3 months, as in the previous study [27]. An established BALB/c suckling mouse model infected with C. parvum in our group [27] was optimized in the present study by increasing the concentration of sodium taurocholate from 0.1% to 1%, and the excystation time from 30 min to 1 h. Briefly, each suckling mouse of the infection group and control group was orally administrated with 1 × 107 oocysts and equal volume of PBS, respectively. Suckling mice in the infection group and the control group were kept in separate cages to avoid contamination. At 1 day post infection (dpi), 2 dpi, 4 dpi, 6 dpi, 8 dpi, 9 dpi, 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi, three animals were randomly selected for the isolation of ileum tissues from both control and infected groups at each time point, respectively. The above-mentioned time points and the range were selected since they represented specific milestones in the infection development. The ileum tissues collected from three animals for each group at each time point were washed in PBS and stored at −80 °C for RNA and protein isolation. Meanwhile, the ileum tissues at the peak of infection (6 dpi), based on RT-qPCR of 18S rRNA gene in mouse ileum tissues, were also stored in 4% paraformaldehyde fix solution for histopathology analysis, and the contents of large intestine at the peak of infection were kept at 4 °C for acid-fast stain analysis. All the experiments were performed thrice. The contents of the large intestine at the peak of infection from both the infected and control groups were analyzed using acid-fast stain to identify C. parvum oocysts, as previously reported [28]. Histopathology analyses of mouse ileum tissues at the peak of infection were conducted as previously reported [29]. Five slides of each mouse ileum tissue sample were randomly selected for calculating villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth. For each slide, five complete intestinal villi with the highest length were analyzed. Furthermore, a C3aR-inhibited BALB/c suckling mouse model infected with C. parvum was established by intraperitoneally injecting animals with C3aR antagonist SB290157 (MedChemExpress, Princeton, NJ, USA) at a dose of 10 mg/kg before C. parvum infection [30]. Collection of ileum tissue, RNA isolation and cDNA synthesis, expression level analysis of Cryptosporidium 18S rRNA gene, and histopathology analyses were conducted to explore the effect of C3a/C3aR signaling on the propagation of C. parvum in the ileum of mice. Extraction of total RNA from mouse ilea and subsequent synthesis of cDNA were the same as previous study [31]. The relative expression levels of Cryptosporidium 18S rRNA gene, C3aR, zo-1, claudin 3, occludin, lgr5, ki67, ifn-γ and tgf-β in mouse ileum tissues were evaluated by qPCR using SYBR Green Fast RT-qPCR Mix (ABclonal, Wuhan, China) according to the manufacturer’s recommendations and with the primers listed in Table 1. The gapdh gene was also included for data normalization (Table 1). Three independent experiments for tissues collected from three animals were performed, with data calculated by applying the 2−ΔΔCt method to assess mRNA expression. The protein expression levels of C3aR in mouse ileum tissues at 2 dpi and 6 dpi were analyzed by Western blotting. Briefly, the mouse ileum tissues were homogenized by grinding, and treated with 1 mL RIPA (ComWin, Beijing, China) containing 1% PMSF (Beyotime, Shanghai, China) for the isolation of proteins. Proteins were separated by SDS-PAGE analysis, and transferred to nitrocellulose membranes pre-activated with methanol. Subsequently, the membranes were blocked with TBST containing 5% non-fat milk for 2 h, and incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:1,000 dilution at 4 °C overnight. The membranes were washed in TBST for 5 min thrice. HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) with 1:5,000 dilution were applied as the secondary antibodies to incubate the membranes for 1 h at room temperature. Then, the membranes were washed in TBST for 5 min thrice and treated with Super ECL Plus (Applygen, Beijing, China) for photography under an automatic gel imaging analysis system (Sage, Beijing, China). The mouse ileum tissues at the peak of infection were analyzed by immunohistochemistry using SP-0023 HistostainTM-Plus Kits (MBL, Beijing, China). Briefly, paraffin sections were made from the ileum tissues at the peak of infection and subsequently incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:200 dilution at 4 °C overnight and HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) for 15 min at room temperature. These were then photographed under a microscope (Olympus, Hataya, Japan). Image-Pro Plus was used to assess the distribution of C3aR on the surface of mouse ileum tissues by calculating the mean optical density value. Accumulative optical density divided by the area of cell distribution equals the mean optical density value. Statistical difference analysis was conducted by GraphPad Prism V.8.0 (https://www.graphpad.com/scientific-software/prism/, accessed on 7 July 2022) using Student’s t test and ANOVA [32]. Significant difference was identified if the p value was less than 0.05. Optimization of the excystation scheme could significantly increase the excystation rate from 40% to over 85%, as shown in Figure 1A (p < 0.05). Compared with the control group, the relative mRNA expression levels of Cryptosporidium 18S rRNA gene in the mouse ilea in C. parvum-infected group increased gradually and reached a peak at 6 dpi, then gradually decreased. The duration period of C. parvum infection in the mouse model reached 29 days (Figure 1B). Acid-fast stain analysis of the large intestine contents at 6 dpi recognized rosy oocysts in the infected group (Figure 1C). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum and the infiltration of the inflammatory cells in the ileum tissues of the infected group (Figure 1D); subsequently statistical analysis indicated C. parvum infection significantly deceased the length of villi and the ratio of villus height to crypt depth, and significantly increased the diameter of villi (Figure 1E). Compared with control group, the mRNA expression levels of C3aR in mouse ileum tissues of the infected group were significantly down-regulated from 1 to 8 dpi, and then up-regulated until 36 dpi (Figure 2A). Western blot analysis also indicated the down-regulation of protein levels of C3aR in mouse ileum tissues of the infected group at both 2 dpi and 6 dpi (Figure 2B, Figures S1 and S2). Further immunohistochemistry analysis indicated C3aR was majorly distributed on the surface of ileal epithelial cells at 6 dpi (Figure 2C), and subsequently statistical analysis also showed significantly decreased distribution of C3aR in mouse ileum tissues of the infected group (Figure 2D). As shown in Figure 3A, the protein level of C3aR in mouse ileum tissues was significantly down-regulated three hours after intraperitoneal injection with SB290157 (Figure 3A and Figure S3). Further studies indicated that inhibition of mouse C3aR significantly up-regulated the relative mRNA expression levels of the Cryptosporidium 18S rRNA gene in mouse ileum tissues during C. parvum infection (p < 0.05). Meanwhile, the infection duration period increased from 29 days to 35 days in the C3aR-inhibited infected group (Figure 3B). Significant differences were found between the relative transcriptional expression of Cryptosporidium 18S rRNA among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 359.612, p < 0.001), 2 dpi (F = 58.443, p < 0.001), 4 dpi (F = 44.112, p < 0.001), 6 dpi (F = 215.622, p < 0.001), 8 dpi (F = 393.826, p < 0.001), 9 dpi (F = 114.706, p < 0.001), 10 dpi (F = 427.847, p < 0.001), 29 dpi (F = 41.950, p < 0.001), 30 dpi (F = 55.628, p < 0.001), 35 dpi (F = 25.905, p < 0.001) and 36 dpi (F = 13.106, p = 0.006). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum in the ileum tissues in the infected group and the C3aR-inhibited group (Figure 3C). Subsequent statistical analysis indicated that C. parvum infection in C3aR-inhibited group further decreased the length of villi and the ratio of villus height to crypt depth, and increased the diameter of villi and mucosal thickness compared with the normal mouse in the infected group (Figure 3D), suggesting that C3aR likely alleviated the intestinal injury caused by C. parvum. Significant differences were found between the relative transcriptional expression levels of the zo-1 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 4 dpi (F = 253.975, p < 0.001), 6 dpi (F = 434.805, p < 0.001), 8 dpi (F = 21.897, p = 0.002), 9 dpi (F = 164.639, p < 0.001), 10 dpi (F = 18.990, p = 0.003), 29 dpi (F = 9.539, p = 0.014), 30 dpi (F = 14.252, p = 0.005), 35 dpi (F = 7.212, p = 0.025) and 36 dpi (F = 14.015, p = 0.005), while differences at 1 dpi (F = 3.316, p = 0.107) and 2 dpi (F = 0.146, p = 0.867) were not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the zo-1 at 4 dpi and down-regulated at 6 dpi, 8 dpi, 9 dpi, 30 dpi and 35 dpi. Inhibition of C3aR led to dynamic changes in the zo-1 gene during C. parvum infection, reflected by the down-regulation at 8 dpi, 9 dpi, 10 dpi and 29 dpi, and up-regulation at 4 dpi, 6 dpi, 30 dpi, 35 dpi and 36 dpi (Figure 4A). Significant differences were found between the relative transcriptional expression of the claudin 3 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 2 dpi (F = 29.079, p = 0.001), 4 dpi (F = 89.677, p < 0.001), 6 dpi (F = 10.677, p = 0.011), 8 dpi (F = 37.588, p < 0.001), 9 dpi (F = 119.767, p < 0.001), 10 dpi (F = 13.474, p = 0.006), 29 dpi (F = 210.694, p < 0.001), 30 dpi (F = 380.076, p < 0.001), 35 dpi (F = 52.630, p < 0.001) and 36 dpi (F = 60.535, p < 0.001), while the difference at 1 dpi (F = 3.593, p = 0.094) was not significant. Compared with the control group, the mRNA expression levels of the claudin 3 gene of mouse ileum tissues in C. parvum-infected group were significantly down-regulated at 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi, and up-regulated at 4 dpi, 8 dpi and 9 dpi. Inhibition of C3aR led to dynamic changes in the claudin 3 gene during C. parvum infection, reflected by the down-regulation at 6 dpi, 8 dpi, 9 dpi, 29 dpi and 30 dpi, and up-regulation at 2 dpi, 10 dpi, 35 dpi and 36 dpi (Figure 4B). Significant differences were found between the relative transcriptional expression of the occludin gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 15.306, p = 0.004), 4 dpi (F = 13.151, p = 0.006), 6 dpi (F = 257.643, p < 0.001), 8 dpi (F = 33.276, p = 0.001), 9 dpi (F = 29.416, p = 0.001), 10 dpi (F = 62.741, p < 0.001), 29 dpi (F = 219.563, p < 0.001), 30 dpi (F = 28.790, p = 0.001), 35 dpi (F = 67.671, p < 0.001) and 36 dpi (F = 13.095, p = 0.006), while the difference at 2 dpi (F = 4.718, p = 0.059) was not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the occludin gene at 9 dpi, and down-regulated at 1 dpi, 6 dpi, 8 dpi, 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi. Inhibition of C3aR led to significant up-regulation of the occludin gene at 1 dpi, 35 dpi and 36 dpi, but down-regulation from 4 dpi, 6 dpi, 8 dpi, 9 dpi, 10 dpi and 29 dpi (Figure 4C). Significant differences were found between the relative transcriptional expression levels of the lgr5 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 115.620, p < 0.001), 2 dpi (F = 8.921, p = 0.016), 4 dpi (F = 20.221, p = 0.002), 6 dpi (F = 41.482, p < 0.001), 8 dpi (F = 15.293, p = 0.004), 9 dpi (F = 6.992, p = 0.027), 10 dpi (F = 16.161, p = 0.004), 29 dpi (F = 23.714, p = 0.001), 30 dpi (F = 35.947, p < 0.001), 35 dpi (F = 11.568, p = 0.009) and 36 dpi (F = 25.923, p = 0.001). Compared with the control group, the mRNA expression levels of the lgr5 gene of mouse ileum tissues in the C. parvum-infected group were significantly down-regulated at 1 dpi, 2 dpi, 6 dpi, 8 dpi and 10 dpi, and up-regulated from 30 dpi, 35 dpi and 36 dpi. Inhibition of C3aR led to significant up-regulation of the lgr5 gene at 1 dpi, 2 dpi and 4 dpi, but down-regulation at 6 dpi, 8 dpi, 29 dpi, 30 dpi and 35 dpi (Figure 5A). Significant differences were found between the relative transcriptional expression levels of the ki67 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 136.699, p < 0.001), 2 dpi (F = 621.666, p < 0.001), 4 dpi (F = 1282.733, p < 0.001), 6 dpi (F = 15.791, p = 0.004), 8 dpi (F = 19.258, p = 0.002), 9 dpi (F = 10.979, p = 0.010), 29 dpi (F = 12.055, p = 0.008), 30 dpi (F = 12.545, p = 0.007), 35 dpi (F = 119.732, p < 0.001) and 36 dpi (F = 69.487, p < 0.001), while the difference at 10 dpi (F = 1.225, p = 0.358) was not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the ki67 gene at 9 dpi, 30 dpi, 35 dpi and 36 dpi, but down-regulated at 1 dpi, 2 dpi, 4 dpi, 8 dpi and 29 dpi. Inhibition of C3aR led to the significant up-regulation of the ki67 at 1 dpi, 2 dpi, 4 dpi, 6 dpi, 29 dpi and 36 dpi, but down-regulation at 9 dpi and 35 dpi (Figure 5B). Significant differences were found between the relative transcriptional expression levels of the ifn-γ gene among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 69.841, p < 0.001), 2 dpi (F = 300.925, p < 0.001), 4 dpi (F = 58.746, p < 0.001), 6 dpi (F = 72.761, p < 0.001), 8 dpi (F = 97.252, p < 0.001), 9 dpi (F = 38.113, p < 0.001), 10 dpi (F = 12.446, p = 0.007), 29 dpi (F = 48.334, p < 0.001), 30 dpi (F = 19.592, p = 0.002), 35 dpi (F = 41.574, p < 0.001) and 36 dpi (F = 45.236, p < 0.001). Compared with the control group, the mRNA expression levels of the ifn-γ gene of mouse ileum tissues in C. parvum-infected group were significantly down-regulated at 1 dpi, but up-regulated at most time points. Inhibition of C3aR led to the significant up-regulation of ifn-γ at 1 dpi, 2 dpi, 8 dpi, 9 dpi and 29 dpi, but down-regulation at 6 dpi, 30 dpi and 35 dpi (Figure 6A). Significant differences were found between the relative transcriptional expression levels of the tgf-β gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 1721.105, p < 0.001), 2 dpi (F = 35.969, p < 0.001), 6 dpi (F = 30.388, p = 0.001), 8 dpi (F = 51.836, p < 0.001), 9 dpi (F = 94.612, p < 0.001), 10 dpi (F = 8.058, p = 0.020), 29 dpi (F = 87.382, p < 0.001), 30 dpi (F = 19.085, p = 0.003), 35 dpi (F = 205.922, p < 0.001) and 36 dpi (F = 8.847, p = 0.016), while the difference at 4 dpi (F = 4.687, p = 0.059) was not significant. Cryptosporidium parvum infection significantly down-regulated the mRNA expression level of the tgf-β at most time points. Inhibition of C3aR led to the significant up-regulation of tgf-β at all time points post infection (Figure 6B). Cryptosporidiosis is an important zoonotic disease caused by Cryptosporidium spp., which greatly threatens the health of humans and animals, especially young and immunocompromised hosts [1,2]. However, there are still no effective drugs and vaccines for the prevention and control of cryptosporidiosis. One of the main reasons for this is a lack of knowledge on the interaction between Cryptosporidium and hosts. Previous studies indicated C3a/C3aR signaling played some important roles in the recovery of intestinal injury [22]. Our previous study showed that C. parvum infection could lead to the up-regulation of C3a and C3aR in mouse ileum tissues [24], but the role of C3a/C3aR signaling during C. parvum infection is still unknown. The present study applied an optimized mouse model with C. parvum infection to explore the function of C3a/C3aR signaling during C. parvum infection, enriching our knowledge on the interaction between Cryptosporidium and hosts. The present study optimized an established suckling mouse model infected with C. parvum in our group by increasing the concentration of sodium taurocholate and the excystation time, and the results indicated that the optimized model significantly increased the excystation of C. parvum. Similar to the previous model, the optimized model also found pathological damage and an inflammatory reaction in the intestines in mice during C. parvum infection [31]. Compared with other infection models, the optimized mouse model in the present study simulated the natural infection process of animals and provided a candidate model for studies on the interaction between Cryptosporidium and hosts without the use of dexamethasone. In the present study, the optimized mouse model found that the up-regulation of C3aR in the ileum of mice during C. parvum infection and inhibition of C3aR significantly aggravated intestinal damage caused by C. parvum infection. Although opposite results were found for the C3aR mRNA expression at 1 dpi and 2 dpi between the present study and our previous work, the mRNA levels of this gene were increased at later stages of infection for both studies [24]. The differences at the early stage of infection may be due to the distinct immunity of animal models. The present study used suckling mice for parasite infection, while older mice aged 3 weeks were used in the previous study. Tight junctions are an important structural basis of the intestinal mucosal mechanical barrier, which plays an important role in maintaining the integrity of the intestinal morphology and resisting the invasion of intestinal pathogens [33]. To explore the mechanisms of C3a/C3aR signaling, the mRNA expression levels of three tight junction proteins, namely zo-1, claudin 3 and occludin, were detected by using qPCR. The results found that C. parvum infection down-regulated the mRNA expression levels of the zo-1, claudin 3 and occludin genes at most time points, indicating that C. parvum likely destroyed the integrity of intestinal barrier by down-regulating tight junctions, which was similar to the C57BL/6 mouse model infected with C. parvum [34]. Meanwhile, Cryptosporidium andersoni infection could also destroy the expression of the ZO-1 in the epithelial cells of humans and cattle [35]. Interestingly, inhibition of C3aR significantly down-regulated the mRNA expression of the occludin gene at half of the time points during C. parvum infection, suggesting that C3a/C3aR signaling likely maintained the integrity of the intestinal barrier by upregulating the expression of the tight junction-related gene occludin, thus reducing the infection and propagation of C. parvum in mouse ilea. Meanwhile, the proliferation and differentiation of intestinal stem cells and the normal renewal of intestinal epithelial cells also play some important roles in maintaining the structure and function of intestinal mucosa [22,36]. Furthermore, the mRNA expression levels of intestinal stem cell marker LGR5 and cell proliferation marker KI67 of ileum tissues during C. parvum infection were detected by qPCR. The results indicated that C. parvum infection down-regulated the mRNA expression levels of the lgr5 and ki67 genes in mouse ileum tissues at half of the time points, reflecting that C. parvum infection possibly retarded the proliferation and renewal of intestinal epithelial cells to prolong the survival time of the parasites in intestinal cells, and then aggravated Cryptosporidium infection in intestine, in accordance with previous reports [37,38]. Inhibition of C3aR led to the down-regulation of the lgr5 gene at most time points, while leading to up-regulation of ki67 at most time points, suggesting that C3a/C3aR signaling likely promoted the regeneration of intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells to inhibit the propagation of C. parvum in the ilea of mice. Early moderate IFN-γ can not only initiate intestinal immunity, but can also promote intestinal cell proliferation and mucosal damage repair. However, abnormal over-activation of the immune system will release a large amount of IFN-γ, and excessive IFN-γ can not only kill pathogenic microorganisms, but also activate the JAK/STAT1 pathway of intestinal epithelial cells, leading to programmed necrosis of intestinal epithelial cells and impairment of intestinal barrier function [36]. Unlike IFN-γ, TGF-β showed a protective function on the intestinal barrier [39]. The aforementioned functions of IFN-γ and TGF-β have also been confirmed in intestinal epithelial cells infected with Cryptosporidium [40,41,42]. Meanwhile, C3a/C3aR signaling can function in resisting pathogen invasion by promoting/inhibiting the expression of IFN-γ and TGF-β [43,44]. To explore the link between C3a/C3aR signaling and the expression of IFN-γ and TGF-β, we used qPCR to detect the mRNA expression levels of the ifn-γ and tgf-β genes in mouse ilea during Cryptosporidium infection. The results revealed that C. parvum infection significantly up-regulated and down-regulated the mRNA levels of the ifn-γ and tgf-β genes, respectively, indicating that C. parvum infection may induce a Th1-type immune response, and inhibit the differentiation and function of Treg cells. Therefore, mice infected with C. parvum were likely in a state of continuous inflammation. However, inhibition of C3aR led to the significant up-regulation of mRNA levels of both ifn-γ and tgf-β in the ileum tissues of infected mice, which was possibly related to the dual anti-inflammatory and pro-inflammatory actions of C3a/C3aR signaling [45], suggesting that C3a/C3aR signaling likely participated in the anti-Cryptosporidium effect by down-regulating the expression of Th1 and Treg cells’ main effect factors; however, these mechanisms need to be explored in future studies. The present study firstly explored the preliminary function of host C3a/C3aR signaling during C. parvum infection, and revealed C3a/C3aR signaling likely inhibited the propagation of C. parvum in the ilea of mice by regulating the gut barrier, cell proliferation, and CD4+ T cell main effectors of hosts, which could contribute to our knowledge on the interaction between Cryptosporidium and hosts.

PMC10000059

Maria Irene Pacini,Maurizio Mazzei,Micaela Sgorbini,Rossella D’Alfonso,Roberto Amerigo Papini

A One-Year Retrospective Analysis of Viral and Parasitological Agents in Wildlife Animals Admitted to a First Aid Hospital

04-03-2023

wildlife,central Italy,viruses,helminths,protozoa,interspecies transmission,zoonotic pathogen

Simple Summary In recent decades, wildlife populations in Italy have continued to expand, and some species are now present in large numbers with a wide geographical distribution. Viral and parasitic agents are an integral part of any wildlife population. The major changes in human land use, the tendency of some wild animals to get closer to urban areas in search of food, the increased interest in visiting protected natural areas, and the hunting of game as a food source increase the possibility of sharing natural areas between wild animals, livestock, pets, and humans. From an epidemiological point of view, these factors also increase the possibility of the exchange of pathogens between these groups. Therefore, wild animals can act as a source of infection for domestic animals and humans. This study represents a retrospective analysis including viral agents and parasites affecting a cohort of wild animals in Italy, providing a comprehensive overview of their health status. Overall, a large number of animals tested positive for at least one pathogen, and many were infected with more than two agents, showing a wide range of pathogens responsible for intra- and interspecific transmission in wild populations living in the study areas. Abstract This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020–2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance.

A One-Year Retrospective Analysis of Viral and Parasitological Agents in Wildlife Animals Admitted to a First Aid Hospital In recent decades, wildlife populations in Italy have continued to expand, and some species are now present in large numbers with a wide geographical distribution. Viral and parasitic agents are an integral part of any wildlife population. The major changes in human land use, the tendency of some wild animals to get closer to urban areas in search of food, the increased interest in visiting protected natural areas, and the hunting of game as a food source increase the possibility of sharing natural areas between wild animals, livestock, pets, and humans. From an epidemiological point of view, these factors also increase the possibility of the exchange of pathogens between these groups. Therefore, wild animals can act as a source of infection for domestic animals and humans. This study represents a retrospective analysis including viral agents and parasites affecting a cohort of wild animals in Italy, providing a comprehensive overview of their health status. Overall, a large number of animals tested positive for at least one pathogen, and many were infected with more than two agents, showing a wide range of pathogens responsible for intra- and interspecific transmission in wild populations living in the study areas. This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020–2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance. In recent decades the incidence of introduced and emerging infectious diseases in humans and animals has increased worldwide [1,2]. Wildlife plays an important role in the epidemiology of infectious and parasitic diseases, resulting in a source of various pathogens [1,3,4] with significant implications on human health, wild and domestic animal health, biodiversity, and economy, as it harbours most of the emerging and re-emerging pathogens and could play a significant role in their spread and persistence in ecosystems [1,5,6,7]. Numerous studies on wildlife have shown that wild animals act as hosts for some known and unknown pathogens that are transmissible to several other species, including domestic animals and humans. Therefore, in recent years, research on wildlife diseases has become an increasing international interest as a crucial aspect of wildlife conservation projects, such as reintroduction and translocation programmes, especially for species of high conservation value, and programs of disease surveillance for domestic animals and humans [1,8,9]. Unfortunately, the elusive and unique nature of these animals makes it difficult to sample and conduct health surveillance actions in these species and to understand their real role in the epidemiology and ecology of diseases [10]. Wildlife rescue centres can play an important sentinel role and represent an underutilised source of information on pathogens circulating in ecosystems, particularly at the wildlife–domestic animal interface [11,12,13]. Due to the increasing proximity of wild animals to urban areas, often in search of food, and the growing public awareness of biodiversity conservation, the number of wild animals admitted to rescue centres is steadily increasing. Each year, rescue centres recover hundreds of animals belonging to many different species, some of which were impossible to sample in the wild. In addition, wildlife rescue centres represent a system in which many injured, diseased, and stressed animals interact and where the risk of introducing, amplifying, and spreading pathogens is considerable. Similarly, the release of rescued animals into the wild habitats can lead to the spread of pathogens in the area, posing a health risk to the local population. It is, therefore, important to gather information on the pathogens that are or could be circulating among hospitalised animals. Bovine viral diarrhoea virus (BVDV), family Flaviviridae, infects a variety of ungulate species and is associated with severe economic losses in livestock production worldwide. Several wild and domestic ruminant species cohabit during the grazing period, and BVDV circulation has been demonstrated in both domestic and wild ruminants and interspecific transmission has already been reported [14,15,16]. In Italy, a serosurvey was carried out in four large alpine ungulates in the High Valley of Susa, and antibodies were detected in chamois, wild boar, and red deer, but all roe deer were negative [15]. A similar result was obtained a few years later by Fernández-Sirera and colleagues. Small Ruminant Lentivirus (SRLV), genus Lentivirus, family Retroviridae, are widespread throughout the world and have a detrimental economic impact on the small ruminant industry due to increased mortality and reduced animal performance [17,18]. Bopivirus is a novel genus of the family Picornaviridae recently discovered in faecal samples from wild and domestic ruminants. To date, genus Bopivirus contains the species Bopivirus A, detected in cattle, the novel Bopivirus B, detected in sheep and goats in Hungary; and the newest, tentatively designed Bopivirus C, detected for the first time in faeces of Australian fallow and red deer [19,20]. Astroviruses (AstV) are small, round, non-enveloped RNA viruses. First discovered in children with diarrhoea, they have been discovered in a variety of domestic and wild animals, both with and without gastroenteritis [21,22,23,24,25,26,27,28,29,30,31]. The wide host range and genetic diversity within the Astroviridae family have made classification difficult, and they are currently divided into two genera, Mamastrovirus (with 19 species) and Avastrovirus (with 3 species), infecting mammalian and avian hosts, respectively. However, numerous unclassified AstVs have yet to be accepted as species [32]. Kobuvirus is a genus belonging to the Picornaviridae family and consists of three species, Aichivirus A (formerly Aichi virus), Aichivirus B (formerly Bovine kobuvirus) and Aichivirus C (porcine kobuvirus). First recognised in humans with gastroenteritis, kobuviruses have recently been detected in a variety of animals in several countries with an ever-increasing number of hosts. In particular, they have been found in domestic and wild carnivores, ruminants, and suids [33,34,35,36,37,38,39,40,41,42]. Canine adenovirus 1 (CAdV-1), family Adenoviridae, genus Mastadenovirus, is the causative agent of infectious canine hepatitis in dogs and is characterised by high pathogenicity due to differential tissue tropism. In addition to the dog, CAdV-1 infection has been documented in several free-ranging wild mammalian carnivores, foxes, wolves, brown bears, striped skunks, and Eurasian otters, causing from subclinical to fatal diseases and sporadic epizootics [43,44,45,46,47]. As far as parasitic agents are concerned, coccidia are protozoa with a direct life cycle that infect the epithelial cells of the intestinal tract of their hosts and that usually have high host-specificity. The following Eimeria species are found in roe deer: Eimeria capreoli; Eimeria catubrina; Eimeria panda; Eimeria patavina; Eimeria ponderosa; Eimeria rotunda; and Eimeria superba [48]. Instead, Isospora canis, Isospora canivelocis, Isospora ohioensis, Isospora pavlodoratica, Isospora vulpina, and Isospora vulpis may occur in red foxes, though it must be considered that coccidia of canids is an exceptionally confused group of coccidia [49]. Eimeria melis and Isospora melis have been reported in badgers [50]. Gastrointestinal strongyles (GSI) are very common among wild ruminants and always occur in mixed infections with different nematode species whose eggs are morphologically indistinguishable from one another. Infections with helminths belonging to the genera Bunostomum, Chabertia, Haemonchus, Nematodirus, Oesophagostomum, Ostertagia, and Thychostrongylus have been reported in the cervids [51]. Hosts get infected by direct ingestion of third-stage larvae whilst grazing. Capillaria bovis and Capillaria bilobata have been reported in roe deer [52,53]. This intestinal nematode of ruminants has a direct life cycle and is considered to be of low pathogenic significance. Dictyocaulus capreoli has previously been reported as a parasitic agent of bronchopulmonary infection in roe deer [54,55]. In particular, this lungworm is a specific parasite for C. capreolus, as the name suggests, and Alces alces (moose) [54]. The life cycle of the Dictyocaulus species is direct. Adult lungworms live in the bronchi and bronchioles of their hosts. Females are ovoviviparous. First-stage larvae hatch in the respiratory or intestinal tract and are expulsed with the faeces into the environment. Hosts get infected by ingesting L3 larvae whilst grazing. Intestinal worms (Toxocara canis, Trichuris vulpis, Ancylostomatidae), lungworms (Capillaria spp.), and heartworms (Dirofilaria immitis, Angiostrongylus vasorum) can be found in red foxes. Their life cycle can be direct (T. vulpis, Capillaria spp.), indirect (D. immitis, A. vasorum), or both (T. canis, Ancylostomatidae), depending on the species. Within the family Ancylostomatidae, both Ancyclostoma caninum and Uncinaria stenocephala have been reported in V. vulpes [56]. Among Capillaria species, both Capillaria aerophila (syn. Eucoleus aerophilus) and Capillaria boehmi (syn. Eucoleus boehmi) are commonly found in the respiratory system of fox populations [57,58]. Overall, within the family Ancylostomatidae, four species belonging to the Uncinaria or Tetragomphius genera [59] and unidentified species belonging to the Ancylostoma genus [60] have been reported in badgers. Uncinaria criniformis is the species widely reported in European badger populations [59,61,62,63]. Lungworm species reported in Meles include Crenosoma melesi [63] and Crenosoma vulpis [64], as well as C. aerophile [60]. Crenosoma spp. has an indirect life cycle with terrestrial gastropods as intermediate hosts, while C. aerophila has a direct life cycle. Strongyloides is a genus of small parasitic nematodes infecting the small intestine of mammals, characterised by an unusual life cycle involving one or more generations of free-living adult nematodes. Strongyloides sp. infections have been reported in European badgers [61,62]. Results of phylogenetic analysis in different carnivorous hosts suggested that badgers can be infected with a distinct species of Strongyloides [65]. The European badger may also be a definitive host for three species of heartworms. These include D. immitis as well as A. vasorum and Angiostrongylus daskalovi, which was considered a relatively rare species [61,62,66,67,68]. Angiostrongylus spp. has snails and slugs as intermediate hosts, while D. immitis is transmitted by mosquito bites. Reported prevalence values in badgers are 0.87% for D. immitis, 6.4 to 24% for A. vasorum, and 72% for A. daskalovi [61,62,66,67]. Neospora caninum, an apicomplexan protozoan, is among the main causes of abortion in cattle and has both horizontal and vertical transmission patterns. The existence of a sylvatic life cycle of N. caninum has been supported by some authors [69]. Giardia duodenalis is a flagellate protozoan with a direct orofecal life cycle. Molecular investigations have revealed that G. duodenalis is a complex species, including eight morphologically indistinguishable genot ypes or assemblages with different host ranges classified as A to H. Genotypes A and B infect humans but can also be found in animals with their respective sub-assemblages [70,71]. The Veterinary Teaching Hospital (VTH) of the Department of Veterinary Sciences of the University of Pisa has been providing a 24-h emergency service for wild mammals rescued in the Pisa area since 2010. The aim of the present study was to retrospectively investigate the presence and frequency of different virological and parasitic agents in a cohort of 50 injured wild mammals admitted to the VTH from September 2020 to August 2021. In addition, the study area was divided into three zones based on the different levels of urbanisation, population density, and land use and differences in the prevalence of viral pathogens concerning the rescue area were also investigated. Tuscany is the region of the Italian peninsula with the highest number of wild ungulates, with about 400,000 animals in constant increase, and it is estimated that 40% of roe deer, 45% of fallow deer, and 30% of wild boar of the entire country live there [72]. The province of Pisa, composed of 39 municipalities, covers an area of 2450 km2; 25% of the territory is made up of plains, with most of the inhabited centres, and the rest of the territory consists of hills and mountains suitable for the development of numerous and diverse wildlife populations [73]. Despite this, the data on land use shows that almost the entire territory of the province is used for industrial or agricultural purposes and that unused part is very limited [73]. Therefore, the uncontaminated and uninhabited areas are very limited, and wildlife habitats are fragmented and interrupted by an extensive network of roads and urban areas with many opportunities for contact and interaction between wildlife, domestic animals, and humans. Moreover, the territory of the province of Pisa is very diverse due to its considerable size, with the main urban settlements located in the northern area, where, in addition to the city of Pisa, some of the most important inhabited centres are located. For this study, the province was divided into three zones of similar size, characterised by different demographic densities, road networks, and land use (Figure 1, Figure 2, Figure 3 and Figure 4). The zones have been named Zone A, B, and C (Figure 1). Data on zone extension, number of inhabitants, population density, and forest cover, expressed as its relationship with the total area surface, are shown in Table 1 [73,74]. In this study, a cohort of 50 wild animals, injured and hospitalised during a one-year period (between September 2020 and August 2021), was retrospectively assessed for the presence and frequency of different virological and parasitic agents. The enrolled animals were older than 12 months (based on general conformation and teeth eruption), were of both sexes, and belonged to the following species: 23 roe deer (Capreolus capreolus); 4 fallow deer (Dama dama); 12 red foxes (Vulpes vulpes); 6 badgers (Meles meles); 4 porcupines (Hystrix cristata); and 1 pine marten (Martes martes). For each animal, information on the place and the cause of the rescue, as well as on age, sex, and species, was collected from the person who brought the animals (volunteers, citizens, local authorities) and during the first clinical examination and recorded in the hospital database. Data on the species and sex of the enrolled cohort are presented in Table 2. All the animals enrolled were hospitalised because they were injured. Overall, 34/50 (68%) of animals were injured in car accidents, 3/50 (6%) by predators, and 2/50 (4%) by fighting with conspecifics. In 11/50 (22%) animals, the cause for injuries could not be readily determined. Upon admission to the VTH, during the first clinical examination, a blood sample was usually collected from the jugular or cephalic vein of each animal under sedation or general anaesthesia to reduce the stress and handling time [75]. The blood sample was immediately used for routine laboratory analyses in order to perform a rapid diagnosis, while an aliquot was collected separately in a serum tube and centrifuged within 30 min after collection at 1000 rpm for 10 min. The serum sample obtained was stored at −20 °C to be used for future analyses. A faecal sample was also usually collected from each animal after natural voiding; an aliquot of the faecal sample was immediately stored at +5 °C and examined within 12 h for routine parasitological examinations (passive flotation, Baermann technique, coproantigen detection) while an aliquot was stored at −20 °C pending further analyses. Therefore, all the serological and molecular analyses on serum and faecal samples were performed after the period of hospitalisation and on stored frozen samples. A species-specific serological and molecular analysis panel was designed in agreement with previously reported data in the available literature on the most commonly detected viral agents in the enrolled species [28,33,76,77,78,79,80,81,82,83,84,85]. Molecular analyses were performed on faecal samples. Roe deer (n = 23) and fallow deer (n = 4) sera were tested for the detection of antibodies against Bovine Viral Diarrhea Virus (BVDV) and Small Ruminant Lentivirus (SRLV), and all animals’ (n = 50) sera were tested for antibodies detection against Hepatitis E virus (HEV). BVDV and SRLV antibodies were detected using the BVDV p80 Ab (IDEXX, Westbrook, ME, USA) and by Eradikit® SRLV Genotyping Kit (IN3 diagnostic, Grugliasco, Italy), respectively, after adaptation. The SRLV kit is suitable for the detection and genotyping of SRLV antibodies as it can discriminate whether a positive sample belongs to genotype A (Maedi Visna-like), B (CAEV-like), or E (Roccaverano strain). HEV antibodies were tested in all enrolled animals using the HEV Ab version ULTRA kit (DIA.PRO, Sesto San Giovanni, Italy). Optical density was measured using a plate reader (Multiscan FC; Thermo Scientific, Waltham, MA, USA) at a wavelength of 450 nm. The procedures and interpretation analysis of the analyses were carried out according to the manufacturer’s instructions. Species-specific panels for molecular analysis are summarised in the Supplementary Material (Table S1). For species with little data in the literature about previous virological studies, such as porcupine and pine marten, large panels were developed that included viruses identified in related species. Due to the multiple species tested in this study and the paucity of data on virus circulation in wildlife, broad-spectrum PCR protocols with consensus primers covering as many viral species as possible were used. Molecular analyses were performed on DNA and RNA extracted from 200 mg of faecal samples using the AllPrep PowerFecal DNA/RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA and RNA were eluted in 30 µL of Buffer AE and RNase-free water, respectively, and stored at −80 °C until analysis. PCR for the detection of Canine bocavirus-2 (CboV-2), Feline/canine parvovirus (FPV/CPV), Torque teno virus 1 and 2 (TTV-1, TTV-2), and Canine adenovirus (CadV), and both rounds of nested PCR for CboV-1 and 3 were performed on DNA using the HotStarTaq® Plus Master Mix Kit (Qiagen, Germany). RT-PCR for the detection of Canine distemper virus (CDV), Bovine viral diarrhea virus (BVDV), Bopivirus (BoPV), and Kobuvirus (KoV), and first rounds of the nested RT-PCR for Coronavirus (CoV) and Astrovirus (AstV) were performed on RNA using the OneStep RT-PCR Kit (Qiagen, Germany). Secondary rounds were performed using the HotStarTaq® Plus Master Mix Kit (Qiagen, Germany). The protocol used for CadV detection is reported to discriminate between CadV 1 and CadV 2, resulting in distinct amplicons of 508 bp and 1030 bp, respectively [86]. Samples positive at molecular analysis were subjected to sequence analysis (BMR genomics, Padova, Italy) to confirm PCR results and obtain phylogenetic information. The primer sets used in the PCR and rt-PCR were included as Supplementary Data (Table S2) [20,77,85,86,87,88,89,90,91,92,93,94,95]. PCR was performed according to the manufacturer’s instruction. A passive flotation technique was carried out on the refrigerated aliquots to detect nematode eggs and coccidian oocysts, using a commercial saturated solution of sodium chloride (specific gravity = 1.20) as flotation fluid (Coprosol®, Candioli Farmaceutici, Beinasco, TO, Italy). The parasite burden was estimated by determining the number of eggs per gram (EPG) and oocysts per gram (OPG) of faeces by the modified McMaster method, with the lowest detection limit of 50 EPG/OPG per gram. In addition, faecal samples were examined via the Baermann technique to detect bronchopulmonary and cardiopulmonary nematode larvae. The number of larvae per gram (LPG) of faeces was estimated as above. The mean and range of OPG, EPG and LPG were calculated. A panel of endoparasite infections was investigated by rapid in-clinic tests in a different range of species. In particular, a rapid diagnostic test to detect both Cryptosporidium and Giardia coproantigens (Fastest® Crypto-Giardia Strip, Megacor Diagnostik, Austria) was performed on all 47 faecal samples examined for parasitological infections. Moreover, sera from foxes (n = 10) and badgers (n = 5) were screened for the presence of antibodies against Leishmania infantum (Snap Leishmania, Idexx, USA), Angiostrongylus vasorum (Angio Detect Test, Idexx, Westbrook, ME, USA), and Dirofilaria immitis (Snap Heartworm Antigen Test, Idexx, Westbrook, ME, USA). Screening of seropositivity for Neospora caninum (Fastest® Neospora caninum test kit, Megacor Diagnostick, Gemeinde Hörbranz, Austria) was carried out on foxes, badgers, roe deer (n = 23), and fallow deer (n = 4). All the rapid in-clinic tests were performed according to their respective manufacturer’s instructions. Finally, serum samples from all animals were screened for Toxoplasma gondii infection by a commercial ELISA kit (ID Screen® Toxoplasmosis Indirect Multi-species, IDVET, Montpellier, France) according to the manufacturer’s protocol. In 4/13 (30.8%) roe deer that tested positive for lungworm larvae via the Baermann technique (see above), a transtracheal wash (TTW) was performed immediately after humane euthanasia due to severe injuries. Briefly, a polyurethane catheter (14G) (Terumo, Roma, Italy) was introduced 5 cm distal to the larynx at 45° to the skin between two tracheal rings identified by palpation. Once the catheter entered the tracheal lumen, the needle was retracted, and the stylet advanced. Then a catheter (1.3 mm × 50 cm) (Buster dog catheter, Kruuse, Denmark) was passed through the stylet, down the trachea, and towards the lungs, until resistance was found. A bolus of 30 Ml of sterile saline was injected using a 60 Ml syringe and the fluid was immediately aspirated to recover at least 10 Ml of fluid [96]. When the aspiration of fluid was complete, the catheter and stylet were withdrawn simultaneously. The TTW fluid was split into two 4 Ml EDTA tubes for parasitological examination. When lungworm specimens were detected in the amount of fluid recovered, they were stored for 24–48 h in physiological saline at 4 °C before the morphological examination and then stored in 70% alcohol pending molecular procedures for identification to species level. Molecular investigation of G. duodenalis and Cryptosporidium was performed on faecal samples testing positive for coproantigens test (Fastest® Crypto-Giardia Strip, Megacor Diagnostik, Gemeinde Hörbranz, Austria). Genomic DNA extraction was performed by QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Giardia genotyping was determined through the amplification of tpi and β-giardin genes. In detail, a 530 bp fragment of the tpi gene was obtained via a two-step nested PCR protocol using AL3543/AL3546 primers for primary PCR and AL3544/AL3545 for secondary PCR [97]. A 384 bp fragment of the β-giardin gene was amplified with a forward primer G7, a forward inner primer G376, and a reverse primer G759 [98]. Cryptosporidium genotyping was determined through the amplification of 800–850 bp of the GP60 gene. In a two-step nested PCR protocol, AL3531/AL3535 primers were utilized for primary PCR and AL3532/AL3534 for the secondary PCR [99]. All amplicons obtained were purified using the mi-PCR Purification Kit (Metabion International AG, Planegg, Germania), and sequencing was performed in the laboratory of Bio-Fab Research (Rome, Italy). In order to check the quality of the sequences, the resulting chromatograms were manually analyzed using Finch TYV1.4 software (Geospiza, Inc, Seattle, WA, USA). Consensus sequences were then compared with those available in the GenBank database by using the Standard Nucleotide BLAST search and aligned by Clustal Omega implemented in MEGA X with representative sequences of tpi, bg, and GP60 loci and used as references. Adult lungworms collected by TTW were identified to species level by PCR technique. Molecular analyses were performed by BMR Genomics (Padua, Italy, https://www.bmr-genomics.it/ accessed on 7 January 2022). Genomic DNA was extracted from the stored adult lungworms using the commercial PCRBIO Rapid Extract Lysis Kit (PCR Biosystems) following the manufacturer’s instructions. A fragment (about 700 base pairs in length) of the mitochondrial gene for cytochrome C oxidase subunit 1 (COX1) was used as a DNA barcoding system and amplified. The PCR amplification was carried out in a final mixture containing 12.5 μL of AmpliTaq Gold™ 360 Master Mix (Thermo Fisher), 2 μL of gDNA extracted, 1 μL (10 μM) of LCO1490 primer (5′-GGTCAACAAATCATAAAGATATTGG-3′), 1 μL (10 μM) of HCO2198 primer (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′), and deionized water, reaching a total reaction volume of 25 μL. The PCR reactions were subjected to the following conditions in a thermal cycler (Mastercycler®, Eppendorf): 95 °C × 2 min, then 5 cycles (95 °C × 40 s, 45 °C for 90 s, and 72 °C for 90 s), followed by 35 cycles (95 °C for 40 s, 50 °C for 90 s, and 72 °C for 60 s), and finally, 72 °C for 10 min. The amplification products were visualized after electrophoresis on 1.5% agarose gel. PCR products were purified with ExoSAP-IT™ (Thermo Fisher Scientific, Massachusetts, MA, USA), sequenced, and aligned via BLAST analysis to detect their identity by retrieving similar sequences deposited in NCBI’s GenBank database [100,101]. The prevalence of virological and parasitic agents was determined as the number of positive animals/numbers of examined animals X 100. The Fisher test was used to determine statistical differences between rescue areas on positivity/negativity for Astrovirus, Kobuvirus, Bopivirus, and Adenovirus. One out of 23 roe deer (4.3%) scored positive for antibodies against BVDV and one for SRLV, genotype A Maedi Visna-like, while all the fallow deer were serologically negative for both viruses. The positive roe deer was a male rescued in zone B. All the animals (50/50, 100%) scored negative for HEV antibodies. All animals tested negative for CBoV, FPV, CPV, TTV, BVD, CDV, and CoV. Animal distribution in the three-rescue area and results of virological molecular analysis presented by viruses, species and rescue area are reported in Table 3 and Table 4. Six out of 50 animals (12%) tested positive for KoV: 1/23 roe deer; 2/12 foxes; 2/4 porcupines; and 1/6 badgers, respectively. All positive roe deer, badger, foxes, and one porcupine were male; the other porcupine was female. All positive animals belonged to zone A; thus, no statistical comparison among zones was performed. Eight out of 50 (16%) animals tested positive for AstV: 3/23 roe deer (1 male and 2 female) and 5/12 foxes (4 male and 1 female). Four out of 8 (50%) animals belonged to zone A (3 foxes and 1 roe deer), 3 (37.5%) animals belonged to zone B (2 foxes and 1 roe deer) and 1 (12.5%) roe deer belonged to zone C. No differences were found between zone A vs. B (p = 0.626), zone A vs. C (p = 0.536), or zone B vs. C (p = 0.779). Foxes (n = 12), badgers (n = 6), and pine martens (n = 1) were tested for CAdV, and 5 out of 19 (26%) animals tested positive for CAdV type 1. Three out of 12 foxes and 2/6 badgers were positive; all the positive foxes were male, 2 from zone B and 1 from zona A. Both positive badgers were male, 1 from zone A and 1 from B. No animals tested for CAdV have been rescued in zone C. No statistical differences were found in CAdV type 1 prevalence between zones A vs. B (p = 0.161). Roe deer (n = 23) and fallow deer (n = 4) were tested for BoPV, and 1 out of 27 (3%) tested positive for BoPV. This was a female fallow deer from zone A; thus, no statistical comparison among zones was performed. Table S3 shows the accession numbers of the sequences entered in the GenBank database, along with the results of their analysis with Blast software. For each sequence in this study, the analysis identified a reference sequence and determined the per cent of nucleotide identity and E value obtained from their comparison. Overall, parasitological analyses were performed in a total of 47/50 (94%) animals, including 10 foxes, 5 badgers, 4 porcupines, 23 roe deer, 4 fallow deer, and 1 marten, as faecal and serum samples from 2 foxes and 1 badger were not available. Results of faecal examinations by flotation and Baermann techniques are shown in Table 5. Roe deer. Overall, 19/23 (82.6%) roe deer were found to be coprologically positive by flotation and/or Baerman techniques. Ten out of 23, 8/23, and 1/23 roe deer showed two, one, or three parasitological infections, respectively. Eggs of gastrointestinal strongyles (mean = 7, range ≤ 50–100 EPG), Eimeria oocysts (mean = 250, range ≤ 50–>1000 OPG), and Capillaria spp. eggs (mean ≤50 EPG), were found in 14/23, 5/23, and 2/23 roe deer, respectively. Lungworm larvae (mean = 23, range ≤ 50–150 LPG) consistent with first-stage larvae of Dictyocaulus sp. were detected in 13/23 (56.2%) roe deer. Red foxes. All foxes (10/10, 100%) were found to be parasitised. Two, three, four, and even six parasitic co-infections were detected in 5/10, 2/10, 1/10, and 1/10 foxes, respectively. Therefore, only one fox out of 10 showed a single parasitic infection with coccidia oocysts. Parasite prevalence was as follows: Ancylostomatidae eggs (mean = 344, range ≤ 50–>1000 EPG) were found in 8/10 foxes; Toxocara canis eggs (mean ≤50 EPG) in 5/10; Capillaria spp. eggs (mean = 75, range ≤ 50–300 EPG) in 4/10; Trichuris vulpis eggs (mean ≤50 EPG) in 3/10; and coccidia oocysts (mean = 600, range = 200–>1000 OPG) in 2/10. In addition, the spurious passage of Hymemolepis eggs (<50 EPG) was detected in 1/10 foxes. Larvae of A. vasorum (mean = 275, range < 50–>1000 LPG) were detected in stool samples from 4/10 foxes. Badgers. All badgers (5/5, 100%) harboured parasites. Infections with two and four parasitic agents or only a single infection were detected in 3/5, 1/5, and 1/5 of them, respectively. Ancylostomatidae eggs (mean = 80, range ≤ 50–300 EPG) and larvae of Crenosoma sp. (mean = 137, range ≤ 50–400 LPG) were found in stool samples from 5/5 and 4/5 badgers, respectively, while Strongyloides eggs (<50 EPG), Capillaria spp. eggs (<50 EPG), and coccidia oocysts (<50 OPG) were detected in 1/5 each. All the fallow deer, all the porcupines, and the marten were found negative for both the flotation and Baermann techniques. Results of the rapid in-clinic tests and the ELISA kit are summarized in Table 5. Briefly, eleven, six, and two samples from different host species showed positive results for N. caninum, G. duodenalis, and C. parvum, respectively. Six and four samples from foxes tested positive for A. vasorum and D. immitis, respectively, and one sample from badgers tested positive for D. immitis. No samples tested positive, either for L. infantum or T. gondii. Four adult lungworms were detected in the TTW fluid from one of the four (25%) roe deer found positive for Dictyocaulus sp. larvae via the Baermann technique. The specimens measured on average about 5 cm in length and 0.4 mm in width at the mid-body and had an elongated oral opening, dorso-ventrally flattened. Based on the length of the body and the shape of the oral opening, the lungworms were presumptively identified as Dictyocaulus sp. A total of six samples for G. duodenalis and two for Cryptosporidium spp., carried out positive by the coproantigens test, were tested by the two-step nested PCR protocols. By sequencing at the tpi locus, 1/6 (16.7%) sample from a roe deer and 1/6 (16.7%) from a porcupine were assigned to Assemblage A-AI and Assemblage B-BIV, respectively. Sequences obtained in this study were deposited in the GenBank database and are available under the accession numbers OP946512 and OP946513, respectively. The BLAST search results showed that COX1 of adult lungworms collected from a euthanized roe deer had 99–100% DNA homology with sequences of Dictyocaulus capreolus available in the GenBank database. The sequence obtained in this study was deposited in the GenBank database and is available under the accession number: OQ209836. To the best of our knowledge, this is the first molecular identification of D. capreolus in roe deer in Italy. Serological surveys in wildlife are a diagnostic tool to demonstrate the presence of antibodies against specific pathogens and to provide epidemiological data on pathogens; however, serological sampling in wild animals is not always feasible and can be very stressful for the animals themselves. In the present study, the hospitalisation of wild animals allowed blood sampling during clinical practice, and we decided to investigate by serological analysis a panel of pathogens for which direct virological diagnosis on faecal samples is not applicable. The detection of BVDV antibodies indicates the presence and circulation of the virus in wild animals; moreover, the absence of vaccination plans for wildlife removes doubts about possible vaccine seropositivity. In our survey, one roe deer out of 21 was serologically positive. This result is in agreement with data from European studies, which reported the absence of seropositive animals (Spain and England) up to 12% of seroprevalence (Norway) [14,102,103,104,105]. In Italy, a serosurvey was carried out in four large alpine ungulates in the High Valley of Susa, and antibodies were detected in chamois, wild boar, and red deer, but all roe deer were negative [15]. A similar result was obtained a few years later when Fernández-Sirera and colleagues tested domestic ruminants, cattle, sheep, wild ruminants, chamois, and roe deer in the same research area for the presence of antibodies against BVD. High prevalence was found in both domestic ruminants, 25% in cattle, 17% in sheep, and 42 % in chamois, but none of the 213 roe deer tested positive [16]. The results of the Italian studies and the low seroprevalence found throughout Europe, even in areas with regular contact with domestic animals with high prevalence, suggest that roe deer play a marginal role in the epidemiology of the virus and that they are mostly an occasional guest and does not represent a major risk to domestic ruminants. The low prevalence of the virus among the European roe deer populations compared to other wild deer, especially the chamois, is explained by the different habits of the roe deer. The roe deer is mainly a solitary animal that prefers bushy feeding areas to open pastures where it would be easy to encounter domestic ruminants [15]. In addition, it is possible that this species has low susceptibility to common pestiviruses. Indeed, Fischer and colleagues have discovered a new pestivirus subtype in roe deer [106], and this may influence the serological results. However, as the presence of BVDV infection in roe deer is rarely detected, it would be advisable to conduct further surveys in the study area to increase the number of samples and obtain a more reliable estimate of prevalence. In addition, 1 roe deer tested positive for SRLV, genotype A Maedi Visna-like. Although SRLV were originally thought to be species-specific pathogens, several lines of evidence showed that lentiviruses can efficiently cross the species barrier and adapt to new hosts [107,108]. Several studies reported the detection of SRLV antibodies in wild ruminants: Rocky Mountain goats; red deer; roe deer; and mouflon [109,110,111]. Therefore, it is possible that contact between wild and domestic small ruminants could result in cross-transmission of SRLV. Regarding viral molecular analysis, positivities in foxes (CAdV-1, KoV, AstV), roe deer (KoV, AstV,) fallow deer (BoPV), badgers (CAdV-1, KoV), and porcupine (KoV) have been detected. A recent investigation in Italy revealed the presence of bopiviruses in sheep, roe deer, chamois, and Alpine ibex, with sequences clustering with bopivirus strains previously detected in goats and sheep from Hungarian farms and in red deer with sequences closely related to Bopivirus C sequences identified in fallow and red deer in Australia [112]. In our study, one fallow deer sample resulted positive for Bopivirus, whereas all samples from roe deer were negative. According to BLAST analysis, the sequence shared 94–92% nucleotide (nt) identity with deer Bopivirus strain (Bopivirus C), 88–85% with bovine Bopivirus strain (Bopivirus A), and 72–70% with ovine–goat Bopivirus strain (Bopivirus B). The sample, therefore, showed the highest nucleotide identity to Bopivirus C previously identified in Australian fallow deer and Italian red deer. Although numerous viruses of the Picornavidae family are responsible for diseases with relevant clinical forms in humans and animals, the pathogenicity of Bopivirus in deer remains undetermined. The population In our study, as in previous studies, was apparently healthy or not affected by symptoms attributable to an infectious disease. Astroviruses were detected in foxes and roe deer representing, to our knowledge, the first report of astrovirus in these two species in Italy. Astroviruses have already been identified in foxes in the Netherlands and Australia using metagenomic techniques. In the first study, the sequences were mainly identified as two novel astroviruses called Fox Astrovirus F5 and F4, in the second, sequences clustered with feline astrovirus with high nt identity. Little research has been reported on AstVs in deer, but astroviruses have been identified in European roe deer in Denmark in 2010 [113] from animals with gastrointestinal disease, and in Slovenia from healthy hunted animals [114]. The following sequence analysis identified a new species of Mamastrovirus, the Capreolus capreolus Astrovirus (CcAstV strain 1 and 2), related to other bovine, porcine, yak, porcupine, and dromedary AstVs strains, possibly forming a group of currently unassigned sequences that are distantly related to mAstV genogroups I and II. Our sequences, both from foxes and roe deer, were obtained using broad-range primers amplifying a portion of RdRp without phylogenetic information; however, BLAST analysis of the amplicons confirms that this virus clustered with other mammalian-associated viruses within the mamastrovirus genera. In general, AstV infection is associated with mild to severe gastrointestinal tract disease, typically with diarrhoea and vomiting [115], but has also been associated with other diseases, such as shaking syndrome in mink, neurological disease in cattle and sheep, and encephalitis in humans [116,117,118,119]; infection may be asymptomatic [115]. There is no information on the symptoms in foxes, although it is likely that CcAstV can cause gastroenteritis in roe deer. In our study, the sampled animals did not have gastrointestinal disease or other disorders attributed to astroviruses. In Italy, kobuviruses have been detected in dogs, foxes, wolves, roe deer, goats, swine, cats, and cattle [33,38,39,40,76,120]. In this study, kobuviruses were detected in two foxes, one roe deer, one badger, and two porcupines, and none of the positive animals showed symptoms suggestive of enteritis. In foxes, Canine kobuvirus (CaKoV) has been detected in free-ranging foxes in Italy with a prevalence of 14.7%. To date, the known host range of CaKoV includes several species of Canidae (domestic dog, red fox, wolf, golden jackal, and side-striped jackal), and the virus has been detected in both diarrhoeic and asymptomatic animals, so the pathogenic potential of kobuviruses in carnivores remains to be elucidated. Our sequence of RdRp partial region from fox shared the highest identity with the CaKoV strains from Italian foxes with an nt sequence identity of 89–94% showing E-value ranging from 2e−30 to 9e−39. CaKoV is often found in co-infections as a potential secondary infection after primary infections by mainly viral agents responsible for immunosuppression, such as canine distemper virus or parvovirus [121,122,123]. In the studies of Di Martino and colleagues, both a fox and some dogs were co-infected by Kobuvirus and cCoV [40]. In our study, all foxes were negative to molecular analysis for CPV, CDV, and cCoV, but two foxes positive for kobuvirus were also PCR-positive for astrovirus, and one also for adenovirus; both were responsible for clinical gastroenteritis. Coinfection with CaKoV and cAstV and with CaKoV and cAdV has been described in diarrhoeic dogs in China and Korea, respectively [124,125]. In roe deer, kobuvirus RNA was found in 6.6% of rectal swabs collected from healthy roe deer in northern Italy between 2012 and 2014; most of these strains showed high homology with bovine kobuvirus, and others with caprine kobuvirus [76]. In our study, kobuvirus has been detected in 1/23 roe deer with a prevalence in line with previous studies. BLAST analysis of the partial 3D gene sequence showed 75% and 71% of nucleotide identity, respectively, with bovine and caprine kobuvirus detected in Italian roe deer, while the highest identities were shared with Italian fox kobuvirus. This result contrasts with what resulted in the previous study and it cannot be excluded that the roe deer may have ingested food contaminated with faecal contents of other species. However, it is important to consider that this is the second study to report Kobuvirus in wild ruminants and that very little is still known about which strains circulate among the populations of these animals and about the possibility of transmission of strains between different species. Kobuvirus has only been identified in one member of Mustelidae, a domestic ferret in the Netherlands, using a metagenomic approach, and the almost complete kobuvirus genome clustered with bovine and ovine kobuvirus (Aichivirus B) possibly indicates a past cross species transmission events [126]. No data is currently present about the circulation of kobuvirus in free-ranging mustelids that share the ecosystem with other wild animals susceptible to infection. In the present study, kobuvirus has been identified in 1/6 badger representing its first detection in this species. The badger resulted also positive for adenovirus. Our sequence clustered with sequences of CaKoV detected in wild and domestic carnivores with an nt sequence identity of 85–87% and an e value of 3 e−80–1 e−65. Finally, in our study, kobuvirus was also identified in two porcupines representing its first detection in this species. To date, kobuvirus has been detected in other rodents, such as rabbits, grey squirrels, and some species of mice (Peromyscus crinitus, Apodemus agrarius, Rattus norvegicus, Rattus losea, and Rattus argentiventer, Mus musculus, Rattus losea, Rattus tanezumi, and Rattus norvegicus) [30,36,127,128,129]. The BLAST analysis of our sequence showed a high homology both with both CaKoV (90–95% of nt identity, e value of 9 e−98–2 e−42), and murine kobuvirus (85–90% of nt identity, e value of 6 e−81–1 e−64), both belonging to the same genetic cluster within Aichivirus A, and more in-depth phylogenetic studies would be needed to better classify the virus. Interestingly, a high percentage of nt identity resulted between all our sequences (87–95%). This evidence, together with the common origin of all positive animals from zone A, suggests the possibility that a similar kobuvirus could infect different animal species sharing the same habitat. Moreover, three foxes and two badgers tested positive for cAdV-1. In foxes, cAdV was first identified in 1925 as the causative agent of severe neurologic disease, the epizootic fox encephalitis [130]. Subsequently, evidence of cAdV circulation in foxes has been reported in several geographical areas, with serological prevalence in the European population ranging from 3% in Germany to 64.4% in the UK [131,132]. In Italy, and more specifically in the Pisan area, the virological prevalence reported in previous studies is around 28% [132], which is in line with the value of 25% obtained in our study. All the positive animals were traumatised, and none showed clinical signs attributable to infectious diseases. Our data, together with the high prevalence values reported worldwide [44,45,132,133,134] and the identification of the virus in healthy animals reported in previous studies [132,134], suggest that red foxes may play a role in maintaining cAdV-1 in the territory and may be a source of viral spread in wild ecosystems. In addition, although cAdV-1 can be controlled in domestic dogs by vaccination, cases of infectious canine hepatitis in domestic dogs have been diagnosed in Italy in recent years [135], and the presence of infected foxes may also be a source of infection for domestic dogs, especially foxes living in peri-urban areas [135]. Adenovirus has previously been detected in Mustelidae, in a young striped skunk with hepatitis, in a seriously diseased Eurasian otter in the Seoul Grand Park Zoo, and in several pine martens and Eurasian otters in the UK, with prevalences of 33% and 88%, respectively [80,136,137]. However, our evidence is the first detection of adenovirus in badgers. Both fox and badger viral sequences clustered with Canine adenovirus 1 with a high percentage of nt identity (94–98%). The presence of cAdV-1 in badgers was expected as specific adenoviruses have also been identified to date in otters and pine martens (marten adenovirus and lutrine adenovirus) and it is already known that mustelids are susceptible to CAdV-1 infection [80]. Moreover, the home ranges of mustelids and other predators such as red foxes (Vulpes vulpes) may overlap and there is potential for cross-infection with pathogens through indirect contact with urine, faeces, and other infected fomites. Moving on to discuss the parasitological findings, although coccidiosis was presumably harmless for the animals examined in this study, coccidia may pose a major threat to some species of wildlife; for instance, juvenile coccidiosis may be associated with impaired growth and increased mortality in badger cubs [50]. In this study, oocysts of Eimeria were detected in fecal samples from roe deer, red foxes, and badgers, but identification to species level could not be reached. Normally GSI are of little clinical significance in wild ruminants, but if combined, they can cause profuse and watery diarrhea, anaemia of varying degrees, edema under the lower jaw extending along the abdomen, progressive weight loss, rough hair coat, anorexia, hypoproteinemia, reduced growth, and even death in severe cases [138]. Since a much higher prevalence of C. bovis (26.6%) than C. bilobata (6%) has been detected in C. capreolus [52,53], it is likely that C. bovis was the Capillaria species involved in roe deer of this study. Adults of D. capreoli were detected in the transtracheal wash fluid from a roe deer, as shown by the results of the molecular analysis. D. capreoli has been reported to cause anorexia, poor haircoat quality, delayed haircoat change, diarrhea, small apical atelectatic areas, peri-bronchial inflammation, and total bronchopneumopatia in roe deer [55]. In agreement with previous studies carried out in Italy [56,57], combining results of coprological analyses and commercial assays, intestinal worms, lungworms, and heartworms were found in red foxes. It has been reported that the infection with intestinal helminths, especially T. canis, and the number of helminth species per individual may negatively affect the host body condition in red foxes [139,140]. All parasites of foxes are shared with dogs [141], and some species can be zoonotic agents [142]. In particular, foxes may be considered an important source of infections with cardiopulmonary nematodes, which may be responsible for overt clinical disease in dogs [143,144]. The eggs of A. caninum and U stenocephala cannot be reliably distinguished microscopically. Likewise, eggs of C. aerophila and C bohemi are microscopically indistinguishable from one species to another because all Capillaridae eggs are morphologically characterized by a similar barrel shape with polar plugs [134]. Therefore, it was not possible to determine whether the foxes examined in this study were infected with A. caninum, U. stenocephala, or both and with C. aerophila, C. boehmi, or both. In addition, the spurious expulsion of Capillaria hepatica (syn. Calodium hepaticum) eggs, after preying or scavenging on infected hosts, cannot be excluded. Indeed, foxes are known to scavenge on carcasses and eat a wide range of rodents, including mice and rats, which commonly show a very high prevalence of infection with C. hepatica [145]. In this study, the spurious expulsion of typical eggs of Hymenolepis spp. was detected. Hymenolepis is a genus of zoonotic cestodes that commonly infect rodents, mostly mice and rats, with usually a high prevalence [146]. This suggests that at least some of the foxes examined were feeding on rodents at the time of sampling and that foxes may act as carriers for the dispersal of the eggs of Hymenolepis species into the environment. Conversely, though Capillaria plica may be highly prevalent in foxes in central Italy [147], shedding of C. plica eggs in faeces was excluded because adult parasites of C. plica live in the bladder of definitive hosts, and eggs are expulsed with urine. Within the family, Ancylostomatidae U. criniformis is the species widely reported in European badger populations [59,61,62,63]. Therefore, it may be suggested that U. criniformis was the species infecting badgers examined in this study. Lungworm infections in M. meles can be identified based on the typical morphology of L1 larvae (Crenosoma spp.) and eggs (C. aerophila). However, the limited information available on the morphological differences between L1 larvae of C. melesi and C. vulpis did not allow differentiation from one species to another. Therefore, it is likely that C. aerophila was the Capillaria species involved in the badgers of this study, but it was not possible to determine whether C. melesi, C. vulpis, or both were infecting the badgers examined. Similarly, to foxes, the shedding of spurious C. hepatica eggs, merely passing through the intestinal tract, cannot be completely excluded. Indeed, C. aerophila and C. hepatica eggs are hardly distinguishable by microscopy, while badgers are known to be scavenger animals and to eat small mammals, including mice and rats (often heavily infected by C. hepatica, as aforementioned). Oval, thin-shelled eggs with a fully developed coiled larva inside (typical of rhabditiform nematode parasites) were found in a badger faecal sample and were identified as eggs of Strongyloides sp. To our knowledge, the identification of Strongyloides species infecting the European badger is still unknown since Strongyloides specimens recovered in M. meles have not been identified to the species level [61,62]. Strongyloides specimens isolated from the Japanese badger (Meles anakuma) are closely related to Strongyloides procyonis on a phylogenetic tree and are morphologically similar to S. procyonis and Strongyloides martis [65]. However, the results of molecular phylogenetic analyses using mitochondrial genome sequencing as a marker suggested that they belonged to a species genetically separate from S. procyonis [65] Concerning Leishmania and Toxoplasma, it cannot be excluded that the negative results found in this study may be due to limitations (uncertain sensitivity) attributable to the screening tests. A rapid immunochromatographic test (marketed for dogs) has been previously used to determine the seroprevalence of anti-Leishmania antibodies in wild canids, such as red foxes and coyotes [148]. Likewise, a commercial indirect multi-species ELISA (marketed for ruminants, swine, dogs, and cats) has been previously used to determine the seroprevalence of anti-Toxoplasma antibodies in free-ranging and captive large African carnivores [149]. Therefore, at present, it can be assumed that these two commercial assays may be suitable for wildlife species, but they have not yet been validated for the diagnosis of Leishmania and Toxoplasma infections in the species examined in this study. Neospora caninum-antibodies were detected in 3/4 fallow deer, 7/23 roe deer, and 1/5 badgers. These findings agree with those of other studies where the reported seroprevalence values for Neospora were 11% in fallow deer [150] and 28% in roe deer [151], while 10.9% of badgers were found positive for Neospora DNA in brain, liver, or neck muscles [152]. Red foxes are considered natural intermediate hosts for N. caninum, with reported prevalences of 10.7% by the PCR [153] to 69.8% by the agglutination test [154]. However, no foxes tested positive for Neospora in this survey. The possible existence of a sylvatic life cycle of N. caninum has been postulated [69]. Therefore, the present results suggest that fallow deer, roe deer, and badgers may contribute to the sylvatic life cycle of N. caninum, if any, in some areas of central Italy. Further investigations are needed to elucidate the role they may play in maintaining the circulation of the parasite in nature. For the detection of A. vasorum infection in red foxes and badgers, the Angio Detect test was used in combination with the typical recovery of cardiopulmonary larvae by the Baermann technique. Angio Detect is commercialized as an in-clinic assay to detect A. vasorum circulating antigen in dogs but it has also been successfully used in other host species, such as red foxes and badgers [155,156]. Likewise, commercially available antigen detection tests, commercialized for use in dogs, have been successfully used to identify D. immitis infection in other host species, such as wolves and red foxes, coyotes, and island foxes [157,158,159]. In this study, the use of the Angio Detect rapid test enhanced the global sensitivity of the diagnostic procedure in foxes as it allowed the detection of two additional positive subjects concerning the traditional Baermann apparatus. It is likely that these two A. vasorum-antigen positive/Baermann negative foxes were in the prepatent period that characterises the early phase of any parasitic infection. The European badger may be a definitive host for the cardiopulmonary nematodes D. immitis, A. vasorum, and A. daskalovi [61,62,66,67,68]. In this study, D. immitis circulating antigen was detected in 1/5 badgers by the in-clinic test, but no one was found positive either for circulating antigen or larvae of Angiostrongylus spp. In this study, the molecular analysis of G. duodenalis was successfully amplified at the tpi locus in one roe deer and in one porcupine. Among isolates of G. duodenalis from C. capreolus, assemblage A was previously identified in the Netherlands and Poland, while assemblage B was detected in Poland [160,161,162]. Identification to sub-assemblage level was not carried out in those investigations. G. duodenalis sub-assemblage AII was reported in roe deer in Spain [163]. In the present study, sub-assemblage AI was identified. To the best of our knowledge, this is the first report of G. Duodenalis sub-assemblage AI in C. capreolus in Italy. This sub-assemblage has previously been isolated in other wild species in the same geographical area [164]. Likewise, the finding of G. duodenalis sub-assemblage BIV in a porcupine is consistent with data previously reported for the same host species in the same geographical area [165]. G. duodenalis sub-assemblages AI and BIV are mainly found in animals and, to a lesser extent, in humans. Therefore, they are considered potentially zoonotic [70]. The results highlight the widespread presence of pathogens in the population studied, confirming the role of wildlife in the epidemiology of several viruses and parasites. Although the impact on wild species’ health is not yet known for all the pathogens identified, they can pose a threat to wildlife and can potentially have important effects on the population dynamics of their hosts, especially when they are experiencing the cumulative effects of other pathogens and stressors. Moreover, some of the parasitic agents found can be transmitted to domestic animals, and some of them are zoonotic. Therefore, they can affect the health status of livestock or pets and can have public health implications, especially in a highly urbanised area such as the study area. The study highlights the key role that structures admitting wild animals can play as focal points for collecting information on the health of wildlife populations and monitoring the health of the environments in which they live.

PMC10000066

Yansheng Guo,Feifei Wang,Yongxia Mao,Weiyi Kong,Jiandong Wang,Guijie Zhang

Influence of Parturition on Rumen Bacteria and SCFAs in Holstein Cows Based on 16S rRNA Sequencing and Targeted Metabolomics

21-02-2023

parturition,dairy cows,rumen bacterial communities,short-chain fatty acids

Simple Summary The depressed appetite at parturition might induce the changes in the composition and quantity of rumen microbiota. At present, little is known about the influence of parturition on the levels of rumen microbiota and their fermentation ability in dairy cows. The objective of this study was to evaluate the effects of parturition on composition and quantity of bacterial communities and concentrations of short-chain fatty acids (SCFAs) with 16S rRNA high-throughput sequencing and targeted GC–MS/MS metabolomics. The results showed that parturition altered the levels of rumen bacteria and their fermentation ability. The findings provide a better understanding of the effect of parturition on rumen digestive function. Abstract The rumen fluids from ten cows at Day 3~5 before calving and Day 0 after calving were collected to analyze the composition and quantity of bacterial communities and concentrations of SCFAs. The results showed that the relative abundances of unidentified Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus genera were significant increased (p < 0.05), while that of unidentified-Prevotellaceae was notably decreased after calving (p < 0.05). In addition, the concentrations of acetic acid, propionic acid, butyric acid, and caproic acid obviously decreased after calving (p < 0.01). Our findings show that parturition altered the rumen microbiota and their fermentation ability in dairy cows. This study defines a rumen bacteria and metabolic profile of SCFAs associated with parturition in dairy cows.

Influence of Parturition on Rumen Bacteria and SCFAs in Holstein Cows Based on 16S rRNA Sequencing and Targeted Metabolomics The depressed appetite at parturition might induce the changes in the composition and quantity of rumen microbiota. At present, little is known about the influence of parturition on the levels of rumen microbiota and their fermentation ability in dairy cows. The objective of this study was to evaluate the effects of parturition on composition and quantity of bacterial communities and concentrations of short-chain fatty acids (SCFAs) with 16S rRNA high-throughput sequencing and targeted GC–MS/MS metabolomics. The results showed that parturition altered the levels of rumen bacteria and their fermentation ability. The findings provide a better understanding of the effect of parturition on rumen digestive function. The rumen fluids from ten cows at Day 3~5 before calving and Day 0 after calving were collected to analyze the composition and quantity of bacterial communities and concentrations of SCFAs. The results showed that the relative abundances of unidentified Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus genera were significant increased (p < 0.05), while that of unidentified-Prevotellaceae was notably decreased after calving (p < 0.05). In addition, the concentrations of acetic acid, propionic acid, butyric acid, and caproic acid obviously decreased after calving (p < 0.01). Our findings show that parturition altered the rumen microbiota and their fermentation ability in dairy cows. This study defines a rumen bacteria and metabolic profile of SCFAs associated with parturition in dairy cows. Parturition is a complex physiological process modulated by the endocrine, nervous, and immune systems, and other factors, during which the metabolism of hormones, sugars, proteins, and lipids is severely disturbed. Among these factors, alterations in hormones play a crucial role in the loss of appetite during late gestation in dairy cows [1,2]. On Days 3~5 before parturition, DMI is reduced sharply so that the daily feed intake is only approximately 8~9 kg per dairy cow [3]. Due to the limited feed intake, rumination time has been observed to be largely decreased about 8 h before delivery and gradually recovered about 6 h later [4]. Hence, rumination time has been well recognized as a means of detecting parturition in dairy cows [5]. Rumen microbiota, consisting of anaerobic bacteria, archaea, protozoa and fungi, ferment fibrous and nonfibrous sources of carbohydrates in the feed into SCFAs to supply energy [6]. Rumination is universally acknowledged as the primary means by which the size of feed particles consumed is decreased in dairy cattle [4]. More sites for microbial attachment are exposed when the feed particle is ruminated. During rumination, saliva is added and the accumulated CO2 and VFA are released via chewing, which can make the microenvironment more beneficial to bacterial growth [7]. Although the importance of rumination for microbial activities by reducing particle size is well recognized, the detailed effect of parturition on the quantity and fermentation activity of rumen microbial communities are not well reported and require further study. High-throughput sequencing is a category of powerful technologies for obtaining high-coverage information on the classification and diversity of microbial communities without isolation and culture [8], among which 16S rRNA sequencing has been commonly applied to explore the composition of microbial communities in the rumens and intestines of dairy cows [9,10]. Targeted GC–MS/MS metabolomics are suitable for identifying and quantifying small molecule acids, amino acids, sugars, and fatty acids, and have also been successfully used to check the concentrations of these metabolites in the serum, feces, and rumen fluid of ruminants [11,12,13]. Consequently, we collected the rumen fluids from ten cows on Days 3~5 before calving and Day 0 after calving to reveal the influence of parturition on rumen bacterial communities and SCFAs using 16S rRNA high-throughput sequencing and targeted GC–MS/MS metabolomics, attempting to provide more information on physiological process of parturition in Holstein dairy cows. The experimental scheme was approved by the Animal Ethics Committee of Ningxia University (authorization number: 025/22) and complied with the international guidelines for animal experiments. At a large modern enterprise around the Yinchuan city of China, twenty healthy Holstein cows on Days 3~5 before calving (2~3 parity and 3.2~3.5 BCS) were selected to collect rumen fluids. The cows were fed three times each day with TMR diet (Supplemental Table S1) and DMI was 9.2 ± 0.6 kg·d−1 during calving. The concentrate-to-forage ratio of TMR diet was 3.6:1. Their body temperatures were normal and blood ketones values were around 1.0. Each cow had taken rumen fluid at 1 h after their second meal. Ultimately, the rumen fluids from ten cows were retained as “samples before parturition” because the calving date of the ten cows coincided with expected calving date. Then, the rumen fluids as “samples after parturition” were taken from the same ten cows at 1 h after calving without eating. Rumen fluids before parturition were labeled as E1~E10 (Group E), and those after parturition were marked as A1~A10 (Group A). The rumen fluids were taken using special collectors equipped with a metal filter at one end and a 50 mL syringe at the other end. Discarding the first tube of rumen fluid and the second tube of rumen fluid was used as the test sample. The rumen fluids were filtered by four layers of sterilized gauze, transferred to cryopreservation tubes and stored at −80 °C. The rumen fluids were well mixed in maximum vortex frequency after slowly thawing at 4 °C. Fifty microliters of rumen fluid was vortexed with 100 μL of 36% chromatographic grade phosphoric acid solution for approximately 3 min in the eppendorf tube and then vortexed with 150 μL of chromatographic grade MTBE (methyl tertiary butyl ether) solvent containing internal standard to extract SCFAs from rumen samples with ultrasonication for approximately 5 min in an ice bath. The extracted solution was centrifuged at 12,000× g r/min and 4 °C for 10 min, and 90 μL of supernatant was absorbed into the sample bottle with a glass liner for later targeted GC–MS/MS analysis. An Agilent 7890A–5975C gas chromatograph-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a quadrupole analyzer was used for the qualitative and quantitative analysis of rumen SCFAs. According to previous literature [14], the GC–MS/MS conditions were developed (Supplemental Table S2). The high reliability of the GC–MS/MS conditions was confirmed with intraday precisions, interday precisions, and recovery rates of SCFAs with different concentrations of QC samples (Supplemental Table S3), and good stability of the instrument was observed through the total ion current [15] overlap of QC sample mass spectrometry (Supplemental Figure S1). The chromatographic peaks representing acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and caproic acid were ensured according to the ion pair information, secondary spectrum and retention time (Supplemental Figure S1). The linear regression equations of the seven SCFAs were established based on the peak intensities of SCFA standard solutions with serial concentrations (Table 1). The concentrations of SCFAs in rumen fluid were calculated based on linear regression equations (Supplemental Table S4). The file in the csv format containing names of SCFAs, concentrations and samples information was imported into MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/, accessed on 8 October 2021) to carry out the following metabolomics analysis. Principal component analysis (PCA) was used to visualize the changes in the metabolic profile of the seven rumen SCFAs before and after parturition. Variable importance in projection (VIP) values of the seven SCFAs were obtained by partial least squares-discriminant analysis (OPLS-DA). The p value and fold change (FC) were also acquired by t test. SCFAs with VIP ≥ 1, FC ≥ 1.5, and p < 0.05 were selected as biomarkers for differentiating dairy cows before and after parturition. The SDS method was used to extract the genomic DNA of rumen bacterial communities, and the purity and concentration of genomic DNA were evaluated with 1% AGE. Next, 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) primers with barcodes were designed to amplify the V3~V4 hypervariable regions of the rumen bacterial 16S rRNA gene [16,17]. The PCRs were carried out using a thermal cycle PCR system (Gene Amp 9700, ABI, Waltham, MA, USA) according to the published literature [17]. The amplified products were validated using 2% agarose gel electrophoresis and further purified using the Qiagen gel extraction kit (Qiagen, Hilden, Germany). After amplification, DNA libraries were constructed with a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and quantified with Qubit and Q-PCR methods. The qualified libraries were subsequently sequenced with a PE250 strategy via the NovaSeq6000 platform (Illumina Inc., San Diego, CA, USA). The reads of each sample were separated after removing the barcode and the primer sequences and then spliced into raw tags using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/ accessed on 9 January 2023) [18]. The raw tags were quality-filtered to obtain high-quality tags (clean tags) [19]. The clean tags were truncated and filtered via QIIME (V1.9.1, http://qiime.org/scripts/split_libraries_fastq.html accessed on 9 January 2023) [20], and chimeras were further removed with VSEARCH (https://github.com/torognes/vsearch/ accessed on 9 January 2023) [21]. The effective tags of all samples were finally obtained after the above data processing and then clustered into operational taxonomic units (OTUs) at 97% identity using UPARSE (v7.0.1001, http://www.drive5.com/uparse/ accessed on 9 January 2023) [22]. Meanwhile, the representative sequence of OTUs was selected according to the UPARSE algorithm. Species annotation and taxonomic analysis of each 16S rRNA gene sequence was performed with Mothur and the SSUrRNA database of SILVA132 (http://www.arb-silva.de/ accessed on 9 January 2023) with a threshold of 0.8~1 [23]. The bacterial community composition of each sample was assessed at the levels of kingdom, phylum, class, order, family, genus, and species. Alpha diversity indices of the groups, including Shannon, Simpson, Chao1, and ACE, were calculated with QIIME after homogenizing the data of each sample. The program R (Version 2.15.3) was used to generate the rarefaction curve and t test of alpha diversity between groups. Principal coordinate analysis (PCoA) based on unweighted UniFrac distance was selected to visualize beta diversity using WGCNA, stats, and ggplot2 packages in R. Anosim analysis based on Bray–Curtis distance were used to determine the difference in bacterial communities between two groups with the anosim function of the vegan package in R. The t test in R was used to search and visualize differential species between the two groups at the genus level. LEfSe (LDA Effect Size) software was used to find biomarkers for the two groups and generate an LDA score cladogram. Spearman correlation analysis between rumen bacterial genera with the top 30 abundances and SCFAs was carried out using MicrobiomeAnalyst 2.0 (https://www.microbiomeanalyst.ca/ accessed on 9 January 2023). The 2D scatter plots of PCA and OPLS-DA exhibited a clear separation of the metabolic profiles of the seven SCFAs between Groups E and A (Figure 1a,b), indicating that rumen fermentability in dairy cows obviously changed during parturition. Acetic acid, propionic acid, butyric acid, and caproic acid could be metabolic biomarkers for differentiating the rumen fluids before and after calving according to their VIP values (≥1), FC (≥1.5) p values (<0.05), and their concentrations in rumen fluid presented a significant decreasing trend during parturition. The average concentrations, pH of rumen liquids, p values, FC, and VIP values are shown in Table 1. An average of 1,399,432 raw tags were detected by 16S rRNA gene sequencing from the 20 rumen fluid samples of dairy cows before and after parturition, and 837,879 effective tags with an average length of 413 bp were ultimately screened after quality control and filtration (Supplemental Table S5). A total of 2590 OTUs were acquired by clustering at 97% identity, of which 1965 OTUs were shared by both groups. The species accumulation curve was prone to be flat when the number of samples were higher than 20, showing that the number of samples in this study was reliable for estimating species richness (Figure 2a). The rarefaction curve of alpha diversity tended to be flat as the sequencing depth increased, indicating that the sequencing results could reflect the diversity of bacterial communities in rumen fluid samples and that there would be no large number of new OTUs to appear even if the sequencing depth was further increased (Figure 2b). The Shannon, Simpson, Chao1, and ACE indices of alpha diversity reflecting the within-group diversity and abundance of the bacterial community are listed in Table 2. The Shannon, Chao1, and ACE indices in Group A were significantly higher than those in Group E (p < 0.05). The 2D scatter plot of PCoA by unweighted UniFrac distance of beta diversity showed a visible separation between Groups A and E (Figure 3a). As shown in Figure 3b, the nonparametric test of anosim by the Bray–Curtis distance proved that the difference between groups was significantly greater than that within groups (R = 0.399, p = 0.001). Five kinds of bacterial communities from phylum to species were identified as biomarkers for Groups E and A with LEfSe analysis, including f_Methanobacteriaceae, o_Methanobacteriales, c_Methanobacteria, f_Prevotellaceae, and f_Lachnospiraceae (Figure 4). Furthermore, genera with the top 30 abundances in the two groups were selected for the t test, and eighteen rumen bacterial genera were ascertained to be significantly different in relative abundance between the two groups (Figure 5). Eight major abundant genera (accounting for 0.05% of the total sequences in at least one sample) with differences multiple ≥2 were further confirmed as differential species between the two groups (Table 3). Among these eight genera, the relative abundances of unidentified_Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus in Group A were significantly higher than those in Group E. In contrast, the relative abundance of unidentified_Prevotellaceae in Group A was significantly lower than that in Group E. Correlation analysis was performed on rumen bacterial genera in group A and group E. The bacterial genera and SCFAs with |r| > 0.4 and p ≤ 0.05 were listed in Table 4. The results showed that acetic acid was significantly negatively correlated with Syntrophococcus, Acetitomaculum, Lactobacillus, unidentified_Enterobacteriaceae, and Methanobrevibacter (p < 0.05 or p < 0.01). Propionic acid was significantly negatively correlated with Olsenella, unidentified_Lachnospiraceae, Desulfobulbus, unidentified_ Enterobacteriaceae, and Lachnospira (p < 0.05 or p < 0.01), and significantly positively correlated with unidentified_ Prevotellaceae (p < 0.05). Butyric acid was significantly negatively correlated with Subdoligranulum, Lactobacillus, Acetitomaculum, Syntrophococcus, Solobacterium, Desulfobulbus, and unidentified_ Enterobacteriaceae (p < 0.05 or p < 0.01). The current study investigated the influence of parturition on the phenotype composition and quantity of bacteria in ruminal liquid of dairy cows using 16S sequencing technique. At present, either liquid or solid fractions in rumen are widely used for microbiome analysis. The two fractions have differentiated ecological niches [24], however, the high degree of similarity in microbial community composition, diversity, and relative abundance profiles between the two fractions in cattle and sheep have also been affirmed by a series of studies [25,26,27,28]. Considering this, the ruminal liquid is selected for the current study, which is easy to collect and poorly contaminated. Ruminal SCFAs, originating from microbial fermentation of feed carbohydrates, are absorbed by the rumen epithelium as a dominant source of energy for ruminants. Acetic acid is the most-produced compound, accounting for approximately 70~75% of the total production of SCFAs and supplying energy through the tricarboxylic acid cycle [29]. Propionic acid generates approximately 50~60% of glucose via hepatic gluconeogenesis [30]. Butyric acid accounts for 10~20% of the total production of SCFAs and is transformed into β-hydroxybutyric acid to provide energy for muscle tissue [31], and it also plays an important role in the regulation of innate and adaptive immune cell generation and function [32]. An intense negative energy balance at parturition is most likely induced by depressed appetite and the initiation of milk synthesis in dairy cows [33]. Although dairy cows can mobilize adipose tissue into fatty acids to remedy the energy deficit, excessive lipolysis heightens the risk for metabolic and inflammatory diseases [34]. The parturition period is critical for determining the potential of the cow mammary gland to synthesize milk, and the degree of that potential depends on how much nutrients are received by the gland [35]. In this study, the concentrations of acetic acid, propionic acid, and butyric acid in the rumen fluid after parturition were significantly lower than those before parturition, indicating that parturition probably aggravated negative energy balance, which in turn negatively affected milk production performance and increased the risk for postpartum metabolic and inflammatory diseases. Bacteria population acts as a key role in digestive and metabolic activities of rumen, obtaining energy from fiber, starch, sugars, and protein of feed. In our study, alpha diversity and beta diversity of rumen bacteria are distinctly altered before and after calving, indicating rumen bacterial community composition and its digestive and metabolic activities were affected by parturition. The relative abundances of rumen bacteria in genera level was further observed in our study to characterize their change feature during parturition, showing that seven bacteria abundances increased including Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira and Lactobacillus, and Prevotellaceae abundances decreased after calving. Prevotellaceae is the dominant flora, accounting for approximately 60%~70% of rumen microbes of dairy cows, which can decompose the protein and carbohydrates in the feed into propionic acid, lactic acid, and succinic acid [36]. Approximately 90% of glucose in dairy cows is generated by gluconeogenesis, of which 50–60% originates from propionic acid through hepatic gluconeogenesis [37]. In this study, propionic acid shows a positive correlation with Prevotellaceae in concentration before and after calving. Numerous studies have shown that dairy cows are prone to negative energy balance after calving [38]. Therefore, the decreasing in Prevotellaceae abundance is most likely be one of reasons for the negative energy balance in postpartum dairy cows. In the rumen, methane production is beneficial to the microbial growth and digestion by regulating the partial pressure of hydrogen [39]. Methanobrevibacter can utilize some metabolites, such as acetic acid, propionic acid, and H2, to generate methane [40], and a higher levels of methane emissions is correlated with high abundance of Methanobrevibacter [41]. In this study, the abundance of Methanobrevibacter is lower before than after calving, which is speculated to be related to the inhibition of rumination during parturition. As the abundance of Methanobrevibacter decreases, several acetic acid-producing bacteria (Lachnospiraceae, Acetitomaculum, Lactobacillus, Olsenella, Syntrophococcus, and Lachnospira) have declined before calving in this study. Family Lachnospiraceae has been verified as the predominant acetogen in the rumen fermentation system of dairy cows [42]. The genus Acetitomaculum exhibits a significant positive association with lower feed efficiency [25], which can ferment monosaccharides into acetic acid [43]. Lactobacillus can hydrolyze starch and other sugars to produce acetic acid, butyric acid, and lactic acid [44]. Olsenella has been found in GIT of human and animals, fermenting starch and glycogen substrates and producing lactic, acetic, and formic acid [15]. Syntrophococcus produces acetate only from pyruvate and various carbohydrates although its role is not well-understood in the rumen metabolism [45]. Lachnospira can produce pectin lyases and release into extracellular environment to decompose pectin to oligogalacturonides, which are metabolized as acetic acid within the cell [46]. However, we find that the concentrations of acetic acid (including butyrate) in rumen are significantly higher before calving than after calving, which is inconsistent with these bacterial changes. The reason for this inconsistency is most likely related to the large amount of energy required for postpartum lactation initiation in dairy cows. Our study investigated the composition of the bacterial communities and the concentrations of SCFAs in the rumen of dairy cows before and after calving with 16S rRNA high-throughput sequencing and targeted GC–MS/MS metabolomics. Distinct alteration in the composition of rumen bacteria before and after calving could attributed to the sharply decreasing feed intake during parturition. As a dominant flora, the decreasing in levels of Prevotellaceae of propionic acid-producing bacteria are most likely be one of the reasons for the negative energy balance in postpartum dairy cows. Lower Methanobrevibacter abundance before calving may be related to the inhibition of rumination during parturition. On the other hand, the change in abundance of several acetic acid-producing bacteria is inconsistent with that of acetic acid before and after calving, which is most likely related to the large amount of energy required for postpartum lactation initiation in dairy cows. Our current study only focused on rumen bacterial populations; it provided a more comprehensive understanding of parturition on the dairy cow microbiota and as to how scientists would investigate all major microbial populations within therumen, including protozoa and fungi.

PMC10000067

Yang Liu,Xiaomin Wang,Xiaoping Luo,Rui Wang,Bintao Zhai,Penglong Wang,Junyan Li,Xiaoye Yang

Transcriptomics and Proteomics of Haemonchus contortus in Response to Ivermectin Treatment

03-03-2023

ivermectin,Haemonchus contortus,transcriptomics,proteomics

Simple Summary Haemonchus contortus has a serious impact on the gastrointestinal health of ruminants and the economic sustainable development of animal husbandry. In this study, we examined transcriptomic and proteomic differences in ivermectin-resistant and -susceptible strains of H. contortus before and after Ivermectin (IVM) treatment. The results of two omics association analyses showed that UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in H. contortus drug resistance. Our study may provide useful data and new targets for research on the resistance response of H. contortus. Abstract A major problem faced by the agricultural industry is the resistance of Haemonchus contortus to anthelmintic drugs. For a better understanding of the response of H. contortus to IVM and for the screening of drug-resistance-related genes, we used RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) technology to detect the transcriptomic and proteomic changes in H. contortus after ivermectin treatment. An integrated analysis of the two omics showed that the differentially expressed genes and proteins were significantly enriched in the pathways of amino acid degradation, the metabolism of xenobiotics by cytochrome P450, the biosynthesis of amino acids, and the tricarboxylic acid cycle. We found that the upregulated UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in drug resistance in H. contortus. Our work will help in the understanding of the transcriptome and proteome changes in H. contortus after IVM and will facilitate the discovery of genes related to drug resistance. This information can be further applied to increase the understanding of the response of IVM in relation to H. contortus.

Transcriptomics and Proteomics of Haemonchus contortus in Response to Ivermectin Treatment Haemonchus contortus has a serious impact on the gastrointestinal health of ruminants and the economic sustainable development of animal husbandry. In this study, we examined transcriptomic and proteomic differences in ivermectin-resistant and -susceptible strains of H. contortus before and after Ivermectin (IVM) treatment. The results of two omics association analyses showed that UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in H. contortus drug resistance. Our study may provide useful data and new targets for research on the resistance response of H. contortus. A major problem faced by the agricultural industry is the resistance of Haemonchus contortus to anthelmintic drugs. For a better understanding of the response of H. contortus to IVM and for the screening of drug-resistance-related genes, we used RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) technology to detect the transcriptomic and proteomic changes in H. contortus after ivermectin treatment. An integrated analysis of the two omics showed that the differentially expressed genes and proteins were significantly enriched in the pathways of amino acid degradation, the metabolism of xenobiotics by cytochrome P450, the biosynthesis of amino acids, and the tricarboxylic acid cycle. We found that the upregulated UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in drug resistance in H. contortus. Our work will help in the understanding of the transcriptome and proteome changes in H. contortus after IVM and will facilitate the discovery of genes related to drug resistance. This information can be further applied to increase the understanding of the response of IVM in relation to H. contortus. Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes infecting small ruminants worldwide. It feeds on the blood of ruminant abomasum, especially that of sheep and goats. Infection by this blood-sucking nematode causes the symptoms of haemonchosis, which include anemia, diarrhea, weight loss, and even death in cases of severe infection [1]. Furthermore, milk and meat production can be reduced in infected animals, causing tremendous economic losses to the agricultural industry worldwide. However, no vaccines protecting against these parasites are currently available; the primary form of nematode control is the use of anthelmintic drugs [2,3,4,5]. Ivermectin (IVM) is the first commercially available macrocyclic lactone endectocide. Since its introduction into the agricultural market in the early 1980s, it has been used to treat a wide variety of nematode parasites, including H. contortus, and it has quickly proven to be a very effective drug [6,7,8]. However, despite its benefits, the widespread usage of IVM has resulted in serious problems related to drug resistance worldwide, especially in countries where livestock husbandry is the dominant industry. In a vicious cycle of use and resistance, as IVM use increases, parasite resistance increases accordingly, which necessitates the use of yet more drugs [9,10,11]. Therefore, the problem of drug resistance has become an international issue that urgently requires a solution. Drug resistance is the tolerance of a parasite to a given drug. This problem arose in conjunction with IVM five years after its initial use in the control of parasitic diseases [12]. It can therefore be inferred that the development of drug resistance is a gradual process. According to reports, IVM can be used as an agonist of glutamate to enhance the opening frequency of glutamate-gated chloride (GluCl). A low concentration of ivermectin can enhance the action of neurotransmitters, and a high concentration of ivermectin can enhance cell membrane permeability to chloride ions, leading to blocked nerve conduction and muscle relaxant paralysis. After the peristalsis of the pharyngeal muscles of H. contortus is blocked, the feeding of H. contortus is disturbed or hindered, which eventually leads to the starvation and death of H. contortus [13,14,15]. Many studies have shown that nematode ATP-binding cassette (ABC) transport proteins, including P-glycoproteins (Pgps), play an important role in anthelmintic resistance [16,17,18]. One study showed that Pgps act as efflux pumps to expel hydrophobic xenobiotics from cells [19,20]. According to the report, the ability of multidrug-resistant Teladorsagia circumcincta to survive IVM exposure may be associated with the increased expression of Pgp-9 and gene sequence polymorphism [21]. Similarly, the expression levels of Pgps in Caenorhabditis elegans after exposure to IVM and the sensitivity of Pgp knock-out strains of C. elegans to IVM are both increased [2,22]. Furthermore, Pgp-9.2 may be one of the most relevant candidates contributing to the multi-genic nature of the IVM resistance trait [23]. Cwiklinski et al. (2013) found that glc-3 of Cylicostephanus goldi is one of the primary targets of macrocyclic lactone anthelmintics through a transcriptome analysis [24]. Anthelmintic resistance can be inherited, as its development requires the existence of resistance genes. The study of these drug-resistant genes is therefore the first step in understanding parasite resistance. Drug resistance is the result of the common regulation of multiple genes, rather than being attributable to a single gene [25,26,27]. Studying drug resistance is therefore vital for monitoring and controlling its further evolution and for delaying the accumulation of drug-resistance-related genes. However, the study of drug resistance is still in relatively early stages; drug-resistance-related genes are not comprehensively understood, and there are many such genes that need to be explored. It is urgent to identify genes that are potentially related to drug resistance. The objective of the present study was to use high-throughput sequencing combined with a bioinformatics analysis to investigate the changes in gene expression in H. contortus in both resistant and sensitive strains before and after IVM treatment. This study shows that IVM can cause transcriptional and proteomic changes in H. contortus. The study design was reviewed and approved by the Animal Ethics Committee of Ningxia University (Permit No. 22-031). The procedures involving animals were carried out in accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. All efforts were made to minimize suffering and to reduce the number of sheep used in the experiment. The IVM-susceptible and -resistant H. contortus strains were isolated in our laboratory at Inner Mongolia Agricultural University, where they have been maintained for several years [28]. All experimental sheep were newborn lambs from our laboratory in Hohhot. Each animal was housed in a single pen and had free access to food and water. Fecal samples were collected and examined using the McMaster technique at regular intervals to ensure that the nematode egg counts of all sheep showed negative values (mean fecal egg count = 0 eggs per gram). After a week of feeding, the sheep were infected with approximately 104 of either susceptible or resistant H. contortus in the L3 stage. The health of all infected sheep was monitored closely. After 20 days, feces were collected from each infected animal and placed into corresponding boxes with small holes, which were marked with the collection date and strain number. For the recovery of H. contortus, the boxes were incubated at 27 °C for approximately one week, and the fecal samples were slightly moistened with tap water as necessary (e.g., under dry conditions). After approximately one week, H. contortus at the L3 stage were collected with a self-made funnel separator, rinsed thoroughly with deionized water, and stored at 15 °C for further use. The two treatment groups were susceptible larvae treated with IVM (S1) and resistant larvae treated with IVM (R1). The two control groups, S0 and R0, were L3 larvae from susceptible and resistant strains, respectively, that had not been treated with IVM. The final concentration of IVM in the treatment groups (S1 and R1) was 0.28 μM, while the control groups (S0 and R0) were treated with a fresh medium without IVM. Each group included three biological replicates. All samples were cultured for 24 h and then harvested and stored in liquid nitrogen until used for RNA extraction, RNA-seq, and iTRAQ. Lysis Buffer (600 µL) was added to the sequencing sample, and then the total RNA was individually extracted from each sample using a mirVana miRNA Isolation Kit (Ambion, Shanghai, China) following the manufacturer’s protocol (Supplementary Materials Table S1). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with an RNA integrity number ≥ 7 were subjected to subsequent analyses. Libraries were constructed using a TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The libraries were sequenced, performed on an Illumina sequencing platform (HiSeqTM 2500, Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated. Raw data were processed using Trimmomatic [29] to remove low-quality reads and those containing ploy-N in order to obtain clean reads. The clean reads were assembled into expressed sequence tag clusters (contigs) and assembled de novo into transcripts with Trinity [30] using the paired-end method. The longest transcript was chosen according to similarity for subsequent analyses. All downstream analyses were based on the clean, high-quality reads. Blastx [31] was used to annotate the unigenes by aligning these with the following NCBI databases: nonredundant protein (NR), SwissProt, and Clusters of Orthologous Groups (COG) for C. elegans complete genomes. In addition, the proteins with the highest number of unigene hits were assigned functional annotations. Based on the SwissProt annotation, Gene Ontology (GO) classification was performed by mapping the associations between the SwissProt sequences and the GO terms; to annotate potential metabolic pathways, the unigenes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [32]. Fragments per kilobase of exon per million fragments mapped (FPKM) [33] and the read count values of each unigene were calculated using Bowtie 2 [34] and eXpress [35]. The differentially expressed genes (DEGs) of each group (three replicates per group) were identified using the DESeq [36] R package functions estimateSizeFactors and nbinomTest. A gene with a p-value < 0.05 and log2foldchange ≥ 1 or ≤−1 was considered to be differentially expressed. GO and KEGG pathway enrichment analyses of DEGs were performed using R based on the hypergeometric distribution. RNA-seq, read alignment, and DEG identification were carried out at OE Biotech (Shanghai, China). The frozen samples were removed and ground thoroughly in the presence of liquid nitrogen. A mixture of phenol extraction solution and PMSF (600 µL) was added to attain a final concentration of 1 mM. The samples were further lysed via sonication (1 s ntervals, 3 min, 80 W). A phenol-Tris-HCl (pH 7.8) saturated solution was added and shaken (30 min, 4 °C). The mixtures were centrifuged (7100× g, 10 min, 4 °C) to collect phenol supernatants. The supernatants were added to five volumes of 0.1 M cold ammonium acetate–methanol buffer and precipitated at −20 °C overnight. The precipitate was washed with five volumes of cold methanol and centrifuged again (12,000× g, 10 min, 4 °C) to remove more precipitate. This process was then repeated. Methanol was replaced with acetone, and the wash step was performed twice. The samples were centrifuged (12,000× g, 10 min, 4 °C) to collect the precipitate, which was dried at room temperature and dissolved in lysis buffer for 3 h. The samples were centrifuged, and the supernatants were collected. The supernatants were centrifuged again to remove precipitates completely. Protein concentration was determined using the BCA method [37], and the proteins were then stored at −80 °C. Additionally, 7 μg samples were subjected to 12% SDS-PAGE, visualized, and scanned according to Candiano’s protocol [38]. The FASP method [39] was adopted for the enzymatic hydrolysis of the proteins (100 μg), and the labeling peptide solutions were lyophilized and stored at −80 °C. Reversed-phase liquid chromatography was performed on an 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). The elution buffer was collected every 1 min and placed in turn into a 1–15 centrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA); samples were harvested from 8 min to 60 min. After collection, the samples were vacuum freeze-dried and cryopreserved for MS detection. The samples were loaded using a capillary C18 trap column (3 cm × 100 µm) and separated using a C18 column (15 cm × 75 µm) on an Eksigent nanoLC-1D Plus System (SCIEX, Framingham, MA, USA). An analysis was performed using a TripleTOF 5600 mass spectrometer (SCIEX, Framingham, MA, USA) equipped with a Nanospray III source (SCIEX, Framingham, MA, USA). All raw LC-MS/MS data were searched against the sample protein database using Proteome DiscovererTM 2.2 software (Thermo, USA). At least two peptides are required for a peptide group to be considered for the purpose of quantification; the false positive rate of peptide identification was controlled below 1%. The expressions of DEGs in different groups were detected using quantitative real-time PCR (q-PCR) to confirm the RNA-seq-based transcriptional response of the susceptible and resistant strains of L3-stage H. contortus before and after IVM treatment. Genes that were upregulated or downregulated were identified by performing a sequencing analysis. The RNA samples were reverse-transcribed to single-stranded cDNA using a PrimeScriptTM RT Reagent Kit (TaKaRa, Dalian, China). The same samples were used for sequencing and q-PCR. β-tublin was used as the reference gene, and the DEGs used for q-PCR verification were randomly selected. TB Green® Premix Ex Taq™ II (TaKaRa, Dalian, China) was used to perform q-PCR according to the manufacturer’s instructions. The selected genes were analyzed in triplicate; the forward (F) and reverse (R) primers used in q-PCR are listed in Table S2. The q-PCR cycling was performed under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, 95 °C for 10 s, and a melting curve analysis ranging from 65 °C to 95 °C. The 2−ΔΔCT relative expression method was used to calculate the expression of each gene. We used the Illumina HiSeqTM 2500 platform with the cDNA libraries from IVM-treated H. contortus and obtained over 48,000,000 raw reads from each sample and more than 47,000,000 clean reads after processing (Supplementary Materials Table S3). A total of 69,728 unigenes were spliced, with a total length of 62,243,154 bp and an average length of 892 bp. The correlation coefficient of the unigene expression level among the different samples was close to 1, which indicates a high similarity of expression patterns between the samples (Supplementary Materials Figure S1). The sequencing data determined in this work have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra, accessed on 3 April 2022) under accession no. PRJNA663203. The transcriptome analysis detected 3301 upregulated and 1227 downregulated genes in the susceptible H. contortus strain after IVM treatment (S0-vs-S1), while 1606 upregulated and 1432 downregulated genes were detected after IVM treatment in the resistant strain (R0-vs-R1). Additionally, 2058 upregulated and 2757 downregulated genes were detected in the non-treated resistant strain compared with the non-treated susceptible strain (S0-vs-R0), while 1406 upregulated and 3493 downregulated genes were detected in the IVM-treated resistant strain compared with the IVM-treated susceptible strain (S1-vs-R1; Figure 1). Regarding proteomics, a total of 1549 proteins were identified, of which 354 (226 upregulated and 128 downregulated), 89 (11 upregulated and 78 downregulated), 655 (438 upregulated and 217 downregulated), and 236 (81 upregulated and 155 downregulated) proteins were differentially expressed in the four comparison groups, (S0-vs-S1, R0-vs-R1, S0-vs-R0, and S1-vs-R1, respectively; Figure 2). We also found that the total number of DEPs was much lower than the total number of DEGs. The expressions of the genes obtained through the RNA-seq were confirmed using qPCR, and the validation results are shown in Figure 3. To identify the genes and proteins associated with drug resistance, we integrated the differentially expressed transcripts and proteins. As shown in Figure 4, almost all of the log2 mRNA:log2 protein ratios are concentrated in the center of the plot; these genes and proteins were filtered out, while the DEPs and DEGs were left in place. An integrated analysis of the transcriptome and proteome data revealed that the expressions of one gene and the corresponding protein (TRINITY_DN33068_c0_g1_i6_4) were upregulated and that two other genes (TRINITY_DN29476_c0_g1_i7_3 and TRINITY_DN42604_c4_g1_i6_1) were downregulated in the S0-vs-S1 group; one gene and the corresponding protein (TRINITY_DN35814_c0_g1_i8_2) were downregulated in the R1-vs-R0 group; one gene and the corresponding protein (TRINITY_DN43412_c0_g1_i1_3) were upregulated and nine were downregulated in the R0-vs-S0 group; and three genes and the corresponding proteins (TRINITY_DN30909_c0_g1_i3_1, TRINITY_DN38401_c0_g1_i1_1, and TRINITY_DN3919_c0_g1_i1_1) were downregulated in the R1-vs-S1 group (Supplementary Materials Figure S2). The GO enrichment analysis showed dynamic differences in the biological processes, cellular components, and molecular functions of H. contortus before and after IVM treatment. Based on the −log10 p-value, we list the top 30 GO terms for DEGs and DEPs of different comparison groups (Figure 5 and Figure 6). Most of the GO terms in the biological process category for DEGs were associated with metabolic processes and catalytic activity, while those for DEPs were mainly involved in redox and catabolism. In the cellular component category, DEGs showed significant enrichment in the extracellular region (GO:0005576), ribosome (GO:0005840), and cytosolic large ribosomal subunit (GO:0022625), while DEPs were mainly classified in the intracellular organelle region (GO:0044446), cytoplasm (GO:0005737), and mitochondrial membrane (GO:0044455). In the molecular function category, DEGs were enriched in metalloendopeptidase activity (GO:0004222), structural constituents of ribosomes (GO:0003735), and cysteine-type peptidase activity (GO:0008234; Figure 5), while enriched DEPs were mainly related to oxidoreductase activity (GO:0016635), structural molecule activity (GO:0005198), and structural constituents of ribosomes (GO:0003735; Figure 6). The other enriched terms shared by DEGs and DEPs (not shown in the figure) were response to drug (GO:0042493), drug transmembrane transport (GO:0006855), negative regulation of response to drug (GO:2001024), drug binding (GO:0008144), and drug transmembrane transporter activity (GO:0015238). In summary, the GO enrichment analysis further showed that the orderly cooperation of biosynthesis, decomposition, metabolism, and transmembrane transport collectively maintained the metabolism and homeostasis of H. contortus. The KEGG enrichment analysis of the DEGs and DEPs revealed 4528 DEGs in the 186 KEGG pathways of the S1-vs-S0 group; 3038 DEGs were enriched in the 170 KEGG pathways of the R1-vs-R0 group; 4815 DEGs were enriched in the 231 KEGG pathways of the R1-vs-R0 group; and 4899 DEGs were enriched in the 230 KEGG pathways of the R1-vs-R0 group. We selected the 20 most significantly enriched KEGG pathways according to the enrichment scores. These DEGs were significantly enriched in the pathways related to xenobiotic metabolism by cytochrome P450, amino acid degradation, the biosynthesis of amino acids, drug metabolism–cytochrome P450, carbon metabolism, and the tricarboxylic acid (TCA) cycle (Figure 7). The DEPs were significantly enriched in the pathways related to carbon metabolism, the TCA cycle, endocytosis, ABC transporters (MRPs, Pgps), and drug metabolism–other enzymes (UGT, GST) (Figure 8). Overall, these DEGs and DEPs were enriched in pathways related to the decomposition, metabolism, and synthesis of substances; this result is consistent with that of the GO enrichment analysis. These genes and proteins may play an important role in the response to the metabolism of anthelmintics in susceptible and resistant strains. Drug resistance is usually defined as the ability of an organism to survive a given dose of drugs. Partly because of its large impact on economic development in most parts of the world, H. contortus is the most widely studied nematode in terms of drug resistance. The focus on this topic in this species has led to an upsurge in research into drug resistance. At the same time, due to its biological and physiological factors, such as its high fecundity, relatively large body size, and simple larval storage conditions, H. contortus is regarded as a good experimental model [40,41,42]. Omics has become an important research tool for exploring the molecular mechanisms related to drug resistance and for identifying genes related to drug resistance [43]. We therefore used omics sequencing techniques to analyze and investigate the expressions of different genes and proteins in resistant and sensitive strains of H. contortus. In this study, transcriptomic and proteomic sequencing techniques were used to evaluate the global transcriptomic and proteomic changes in H. contortus after IVM treatment. We found that 4528 genes in susceptible strains and 3038 genes in resistant strains were significantly regulated after IVM treatment (Figure 1), which indicates that IVM had significant effects on the gene and protein expressions in H. contortus, especially in susceptible strains. Conversely, 354 and 89 proteins were significantly regulated in the susceptible and resistant strains, respectively, after IVM treatment. The apparent quantitative contrast between the genes and proteins reflects the proteome–transcriptome complexity, and the result of this comparison may be due to the fact that most of the genes encoded either hypothetical or non-functional annotated proteins. Thus, there is still a large knowledge gap in our understanding of transcriptional responses under IVM treatment. GO and KEGG pathway enrichment analyses are downstream procedures that are commonly used to interpret differential expression results [44]. Some of the genes we were interested in were upregulated or downregulated; we used annotation as part of our GO and KEGG analyses in order to determine which terms or pathways were significantly enriched [45]. The research objectives of the two omics are the same, and there must be a certain correlation between the groups. The transcriptome and proteome association analyses showed that the genes and proteins that are associated with GTP, RNA, proteolysis, synthesis, and catabolism terms were upregulated under IVM treatment, indicating that the energy and protein production rates were increased in H. contortus after IVM treatment. It has been reported that the genes involved in these functions are also upregulated in H. contortus after albendazole (ABZ) treatment [46] and in Acinetobacter baumannii after antibiotic treatment [47]. This phenomenon may be caused by the drug delivery screening that the organisms are subjected to in determining resistant strains, with selective pressure leading to the upregulation of resistance-related gene expression in the presence of the drug. In addition, we found that a single drug-resistant gene did not necessarily lead to IVM resistance in H. contortus. An extensive network of resistance-related genes, such as MRPs and Pgps, play a protective role in response to the efflux of anthelmintics in susceptible and resistant strains. In this study, these genes and proteins were significantly regulated in both susceptible and resistant strains. The transcriptome and proteome KEGG enrichment analyses showed that some pathways associated with drug metabolism, such as that of xenobiotics by cytochrome P450 (cel00980) and drug metabolism–cytochrome P450 (cel00982), were activated after IVM treatment. Cytochrome P450 (CYP) is involved in a variety of biosynthetic, catabolic, and xenobiotic detoxification functions [48]. The relationship between CYP expression and drug resistance has been demonstrated in insects. It has been reported that multi-insecticide resistance in Drosophila simulans is associated with the overexpression of CYP6g1 [49,50] and that pyrethroid resistance is associated with the overexpression of CYP6P9 in Anopheles funestus [51]. Additionally, some studies suggest the opposite. For example, some proteins of C. elegans, especially members of the CYP35 family, have been shown to be inducible by exogenous organisms [52,53]. ABZ can induce the expression of multiple CYP genes in C. elegans [54], while the inhibitor of CYP, piperonyl butoxide (PBO), increases the toxicity of the insecticide rotenone to H. contortus larvae and adults [55]. In this study, a comparative analysis showed that the CYPs of H. contortus were significantly regulated after IVM treatment. Differences at the molecular level in susceptible strains before and after IVM treatment play a role in the study of drug resistance. The transcriptome and proteome association analysis of the expressions of differential genes and proteins in the S1-vs-S0 group revealed that the expression of only one gene, glutathione S-transferase (GST; TRINITY_DN33068_c0_g1_i6_4), in the IVM-treated susceptible strains showed the same trend (upregulation) as the corresponding protein (Supplementary Materials Figure S2). This gene was enriched in the pathways of xenobiotic metabolism by cytochrome P450 (cel00980), drug metabolism–cytochrome P450 (cel00982), and glutathione metabolism (cel00480). It has been reported that the activity of glutathione S-transferase (GST) is 1.5–1.8 times higher in the cambendazole-resistant strains of H. contortus than in susceptible strains [56]. The activity and expression of GST genes are upregulated in a dose-dependent manner in Helicoverpa armigera larvae after pesticide exposure [57]. Pugazhendhi et al. (2017) found that the antibiotic-induced GST activity of bacteria from a poultry litter was 3–4 times higher than that of the control, which led to the speculation that GST plays an important role in antibiotic resistance [58]. In addition, GST is involved in drug resistance in organisms such as Musca domestica, Bombyx mori, and Aedes aegypti [59,60,61,62]. In this study, the expression of GST in the drug-resistant strains was three times higher than that in the susceptible strains. This gene upregulation may enable resistance to the pressure of drug selection before the susceptible strain becomes resistant to the drug. In addition, we found that the UGT (TRINITY_DN44820_c0_g1_i7_2) gene, which is related to detoxification in organisms, was significantly enriched in transcriptome and proteome association pathways. According to one study, the UDP-glycosyltransferases (UGT) inhibitor chrysin reduces ABZ biotransformation in C. elegans [63]. Matoušková et al. (2018) found that the expression of UGT in drug-resistant strains of H. contortus was significantly higher than that in susceptible strains [64]. In this study, the expression of UGT was upregulated in drug-resistant strains before and after treatment and in both strains after IVM treatment; the expression was higher in resistant strains than in susceptible strains. The significant differences in the transcription and protein levels support the possibility of important roles for UGT and GST in general drug resistance; however, the role played by this upregulated transcription is currently unknown. Additionally, it remains to be elucidated whether the UGT and GST genes are involved in the biotransformation of IVM anthelmintics and how significant a role they play in resistance. The genes and pathways that we identified here may serve as therapeutic targets to control the further development of drug resistance. Further research will provide insights into the function of the various DEGs, elucidating the mechanisms of drug resistance, thereby potentially enabling these to be overcome through targeted therapy. In this study, we identified and evaluated the genes and pathways related to IVM resistance in H. contortus using an integrated transcriptomic and proteomic analysis. We found that 1432 and 1227 genes were downregulated in the IVM-treated resistant and susceptible strains, respectively, suggesting that IVM inhibits the expression of some of the genes in H. contortus. Among the many upregulated genes that we uncovered, we focused on the changes in UGT, GST, and the CYP genes. These DEGs and their associated DEPs were significantly enriched in RNA, proteolytic synthesis, catabolic functions, and some metabolism-related pathways. To sum up, our study provides useful information for a better understanding of the response of H. contortus to IVM and for the screening of genes that may be associated with drug resistance.

PMC10000068

Xuejia Zhai,Ling Mao,Min Wu,Jie Liu,Shicang Yu

Challenges of Anti-Mesothelin CAR-T-Cell Therapy

21-02-2023

chimeric antigen receptor T cells (CAR-T cells),mesothelin,clinical trial,solid tumor,immunotherapy

Simple Summary In recent years, chimeric antigen receptor (CAR)-T-cell therapy has achieved good results in hematological malignancies. Clinical trials on anti-MSLN CAR-T cells have shown that they have a high safety profile but limited efficacy. This article reviews the clinical research status, obstacles, progress and challenges of anti-MSLN CAR-T-cell therapy and summarizes the relevant strategies to improve the efficacy and safety of anti-MSLN CAR-T-cell therapy. Abstract Chimeric antigen receptor (CAR)-T-cell therapy is a kind of adoptive T-cell therapy (ACT) that has developed rapidly in recent years. Mesothelin (MSLN) is a tumor-associated antigen (TAA) that is highly expressed in various solid tumors and is an important target antigen for the development of new immunotherapies for solid tumors. This article reviews the clinical research status, obstacles, advancements and challenges of anti-MSLN CAR-T-cell therapy. Clinical trials on anti-MSLN CAR-T cells show that they have a high safety profile but limited efficacy. At present, local administration and introduction of new modifications are being used to enhance proliferation and persistence and to improve the efficacy and safety of anti-MSLN CAR-T cells. A number of clinical and basic studies have shown that the curative effect of combining this therapy with standard therapy is significantly better than that of monotherapy.

Challenges of Anti-Mesothelin CAR-T-Cell Therapy In recent years, chimeric antigen receptor (CAR)-T-cell therapy has achieved good results in hematological malignancies. Clinical trials on anti-MSLN CAR-T cells have shown that they have a high safety profile but limited efficacy. This article reviews the clinical research status, obstacles, progress and challenges of anti-MSLN CAR-T-cell therapy and summarizes the relevant strategies to improve the efficacy and safety of anti-MSLN CAR-T-cell therapy. Chimeric antigen receptor (CAR)-T-cell therapy is a kind of adoptive T-cell therapy (ACT) that has developed rapidly in recent years. Mesothelin (MSLN) is a tumor-associated antigen (TAA) that is highly expressed in various solid tumors and is an important target antigen for the development of new immunotherapies for solid tumors. This article reviews the clinical research status, obstacles, advancements and challenges of anti-MSLN CAR-T-cell therapy. Clinical trials on anti-MSLN CAR-T cells show that they have a high safety profile but limited efficacy. At present, local administration and introduction of new modifications are being used to enhance proliferation and persistence and to improve the efficacy and safety of anti-MSLN CAR-T cells. A number of clinical and basic studies have shown that the curative effect of combining this therapy with standard therapy is significantly better than that of monotherapy. The chimeric antigen receptor (CAR)-T-cell technique is a kind of cancer immunotherapy that has attracted much attention in recent years. It has achieved good curative effects in hematological malignancies, but many obstacles remain in the treatment of solid tumors. Mesothelin (MSLN) is a tumor-associated antigen (TAA) that is usually expressed only on the mesothelial surface of the body, but is significantly overexpressed in most solid tumors. This article reviews the clinical research status, difficulties and challenges of anti-MSLN CAR-T cells to provide new ideas for their application in the treatment of solid tumors. CAR is a protein composed of three parts: an extracellular antigen-binding domain, an intracellular signaling domain and a hinge region. Extracellular single-chain variable fragment (scFv) antibodies that specifically identify the surface antigen of cancer cells make up the extracellular antigen-binding domain. The target-binding domain of the CAR is composed of changeable heavy and light chains that are joined together by adaptable peptide linkers in the scFv fragment. The hinge region connects the scFv fragment to intracellular components. Through its glycine and serine sequences, the linker residue’s hydrophilicity improves flexibility, while the intermittent glutamine and lysine sequences improve solubility. The signal transduction components of T cells or natural killer cells, such as 4-1BB and CD28, which transduce extracellular binding signals to start downstream cascade stimulation signals are often the source of intracellular signaling domains. The immunoreceptor tyrosine-based activation motif (ITAM) found in the cytoplasmic CD3 domain is required for T-cell activation [1,2] (Figure 1). Four generations of CARs are currently undergoing experimental and clinical research. The difference lies in their intracellular signaling domains linked to the scFv receptor. Theoretically, third-generation CAR-T cells should have more activation and killing capacities than second-generation CAR-T cells. In addition, because of the heterogeneity of tumor cells, some tumor cells do not have antigens that can be specifically recognized by T cells and cannot be recognized and cleared by traditional CAR-T cells. This problem may be solved by fourth-generation CAR-T cells, which can recruit immune cells other than T cells to the tumor area [1,2,3] (Figure 2). To exert antitumor effects in vivo, CAR-T cells must be persistent, proliferative and able to infiltrate tumor tissue. In general, antigens and costimulatory signals in major histocompatibility complex (MHC)-dependent complexes, which involve the recruitment of T-cell surface CD28 and the costimulatory molecules CD80 or CD86 on antigen-presenting cells (APCs), are required for T-cell-mediated immune responses. CAR-T cells recognize specific tumor antigens independent of MHC molecule restriction to perform their antitumor functions. Once CAR binds specifically to TAA, ITAM phosphorylation activates CAR-T cells and induces cytokine secretion, CAR-T-cell proliferation and cytotoxicity. CAR-T cells exert cytotoxic effects by secreting perforin and granzymes and activating death receptor signaling through Fas/Fas ligand (FasL) or TNF/TNF-α [4,5]. MSLN is a glycosylated phosphatidylinositol-anchored protein that is usually expressed in small amounts on the surface of mesothelial cells in the pleura, pericardium, peritoneum and sheath (in males). However, it has been found that MSLN is overexpressed in a variety of cancers [6,7], including malignant mesothelioma [7,8,9,10,11], ovarian cancer [8,11,12,13], breast cancer [14,15,16], pancreatic cancer [8,17,18,19,20], lung cancer [21,22,23], gastric cancer [24,25,26,27], cervical cancer [28], uterine serous cancer [29] and cholangiocarcinoma [30,31]. The overexpression of MSLN in triple-negative breast cancer (TNBC) [14,15], ovarian cancer [12], lung adenocarcinoma [21,22], cholangiocarcinoma [31] and pancreatic cancer [17,18] is related to poor prognosis. MSLN is related to chemotherapy resistance, and downregulation of MSLN can restore cell sensitivity to cisplatin in malignant pleural mesothelioma [32,33]. The MSLN precursor is a 71-kDa glycoprotein that is cleaved by enzymes to release the 31-kDa megakaryocyte-enhancing factor (MPF) and 40-kDa mature MSLN. Mature MSLN can generate soluble mesothelin-related peptide (SMRP). The biological effect of SMRP is limited, but it can be quantified by detection in serum and pleural effusion. At present, some clinical studies have identified it as an observation index [33]. The extracellular domain of MSLN consists of region I (N-terminal region; residues 296–390), region II (residues 391–486) and region III (C-terminal region; residues 487–598) (Figure 3). Region I is the membrane-distal region (MDR), which can bind to the mucin MUC16 (also known as CA125). The mucin MUC16 is also expressed in most malignant mesothelioma cells and is associated with tumor aggressiveness. Compared with region I, region III mediates stronger T-cell activation and cytotoxicity and is a better target [34]. The mechanism may be that anti-MSLN CAR-T cells targeting MSLN region I must compete with CA125/MUC16 for the MSLN antigen interaction, which may weaken the binding and function of anti-MSLN CAR-T cells. However, MSLN region III bridges the extracellular domain and transmembrane region of MSLN, which might have a rigid structure or be responsible for a specific function, to provoke a stronger antitumor response [35]. As of December 2022, a total of 41 clinical trials have been registered on the clinicaltrials.gov website (https://clinicaltrials.gov/). Six studies (NCT02159716, NCT01355965, NCT01897415, NCT02414269, NCT03545815 and NCT01583686) have been published with all but one obtaining clinical outcome events. NCT01583686 did not enter phase II because of the 15 patients enrolled only 1 achieved stable disease (SD) with the remaining patients having progressive disease (PD) or succumbed to their disease [34]. (Table 1). Pretreatment with radiotherapy and chemotherapy prior to CAR-T-cell therapy can alter the tumor microenvironment (TME) and host immune response through several potential mechanisms: immunogenic cell death, decreased regulatory T cells (Tregs) [34], localized T-cell infiltration and activation of various proinflammatory factor pathways. Common medications used in preclinical studies include cyclophosphamide, oxaliplatin, fludarabine and albumin-bound paclitaxel before CAR-T-cell therapy [36]. Preclinical studies showed that pretreatment using oxaliplatin combined with cyclophosphamide (Ox/Cy) could improve the migration of CAR-T cells to the tumor, increase the infiltration of CAR-T cells into the tumor and enhance the sensitivity of the tumor to immune checkpoint blockade [37]. The mechanism is as follows: Ox/Cy activates multiple proinflammatory pathways, including the expression of T-cell recruitment chemokines in multiple cells in the TME, which helps CAR-T cells recruit tumors expressing chemokine receptor 5 (CCR5) and C-X-C chemokine receptor type 6 (CXCR6). Upon CXCR3-dependent CAR-T-cell recruitment into tumors, infiltrating CAR-T cells release interferon-γ (IFN-γ), modify the TME to activate M1 macrophages that express the CXCR3 ligands CXCL9 and CXCL10, and then initiate a positive feedback loop. Cyclophosphamide combined with fludarabine (Cy/Flu) is another common pretreatment used in preclinical studies [38]. Adding fludarabine can improve the proliferation of CAR-T cells and the disease-free survival rate of acute B-cell lymphoblastic leukemia [39]. In neuroblastoma, Cy/Flu-induced lymphoid depletion increases the circulation level of the steady-state cytokine interleukin-15 (IL-15) and increases CAR-T-cell expansion by up to 3 logs [40]. Currently, in clinical trials of anti-MSLN CAR-T cells, most of the conditioning regimens are cyclophosphamide alone (NCT03608618 and NCT02414269) and cyclophosphamide combined with fludarabine (NCT03814447, NCT03799913, NCT01583686 and NCT05531708). One clinical trial (NCT02159716) [41] evaluated the feasibility and safety of anti-MSLN CAR-T cells with and without cyclophosphamide pretreatment in 15 patients, and the results showed that the maximum tolerable dose of anti-MSLN CAR-T cells was 3 × 108 cells/m2 and that there were no targeted toxic reactions, such as pleurisy, pericarditis, and peritonitis. However, CAR-T cells did not exert significant clinical efficacy on SD. There is strong evidence suggesting that the PD-1/PD-L1 interaction between T cells and tumor cells causes the inhibition of T-cell function [42] and depletion of T cells [43]. Therefore, combination with PD-1 or PD-L1 monoclonal antibodies or editing out PD-1 (PD-1 KO) may noticeably enhance the antitumor activity of cytotoxic T lymphocytes (CTLs), enable T cells to recognize tumor cells, promote tumor cell eradication and reduce T-cell exhaustion. Some studies have shown that PD-1KO CTLs are more effective in killing tumor cells than normal CTLs in vitro and in vivo [42,44]. A mouse xenograft tumor model was used to confirm the anticancer activity of PD-1KO CTLs [42]. The therapeutic impact of CAR-T cells may be enhanced by PD-1/PD-L1 inhibition, particularly in the treatment of solid malignancies. Mechanistically, PD-1KO can enhance the antitumor activity of CAR-T cells by blocking PD-1/PD-L1 and PD-1/PD-L2 signaling in these gene-edited T cells. As a technology to destroy the PD-1/PD-L1 interaction, gene modification has certain advantages in knocking out the PD-1 gene in T cells [42]. The question of whether PD-1 gene editing in host T cells is better than or equally effective to PD-1 mAb treatment is still unanswered due to a lack of solid evidence. However, from recent research results, gene editing of T cells for intracellular intrinsic immune checkpoint blockade may be safer than systemically injecting blocking antibodies into the body [45]. Hu et al. [46] employed CRISPR—Cas9-mediated gene editing to interfere with the PD-1 gene locations in anti-MSLN CAR-T cells to circumvent the inhibitory activity of PD-1 on CAR-T cells. Compared to control CAR-T cells, the release of cytokines (IFN-γ and IL-2) was significantly increased in PD-1 KO CAR-T cells. The results from experiments conducted in vitro and in vivo demonstrated that PD-1 KO CAR-T cells had potent anticancer efficacy against TNBC. The anticancer effect of CAR-T cells was improved in this model by PD-1 KO over PD-1 antibody. The aforementioned preclinical trials demonstrated the advantages of PD-1 inhibition and CAR-T-cell treatment in combination. To date, the results are very promising and have spurred a growing number of clinical studies. However, a study showed that PD-1 knockout can be problematic, with PD-1 KO T cells exhibiting advantageous short-term proliferation and cytotoxicity, but lacking long-term tolerance and showing vulnerability to T-cell depletion [47]. The therapeutic potential of PD-1 KO or PD-1 antibodies must therefore be completely clarified by additional, carefully conducted follow-up research and clinical studies [47]. In patients with solid tumors, PD-1 inhibition in combination with anti-MSLN CAR-T cells is being studied in terms of both safety and effectiveness. The relevant clinical trials are as follows: NCT05089266, NCT04503980, NCT04489862, NCT03615313, NCT03030001, NCT04577326, NCT03747965, NCT03545815, NCT03182803, NCT02414269 and NCT05373147. Of all these registered trials, only two have been published. Wang et al. [48] used CRISPR—Cas9 technology to knock down PD-1 and T-cell receptor (TCR) in anti-MSLN CAR-T cells, thereby affecting the TME, in order to treat MSLN-positive solid tumors. A total of 15 patients were included, and CAR-T cells were identified by qPCR in biopsy specimens, which proved that CAR-T cells could effectively infiltrate tumors. The median progression-free survival (PFS) of the seven patients with SD was 7.1 weeks, and most of the eight patients with PD died within 2 months after CAR-T-cell therapy. Adusumilli et al. [49] studied 23 patients who received cyclophosphamide combined with anti-MSLN CAR-T cells followed by at least three doses of pembrolizumab, and the overall survival (OS) following CAR-T-cell therapy was 23.9 months (95% CI, 14.7 months to NE). The one-year OS rate was 83% (95% CI, 68–100%). Among the combined immunotherapy patients (N = 16) with mRECIST measurable disease, two patients (12.5%) had partial response (PR), nine patients (56.3%) had SD, and five patients (31.3%) had PD. The application of combined CAR-T cells and an anti-PD-1 antibody in solid tumors is supported by these data. From this, a phase II study is now underway with a fixed dose of anti-MSLN CAR-T-cell infusion (6×107 CAR-T cells/kg) and pembrolizumab administration four weeks after CAR-T-cell infusion. After infusion, CAR-T cells can proliferate in the host and further differentiate into memory cells, potentially lasting up to 4 years [50]. However, the presence of these memory cells also increases the likelihood of autoimmune disease. To switch off potential toxicities of PD-1 KO CAR-T-cell therapy, suicide genes can be introduced into the CAR-T structure. Adusumilli et al. [49], Minagawa et al. [51] and Monica et al. [52] linked inducible suicide genes (icaspase9, TK suicide gene) to MSLN-CAR, CD33-CAR, CD44v6-CAR, CD19-CAR and CEA-CAR in their experiments. The barrier effect of the TME and extracellular matrix (ECM) limits the tumor invasion rate of CAR-T cells. Local administration can cause CAR-T cells to directly enter tumor cells, which greatly improves the tumor infiltration rate. Mayor et al. and Adusumilli et al. [53,54] showed that intrapleural injection of anti-MSLN CD28 costimulatory (M28z) CAR-T cells eradicated established pleural tumors, even at doses that were lower than intravenous injection of CAR-T cells by 30 times. Furthermore, locally injected CAR-T cells also demonstrated systemic, long-lasting antitumor immunity and were capable of easily traveling from the thoracic cavity to the flanking and peritoneal tumor locations. T-cell imaging revealed that local administration resulted in the accumulation of more CAR-T cells in the tumor at earlier time points. Several clinical trials (NCT04577326, NCT03608618, NCT03323944, NCT03267173, NCT03198052, NCT03054298, NCT02959151, NCT02706782 and NCT02414269) have been conducted to assess the effectiveness and safety of anti-MSLN CAR-T cells in cancer patients with positive results for MSLN administered via the intrathoracic, intraperitoneal, intratumoral or vascular routes. Although anti-MSLN CAR-T-cell therapy offers new hope for patients with solid tumors, many challenges remain. Next, we identify solutions from two aspects: toxicity and technical challenges. The target antigen MSLN is not specifically expressed on the surface of tumor cells, which may lead to off-target effects. Anti-MSLN CAR-T cells recognize MSLN target antigens and are activated to release cytokines or trigger macrophages to release inflammatory cytokines [55], which may lead to cytokine release syndrome (CRS), neurotoxicity and other adverse reactions. In a phase I/II clinical trial (NCI-09-C-0041), a patient experienced respiratory discomfort and a decline in blood oxygen saturation within 15 min after completing HER2-CAR-T-cell infusion. Approximately 40 min later, chest X-ray showed pulmonary edema, and he died. It is believed that after the first clearance of HER2-CAR-T cells in the lungs, inflammatory cytokines are subsequently released once the body detects HER2 expressed by healthy lung cells, causing lung toxicity and edema that leads to multiorgan dysfunction and death [56]. MSLN is also expressed in normal mesothelial tissue. While targeting tumor cells, anti-MSLN CAR-T cells may also kill normal tissue cells expressing MSLN, resulting in off-target effects. However, from the results of the five published clinical studies of anti-MSLN CAR-T-cell therapy, no obvious off-target effects have been observed [41,48,49,57,58]. Of course, this may be related to the small number of patients, and it remains to be further investigated. In addition, researchers have developed a variety of methods to reduce off-target effects: (1) The construction of bispecific antibodies [59]. (2) Trans-signaling CARs, which means that the two CARs target different TAAs. CAR1 contains only the CD3ζ signaling domain, and CAR2 contains only the CD28 or other costimulatory factor signaling domain. They are transduced to construct T cells that coexpress two CARs. These CAR-T cells only target cells expressing both TAAs [60,61,62,63]. (3) T cells designed using the synthetic Notch (synNotch) receptor, a new class of synthetic receptors based on the Notch receptor. Antigen A is on the synNotch receptor, and antigen B is on the CAR. Only cells expressing both antigens can be targeted [63,64,65]. (4) Simultaneous introduction of two CARs, one targeting TAA and one targeting inhibitory receptors for antigens present on normal cells rather than tumor cells. CAR-T cells express inhibitory signals when they bind to normal cells and have tumor-killing effects when they bind to tumor cells [63,66]. The above strategies can also be considered in the anti-MSLN CAR-T cell process in the future. CRS is one of the serious adverse reactions of CAR-T-cell therapy. At present, most studies on CRS are focused on hematological malignancies. Fever, weariness, headache, rash, joint discomfort and myalgia are some of the milder signs of CRS. Hypotension and a high fever are serious symptoms that can worsen and lead to circulatory shock, vascular leakage, disseminated intravascular coagulation and multiple organ failure [67]. An anti-IL-6 receptor antibody (tocilizumab) and symptomatic support therapy (high-dose steroids, vasopressors, ventilatory support, etc.) are all included in the treatment [68]. Some studies suggest that blocking IL-1 may be a new method for treating CRS [69]. The pathogenesis of CRS remains unclear. It has been reported [70] that CAR-T cells release granzyme B, activate caspase 3 and cleave GSDME in target tumor cells, leading to pyroptosis, thereby activating caspase 1 and GSDMD in macrophages and triggering CRS. Elevated GSDME levels in cancer patients were positively correlated with CRS severity. The type of therapy, the underlying condition and the features of the patient all impact the risk of CRS [67]. The degree of T-cell growth and T-cell activation were linked with CRS severity [71]. The nature of the CAR structure affects the clinical presentation, severity and occurrence time of CRS [72]. The incidence rates of CRS in CAR-T-cell therapies containing CD28 and 4-1BB were 93% and 57%, respectively [73,74]. The development of CRS is impacted by lymphoid depletion before CAR-T-cell injection. Following cyclophosphamide or fludarabine lymphodepletion, the likelihood of developing CRS increases [75]. This may be a result of the increased rate of CAR-T-cell proliferation due to the more significant lymphoid depletion achieved by the combination therapy [67]. Fortunately, no severe CRS has occurred in any published clinical trials of anti-MSLN CAR-T-cell therapy [41,48,49,57,58], which may be related to the small number of published clinical trials. The observations of follow-up clinical trials are highly anticipated. Similar to CRS, mild to severe neurological dysfunction within days and weeks following CAR-T-cell infusion is termed CAR-T-cell-induced neurotoxicity, which often includes epileptic activity as well as specific deficits such as aphasia, altered eyesight, shaking and facial drooping. Symptomatic support therapy is the main treatment, except for the prophylactic use of levetiracetam during CAR-T-cell infusion, and data on other interventions are limited [76]. At present, little is known about the mechanism of neurotoxicity. According to some studies [77], inflammatory mediators released by macrophages trigger the release of von Willebrand factor and angiopoietin-2 from the Weibel–Palade bodies of endothelial cells in the central nervous system. This replacement of angiopoietin-1 results in the inhibition of TIE receptor tyrosine kinase signal transduction. The integrity of the blood-brain barrier is compromised by endothelial cells, which also become more porous. Coagulopathy is caused by high-molecular-weight von Willebrand factor. As cytokines and activated inflammatory cells continue to cross the blood-brain barrier, this positive feedback loop continues. Because of the pathophysiology’s resemblance to thrombocytopenic purpura, plasmapheresis is being studied as a treatment for neurotoxicity. It has also been postulated [78] that the mechanism may involve NK-cell populations. NK cells secrete IL-2 and IL-15, both of which were found to be elevated in patients with neurotoxicity. This high level of NK cell activation triggers the activation of microglia and mediates a strong pathogenic inflammatory environment in the central nervous system. Fortunately, no serious neurotoxicity has been observed in published clinical trials of anti-MSLN CAR-T-cell therapy [41,48,49,57,58]. Of course, the results of more clinical studies are awaited. The chimeric antibody substitutes the mouse constant region with the constant region of the human antibody-producing gene, which greatly reduces the immunogenic reaction produced by the mouse-derived antibody so that 70% of the antibody components are human components. The murine gene is still present in the chimeric antibody, even though it only makes up a very minor portion of the antigen-recognition sequence of the variable region of the antibody. Clinical trials have shown that chimeric antibodies can also generate HAMA immune responses when applied. The therapeutic efficacy of murine antibodies in humans are constrained by their immunogenicity. The use of murine-derived CAR limits the persistence of CAR-T cells in humans and raises the possibility of allergic responses. The construction of a fully human scFv CAR is one solution. A clinical trial (NCT01355965) conducted by Maus et al. [79] included a total of four patients who received multiple intravenous infusions of MSLN-targeted mRNA transiently transduced second-generation CAR-T cells. One patient developed anaphylaxis during treatment; he received anti-MSLN CAR-T-cell infusions on days 0, 7 and 49, and anaphylaxis occurred within minutes of infusion on day 49. After exclusion, anti-MSLN CAR-T cells most likely triggered allergic responses by inducing murine antibody sequence-specific IgE antibodies present in CAR-T cells. It is suggested that a single infusion of CAR-T cells is sufficient to achieve efficacy. In this case, with continuous exposure to the product, CAR-T-cell infusion would not induce IgE antibodies. At present, the results of two clinical trials (NCT02414269 [49] and NCT03545815 [48]) of fully human scFv anti-MSLN CAR-T cells have been published, and no new allergic reactions have been found. The technology related to CAR-T-cell therapy has been continuously improved, but there are still many obstacles that limit its further clinical promotion and application. In the following subsections, we introduce and summarize the corresponding solutions proposed by relevant basic research from the aspects of an immunosuppressive TME, insufficient transport into the tumor, target antigen heterogeneity, proliferation and persistence. The glycolytic metabolism of tumor cells induces hypoxia within the TME, and the accumulation of metabolic waste leads to a decrease in pH and low nutrient content, resulting in oxidative stress [80]. The TME can upregulate immune checkpoint molecules (e.g., PD-1, CTLA-4, TIM3 and LAG3), thereby limiting T-cell function. The TME contains a large number of stromal cells, such as cancer-associated fibroblasts (CAFs) and immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) [81], tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), mast cells and Tregs [82]. M2 TAMs and Tregs can produce transforming growth factor-β (TGF-β) [83] and prostaglandin E2 (PGE2) [84] to protect tumors from immune surveillance and attack. CAR-T cells are sensitive to immunosuppressive mechanisms in the TME. An immune checkpoint molecular blockade can enhance the antitumor activity of CAR-T cells and is expected to enhance their functional persistence in solid tumors. At present, clinical trials mainly involve knockout of PD-1 by CRISPR–Cas9, PD-1 antibody and anti-PD-1 nanoantibody to combat this immunosuppressive mechanism. Preclinical studies have reported improvement in the activity of anti-MSLN CAR-T cells by knocking down TIM3 via shRNA [85] or by applying TIM3 immune checkpoint inhibitors [86]. TIM3 blockade combined with anti-MSLN CAR-T cells significantly improved their killing potency, cytokine secretion and proliferation. In addition, simultaneous downregulation of the inhibitory receptors PD-1, TIM-3 and LAG-3 on CAR-T cells can enhance the antitumor ability of anti-HER-2 CAR-T cells by upregulating the expression of CD56 [87]. However, this approach has not been applied in anti-MSLN CAR-T cells. In addition to immune checkpoint inhibitors, oncolytic adenoviruses (OAds) are another immunotherapeutic approach for the treatment of solid tumors. The combination of OAd therapy and CAR-T-cell therapy is a new development direction. OAds expressing tumor necrosis factor-α (TNF-α) and IL-2 enhance and maintain T-cell function, promote the infiltration of anti-MSLN CAR-T cells in tumors, overcome the heterogeneity of tumor target antigen expression and reduce tumor immunosuppression, thereby improving the efficacy of anti-MSLN CAR-T cells in pancreatic cancer [88]. In addition, a study [89] evaluated the combination of anti-MSLN CAR-T-cell and OAd therapies in a TNBC model. It was found that OAds targeting TGF-β could directly lyse tumor cells, with an obvious antitumor response in the early stage and weakened antitumor activity in the later stage. However, anti-MSLN CAR-T-cell therapy has a sustained antitumor effect, with a stronger antitumor response detectable in the later stage. The combination of the two treatments produced a stronger antitumor response. CD40 is mainly expressed on APCs, including dendritic cells (DCs) and macrophages. CD40L expressed on CD4+ T cells plays a key role in the immune response of DCs and activation of antitumor CD8+ T cells [90]. Studies have demonstrated that CD40 is expressed on activated CD8+ T cells and that CD40+CD8+ T cells can communicate with CD4 through CD40. The direct interaction between CD4+ T cells and CD8+ T cells can encourage CD8+ T-cell cytokine release and cell proliferation [91]. CAR-T cells targeting MSLN region III (MSLN3) were designed to secrete anti-CD40 antibodies. The ratio of cytokines and central memory T cells (TCM) secreted by MSLN3 CD40 CAR-T cells was greater than that secreted by MSLN3 CAR-T cells. MSLN3 CD40 CAR-T cells elicited a stronger antitumor response in vitro and in vivo [92]. To avoid immunosuppressive effects, genetically modifying the CAR can help increase the resistance of T cells to immunosuppression. The introduction of costimulatory molecules and inhibitory cytokines into the CAR can help T cells develop stronger resistance to Tregs, TGF-β and other related immunosuppressive molecules [93,94]. Knockdown of TGF-β receptor II (TGFBR2) by CRISPR–Cas9 technology enabled anti-MSLN CAR-T cells to withstand the negative effects of TGF-β signaling [95]. Adenosine and PGE2 can inhibit the immune system by triggering protein kinase A (PKA). Targeting the adenosine 2A receptor (A2AR) can diminish the inhibitory action of adenosine in vitro. Blocking the localization of PKA to the immune synapse increased the migration of anti-MSLN CAR-T cells to the tumor and enhanced the antitumor effect in a mouse model of melanoma [96]. Additionally, knockdown of A2AR by shRNA resulted in enhanced proliferation, cytokine production and cytotoxic function of anti-MSLN CAR-T cells in a simulated TME [97]. In addition, CAR-T cells can be further modified to express cytokines such as IL-2, IL-12, TNF-α and IFN-γ to escape immunosuppression [98,99]. Tumor cells and CAFs form the ECM, which is essential for the progression of cancer. The physical barrier that prevents certain anticancer medicines from penetrating tumor cells is represented by the ECM. In addition, ECM collagen fibers around the tumor restrict T cells from entering the tumor [100,101]. One method to increase the effectiveness of CAR-T-cell treatment is to use matrix degraders. CAR-T cells were engineered to express heparanase (HPSE), which can degrade heparan sulfate proteoglycans, allow CAR-T cells to better infiltrate tumors and increase antitumor activity in mouse models [102]. Another strategy is to exploit the ability of macrophages to secrete matrix metalloproteinases (MMPs) to remodel the ECM so that CAR-T cells can infiltrate tumors [103]. The ECM contains hyaluronic acid (HA), which is broken down by hyaluronidase. The membrane protein PH20, which is naturally expressed by human sperm, has high hyaluronidase activity. Because the PH20 protein has a brief half-life, the IgG2 Fc fragment was incorporated to stabilize the structure of the protein. CAR-T cells expressing sPH20-IgG2 were constructed and showed a strong ability to degrade HA and inhibit tumor growth in a mouse model of xenogeneic gastric cancer [104]. The CAR-T-cell trafficking process requires chemokines secreted by tumor cells to interact with chemokine receptors on CAR-T cells. Chemokines are involved not only in leukocyte recruitment, but also in tumor angiogenesis, cell multiplication and metastasis. It is possible to stimulate more chemokine receptors to be expressed on CAR-T cells; for example, anti-MSLN CAR-T cells can be genetically modified to express the chemokine receptor CCR2 combined with a CXCR4 antagonist. The chemokine receptor CCR2b was introduced into anti-MSLN CAR-T cells to enhance the transport of CAR-T cells to the tumor. Functional CCR2b on anti-MSLN CAR-T cells can significantly increase the number of T cells in tumors and improve the antitumor effect in vitro and in vivo [33]. In addition, CCR2b and CCR4 are receptors for serum monocyte chemotactic protein 1 (MCP-1) and are expressed at low levels on activated T cells. Anti-MSLN CCR2b CAR and anti-MSLN CCR4 CAR-T cells have increased migration rates into tumor supernatants expressing high levels of MCP-1 in vitro. In a mouse model of non-small cell lung cancer, anti-MSLN CCR2b CAR-T cells had better tumor tissue infiltration and antitumor function [105]. Local administration allows the drug to be directly injected into the tumor through the barrier, which can alleviate the obstacle of drug transport into the tumor. This was discussed in detail in Section 2.3. In view of the good efficacy of CAR-T-cell therapy in early clinical trials of hematological malignancies, the FDA approved four anti-CD19 CAR-T cells, namely Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Tecartus (brexucabtagene autoleucel) and Breyanzi (lisocatagene maraluecel) [106]. In addition, idecabtagene vicleucel (ide-cel), which targets BCMA, is awaiting FDA approval. However, subsequent follow-up showed that acute B-lymphocytic leukemia (B-ALL) had a high relapse rate after CAR-T-cell therapy [107]. The results of a clinical study of anti-CD19 CAR-T cells (CTL019) revealed that among the 59 patients enrolled, 55 (93%) achieved CR at 1 month. However, during the 12-month follow-up, the recurrence-free survival rate was only 55% [108]. Loss of target antigens, downregulation of target antigen expression and heterogeneous expression of target antigens are the main causes responsible for relapse after CAR-T-cell therapy [109]. Target antigen heterogeneity is mainly manifested in two aspects, time and space, which we call temporal expression heterogeneity and spatial expression heterogeneity. Among them, the temporal expression heterogeneity of target antigens refers to the fact that antigen expression on the surface of tumor cells can change significantly over time, mainly in the form of antigen loss and antigen expression downregulation [110]. The first published phase I trial of CD19-4-1BB-CD3ζ CAR-T cells for B-ALL at Children’s Hospital of Philadelphia (CHOP) (NCT01626495 and NCT01029366) showed that 3 of 27 CR patients (11%) relapsed due to loss of CD19 in leukemia cells [111]. Another clinical trial of CAR-T cells in the treatment of B-ALL (NCT02315612) showed that 8 of 12 patients (67%) who had CR after CD22 CAR-T-cell therapy relapsed, and 7 of these patients had downregulated CD22 expression. Downregulation of CD22 expression occurs at the posttranscriptional level, and CD22 CAR-T cells cannot effectively eliminate cells with downregulated CD22 expression [112]. This has also been demonstrated in glioblastoma [113,114,115], non-Hodgkin lymphoma [116] and multiple myeloma [117]. However, there is no research on the target antigen MSLN thus far. Heterogeneity in the spatial expression of target antigens refers to the inconsistent expression of antigens on the surface of different tumor cells in the same patient [110,118]. Rabilloud et al. [119] performed single-cell sequencing analysis of leukemia cells from a patient with relapsed B-ALL, found that CD19-negative leukemia cells were present before CAR-T-cell therapy and confirmed that relapse was due to the cloning of these CD19-negative leukemia cells. A phase I clinical study conducted by Haas et al. [41] investigated the safety and activity of anti-MSLN CAR-T cells in patients with malignant pleural mesothelioma, ovarian cancer and pancreatic ductal adenocarcinoma, (NCT02159716) and found that in only 3 of 15 patients the expression of MSLN on tumor cells was >75%. Thus, clinical trials focusing on efficacy are recommended to prospectively screen MSLN expression to reduce recurrence in the future. By downloading immunohistochemical (IHC) staining data from The Human Protein Atlas website (https://www.proteinatlas.org/ (accessed on 18 August 2022)) to quantitatively analyze the IHC results of MSLN expression in various solid tumors, we found that there were some malignant cells with negative MSLN expression in solid tumors defined as having positive MSLN expression. In different IHC samples, the positive rates of MSLN expression were approximately 0–99%. In addition, we analyzed the single-cell sequencing results of TNBC (GSE75688 and GSE118389), ovarian cancer (GSE118828) and pancreatic cancer (GSE111672) downloaded from the Gene Expression Omnibus (GEO: https://www.ncbi.nlm.nih.gov/gds/ (accessed on 28 September 2022)) database and found that approximately 70–97% of malignant cells had negative MSLN expression (Figure 4). The heterogeneous expression of MSLN significantly affects the efficacy and recurrence rate of solid tumors after treatment with anti-MSLN CAR-T cells [109]. In view of this, a variety of measures against target antigen heterogeneity have been developed. Among them, enhancing the “bystander effect” of tumor cells is an important strategy. The bystander effect suggests that CAR-T cells can induce tumor killing, even though these malignant cells negatively express the target antigen. To dissect the mechanism of the bystander effect, Upadhyay et al. [120] used CRISPR—Cas9 technology to screen and determine the essential role of Fas-FasL in antigen-specific T-cell killing. They discovered that Fas-FasL mediated the off-target bystander killing effect on antigen-negative malignant cells: the killing effect of CD19 CD3 ζ-CD28 CAR-T cells on target cells showed moderate Fas dependence, and Fas-expressing mice were more sensitive to bystander killing effects. An analysis of pretreatment tumor RNA-sequencing data from the ZUMA-1 trial (NCT02348216), which included patients with CAR-T-refractory diffuse large B-cell lymphoma, showed that patients with a durable response to treatment had significantly increased tumor Fas expression. Similarly, an analysis of a diffuse large B-cell lymphoma patient cohort receiving standard treatment in the TCGA database showed that tumor patients with high expression of Fas had a poorer prognosis; in contrast, patients with high expression of Fas treated with CAR-T cells had significantly longer survival times. This mechanism also applies to anti-MSLN CAR-T cells. Klampatsa et al. [121] designed a model in which anti-MSLN CAR-T cells could cure xenografts in mice inoculated with 100% MSLN-positive tumor cells. However, even if the positivity rate of MSLN dropped to 90%, anti-MSLN CAR-T cells could not cure the tumor. Nevertheless, up to 25% of MSLN-negative xenografts could be cured if pretreatment with a nonlymphoid-depleting dose of cyclophosphamide prior to CAR-T-cell therapy induces a bystander effect. Therefore, exploring the application of multidrug combinations and the application of small-molecule Fas signaling modulators may solve the poor curative effect caused by target antigen heterogeneity to a certain extent. Park et al. [122] introduced a new method for the exogenous introduction of a homogeneous antigen. Oncolytic vaccinia virus (OV19t) encoding CD19t was used to infect various solid tumor cells. Before the virus-infected tumor is lysed, its cell surface produces new CD19. The expression of CD19t in tumors is encouraged by CAR-T-cell-mediated tumor lysis, which can also cause the release of the virus from tumor cells that are on the verge of death. At this point, all tumor cells are loaded with the new target antigen CD19, potentially extending the application of clinically approved anti-CD19 CAR-T cells to the treatment of solid tumors and overcoming the problem of target antigen expression heterogeneity. In addition, to address target antigen heterogeneity, multiantigen-targeted CAR constructs are currently being developed, thereby reducing the risk of tumor recurrence. There are numerous methods for creating CARs that target several antigens [123]: (1) dual-targeted CAR-T cells, including bicistronic transgenes [124] and cotransduction and tandem single-chain antibodies [125,126,127,128]; (2) coadministered monospecific CAR-T cells [129]; and (3) sequential monospecific CAR-T cells [129] (Figure 5). In addition, some studies have designed aptamer CAR-T-cell technology. The construction of universal CAR-T cells expressing nontumor-associated antigens, such as fluorescein isothiocyanate (FITC) CAR-T cells, targets multiple target antigens on the surface of tumor cells through a mixture of bispecific aptamers [130] (Figure 6). Kailayangiri et al. [131] found that inhibition of enhancer of zeste homolog 2 (EZH2) could induce the surface expression of GD2 in Ewing sarcoma cells, thereby enhancing the killing ability of anti-GD2 CAR-T cells against Ewing sarcoma cells. These studies suggest that our similar mechanism may also apply to anti-MSLN CAR-T cells. In a tissue microarray of 107 excised pancreatic cancers, Tholey et al. [132] stained tissues with antibodies against MUC1, MSLN or both. At the protein level, they verified that when MUC1 and MSLN were labeled separately and simultaneously, the percentage of tumor cells with a high labeling pattern (2+ or 3+) increased from 56% and 62% to 82%. To increase the effectiveness, infiltration, persistence and proliferation of CAR-T cells in ovarian cancer, Liang et al. [126] created a unique tandem CAR expressing an anti-FOLR1 scFv, an anti-MSLN scFv and two peptide sequences of IL-12. KRAS mutations were shown to be positively associated with MSLN expression in pancreatic cancer and lung cancer, as demonstrated by Fukamachi et al. [133] and Bauss et al. [134]. It is reasonable to infer that the use of bispecific CAR-T cells (targeting both MSLN and MUC1 or targeting both FOLR1 and MSLN) and CAR-T-cell therapy combined with mutated gene targeting drugs could improve the efficacy of anti-MSLN CAR-T-cell therapy. However, these therapies are still in the preclinical stage, and whether they can solve the issue of tumor recurrence caused by the heterogeneity of the target antigen requires further observation. There are four factors that affect the proliferative capacity and persistence of CAR-T cells: preconditioning, the intracellular signaling domain of CAR-T cells, the immunogenicity of CAR-T cells, and T-cell depletion. The current commonly used conditioning regimens are cyclophosphamide and cyclophosphamide combined with fludarabine. A clinical trial (NCT02159716) [41] demonstrated that preconditioning with cyclophosphamide before CAR-T infusion can increase the proliferation of CAR-T cells, but has little effect on durability. Preclinical and clinical studies conducted by Goto et al. [135] and Pang et al. [136] showed that the secretion of IL-7 and CCL19 promoted the proliferation and persistence of anti-MSLN CAR-T cells in vivo. CD28 and 4-1BB are commonly used intracellular signaling domains of CAR-T cells, but their functions are quite different. CD28 CAR-T cells have a better tumor-killing ability, while 4-1BB can better prolong the persistence of CAR-T cells. The reason may be that CD28 CAR-T cells have higher basal activity of nuclear factor of activated T cells (NFAT) and unique sensitivity to PD-1/PD-L1-mediated checkpoint inhibition, while 4-1BB CAR-T cells have stronger nuclear transcription factor kappa B (NF-κB) activity and are not affected by the PD-1/PD-L1 checkpoint [137,138]. Guedan et al. [139] suggested that a single amino acid residue in CD28 causes T-cell depletion and may hinder the persistence of CD28-based anti-MSLN CAR-T cells. Another preclinical study demonstrated that the conversion of asparagine to phenylalanine enhanced the in vivo persistence of anti-MSLN CAR-T cells containing the CD28 costimulatory domain, thereby enhancing their antitumor efficacy [140]. CAR is a protein that also has immunogenicity, so an immune response against CAR is generated in the body, thereby reducing the persistence of anti-MSLN CAR-T cells. Clinical trials (NCT01355965 [57,79], NCT02159716 [41] and NCT01897415 [58]) have reported the detection of human anti-chimeric antibody (HACA) during CAR-T-cell infusion. Constructing a CAR fully targeting human scFv MSLN can reduce the immunogenicity of anti-MSLN CAR-T cells. The scFv in the CAR structure is unstable and has an inherent tendency to self-aggregate, which may lead to depletion of anti-MSLN CAR-T cells in vivo, thereby reducing their persistence. Studies have shown [109,133] that replacing the scFv with a fully human VH domain or linking the TCR constant region to the heavy and light chain variable regions of monoclonal antibodies produces synthetic T-cell receptors and antigen receptors (STAR). This approach can reduce T-cell exhaustion and results in better or equal cytotoxicity, proliferation and persistence than CAR-T cells. At present, for anti-MSLN CAR-T-cell therapy, the above four methods are mainly used to improve proliferation and persistence, which are key factors affecting efficacy. In addition, local administration and induction of T-cell costimulators to improve the structure of CAR contribute as well [34]. Clinical trials of CAR-T-cell therapy in solid tumors have begun in recent years as a result of the approval of the treatment for hematological malignancies. The present state of anti-MSLN CAR-T-cell treatment in patients with solid tumors was discussed in this study. Its toxicity, including off-target effects, CRS, neurotoxicity and immune response, was analyzed. Finally, technical hurdles that may affect its safety and efficacy, including the immunosuppressive TME, trafficking into tumor tissue, target antigen heterogeneity, proliferation and durability, were defined. At present, the persistence and efficacy of the intravenous infusion of anti-MSLN CAR-T cells still has much room for development. Novel strategies of combination therapy with immune checkpoint inhibitors and/or chemotherapeutic drug pretreatment have improved the antitumor ability of anti-MSLN CAR-T cells to some extent. The application of topical, fully human anti-MSLN scFv, which has entered clinical trials, is also expected to improve the efficacy and reduce the toxicity of anti-MSLN CAR-T-cell therapy. The next steps in the development of new CAR-T-cell therapies for solid tumors will involve multidisciplinary collaboration, focusing on combination therapy and new clinical study designs, and anti-MSLN CAR-T-cell therapy is expected to impact clinical outcomes in patients with various solid tumors.

PMC10000071

I-Ping Yang,Kwan-Ling Yip,Yu-Tang Chang,Yen-Cheng Chen,Ching-Wen Huang,Hsiang-Lin Tsai,Yung-Sung Yeh,Jaw-Yuan Wang

MicroRNAs as Predictive Biomarkers in Patients with Colorectal Cancer Receiving Chemotherapy or Chemoradiotherapy: A Narrative Literature Review

21-02-2023

microRNAs,colorectal cancer,chemoresistance,radioresistance,predictive biomarkers

Simple Summary Nearly two decades would be required for a cancer lesion to develop from normal colon mucosa, but most colorectal cancer (CRC) patients are at an advanced stage at presentation. Chemotherapy, targeted therapy, and radiotherapy can improve the prognosis of patients with advanced CRC, but sometimes the therapy resistance occurs, and the 5-year survival rate for patients with locally advanced CRC and with metastatic CRC (mCRC) remain poor. MicroRNAs (miRs) can regulate cancer pathways by inhibiting their target mRNA translation and triggering their degradation. MiRs can serve as predictive biomarkers for the detection of CRC or mCRC or the resistance of chemotherapy or chemoradiotherapy, and miRNA-based therapeutics may finally reach the clinical stages. Abstract Colorectal cancer (CRC) is one of the most common malignancies and is associated with high mortality rates worldwide. The underlying mechanism of tumorigenesis in CRC is complex, involving genetic, lifestyle-related, and environmental factors. Although radical resection with adjuvant FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) chemotherapy and neoadjuvant chemoradiotherapy have remained mainstays of treatment for patients with stage III CRC and locally advanced rectal cancer, respectively, the oncological outcomes of these treatments are often unsatisfactory. To improve patients’ chances of survival, researchers are actively searching for new biomarkers to facilitate the development of more effective treatment strategies for CRC and metastatic CRC (mCRC). MicroRNAs (miRs), small, single-stranded, noncoding RNAs, can post-transcriptionally regulate mRNA translation and trigger mRNA degradation. Recent studies have documented aberrant miR levels in patients with CRC or mCRC, and some miRs are reportedly associated with chemoresistance or radioresistance in CRC. Herein, we present a narrative review of the literature on the roles of oncogenic miRs (oncomiRs) and tumor suppressor miRs (anti-oncomiRs), some of which can be used to predict the responses of patients with CRC to chemotherapy or chemoradiotherapy. Moreover, miRs may serve as potential therapeutic targets because their functions can be manipulated using synthetic antagonists and miR mimics.

MicroRNAs as Predictive Biomarkers in Patients with Colorectal Cancer Receiving Chemotherapy or Chemoradiotherapy: A Narrative Literature Review Nearly two decades would be required for a cancer lesion to develop from normal colon mucosa, but most colorectal cancer (CRC) patients are at an advanced stage at presentation. Chemotherapy, targeted therapy, and radiotherapy can improve the prognosis of patients with advanced CRC, but sometimes the therapy resistance occurs, and the 5-year survival rate for patients with locally advanced CRC and with metastatic CRC (mCRC) remain poor. MicroRNAs (miRs) can regulate cancer pathways by inhibiting their target mRNA translation and triggering their degradation. MiRs can serve as predictive biomarkers for the detection of CRC or mCRC or the resistance of chemotherapy or chemoradiotherapy, and miRNA-based therapeutics may finally reach the clinical stages. Colorectal cancer (CRC) is one of the most common malignancies and is associated with high mortality rates worldwide. The underlying mechanism of tumorigenesis in CRC is complex, involving genetic, lifestyle-related, and environmental factors. Although radical resection with adjuvant FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) chemotherapy and neoadjuvant chemoradiotherapy have remained mainstays of treatment for patients with stage III CRC and locally advanced rectal cancer, respectively, the oncological outcomes of these treatments are often unsatisfactory. To improve patients’ chances of survival, researchers are actively searching for new biomarkers to facilitate the development of more effective treatment strategies for CRC and metastatic CRC (mCRC). MicroRNAs (miRs), small, single-stranded, noncoding RNAs, can post-transcriptionally regulate mRNA translation and trigger mRNA degradation. Recent studies have documented aberrant miR levels in patients with CRC or mCRC, and some miRs are reportedly associated with chemoresistance or radioresistance in CRC. Herein, we present a narrative review of the literature on the roles of oncogenic miRs (oncomiRs) and tumor suppressor miRs (anti-oncomiRs), some of which can be used to predict the responses of patients with CRC to chemotherapy or chemoradiotherapy. Moreover, miRs may serve as potential therapeutic targets because their functions can be manipulated using synthetic antagonists and miR mimics. Colorectal cancer (CRC) is a major public health problem among both sexes and has a worldwide mortality rate of 47.8% [1,2]. According to the annual report of the Health Promotion Administration in USA, each year, more than 1.1 million patients receive diagnoses of CRC worldwide and approximately 608,000 CRC-related mortalities occur, making CRC the third most common cause of death [1]. Approximately 20–30% of patients with stage I–III CRC who undergo a surgical resection eventually develop distant metastasis, which is associated with poor prognoses [3]. Researchers must continue investigating biomarkers that can be used to more accurately identify patients with CRC who are at a high risk of recurrence. Adjuvant or neoadjuvant FOLFOX chemotherapy, which involves the use of 5-fluorouracil (5-FU), leucovorin (LV), and oxaliplatin, is widely used to reduce the risk of recurrence in patients with advanced-stage CRC [4,5,6]. Even after undergoing radical surgical resection or oxaliplatin-based adjuvant chemotherapy, some patients ultimately develop recurrence or metastasis, indicating that current treatments for CRC are insufficient [4,7,8,9]. Genomic and metabolomic analysis of right-sided and left-sided CRC are keystones in the study and treatment of subtypes of CRC [10]. In patients with metastatic CRC (mCRC), two signaling pathways—the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathways—are involved in the proliferation and metastasis of CRC [11]. FOLFIRI (5-FU, LV, and irinotecan) plus anti-EGFR monoclonal antibodies provides a survival benefit to patients with mCRC with distant metastases [12,13]. For patients with right-sided mCRC, FOLFIRI or FOLFOXIRI (5-FU, LV, irinotecan, and oxaliplatin) plus bevacizumab (an anti-VEGF monoclonal antibody) are preferred first-line treatment options, irrespective of the patient’s RAS and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutational statuses [14]. For patients with locally advanced rectal cancer (LARC), neoadjuvant chemoradiotherapy (NACRT) is a standard treatment that can improve the outcomes of radical resection, prevent local recurrence, ensure sphincter preservation and tolerable toxicity levels, and maintain postsurgical quality of life [15,16,17,18]. However, patient responses to NACRT are highly variable [19,20,21], and resistance to chemoradiotherapy (CRT) is a major obstacle in the treatment of patients with LARC [22]. In patients with CRC, epigenetic modifications, including DNA methylation and histone modifications, can be used as clinical biomarkers for diagnosis, prognosis, and the prediction of patient responses to adjuvant or neoadjuvant therapy [23,24,25]. Liquid biopsies employ a wide range of technologies to acquire tumor information, including levels of carcinoembryonic antigen (CEA), circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circular RNAs (circRNAs), and microRNAs (miRs) in body fluids [26]. CEA is widely used as a surrogate biomarker in clinical practice for treatment response prediction and follow-ups in patients with CRC [27,28]. Moreover, the serum levels of CEA and expression levels of EGFR and FGD5-AS1 (FGD5 Antisense RNA 1, an oncogenic long non-coding RNA) have been observed to be significantly elevated in 5-FU-resistant CRC cells [29,30]. Using CTCs as individualized biomarkers can help healthcare providers develop effective treatment strategies for patients with CRC. The persistent presence of CTCs after adjuvant chemotherapy indicates chemoresistance and is often reflected in subsequent recurrence of CRC [31,32]. In addition, the progression of CRC can be modulated via circRNAs, which can sponge and downregulate target miRs [33]. In CRC, miRs have been determined through gene network analysis to act as both oncogenes and tumor suppressors of differentially expressed mRNAs and proteins [34,35,36,37]. MiRs, small noncoding RNAs consisting of approximately 20 nucleotides, can posttranscriptionally regulate the expression of several target genes by directly binding to the 3′ untranslated regions (3′-UTRs) of target mRNAs, thereby triggering mRNA degradation, suppressing mRNA translation, and subsequently regulating the protein expression [38]. The heterogeneity of CRC makes its accurate classification and treatment challenging [39]. The specific panel of miRNAs/mRNAs or miR clusters can contribute to the progression from adenomatous and carcinomatous lesions [40,41]. Four consensus molecular subtypes (CMSs) were identified via the classification of patients with CRC in biologically homogeneous CRC subtypes, and mesenchymal CMS4 presented with a worse prognosis [39]. Plasma miR profiling has been observed to be significantly associated with colorectal cancer CMS and validated for predicting both prognosis and treatment response [42]. For example, the miR-200 family is the most powerful determinant of CMS4-specific gene expression, which is associated with epithelial–mesenchymal transition [39]. The analysis of serum miRNAs is less invasive and still facilitates a real-time analysis of the disease course [42]. To date, researchers have identified at least 250 miRs that may serve as diagnostic biomarkers as well as prognostic indicators of CRC [43]. Therefore, miRs have the potential to serve as biomarkers for cancer detection, prognosis, CMS classification, and treatment response prediction [42,43,44,45,46,47]. MiR panels have been reported to facilitate early detection of relapse in patients with CRC [48,49,50,51]. MiRs are attractive as biomarker candidates because they can be identified through liquid biopsies, which makes them minimally invasive and more convenient for use in the early detection of relevant signals [52]. Several reviews have discussed miRs as biomarkers in CRC [53,54,55], and even textbooks mention the potential cancer-sensitizing agents for CRC chemotherapy [56]. Stiegelbauer et al. (2014) summarized the chemotherapeutic approaches for treating CRC and highlighted the role of miRNAs as predictive biomarkers for chemoresistance in patients with CRC [53]. In their meta-analysis, Masuda et al. concluded that miRs have strong statistical confidence as biomarkers for the early detection of CRC and prediction of prognosis and chemoresistance, but they also indicated that most miR reports were small-scale studies until 2017 [54]. Shirafkan et al. summarized the roles of miRs in CRC by emphasizing their importance in different signaling pathways, such as the EGFR, transforming growth factor beta (TGF-β), and tumor protein (TP53) pathways, and suggested miRs as predictive factors of chemotherapy [55]. Elucidating the mechanisms underlying the development of resistance to chemotherapy and radiotherapy is crucial to the development of more effective cancer treatment strategies. However, no reviews have focused on miRs as predictors of response to chemoradiotherapy in patients with CRC. Numerous studies have explored the activation of regulatory pathways involving miRs in response to chemotherapy, radiotherapy, or antitumor agents, and miRs are attractive candidates for targeted therapy because their functions can be manipulated through the use of synthetic antagonists and miR mimics [43,44,45,46,47]. No miRNA-based drugs are currently on the market; however, many RNA-based therapies, including antisense oligonucleotides, aptamers, small interfering RNAs, miRs, and mRNA, are currently undergoing clinical trials or have already received regulatory approval (including as treatments for liver cancer, lymphoma, and melanoma) [57]. Identifying a reliable biomarker for predicting chemoresistance or radioresistance in patients with CRC may improve survival outcomes [58,59,60]. In this review, we would discuss how these miRs can serve as clinical biomarkers in the early detection of CRC or mCRC and as predictive biomarkers in CRC treatment. We searched PubMed, the Cochrane Central Register of Controlled Trials, and the Cochrane Library for relevant studies. We did not apply any language or regional restrictions. We used the following MeSH terms in our search: (“Colorectal Neoplasm”[Title/Abstract]) AND (MicroRNA*[Title/Abstract]) AND (“Chemotherapy”[Title/Abstract] OR “Chemoradiotherapy”[Title/Abstract] OR “Neoadjuvant Radiotherapy [Title/Abstract] OR “Neoadjuvant Chemotherapy*”[Title/Abstract]). We thoroughly evaluated all the relevant studies and bibliographies to identify additional potentially eligible studies. MiRs may act as oncogenes or tumor suppressors and may serve as biomarkers for the early diagnosis of CRC to facilitate efficient treatment (Figure 1). The expression levels and functions of miRs in various body extracts (including serum, tumor tissues, feces, or urine) have been analyzed in several case–control studies (Table 1). Expression levels of miRs in feces or urine can be used for noninvasive screening for the detection of CRC. Researchers developed a urinary biomarker panel combining miR-129-1-3p and miR-566 that could accurately detect stage 0/I CRC [48]. The expression levels of miR-29a, miR-223, and miR-224 in fecal samples from patients with CRC were all significantly lower than those in fecal samples from healthy volunteers (all p < 0.001) [61]. A systematic review revealed that the expression levels of miR-20a in the feces, serum, or tumor tissues of patients with CRC were upregulated relative to those in the control samples, indicating that miR-20a may serve as an accurate biomarker for CRC detection [62]. MiR-106a and miR-125b are associated with the pathogenesis of CRC and may therefore also be used as significant prognostic markers of early-stage CRC [63]. The overexpression of miR-21 in CRC tumor tissue was significantly associated with advanced CRC, but interestingly, the lower expression levels of serum miR-21 were associated with a higher mortality rate [64,65]. MiRs are used not only in the diagnosis of primary CRC but also in the prediction of early relapse of CRC. In several case–control studies including patients with UICC stage II and III CRC, the miR-29c expression levels in the tumor tissues of the early relapse group were significantly lower than those in the tumor tissues of the non–early relapse group [66]. Tsai et al. reported that serum miR-148a expression in the early relapse group was significantly lower than that in the non–early relapse group [67,68]. In another study, lower miR-148a expression in CRC tissues was positively associated with an advanced TNM stage, poor tumor differentiation, lymph node metastasis, and distant metastasis [69]. Researchers have identified some miRs that can be used to predict the development of metastasis in CRC. Chen et al. reported that plasma miR-96/miR-99b can be used as a biomarker for the early detection of mCRC [72]. In another study, the circulating miR-762 levels of patients with CRC with distant metastasis were higher than those of patients with CRC without distant metastasis [74]. Pidikova and Herichova discovered that the miR-17/92a-1, miR-106a/363, miR-106b/93/25, and miR-183/96/182 clusters were strongly associated with metastasis and poor patient survival [70]. Hoye et al. analyzed various next-generation sequencing data sets of samples of primary CRC and mCRC (liver, lung, and peritoneal metastases) and tumor-adjacent tissues and identified five miRNAs—miR-210-3p, miR-191-5p, miR-141-3p, miR-1307-5p, and miR-155-5p—that were upregulated at multiple metastatic sites [73], which may serve as distinguishing biomarkers of mCRC. Several miR panels have been established on the basis of case–control studies. For example, Wang S. et al. developed a serum panel of miR-409-3p, miR-7, and miR-93 that could accurately discriminate between the plasma samples of patients with CRC and healthy controls [75]. Another serum miR panel comprising miR-203a-3p, miR-145-5p, miR-375-3p, and miR-200c-3p could also discriminate between the plasma samples of patients with CRC and healthy controls with a sensitivity of 81.25% and a specificity of 73.33% [76]. Another panel combining six clinicopathologic factors with six miRs (miR-7, miR-93, miR-195, miR-141, miR-494, and let-7b) could be used to detect early relapsed CRC with a sensitivity of 89.4% and a specificity of 88.9% [49]. Resistance to chemotherapy is one of the most common reasons for treatment failure among patients with CRC, and many patients with advanced CRC are initially responsive to chemotherapy but ultimately develop chemoresistance [77,78]. OncomiRs, which can increase chemoresistance, may reduce the levels of apoptosis proteins and silent apoptosis information and neutralize or reverse antiapoptotic signals [54]. Table 2 lists oncomiRs that can enhance chemoresistance through specific regulatory pathways. The transcription factor p53 is the most thoroughly characterized tumor suppressor gene, and p53 variants appear in approximately 50% of patients with CRC [79]. Loss or mutation of the p53 gene and its related pathways, such as those involving TP53INP1 and TP53INP2 or Bcl-2 and caspase proteins, is positively associated with therapeutic resistance in various cancers [80,81,82,83]. Several oncomiRs, including let-7f-5p [84,85], and miR-96 [86], are overexpressed in patients with chemoresistant CRC (relative to patients with chemosensitive CRC) (Table 2). MiR-34a may serve as a predictor of 5-FU chemosensitivity in CRC, and a combination of miR-34a and 5-FU is effective as a treatment for CRC [87]. Chemoresistance may be related to the AMP-activated protein kinase-mammalian target of the rapamycin (AMPK–mTOR) pathway, and several miRs, including miR-27a, miR-103, and miR-107, are overexpressed in patients with chemoresistant CRC (relative to patients with chemosensitive CRC) [88,89,90,91]. Overexpression of miR-744 may mediate oxaliplatin chemoresistance in CRC by suppressing BIN1 expression [92]. Chemoresistance was correlated with cancer cell stemness in patients with CRC [69,89,90], and patients expressing the CD44 variant appear to present with more aggressive phenotypes of CRC. Moreover, miR-1246 was overexpressed in CD44v6+ cells and was associated with poor overall survival and disease-free survival in patients with CRC [93]. Table 3 presents anti-oncomiRs that can modulate patients’ sensitivity to chemotherapy. Overexpression of miR-141-3p negatively regulates epithelial–mesenchymal transition (EMT) and can restore chemosensitivity; in a previous study, the 5-year overall survival of the high miR-141-3p expression group was superior to that of the low miR-141-3p expression group [94,95,96]. Overexpression of miR-377-3p (miR-154 family) or miR-193b-5p also enhanced chemosensitivity to 5-FU in CRC cells by negatively regulating EMT or the forkhead box M1-ATP-binding cassette subfamily C member 5 (FOXM1-ABCC5/10) signaling pathway and decreasing cell stemness [97,98,99,100]. Xiao et al. demonstrated that miR-1915-3p can improve the chemotherapeutic efficacy of oxaliplatin in CRC cells by suppressing EMT-promoting oncogenes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and ubiquitin specific peptidase 2 (USP2) [101]. The mechanism of 5-FU-induced cytotoxicity in CRC cells involves the inhibition of thymidylate synthase (TS), a 5-FU target enzyme. In one analysis of clinical CRC samples, the miR-375 expression levels in the 5-FU-resistant group were much lower than those in the 5-FU-sensitive group [107]. MiR-375 can enhance 5-FU cytotoxicity in CRC cells by suppressing TS and the Sp1 transcription factor (SP1) [107,108]. High miR-218 and miR-330 expression levels had a positive prognostic value in 5-FU-based treatments for CRC, and miR-218 inhibits TS [105,106]. In one study, lower miR-148a expression was associated with advanced CRC and distant metastasis, and overexpression of miR-148a suppressed the expression of stem cell markers and increased chemosensitivity by regulating the β-catenin signaling pathway [69]. In a study by Huang et al., upregulated miR-148a enhanced chemoradiosensitivity and promoted apoptosis of CRC cells by targeting the MET proto-oncogene, receptor tyrosine kinase (c-Met), in vitro and in vivo [113]. High miR-148a expression levels were associated with more favorable tumor responses to neoadjuvant CRT and survival outcomes [113]. By conducting a Kaplan–Meier survival analysis of a sample of 62 patients, researchers determined that miR-27b-3p expression levels are positively correlated with disease-free survival [102]. The MYC proto-oncogene, bHLH transcription factor (c-Myc), can downregulate miR-27b-3p expression, inducing oxaliplatin resistance in CRC cells by inhibiting autophagy [102]. Diabetes mellitus and hyperglycemia have been demonstrated to affect chemoresistance in and the prognosis of CRC [114]. In one study, miR-488 mimic decreased glucose uptake and increased oxaliplatin/5-FU-sensistivity in CRC cells by targeting the oncogene, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) [109]; the miR-488 expression levels of patients with metastatic/recurrent CRC were significantly lower than those of patients with CRC without metastasis/recurrence; and low miR-488 expression levels were associated with low 3-year survival rates, poor differentiation, and advanced-stage disease [109]. Another study demonstrated that tumor-secreted miR-208b promotes Treg expansion by targeting programmed cell death factor 4 (PDCD4) and may be related to chemoresistance to oxaliplatin in CRC [115]. In one study, the diagnostic accuracy (in terms of area under the curve [AUC]) of CEA and CA19-9 (for distinguishing between oxaliplatin-chemoresistant and oxaliplatin-chemosensitive patients) was 0.542 and 0.686, respectively, whereas a panel containing miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i achieved the highest diagnostic accuracy, with an AUC of 0.825 [116]. Comparing plasma miR levels with CEA and CA19-9 in the follow-up of patients with CRC, CEA, and CA19-9 showed a higher specificity for CRC but a lower sensitivity than miRs in predicting disease recurrence [117]. EGFR expression serves as a prognostic factor in patients with stage III CRC receiving metronomic maintenance therapy [29]. In one study, the inhibition of EGFR by the specific inhibitor erlotinib effectively enhanced the antitumor toxicity of 5-FU though miR-330 directly targeting thymidylate synthase [30]. For patients with mCRC with RAS variations, one therapeutic alternative is targeting and preventing angiogenesis by inhibiting VEGF. FOLFIRI plus bevacizumab can increase sensitivity to and enhance the antitumor effects of 5-FU (Table 3) [118]. Upregulation of miR-1207-5p can suppress bevacizumab resistance in bevacizumab-resistant CRC cells by modulating the expression of ATP-binding cassette subfamily C member 1 (ABCC1) [110]. Lower miR-1287-5p expression levels upregulate the expression of multifunctional Y-box binding protein 1 (YBX1) and are associated with cetuximab resistance in CRC [111,112]. Circulating plasma levels of miR-20b, miR-29b, and miR-155 serve as predictors of bevacizumab efficacy in patients with mCRC (Table 1) [71]. In one study, the serum miR-148a expression levels of patients with mCRC who exhibited partial responses to treatment were higher than those of patients with mCRC with disease progression [103]. MiR-148a decreases angiogenesis and increases the apoptosis of CRC cells by downregulating hypoxia-inducible factor 1 subunit alpha (HIF-1α)/VEGF and the MCL1 apoptosis regulator, BCL2 family member (Mcl-1), and serum miR-148a levels have prognostic and predictive value in patients with mCRC receiving bevacizumab therapy [103]. Radiotherapy induces various DNA lesions and unpaired double-strand breaks; therefore, biomolecules that inhibit DNA repair pathways usually improve radiosensitivity [119,120]. Radiation-induced DSBs principally activate the intrinsic apoptotic pathway, and two principal methods are involved in DSB repair: homologous recombination (HR) and nonhomologous end joining (NHEJ) [121]. MiRs are some of the most important mediators of radiosensitivity in CRC cells and are involved in the HR and NHEJ repair pathways [122,123]. Huang et al. reviewed and discussed the biological functions of miRNAs in the regulation of radiosensitivity in CRC cells [123]. However, some investigations of radioresistance in CRC cells were in vitro studies in which CRC cell lines were transfected with miR mimics or knocked out to modulate the expression of various miRs, such as miR-101-3p [124], miR-185 [125], miR-195 [126], and miR-5197 [127]; these miRs were omitted from Table 4. By analyzing the differentially expressed miRNA profiles of patients with CRC, researchers have identified several oncomiRs that enhance radioresistance as biomarkers of responses to radiotherapy (Table 4). p21 is a tumor suppressor gene that can protect cancer cells from DNA damage [128,129]. The overexpression of several miRs, including miR-106b and miR-222, can inhibit p21 expression, thereby inducing radioresistance through the phosphatase and tensin homolog/phosphatidylinositol-3 kinase/AKT serine/threonine kinase 1 (PTEN/PI3K/AKT) pathway, in vitro and in vivo [104,130]. MiR-93 and miR-106b are categorized into the same family, and upregulated miR-93 induces radioresistance by downregulating forkhead box A1 (FOXA1) and upregulating transforming growth factor beta 3 (TGFB3) [131,132,133]. Exosomes containing miR-93-5p derived from cancer-associated fibroblasts can prevent CRC cells from undergoing radiation-induced apoptosis [131]. MiR-96-5p also reportedly induces radioresistance in rectal cancer cells by inhibiting glypican 3 (GPC3) and abnormally triggering the canonical Wnt signaling pathway (Table 4) [134]. MiR-19b targets the F-box and WD repeat domain containing 7 (FBXW7), thereby promoting CRC stem cell stemness and inducing radioresistance, and in one study, patients with LARC with low miR-19b expression levels had markedly longer overall survival and event-free survival [135,136]. Likewise, high miR-125b expression levels in tissue and serum were associated with a poor treatment response in patients with LARC [137] (Table 5). By analyzing receiver operating characteristic curves, D’Angelo et al. demonstrated that circulating miR-125b levels exhibited greater discriminatory power for treatment responses than serum CEA levels did; therefore, miR-125b may serve as a new noninvasive predictive biomarker in the treatment of LARC [137]. Recent clinical findings have demonstrated that cancer stem cells and their inherent radioresistance are crucial to local control after radiotherapy [138]. Differentially expressed miRs, such as miR-148a and miR-214, were observed in radiated CRC cells. MiR-214 promotes radiosensitivity by inhibiting autophagy-related 12 (ATG12)-mediated autophagy in CRC [139,140]. Comparisons of the serum miR expression levels of patients with CRC before or after radiation therapy have revealed that radiation therapy can significantly reduce serum miR-296-5p expression. Moreover, miR-296-5p overexpression significantly reduced the survival fraction of CRC cells under ionizing radiation (IR) treatment by targeting the insulin-like growth factor 1 receptor (IGF1R) and musashi RNA binding protein 1 (MSI1) [141,142]. Therefore, miRs serve as potential regulators of radioresistance in CRC cells and, in turn, may be a promising therapeutic target in the treatment of CRC. The present study focused on oncomiRs and anti-oncomiRs modulating the sensitivity of chemoradiotherapy, and we noted that studies on chemotherapy- or radiotherapy-related miRs far outnumber those on chemoradiotherapy-related miRs (Table 6). Huang et al. demonstrated that alterations in miR-148a overexpression may enhance chemoradiosensitivity and promote apoptosis by directly targeting c-Met in vivo in both human CRC cells and mice [113]. Moreover, miR-34a attenuates chemoradioresistance in CRC [87,143]. MRX34, a miR-34a mimic, is the first synthetic miR to undergo clinical trials to restore the sensitivity of CRC cells to chemotherapeutic agents [143]. After the discovery of the dysregulation of miRNA expression itself being associated with human disease progress and therapy response, the introduction of a disease suppressor miRNA mimic to restore its functionality is one therapeutic approach under careful consideration [144]. To date, dozens of miRNA molecules are in clinical trials [144], such as: miravirsen (miR-122) for the treatment of hepatitis C virus (HCV) infection, under phase II clinical trials in several countries, Remlarsen (miR-29) for the treatment of different type of fibrosis, under phase I clinical trials, and MRX34 (miR-34a) for the treatment of different types of cancers [144,145]. MRX34 is a synthetic, double-stranded miR-34a mimic that encapsulated in a liposomal nanoparticle for the treatment of different types of advanced solid tumors [145,146]. In the first-in-human Phase 1 study of MRX34-based cancer therapy, Hong et al. (2020) revealed that the need to anticipate toxic effects for this class of miR-based drug and the effective delivery of these RNA constructs also remains an unresolved challenge [145]. As highlighted in this review, miRs have considerable potential as predictive biomarkers in patients with CRC receiving chemotherapy or chemoradiotherapy; therefore, miRs warrant the further investigation in prospective clinical studies. The clinical roles of miRs in the regulation of chemosensitivity and radiosensitivity in CRCs must be further explored. By interacting with other miRs, mRNA, DNA, or proteins, miRs can modulate responses to chemotherapy and radiotherapy. Although miRs have great potential as predictive biomarkers in guiding precision medicine or as therapeutic targets to improve chemosensitivity and radiosensitivity in the treatment of CRC and mCRC, the use of miRs in clinical practice warrants further investigation.

PMC10000074

Xusheng Dong,Xueyan Lin,Qiuling Hou,Zhiyong Hu,Yun Wang,Zhonghua Wang

Effect of Maternal Gradient Nutritional Restriction during Pregnancy on Mammary Gland Development in Offspring

06-03-2023

mammary development,nutritional restrictions,pregnancy,offspring

Simple Summary The embryonic period, together with puberty and pregnancy, are known as the three main stages of mammary gland development. The development of the mammary glands is slowed during the embryonic period due to factors such as inadequate nutrition, which directly affect the development of the mammary glands and lactation after birth. However, the impact of embryonic nutrition on fetal mammary gland development is often unnoticed. We investigate the effect of nutritional intake on embryonic mammary gland development by administering different levels of nutritional restriction to female mice during gestation. Contrary to common belief, we found that mild maternal nutritional restriction contributes to mammary gland development in the offspring. Mammary gland dysplasia is not obvious until maternal nutritional restriction reaches 70% of the normal intake. Further embryonic mammary gland development studies can be performed based on our level of maternal nutritional restriction. In addition, the use of mice as model animals can also provide a reference for dairy farming, where nutrition should not be excessive during the gestation period of the cow; otherwise, it affects the mammary gland development of the offspring. Abstract We aimed to investigate the effect of different levels of nutritional restriction on mammary gland development during the embryonic period by gradient nutritional restriction in pregnant female mice. We started the nutritional restriction of 60 female CD-1(ICR) mice from day 9 of gestation based on 100%, 90%, 80%, 70% and 60% of ad libitum intake. After delivery, the weight and body fat of the offspring and the mother were recorded (n = 12). Offspring mammary development and gene expression were explored by whole mount and qPCR. Mammary development patterns of in offspring were constructed using Sholl analysis, principal component analysis (PCA) and regression analysis. We found that: (1) Mild maternal nutritional restriction (90–70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary-development-related gene expression. In conclusion, our results suggest that mild maternal nutritional restriction during gestation contributes to increased embryonic mammary gland development. When maternal nutritional restriction reaches 70% of ad libitum intake, the mammary glands of the offspring show noticeable maldevelopment. Our results help provide a theoretical basis for the effect of maternal nutritional restriction during gestation on offspring mammary development and a reference for the amount of maternal nutritional restriction.

Effect of Maternal Gradient Nutritional Restriction during Pregnancy on Mammary Gland Development in Offspring The embryonic period, together with puberty and pregnancy, are known as the three main stages of mammary gland development. The development of the mammary glands is slowed during the embryonic period due to factors such as inadequate nutrition, which directly affect the development of the mammary glands and lactation after birth. However, the impact of embryonic nutrition on fetal mammary gland development is often unnoticed. We investigate the effect of nutritional intake on embryonic mammary gland development by administering different levels of nutritional restriction to female mice during gestation. Contrary to common belief, we found that mild maternal nutritional restriction contributes to mammary gland development in the offspring. Mammary gland dysplasia is not obvious until maternal nutritional restriction reaches 70% of the normal intake. Further embryonic mammary gland development studies can be performed based on our level of maternal nutritional restriction. In addition, the use of mice as model animals can also provide a reference for dairy farming, where nutrition should not be excessive during the gestation period of the cow; otherwise, it affects the mammary gland development of the offspring. We aimed to investigate the effect of different levels of nutritional restriction on mammary gland development during the embryonic period by gradient nutritional restriction in pregnant female mice. We started the nutritional restriction of 60 female CD-1(ICR) mice from day 9 of gestation based on 100%, 90%, 80%, 70% and 60% of ad libitum intake. After delivery, the weight and body fat of the offspring and the mother were recorded (n = 12). Offspring mammary development and gene expression were explored by whole mount and qPCR. Mammary development patterns of in offspring were constructed using Sholl analysis, principal component analysis (PCA) and regression analysis. We found that: (1) Mild maternal nutritional restriction (90–70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary-development-related gene expression. In conclusion, our results suggest that mild maternal nutritional restriction during gestation contributes to increased embryonic mammary gland development. When maternal nutritional restriction reaches 70% of ad libitum intake, the mammary glands of the offspring show noticeable maldevelopment. Our results help provide a theoretical basis for the effect of maternal nutritional restriction during gestation on offspring mammary development and a reference for the amount of maternal nutritional restriction. Nutritional challenges that occur during gestation, a critical period for embryonic growth and development, may lead to alterations in the physiological development and metabolism of the offspring after birth [1]. The most possible nutritional challenges during gestation are undernutrition and overnutrition, which can affect the health of both the fetus and the maternal body [2]. In particular, malnutrition during pregnancy, which still exists in underdeveloped regions, as a global problem, has important implications for the healthy development of the mother and the newborn [3]. These nutritional damages can cause permanent adjustments in the embryonic physiological state and organ development by inducing genetic changes in the proliferation/differentiation pathways during embryonic development [4,5]. Current research on nutritional restriction during pregnancy has focused on the placenta, brain and other organs that affect fetal survival [6,7], with little attention paid to fetal mammary gland development. The embryonic period, puberty and pregnancy are known as the three main stages of mammary gland development. The embryonic development of the mammary gland begins in many mammals at mid-gestation [8]; for mice, with a gestation period of 19–21 days, the mammary gland initiates development on day 10 of embryonic life [9]. The embryonic mammary glands are formed by a bilaterally multilayered ectodermal stripe from the forelimb bud to the hindlimb bud on the ventral surface of the embryo, referred to as the mammary line [10]. At day 11.5 of the mouse embryonic stage, these milk lines form five visible pairs of placebos. These placebos then become embedded in the mammary mesenchyme. At day 15–16 of the mouse embryonic stage, primary bud formation invades the secondary mammary mesenchyme and begins to develop a branching morphology [11]. Before birth, the mammary gland consists of a small ductal tree with a dominant duct and 10–15 branches embedded in the nascent fat pad [12]. The basic mammary duct system that forms at this time arises in the absence of hormonal input and remains essentially quiescent until puberty [10]. This basic ductal system forms the framework from which the mammary glands develop further during puberty and pregnancy to form the mature mammary glands [13]. If the development of the embryonic mammary glands is slowed at this period due to nutritional deficiencies and other factors, it directly affects the development of the mammary glands after birth and may even affect the amount of milk produced during lactation [14,15]. Although the embryonic stage is the initiation of mammary gland development, nutritional regulation has remained less studied for this stage of mammary gland development. Since puberty is considered a critical window for nutritional regulation, most nutrition-related research has focused on this period [16,17]. The impact of embryonic nutrition on fetal mammary gland development is often unnoticed. Although the mammary gland that develops during the embryonic period is considered a basic ductal system, it has the ability to produce milk, known as neonatal milk or switch’s milk [10]. This indicates that the mammary gland is already equipped with basic lactation functions after birth, and if there are problems with the development of the mammary gland during the embryonic period, these basic functions are affected. Terminal end buds, an important structure in the extension of the mammary ducts during puberty, form only at the tips of the elongated ducts which are based on branches generated during the embryonic period [9,10]. Multipotent mammary stem cells (MASCs) from embryonic mammary gland formation are the source of MASCs and progenitor cells required for mammary duct development during puberty and alveolar luminal formation during pregnancy [9]. These results all suggest that there is a connection between embryonic, pubertal and gestational mammary development, and that mammary gland damage caused by nutritional fluctuations received during the embryonic period further affect mammary gland development after birth. The embryonic stage is the period of initial mammary gland formation, when the mammary gland gradually begins to expand through proliferation and differentiation in multipotent MASCs [18]. Many genes associated with mammary stem cells during the embryonic period have been shown to influence the future developmental fate of the mammary gland. The Axin2 gene was found to have the ability to allow cell regeneration in mammary gland transplantation assays, and the expression of the Axin2 gene during the embryonic period has been shown to be associated with future development in the ductal cell lineage [18]. During this period, Wnt5a has also been shown to be required for normal development of the mammary ducts [19]. In addition, MASCs marker genes such as Sox10, Procr, ELF5 and Aldh1a1 were identified by knockout studies and regulate key functions of mammary gland development [20,21,22,23]. After birth, MASCs become lineage-restricted with some becoming progenitor cells and contributing to the development of the mammary gland base or lumen [9]. Thus, nutritionally induced changes in embryonic mammary development may continue to affect mammary development in adulthood. The effect of altered nutrient levels on mammary stem cells has been demonstrated in previous studies with cells and adult mice [24,25]. Maria Theresa E. Montales et al. found that angiotensin in food affects the number of mammary cell-like/progenitor cells [24]. Omar M. Rahal et al. speculated that diet-regulated hormonal signaling could influence MASC self-renewal [25]. Studies on the effects of nutritional restriction on mammary stem cells have had mixed results, with one study suggesting that nutritional restriction attenuates mammary stem cell viability and inhibits mammary gland development [26], while another study suggests that nutritional restriction induces the self-renewal of mammary stem cells [27]. These results may be due to differences in the amount of nutrient limitation. Mild nutrient limitation mediates the restoration of stem cell self-renewal capacity through nutrient and energy-sensing pathways [27]. When nutrient limitation exceeds the regulatory level of the cells, apoptosis and necrosis of stem cells can occur due to nutrient deficiency. Compared to nutritional treatment after birth, nutritional treatment for the embryonic period is more difficult and requires nutritional interventions for the maternal body. The most common approach in studies of fetal undernutrition is accomplished through food or caloric restriction of the mother during gestation [28]. The mammalian placenta has evolved mechanisms that help buffer the fetus from short-term fluctuations in maternal diet and energy status [29]. In order to avoid this buffering mechanism, most of the studied protocols reduce maternal nutritional intake to 50–60% of the normal amount, exerting a significant impact on fetal growth and development through high levels of food restriction [7]. Moderate or low levels of food restriction may better mimic the clinical features of malnourished women, but few studies have investigated the effects of moderate food restriction during pregnancy on embryonic development. In addition to maternal nutritional interventions, the smaller size of the embryonic mammary gland presents challenges for the study of mammary gland development. The most visual method of viewing mammary gland development is the whole mount, a method of viewing a three-dimensional overview of the mammary gland, which provides a dense ductal epithelial structure within the complete mammary gland [30]. The whole mount requires the complete mammary gland to be isolated from the skin of the mouse and spread out as naturally as possible, which is more challenging for embryonic and newborn mice. In earlier studies, the results of the whole-mount analysis were difficult to quantify and were only used as a display image in the studies [31]. The complex ducts of the mammary gland in puberty can be evaluated in terms of the area covered and the denseness of the ducts observed visually. However, this unquantifiable observation is difficult to evaluate in the primary mammary gland, which has only 10–15 branches at birth. Jason P. Stanko et al. reported the use of Sholl analysis, an ImageJ plug-in for neuronal analysis, to quantify whole-mount results of the mammary gland [32]. The Sholl analysis creates a series of concentric rings based on a custom center (origin of the mammary duct) and extends to the most distal portion of the branch (enclosing radius). The Sholl analysis plug-in calculates the number of intersections that occur on each ring and then returns a Sholl regression coefficient (k), which is a measure of the rate of decay of the epithelial branches. In Sholl analysis, the sum inters (N) is the number of intersections of multiple concentric circles centered on the primary ducts with the ducts, reflecting the complexity of the mammary gland. The sholl regression coefficient (k) is a measure of the distal mammary branch complexity, which is close to 0, indicating more complex and well-developed distal mammary branches. Branch density is calculated using the formula N/MEA. Sholl analysis provides a valid quantitative measure of mammary branch complexity and has become a reliable method for studying mammary gland development. Mammary gland development in embryonic mice can be evaluated through a combination of fine dissection and whole-mount and Sholl analysis. Different levels of maternal nutritional restriction may have different effects on embryonic mammary gland development due to different maternal nutritional buffering and stem cell responses to nutrition. To investigate this, we established a pattern of nutritional restriction on mammary gland development during embryonic period by setting 100%, 90%, 80%, 70% and 60% diet intakes for female mice during pregnancy. The objective of our study was to investigate the effect of maternal gradient nutritional restriction on mammary gland development in offspring and provide a reference for the amount of maternal nutritional restriction. Sixty female 8-week-old CD-1(ICR) mice were provided by Vital River Laboratory Animal Technology Co., (Beijing, China) and mated with males of similar age. Each male mouse was put in a cage with 1 female mouse. Mating of mice was demonstrated by the presence of vaginal plugs. Female mice were individually housed after the discovery of the vaginal plugs and recorded as day 0 of gestation. All mice in our study were fed commercially available irradiated sterile growth and reproduction diets for experimental mice (SFS9112, Xietong Biotechnology, Yangzhou, China). To reduce the impact of nutritional restriction on early embryonic growth, it began on the ninth day of pregnancy. Pregnant mice were divided into five groups (n = 12): the 100% group was fed ad libitum (control group), and the 90%, 80%, 70% and 60% groups were fed 90%, 80%, 70% and 60% of the ad libitum food weight daily, respectively. The ad libitum group was mated one day earlier and their intake was used as the basis multiplied by 90%, 80%, 70% and 60% as the feed intake for the gradient nutrient restriction. The weight of the mice was recorded daily. The number of litters as well as the weight of the female mice and offspring were recorded on the day of delivery. On the day of delivery, whole body image and body fat percentage were evaluated in vivo using dual-energy X-ray absorptiometry (DEXA) on an InAlyzer (Medikors Co., Seongnam, Republic of Korea). Female mice and female offspring were anesthetized using isoflurane (RWD, Shenzhen, China) and placed on a scanner bed and operated according to the instructions. After in vivo imaging, female offspring mice were euthanized using CO2. The mammary glands were removed immediately after euthanasia, the #4 inguinal mammary glands were placed on slides and immersed in Carnoy’s solution for whole mount, and the other mammary glands were stored at −80 °C for real-time quantitative polymerase chain reaction (qPCR). The mammary glands were fixed in Carnoy’s solution (60% absolute ethanol, 30% chloroform, 10% glacial acetic acid) for 4 h and then placed sequentially in ethanol at 100%, 70%, 50% and 10% concentrations for 15 min each. After soaking in deionized water for 5 min, the mammary glands were stained using carmine alum solution (1 g carmine alum, 2.5 g aluminum potassium sulfate in 500 mL dH2O) for 4 h. The stained mammary glands were soaked for 5 min using distilled water, then sequentially soaked in 70%, 95% and 100% alcohol, each concentration for 15 min. The mammary glands were placed in xylene for 12 h for transparency and then sealed with neutral resin. Whole-mount slices of mammary glands were sectioned for image acquisition using an upright microscope (Nikion, Japan). RNA from offspring mammary glands was extracted using RNA-easy Isolation Reagent (R701-01, Vazyme Biotech, Nanjing, China) according to the instructions. RNA quality was evaluated by 1% agarose gel electrophoresis, while the purity of the total RNA was determined by NanoDrop 2000 (NanoDrop, ThermoFisher Science, Waltham, MA, USA). The genomic DNA was removed from each RNA sample and reverse-transcribed into cDNA using an Evo M-MLV Mix Kit (Accurate Biology, AG11728, Hunan, China). Then qPCR was performed using a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, AG11701, Hunan, China) with a LightCycler 96 Instrument (Roche, Basel, Switzerland). The reaction program was set to pre-denaturation at 95 °C for 30 s, followed by denaturation at 95 °C for 5 s and extension at 60 °C for 30 s, for a total of 40 cycles, with each reaction repeated 3 times. The primer sequences are shown in Table 1. The amplification efficiency and the specificity of the amplified products of each primer pair were verified using standard curves and melting curves, respectively. The mRNA expression of each sample was normalized relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression levels of each target gene were analyzed using the 2−ΔΔct method. We performed Sholl analysis on mammary whole-mount results according to the method described in a previous study [32]. Mammary gland whole-mount analysis was performed using ImageJ 2.1 software, and the Sholl analysis plugin 4.0.1 for ImageJ was used for Sholl analysis. The distance from the primary ducts to the most distal end of the mammary epithelium (enclosing radius) and the mammary epithelial area (MEA) were measured using ImageJ. The Sholl analysis was performed with the primary duct as the center, the enclosing radius as the ending radius and a radius step size of 0.02 mm. Since the mammary ducts in newborn mice are less developed and farther away from the mammary lymph nodes, the area occupied by the mammary lymph nodes was not calculated in Branch density. Body weight, body fat, litter size, Sholl analysis results and gene expression were analyzed using one-way ANOVA in the ad libitum feeding, 90%, 80%, 70% and 60% groups. One-way ANOVA was performed using IBM spss 25 (Armork, NY, USA), with Sidak correction for multiple testing. Body weight, body fat, litter size and gene expression data were presented as the mean ± the standard deviation (SD). Principal component analysis (PCA) was performed on enclosing radius, MEA, sum inters and k from the results of Sholl analysis in all groups. PCA was performed using the FactoMineR and factoextra packages in R4.2.1, and PCA biplot figures were generated. The enclosing radius of each group in the Sholl analysis results were regressed against MEA, sum inters and k. Linear regression analysis of the mammary Sholl analysis was performed using simple linear regression in GraphPad Prism software 9.1.0 (San Diego, CA, USA). After nutrient restriction management, a significant difference in body weight was observed in mice from day ten of pregnancy, and the difference persisted until the end of gestation (p < 0.05; Figure 1A). After parturition, the adult female mice showed a significant decrease in body weight compared to the control group (p < 0.05), except for the 90% group (p > 0.05; Figure 1B). However, there was no significant difference in body fat percentage in adult female mice after parturition (p > 0.05; Figure 1C). When the nutritional intake was only 60% of the normal intake, a significant decrease in litter size was observed compared to the control group (p < 0.05; Figure 2A), while the individual offspring weight was significantly lower than that of the other groups (p < 0.05; Figure 2B). The body fat percentage of the offspring was significantly higher in the control group than in the 80%, 70% and 60% groups (p < 0.05; Figure 2C,D). The mammary whole-mount images are shown in Figure 3A. For the enclosing radius, a significant increase was observed in the control group compared to the 70% and 60% groups (p < 0.05), while a significant increase was observed in the 90% group compared to the 60% group (p < 0.05; Table 2). MEA did not differ in the control, 90% and 80% groups (p < 0.05), while it was significantly lower in the 70% and 60% groups than in the former three groups (p < 0.05; Table 2). Sum inters were significantly higher in the control, 90% and 80% groups than in the other two groups (p < 0.05; Table 2). The k of 70% and 60% were significantly higher than the other three groups (p < 0.05; Table 2). Branching density was not significantly different among the groups (p > 0.05; Table 2). Consistent with the results in Table 2, an identifiable change in mammary gland development was observed from the 80% group to the 70% group in Figure 3B. Figure 3C shows the number of intersections of each concentric circle with the mammary ducts in the Sholl analysis. The control group had the longest duct extension distance. At a radius of 0.5 mm, the control, 90% and 80% groups reached the highest number of intersections with a similar peak, all higher than the 70% and 60% groups. In order to further investigate the reasons for the dramatic decline in offspring mammary development from the 80% group to the 70% group, we performed a PCA (Figure 3D) of the mammary whole-mount results (enclosing radius, MEA, sum inters and k). After dimensionality reduction, the data points in the control, 90% and 80% groups were nearer to each other, forming visible distance differences with the 70% and 60% groups, indicating that a massive reduction in mammary gland development in the offspring occurs when the maternal nutritional limit is reduced from 80% to 70%. The PCA bipartite plot shows the scores and loadings of the first two components (dim1 and dim2), revealing the projection of the observed indicators on a space with dim1 and dim2 as axes. In our study, the indicators of mammary gland development were explained by 79.1% of dim1 and 10.2% of dim2, respectively. The variable with the highest weight in the first principal component is the enclosing radius, indicating that the main reason for the difference in distance from the 80% to the 70% group in the mammary glands was the change in enclosing radius. A positive correlation between enclosing radius and MEA and sum inters and a negative correlation with k are presented in the PCA biplot. To analyze the effect of the enclosing radius, which has the highest weight in PCA, on the pattern of mammary gland development in the offspring, we performed a regression analysis of the whole-mount results (Figure 4). In the regression analysis of the enclosing radius with MEA, the 90% and 60% groups had larger slopes compared to the control group, while the 80% and 70% groups had smaller slopes. In the regression analysis of the enclosing radius versus sum inters, as maternal nutritional restriction increased, the slope first increased in the 90% group, then gradually decreased in the 80% and 70% groups and then showed an increase in the 60% group. In the regression analysis of the enclosing radius versus k, the slope of each group is less than the control group, with the 60% group having the lowest slope. We analyzed the expression of development-related genes (Sox10, Axin2, Elf5, Lgr5, Wnt5a, Aldh1a1, Procr), mammary basal cell marker genes (K5), mammary luminal cell marker genes (K18), estrogen (ERα,ERβ) and progesterone receptor (PR) genes in the mammary glands (Figure 5) by one-way ANOVA followed by a Sidak multiple-comparison test. In the 90% group, Sox10 expression was significantly higher than in the other four groups (p < 0.05), and Elf5 was significantly higher than in the control and 60% groups (p < 0.05). Sox10 was significantly lower in the control group than in the 90%, 70% and 60% groups (p < 0.05), and Axin2 was significantly higher in the control group than in the 60% group (p < 0.05). Aldh1a1 was significantly higher in the 80% group than in the 60% group (p < 0.05). The expression of K5 was significantly higher in the control group than in the 80%, 70% and 60% groups (p < 0.05). In the 60% group, ER1 was significantly lower than in the 90% group (p < 0.05) and ER2 was significantly lower than in the control group (p < 0.05). The expression of other genes did not differ significantly among the groups (p > 0.05). Nutritional deficiencies during gestation cause irreversible effects in fetal organs [33], but nutritional deficiency research on embryonic mammary gland development remains vacant. The impairment of mammary gland development at this phase may directly lead to delayed fetal mammary gland development in adulthood [9]. The small size of the mammary gland, which is difficult to observe, and the buffering through the placenta, which reduces the impact of nutritional fluctuations in the embryo, present challenges for the study of mammary gland development during this period. We performed a quantitative study of the mammary glands using whole mount combined with Sholl analysis and further analyzed the developmental pattern of the mammary gland by PCA and regression analysis. Through maternal gradient nutrient limitation, we established a pattern of offspring mammary gland development and revealed stem cell-related gene expression through a gradient reduction in maternal nutrient intake from 100% to 60% during gestation. The main findings of the study were: (1) Mild maternal nutritional restriction (90–70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary development-related gene expression. Inadequate nutrition during pregnancy can have an impact on maternal and fetal health, most notably in the form of weight loss [34]. In our study, differences in body weight of female mice emerged from the tenth day of gestation after nutritional restriction. After delivery, maternal mice in the 80% group lost significant body weight, while body fat percentage was not affected. For offspring, body fat percentage decreased first when nutritional intake was 80% of ad libitum, and weight loss occurred when it was 60%. Our result is similar to a previous study, which found no significant change in offspring birth weight during gestation for a maternal restriction to 75% ad libitum feeding [28]. A reduction in offspring body weight occurs when nutritional restriction reaches 60% or less of the ad libitum intake [34,35]. It seems that embryonic body fat percentage is more susceptible than body weight when faced with nutritional constraints. Mammary ducts and epithelium need to be embedded in the mammary stroma for growth, which is composed of homogeneous adipose tissue [36]. In studies on obesity, there is a strong association between mammary fat pads and obesity [37]. Although mammary fat pad and body fat have not been studied in studies on nutritional restriction, the possibility exists that a decrease in whole body fat percentage may affect mammary fat pad development. To assess mammary gland development, we performed a Sholl analysis on the mammary glands of offspring with different levels of nutritional restriction. Based on the Sholl analysis reported in the previous study [32], we innovatively performed PCA analysis and regression analysis on the results of the Sholl analysis to explore the developmental pattern of mammary glands. We found a dramatic decrease in mammary gland development when nutritional restriction was dropped from 80% to 70% of ad libitum intake. In contrast, there was no significant difference in the effect of normal feeding versus 90% and 80% of ad libitum feeding on mammary gland development. We hypothesize that the dramatic delay in mammary gland development may be due to the buffering of embryonic nutrients by the placenta as a “nutrient sensor” [29]. For maternal nutritional restriction to 90% and 80% of normal intake, the buffering mechanism in the maternal body mitigates the effect of nutrition on fetal mammary development, and as the intake decreases to 70%, the maternal buffering limit is exceeded, resulting in delayed mammary development. Inconsistently with our results, the nutritional intake of sows being restricted to 70% of ad libitum intake had no effect on the weight of mammary parenchyma, fat content, protein content and DNA content of the offspring [38]. The weight, DNA content and other methods used in their study to evaluate the mammary glands do not provide a complete view of the development of the mammary ducts compared to the whole mount. In addition, the species may also be responsible for the discrepancy between their results and our findings. From the results of the PCA analysis, we determined that the variable with the greatest weight is the enclosing radius. This suggests that the dramatic decrease in mammary development from the 80% to the 70% group was mainly caused by changes in the enclosing radius. To analyze the developmental pattern of the mammary glands, we performed a regression analysis of the enclosing radius with MEA, sum inters and k. Our study found a positive regression relationship between the enclosing radius and MEA and sum inters and an inverse regression relationship with k. This suggests that as the distance of the terminal duct from the primary duct increases, the mammary gland will cover a larger area with more complex branching, while the terminal decay will be slower. Similar to our results, the similar trend of the mammary longitudinal extension distance with the mammary epithelial area was found in a previous study [39]. We found that compared to controls, mild nutritional restriction (90% of ad libitum intake) had larger regression coefficients in regression analyses with MEA and sum inters and smaller regression coefficients with k. This suggests that offspring with mild maternal nutritional restriction have better potential for mammary gland development. When nutrition was restricted to 80% of ad libitum feeding, the regression coefficients of the enclosing radius and MEA reflected reduced mammary epithelial area growth potential, despite no difference in mammary gland developmental indicators compared to the control group. Interestingly, the regression coefficients of MEA, sum inters and k all showed greater absolute values when the nutritional restriction was 60% of the ad libitum intake. At this level of nutritional restriction, the enclosing radius already showed a significant shortening and, therefore, mammary gland development slowed down. Sox10, Axin2 and Elf5 have been shown to function as key genes in embryonic mammary gland development. Sox10 is expressed in fetal mammary gland stem cells during embryonic mammary gland development and plays a central role in mammary gland development [40,41]. Axin2, a target gene of the Wnt/β-catenin pathway, has been used as a marker of functional stem cells in the mammary gland in a lineage-tracing approach [18]. Elf5 is required for the proliferation and differentiation of mammary epithelial cells in embryonic mouse mammary glands [42]. We found that mild nutritional restriction (90% of ad libitum intake) increased the gene expression of Sox10 and Elf5, suggesting a positive effect on mammary gland development. In a previous study on dietary control, alternate-day nutritional restriction was proven to increase the activity of tissue-specific stem cells and had positive implications for life extension [43]. Combined with our regression analysis of whole-mount results, our results suggest that mild maternal nutritional restriction does not impair offspring mammary development and may even increase offspring mammary growth potential by increasing the expression of stem cell-related genes. In addition, 60% of ad libitum feeding reduced Axin2 expression, suggesting that high levels of nutritional restriction inhibit mammary stem cell development and mammary gland development. Consistent with our results, in a study of high levels of maternal gestational nutritional restriction (50% of ad libitum feeding), the ability to differentiate neural progenitor cells was decreased [44]. These results suggest that stem cell activity in the embryonic mammary gland is related to the level of maternal nutritional restriction, with mild nutritional restriction contributing to stem cell-associated gene expression and high nutritional restriction inhibiting them. K5 is a known marker gene in the myoepithelial/basal layer of the mammary gland [45]. Our study shows that a decrease in K5 gene expression occurs in the basal lamina of the mammary glands when nutrition is restricted to 80% of ad libitum feeding. Combined with regression analysis, our results showed that the expansion potential of mammary basal and mammary gland area was affected by maternal nutritional restriction up to 80% of ad libitum feeding, despite no significant difference in the results of whole-mount analysis. Embryonic mammary gland development is considered to be hormone-nondependent, and previous studies have demonstrated that embryonic mammary glands are able to develop in mice lacking estrogen (ER-α and ER-β) and progesterone receptors [9,46,47]. After birth, especially during puberty, the mammary glands are stimulated by these hormones to develop rapidly. Estrogen is required for the branching of the mammary ducts during puberty, and estrogen and progesterone are required for lobuloalveolar development during pregnancy. In our study, ER-β receptor expression appeared to be reduced when nutritional restriction reached 60% of the ad libitum intake. This suggests that high levels of maternal nutritional restriction may affect the development of offspring mammary estrogen receptors whose impairment may have further effects on mammary development during puberty. In conclusion, our results suggest that mild maternal nutritional restriction (90% of ad libitum intake) during gestation contributes to increased embryonic mammary gland development. When nutritional restriction ranges from 80% to 70% of ad libitum intake, mammary gland development decreases dramatically, and changes in developmental patterns occur.

PMC10000075

Tessa Bánki,Jarno Drost,Marry M. van den Heuvel-Eibrink,Annelies M. C. Mavinkurve-Groothuis,Ronald R. de Krijger

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review

21-02-2023

nephrogenic rests,nephroblastomatosis,Wilms tumors,genetic changes,epigenetic alterations

Simple Summary We reviewed all studies investigating molecular changes in nephrogenic rests (NR), the presumed precursor lesions of Wilms tumors (WT) being the most frequent malignant childhood renal tumors, between 1990 and 2022. Only 23 studies were found, reporting 119 pairs of NR and corresponding WT, which may allow the detection of early genetic changes that play a role in tumorigenesis. Two genes, WT1 and WTX, and two chromosomal regions, 11p13 where WT1 is located, and 11p15 harboring the IGF-2 gene, were found to be mutated or show loss of imprinting, respectively, in both nephrogenic rests and WT, suggesting that these could be relevant early genetic events. Abstract Objective: To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). Methods: This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Results: Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Conclusions: Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed.

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review We reviewed all studies investigating molecular changes in nephrogenic rests (NR), the presumed precursor lesions of Wilms tumors (WT) being the most frequent malignant childhood renal tumors, between 1990 and 2022. Only 23 studies were found, reporting 119 pairs of NR and corresponding WT, which may allow the detection of early genetic changes that play a role in tumorigenesis. Two genes, WT1 and WTX, and two chromosomal regions, 11p13 where WT1 is located, and 11p15 harboring the IGF-2 gene, were found to be mutated or show loss of imprinting, respectively, in both nephrogenic rests and WT, suggesting that these could be relevant early genetic events. Objective: To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). Methods: This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Results: Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Conclusions: Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed. Nephrogenic rests (NR) are foci of aberrant embryonal tissue in the kidney, which are still present after 36 weeks of gestation [1,2]. They represent non-obligate precursor lesions of WT and may regress over time or progress to WT due to as yet unknown molecular factors [1,2]. The presence of multifocal or diffuse NR is referred to as nephroblastomatosis [3,4]. NR have been described as either perilobar NR (PLNR) or intralobular NR (ILNR), based on the topographic site in the kidney. Both types can also occur in the same kidney as well as in both kidneys of the same individual. PLNR are located at the periphery of the renal cortex and have been shown to consist mainly of blastemal or immature epithelial components (Figure 1A) [1,5]. ILNR are located within the lobes and are mostly composed of a combination of cystic epithelial elements surrounded by moderately cellular stromal elements (Figure 1B) [1,5,6]. In addition, heterologous components, including fat, may be present. ILNR are considered to arise prior to PLNR, and are usually not as well demarcated as PLNR [1,5,6]. Histologically, according to Beckwith’s criteria [3], NR can be subdivided into different histological subtypes such as dormant rests (especially blastemal cells with minimal proliferation), sclerosing rests (minimal blastemal components and presence of stromal maturation), hyperplastic rests (proliferative signs manifested by blastemal and epithelial cells and increased size), neoplastic rests (usually a discrete spherical nodule), and obsolete rests ((sclerotic) stromal and epithelial components, and minimal blastemal). Any histological subtype of NR has been shown to be able to transition to another NR subtype [1,4]. In infants, microscopic NR are identified in about 1% of autopsies, the majority of which will either remain stable or go into regression [3,7]. The occurrence of NR is partly determined by ethnic and demographic factors [6]. In White American children, 20% reveal WT-associated PLNR compared to 7% in Asian American children, and 2% in Japanese children. ILNR, on the other hand, are more common in Asian American (33%) and Japanese children (25%) relative to the White American children (17%) [8]. As ILNR seem to arise from abnormalities earlier in development, they also occur at a younger age in children, namely at a median age of 23 months, whereas PLNR are usually not discovered until a median age of 36 months [5]. PLNR occur mainly in females and ILNR are more often seen in males [6]. The age at diagnosis also differs between ethnic groups. NR are generally diagnosed earlier in Asian American patients (median age at diagnosis 31 months) than in White American patients (median age at diagnosis 39 months) [8]. NR have been described to lead to the development of WT in a subset of cases [3,5]. WT are the most commonly found malignant renal tumor in children [9]. WT are morphologically heterogeneous embryonic tumors, including epithelial, blastemal, stromal, and sometimes rhabdomyomatous elements [10]. Tumors can be classified in different histological types, which currently grossly determines risk stratification after neoadjuvant chemotherapy treatment [11]. The distinction between NR, mainly PLNR, and WT can be extremely challenging, both radiologically and pathologically, especially if a biopsy is submitted for evaluation. In fact, no single radiological or histological criterion, including size and shape of the lesion, can distinguish NR from WT and a 25% misdiagnosis rate has been reported for radiological assessment (see Figure 1C,D) [12,13]. PLNR are associated with epithelial, blastemal, or mixed type WT and stromal or heterologous elements are limited or absent. In contrast, there is an association with ILNR and stromal type WT and heterologous components are frequent [3]. Most WT occur sporadically and are unilateral, but they can also be familial and bilateral [6]. According to the International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG), kidneys with unilateral WT contain NR in 40% of the cases (25% PLNR, 9% ILNR, 5% both PLNR and ILNR and 1% nephroblastomatosis). Bilateral WT are significantly more frequently associated with NR, as NR have been reported in 94% of the stage V cases [14]. Bilateral WT occur significantly more often in syndromes in the context of which WT recurrently occur [11]. It is beyond the scope of this review to discuss these syndromes, except for their association with NR. Examples are Denys–Drash and WAGR syndrome, which are associated with the presence of ILNR. Patients with these syndromes have a risk of about 30% and 95%, respectively, to develop WT [6]. Patients with these syndromes have a risk of up to 95% and 50%, respectively, to develop WT [15,16]. PLNR occur more frequently in patients with overgrowth syndromes such as Beckwith–Wiedemann syndrome (BWS) [6]. Some of the abovementioned syndromes have a known genetic driver, such as the occurrence of germline WT1 mutations in Denys–Drash and WAGR syndrome [11,17]. In addition, numerous recurrent somatic genetic abnormalities, both gene mutations and copy number variations (CNV) at specific chromosomal locations, as well as hypermethylated regions have been found in non-syndromic, sporadic WTs. These include mutations in WT1, CTNNB1, MYCN, TP53, AMER1, FBXW7, GPC3, MLLT1, DIS3L2, DICER1, DROSHA, DGCR8, SIX1 and SIX2, SMARCA4, ARID1A and chromosomal aberrations such as gain of chromosome arm 1q, and loss of 16q and 1p [11,18]. Although knowledge on tumor-driving changes in WT is increasing, the genetic changes underpinning the development of NR have not been extensively studied. Notably, most NR regress and only some develop into WT [7]. Identifying molecular changes underpinning the transformation of NR to WT may aid the understanding of WT pathogenesis and guide the development of targeted therapies. Therefore, we performed a systematic review of the literature in which molecular analysis was completed in NR or in a combination of NR and WT. This systematic review presents the current knowledge of somatic molecular changes in NR. This review was written according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [19,20] and the protocol was not registered. PubMed, EMBASE and Cochrane were systematically searched for all the available literature on molecular changes in NR in the English language, published from 1990 to 2022. The full search strategy is provided in Table S1. Articles were included if somatic molecular changes in NR were studied, including case reports in which specific analysis for NR was performed. Studies without molecular analysis in NR, non-English literature, animal studies or germline mutation analyses were excluded. Three authors (T.B., A.M., R.K.) assessed all articles independently based on the in- and exclusion criteria. After a comparison and the consensus of the authors on potentially relevant articles, the remaining publications were screened for eligibility criteria, based on their full text. Controversies were resolved by consensus. The “Standards for Reporting Diagnostic Accuracy 2015” (STARD 2015) checklist [20] was used to assess the quality of the included studies individually and is provided in Supplementary Table S2a. The included case reports were assessed using the case report guidelines (CARE checklist 2013), the checklist is presented in Supplementary Table S2b [21]. The included studies were further assessed with the Oxford Centre for Evidence-Based Medicine Levels of Evidence Classification rubric [22], independently by the three authors (T.B., A.M., R.K.), for methodologic quality. In case of disagreement, it was solved by reaching consensus. From the included articles, the number of NR, WT, WT-NR pairs analyzed, mean age at time of diagnosis of WT, sex, histological type of WT and NR and whether a WT was bilateral or hereditary, were obtained. Furthermore, from each study, the genes or chromosomal regions investigated with associated molecular techniques were extracted. Our search in databases PubMed, EMBASE, and Cochrane generated a total of 203 articles. Seventy-three were duplicate articles that were found with more than one search engine and were therefore removed. The 130 remaining articles were screened for title and abstract after which 98 articles were excluded because of the in 2.1 mentioned criteria (Figure 2). We performed full-text screening in 32 potentially relevant studies, critically examining the in- and exclusion criteria. Consequently, 14 more articles were excluded, of which five were abstracts only and nine others were excluded since they did not meet the inclusion criteria. The remaining 18 articles were included in this systematic review. There were five additional articles identified by forward and backward snowballing, resulting in a total of 23 articles. In Figure 2, a flowchart of the search and selection process is presented based on the PRISMA scheme. Quality assessment was completed for all 23 included articles. An overview of the quality assessment of 18 included articles, using the STARD checklist, is provided in Supplementary Table S3a (including the STARD Checklist in Table S2a). The remaining five case reports, using the CARE checklist, are presented in Supplementary Table S3b. All included articles could be classified as Oxford level 2 to 4, as shown in Table 1. Patient characteristics of the 23 included studies are presented in Table 1. In those studies, 221 NR were analyzed, of which 53 were ILNR, 136 were PLNR, 11 were nephroblastomatosis and 21 were not further specified. A total of 119 WT-NR pairs were analyzed with different methods including methylation analysis, sequencing analysis, LOH analysis, expression analysis and in situ hybridization. There were 28 WT-NR pairs reported with concomitant bilateral WT, whereas this was not explicitly reported in the other cases. Sex and age were described in only a few studies. In the eight studies describing sexual gender, there were 42 females and 30 males. The age at diagnosis for WT was variable ranging from 11 months to 144 months and for NR from 10 months to 192 months. Table 2 presents the somatic changes that were found in NR, including the gene or chromosomal region that was involved and the detection method that was used. We identified seven studies in which LOH analysis was used. LOH of 11p15 was found in 13/64 investigated NR (20%), and the same loss was confirmed in the corresponding WT (n = 13) [31,38]. Three studies demonstrated LOH of 11p13, which revealed that this was present in 4/28 WT-NR pairs (14%) all representing ILNR. There was a single WT-PLNR pair that showed LOH of 11p13 in the WT only [35,38,40]. Hoban et al. described a case in which the LOH of chromosome 11 (both 11p13 and 11p15) was observed, where in fact all other maternal chromosomal loci were lost in the NR and WT in a Beckwith–Wiedemann patient [42]. Three studies involved LOH of 16q showing loss in 13/53 WTs (25%). In two studies, loss of 16q was not found in their associated NR [32,38], but Austruy et al. detected loss of 16q in one case of nephroblastomatosis [41]. LOH of 7p was investigated in two pairs of NR-WT, but LOH of 7p was only present in WT, not in the corresponding NR [32,37]. Furthermore, genome-wide comparative genome hybridization (CGH) was performed in two other studies. Vuononvirta et al. investigated 50 PLNR and divided them into three subgroups. The first group showed no copy number changes at all (44%). The second group contained eight cases in which whole chromosome changes were observed (16%). The remaining group, involving 20 cases, presented multiple partial chromosomal gains or losses (40%). In 76%, the NR contained part of the copy number changes seen in the corresponding WT [31]. Steenman et al. also performed whole genome analysis by CGH. This revealed losses of 1p, 7p, 4q and gains of 1q and 12q in nephroblastomatosis and their associated WT. Loss of 11p was found in nephroblastomatosis only. Changes only present in WT were loss of 9p and gain of 8, 10q and 18. Loss of 16q was detected in one case of WT and in one case of nephroblastomatosis adjacent to WT [40]. MdZin et al. found loss of chromosome 22 and showed that the frequency of chromosome 22 loss depended on NR morphology. Dormant, involuted and sclerosing PLNR presented monosomy 22 in 30%, whereas the hyperplastic and adenomatous PLNR showed monosomy 22 in 50%, increasing to a rate of 60–80% in WT [28]. While LOH analysis allows for detecting larger chromosomal regions without exactly identifying the responsible gene(s), candidate gene analysis by targeted sequencing might highlight abnormalities in putative oncogenes and tumor suppressor genes. Seven studies investigated single nucleotide changes in NR compared to WT. Four studies found mutations in the WT1 gene and showed that these can be present in both NR and WT [35,38,43,45]. Using Sanger sequencing analysis, Fukuzawa et al. showed that CTNNB1 mutations occurred only in WT (n = 6) and not in paired NR (n = 6) [35]. Another gene that may play a role in WT tumorigenesis, WTX, presented mutations in both NR (n = 1) and WT (n = 4) [29]. A KRAS gene mutation was found in one case of WT and adjacent NR in the setting of mosaicism [23,24]. In this same patient Slack et. al. [24] also described a somatic FBXW7 mutation in two WT but not in another WT nodule or the associated NR. Two studies reported an absence of mutations of specific candidate genes. In the first study by Wegert et al., EGFR-internal tandem duplications (ITD) and BRAF-internal deletions (ID) were investigated, involving 208 WTs and 12 NR, but no changes in any of these genes were found [26]. In the second study, where PTEN was investigated, no abnormalities were found in the WT and NR included in the study by Grill et al. [30]. Epigenetic changes were described in ten studies, and six of them focused on DNA methylation changes. The different platforms used for methylation profiling in these studies included bisulfate sequencing [34], combined bisulfate and restriction analysis (COBRA) [32,33], pyrosequencing [31], and Illumina BeadChip microarray [25,27]. Charlton et al. [27], using the latter technique, performed a longitudinal study to find differences in DNA methylation to compare NR to NK as well as to WT. For this purpose, differentially methylated regions (DMR) were compared and shown to differ between WT, NR and NK [46]. Hypermethylation was seen in 55% of 629 differentially methylated regions (DMR) in NR as compared to NK [27]. When NR were compared to WT, in paired analysis, two subgroups of WT could be distinguished. In one group, NR and WT grossly harbored the same epigenetic profiles. Yet, the other group of WT showed hypervariability in the methylation profiles in comparison to NR, suggesting that there is a shift in methylation during the development from NK to NR and/or WT [27]. Vuononvirta et al. [31] used pyrosequencing to analyze H19. Hypermethylation of this gene is associated with LOI of IGF-2, which was found in 23/33 (70%) cases and hypermethylation of H19 in 37/40 (93%) of the PLNR cases. Brown et al. analyzed H19 in relation to LOI at 11p13 and 11p15. They found H19 hypermethylation in two WT-NR pairs using COBRA [32]. LOI at 11p13 and 11p15 was found in both WT-NR pairs. LOI of 11p13 leads to the decreased methylation of WT1 antisense regulatory region (ARR), which results in LOI of the noncoding antisense RNA WT1-AS and the alternate coding WT1 transcript AWT1. LOI of 11p15 is responsible for IGF-2 overexpression, another imprinted gene on chromosome 11 [32]. Hypomethylation of WT1 ARR in WT was also studied by Hancock et al. in two WT-NR pairs using bisulfate sequencing, revealing the lowest methylation levels in WT, the highest in NK, and NR showing methylation percentages between fetal kidney (FK) and NK. They also looked at the expression of AWT1 and WT1-AS, and found biallelic expression of AWT1 and WT1-AS in NR and WT, where monoallelic expression was found in the NK [34]. Cui et al. studied RNA expression of the H19 gene in association with IGF-2 expression, using in situ hybridization (ISH) in WT, NR and associated renal medulla. H19 was not expressed in WT and NR, but was present in normal renal medulla [39]. Yun et al. also investigated IGF-2 by in situ hybridization and Northern blotting in NR and WT. Both studies displayed comparable patterns in NR and WT, but there was a variable and heterogeneous level of expression. IGF-2 expression was frequently associated with blastema [39,44]. LOI of IGF-2 was studied by Ravenel et al. and was present in a WT–NR pair with two PLNR [36]. Coorens et al. used Illumina BeadChip microarray to demonstrate the presence of H19 hypermethylation in NK with clonal expansions (58%), while this hypermethylation was not found in NK without clones. One WT–NR pair was included, in which the WT and NR emerged from a similar ancestral clone at different time points, which is indicative of an association between clones, NR and WT all showing H19 hypermethylation [25]. Finally, Chilukamarri et al. investigated the GLIPR1/RTVP-1 gene. Hypomethylation of this gene was shown in 21 out of 24 WTs. There were two associated NR analyzed, which also showed hypomethylation of this gene [33]. We have presented an overview of all molecular studies on nephrogenic rests between 1990 and 2022. As a result of this long-time frame, a wide range of techniques were used to examine chromosomal regions, copy number variations, individual genes and epigenetic changes in NR. A total of 23 studies were found showing loss of chromosomal arms 11p13 and 11p15, 1p, 4q and 11p, and gains in 1q, 7q and 12q, as well as mutations in WT1, WTX and KRAS to occur in both NR and WT, suggesting these are early events (Table 3). Mutations in CTNNB1 and FBXW7 and LOH of 16q and 7p are only present in WT, but not the associated NR, therefore likely representing late events (Table 3). Furthermore, differential methylation levels display a relationship between WT, NR, and NK. Summary of early molecular events that occur in both NR and WT and late molecular events that occur in WT only. Late events are therefore likely not involved in the progression from NR to WT. Little is known about the molecular pathogenesis of WT, including the mechanisms that affect transition from NR to WT, although NR have been recognized as precursor lesions [3]. Studying molecular changes in pairs of NR and WT might shed light on the timing of such events. If the same alteration occurs in NR and the associated WT, this can be considered as an early event. Indeed, LOI and LOH at 11p13 and 11p15 were found in both NR and WT, representing the chromosomal regions where the WT1 and the IGF2 genes are located, respectively [31,32,35,38,40]. No such gene correlations are known for the other chromosomal arms that were recurrently lost (1p, 4q, 7p and 11p), or gained (1q, 7q and 12q) [40]. MdZin et al. found an increasing frequency of monosomy of chromosome 22 from sclerotic/dormant PLNR to hyperplastic/adenomatous PLNR and then finally toward WT, suggesting that loss of chromosome 22 is an early event and that tumor suppressor genes on this chromosome might be involved in WT tumorigenesis [28]. Mutations in WT1 [35,38,43,45] and WTX [29] can also be considered early events, as they were found in both NR and WT. WT1 was first investigated in WT in 1991 [45,47], and WTX was first described in association with WT in 2007 (Figure 3) [48]. Genetic changes that are found in WT only, and not in NR, are likely to be late events and therefore not involved in the transition from NR to WT. For instance, LOH of 16q appears to be a late event [32,38,40,41]. However, in one case, loss of 16q was also found in the associated nephroblastomatosis [41]. Loss of 7p appears to occur later in development as well [32,37], although Steenman et al. found LOH of 7p in a case of nephroblastomatosis [40]. Furthermore, CTNNB1 mutations, which were first described in WT in 1999 (Figure 3), are considered late events in Wilms tumorigenesis [29,35]. No PTEN, EGFR and BRAF mutations or rearrangements were found in both WT and NR, implying that these genes do not seem to contribute to the transition of NR to WT [26,30]. It should be noted that, due to the long time interval, there was large variation between studies with regard to the number and type of polymorphic markers used for LOH or LOI analyses, which may have had an effect on the detection of molecular abnormalities. Likewise, for the candidate gene studies, not all genes have been investigated completely or information on the extent of screening is lacking. In one study, all exons of CTNNB1 were sequenced [35], while Park et al. [43] examined exons 2–10 for detecting mutations in WT1. In studies describing WT1, two studies used primers for only two exons to detect mutations in WT1, not mentioning if all other exons of WT1 were sequenced [35,38]. In one other study that examined both CTNNB1 and WTX, it was not reported which exons were sequenced [29]. Furthermore, many of the genes that have been shown to be involved in WT tumorigenesis, such as SIX1, SIX2, and DROSHA, have not been systematically analyzed in NR [49]. Thus, comprehensive analyses should be performed using WES or WGS to examine all genes and to prevent important genes from not being detected. Regarding epigenetic changes, there appears to be a correlation between the methylation and expression patterns of NR as compared to WT. The similar or increased methylation of WT and NR with respect to NK suggests that this might play a role in tumorigenesis [27]. There was increased methylation of H19 in WT and NR, and also in the clonal expansions of NK, which suggests a transformation from NK to WT in the levels of methylation of H19. It is remarkable that the WT–NR pair included in this study arose from the same clone and that they are therefore phylogenetically related [25]. Together this may indicate that the clonal beds of NK are “primed” to become a WT. LOI at 11p15 and 11p13 and LOI of IGF-2 were detected in WT and NR, and thus seem to be early events [32,36]. The presence of LOI of 11p13 correlates with the decreased methylation of WT1 ARR, suggesting that imprinting defects at 11p13 may be involved in tumorigenesis [32,34]. H19 and IGF-2 are two parentally imprinted genes on 11p15, with opposite regulatory mechanisms: if H19 is silenced, IGF-2 on the other hand is upregulated [50,51]. This explains why there was expression of IGF-2 in both NR and WT, while H19 expression was not detected [39]. Together with ISH studies showing variable IGF-2 expression patterns in NR and WT, these data suggest a role for IGF-2 as an early driver of WT development [39,44]. Likewise, hypomethylation of GLIPR1/RTVP1 was found in WT and NR, suggesting that this specific change might also contribute to WT development [33]. As previously described, NR are present in approximately 40% of unilateral WT and in almost all cases of bilateral WTs [14]. Nephroblastomatosis is more frequently present in bilateral WT [52]. This suggests that not every WT might arise from NR. Conversely, not every NR develops into a WT either. WT and NR both develop from the same embryonic tissue, and both morphologically reflect embryonal renal tissue. Normally, nephrogenesis stops at 34 weeks of gestation and any remaining nephrogenic tissue is considered NR. However, little is known about the underlying factors that lead to such persistence. ILNR are frequent in tumors with stromal histology that are typically associated with aberrations in WT1 on chromosome locus 11p13. PLNR occur more often in blastemal and/or epithelial type WT and are associated with alterations in IGF-2 on 11p15 [18]. These associations have been confirmed in several included studies where a distinction was made between PLNR and ILNR. WT1 mutations and LOH of 11p13 are found in ILNR [35,38,43], and LOI of IGF-2 in PLNR [36]. However, LOH of 11p15 can also be found in ILNR [38]. Interestingly, there is a known association between WT1 and CTNNB1 mutations in WT [53,54]. However, it is notable that CTNNB1 mutations occur as late events and are only present in WT [35], while WT1 mutations can occur in NR and WT [35,38,43,45]. Even though NR are precursors of WT, there are only few studies which focus on and describe the molecular pathogenesis of NR. In this review, all studies between 1990 and 2020 which analyzed molecular changes in NR and used molecular techniques were included, resulting in only 23 relevant articles, i.e., less than 1 per year over the investigated time frame. We chose 1990 as a starting point (Figure 3), as it coincides with the first histopathologic description of NR and with the wider applicability of molecular methods in biomedical research, but even until 2000 few molecular studies were performed. In addition, most techniques required frozen lesional tissue, while frequently only formalin-fixed paraffin embedded (FFPE) material was available, limiting the possibilities of molecular analyses, at least in previous decades. Finally, it is virtually impossible to distinguish NR macroscopically, apart from patients with nephroblastomatosis, requiring expert microscopic detection on FFPE material with its inherent limitations. Throughout this review, we have applied the histological definition of NR and nephroblastomatosis over the radiological definition, which is not 100% superimposable with the histological term of NR. The supplied histological material was used as a reference point. The diagnosis of NR and WT can only be made after histological confirmation. On CT imaging, NR and nephroblastomatosis may show distinct features from WT, mainly with regard to the shape and size of the lesion [13]. However, up to 25% of the NR and WT are still misdiagnosed, with half of nodules radiologically diagnosed as WT, turning out be NR after histological confirmation [12]. It is outside the scope of this review to further discuss the potential a newer imaging technology, such as diffusion-weighted imaging (DWI) MRI for the above distinction. As presented in Table 1, the number of WT and NR examined varied greatly from study to study. Some genes have been studied on less than five tumors. Therefore, firm conclusions cannot be drawn from these studies. To do so in the future, larger-scale studies of WT and corresponding NR would be needed to assign certain genes a significant role in the development of NR to WT. In addition, mutations such as DROSHA and SIX1 and SIX2, which are known driver mutations in WT, have not been examined in NR. These genes, as can be seen in Figure 3, have been discovered from 2010 onwards. Many studies described in this review were completed at a time when these candidate genes were not yet identified and have therefore not been investigated in NR. Until 2000, mRNA in situ hybridization (ISH), sequencing analysis, LOH analysis and CGH analysis were used to analyze NR (Figure 3). From 2006 methylation studies emerged in which the difference in methylation patterns between NR and WT was investigated [25,27,31,32,33,34]. Several methods that were used in studies presented in this review may currently be considered outdated with the advent of new molecular methods, including whole exome and whole genome sequencing, RNA sequencing and also spatial transcriptomics, which allows tissue context while analyzing gene expression profiles. These techniques may allow faster progress and seem particularly relevant for NR analysis, which still relies on morphological identification. It is important to consider tests for the early detection of genetic, epigenetic and somatic changes in NR that may become clinically relevant to the patient before NR develop into a WT. At the moment, not all genetic predispositions have been explored. Therefore, all children with WT or nephroblastomatosis should be referred to a clinical geneticist and advised to undergo whole exome or whole genome sequencing, as there is a high risk (up to 33%) of genetic predisposition, as was recently shown in a Dutch cohort [55]. This certainly applies to children with bilateral WT, in whom an 80% chance of genetic predisposition has recently been revealed [56]. In conclusion, over the past 30 years, several studies have looked at NR with or without associated WT. As a result, much has become known about the genetic changes in NR. LOH of 11p13 and 11p15, expression of IGF-2 and mutations in WT1 and WTX appear to play a role in the early tumorigenesis of WT. LOH of 16q and 7p and mutations in CTNNB1 seem to occur later in development. Methylation patterns of NR in comparison to WT appear to be similar. Due to rapid advances in (genome-wide) molecular techniques, the increased possibilities to use FFPE material and the availability of histologically confirmed frozen material, genetic changes in NR and corresponding WT may be investigated in larger series and might unravel early steps in the progression of WT tumorigenesis.

PMC10000085

Thomas John Lopdell

Using QTL to Identify Genes and Pathways Underlying the Regulation and Production of Milk Components in Cattle

02-03-2023

mammary biology,mammogenesis,lactation,lactogenesis,quantitative trait loci

Simple Summary Milk and other dairy products are commonly consumed in many parts of the world. Dairy cattle, having millions of milk trait records, make excellent model species for understanding the genetics controlling the production of milk. This manuscript gives a summary of the current understanding of the genetic signals for milk production, in terms of the biological pathways they are involved with, and highlights a number of methods that can be used to identify the genes and variants underlying these signals. Knowledge of these variants will improve the ability of farmers and animal breeding companies to increase the rate of genetic gain for milk traits and enable the use of technologies such as gene editing. Abstract Milk is a complex liquid, and the concentrations of many of its components are under genetic control. Many genes and pathways are known to regulate milk composition, and the purpose of this review is to highlight how the discoveries of quantitative trait loci (QTL) for milk phenotypes can elucidate these pathways. The main body of this review focuses primarily on QTL discovered in cattle (Bos taurus) as a model species for the biology of lactation, and there are occasional references to sheep genetics. The following section describes a range of techniques that can be used to help identify the causative genes underlying QTL when the underlying mechanism involves the regulation of gene expression. As genotype and phenotype databases continue to grow and diversify, new QTL will continue to be discovered, and although proving the causality of underlying genes and variants remains difficult, these new data sets will further enhance our understanding of the biology of lactation.

Using QTL to Identify Genes and Pathways Underlying the Regulation and Production of Milk Components in Cattle Milk and other dairy products are commonly consumed in many parts of the world. Dairy cattle, having millions of milk trait records, make excellent model species for understanding the genetics controlling the production of milk. This manuscript gives a summary of the current understanding of the genetic signals for milk production, in terms of the biological pathways they are involved with, and highlights a number of methods that can be used to identify the genes and variants underlying these signals. Knowledge of these variants will improve the ability of farmers and animal breeding companies to increase the rate of genetic gain for milk traits and enable the use of technologies such as gene editing. Milk is a complex liquid, and the concentrations of many of its components are under genetic control. Many genes and pathways are known to regulate milk composition, and the purpose of this review is to highlight how the discoveries of quantitative trait loci (QTL) for milk phenotypes can elucidate these pathways. The main body of this review focuses primarily on QTL discovered in cattle (Bos taurus) as a model species for the biology of lactation, and there are occasional references to sheep genetics. The following section describes a range of techniques that can be used to help identify the causative genes underlying QTL when the underlying mechanism involves the regulation of gene expression. As genotype and phenotype databases continue to grow and diversify, new QTL will continue to be discovered, and although proving the causality of underlying genes and variants remains difficult, these new data sets will further enhance our understanding of the biology of lactation. The composition of milk is complex, featuring an emulsion of fat globules and a colloidal dispersion of casein micelles in an aqueous solution of lactose (and other carbohydrates), whey proteins, and minerals. Although milk from different species contains the same basic constituents, their proportions can vary greatly. In cattle, the typically average percentages (g/100 g) of fat, caseins, whey proteins, and lactose are 3.9%, 2.6%, 0.6%, and 4.6% respectively; in humans, the corresponding percentages are 4.5%, 0.4%, 0.5%, and 7.1% [1]. Even more extreme differences can be seen in other species. Some seal species, for example, producing little to no lactose, resulting in highly concentrated milk with fat percentages of 50% [2]. Less-extreme differences in milk composition are also visible within species. In many cases, the differences in composition among individuals are under partial genetic control. Regions of the genome where the genotypes of genetic variants are associated with phenotypes such as milk composition are known as quantitative trait loci (QTL). When they can be identified, the causative genes underlying these QTL can help elucidate the pathways involved in milk production. The aims of this review were to describe some of the major pathways required for milk production in terms of the QTL and genes that have helped to identify them and to note some of the methods that can be used to identify causative genes underlying QTL. We focus primarily on cattle (Bos taurus) as a model species, though there are some references to QTL identified in sheep; additionally, some comparisons with human milk composition are also presented where there is a major difference between the two species. Many QTL have been observed for both milk yield and milk composition traits in cattle (see Table 1). These QTL have been identified using a number of different techniques, including linkage-based approaches, such as the transmission disequilibrium test (TDT); and association-based approaches, such as genome-wide association studies (GWAS) and transcriptome-wide association scans (TWAS). The genes attributed to these QTL have a variety of functions. Some, such as the hormones prolactin and growth hormone, and their associated signalling pathways, are involved in mammogenesis (the development of the mammary gland during puberty and pregnancy), lactogenesis (the onset of milk secretion), and galactopoiesis (the continued production of milk). Other pathways, such as those for fat and protein synthesis, and ion channels, are involved directly in milk production. The following sections list some of the pathways involved in mammogenesis and lactation, as identified by QTL for milk production, where the candidate causal gene encodes a protein that sits within those pathways (see summary in Figure 1). Methods for identifying candidate causative genes underlying QTL are discussed in Section 3. QTL have been mapped to many milk proteins, i.e., those expressed directly in milk. In cattle, the largest proportion of milk protein content (80% [4]) consists of the four casein proteins, encoded by a cluster of genes mapping to BTA6: casein alpha-S1 (CSN1S1), encoding -casein, casein alpha-S2 (CSN1S2) for -casein, casein beta (CSN2) for -casein, and casein kappa (CSN3) for -casein. The casein proteins aggregate into micelles in the milk, sequestering calcium phosphate as a nutrient source for the neonate. K-casein seems to be particularly important for successful lactation, as knockout mice deficient in -casein fail to lactate, due to destabilisation of the casein micelles [5]. Like -lactoglobulin, the various casein proteins all have a range of coding variants, although, with the exception of CSN1S1*G [6], these have not been intrinsically linked to lower rates of gene expression, such as the -lactoglobulin B variants discussed below. However, there is evidence that the SNP responsible for the A2 variant of -casein is associated with both milk yield and protein yield [7], and QTL for milk protein phenotypes have also been detected at this locus in other studies [8,9]. These QTL can overlap with, but are not in linkage disequilibrium with, other nearby QTL assigned to the GC gene [8,10,11]: this gene encodes the protein group-specific component (vitamin D binding) and maps around one megabase from the casein gene cluster. Outside the mammary gland, casein expression in human CD14+ monocytes has been observed to upregulate expression of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), and IL-6 via the p38–MAPK pathway [12,13]. This suggests a possible role for caseins in regulating the innate immune response in the neonate intestine following milk consumption. The major whey protein in cattle, accounting for around 50% of whey protein [4] and 10% of total protein, is -lactoglobulin, which is encoded by the progestagen-associated endometrial protein (PAEP) on BTA11. A QTL has been detected to map to this gene. It has pleiotropic effects on milk fat yield, protein yield, and volume [8,14]. Other studies looking at individual milk proteins have also shown that genetic variants associated with low -lactoglobulin concentrations yield high concentrations of -, -, and -caseins [15]. Variation in milk -lactoglobulin concentrations are primarily driven by differences between the A and B protein variants [16,17]. The B variant showed lower expression than the A variant. Other variants mapping to the B variant background, such as B [18] and B [19], cause further reductions in -lactoglobulin expression. Milk with low -lactoglobulin concentrations has potential uses in infant formula (as human milk lacks this protein), and the associated higher concentrations of caseins would also be expected to provide better properties for cheese making. The dominant whey protein in humans, which is second in cattle (18.5% of total whey protein [4]), is -lactalbumin. It is encoded by the gene lactalbumin alpha (LALBA) on BTA5. The QTL associated with milk protein concentration have been identified at this locus in several dairy populations and breeds [20,21,22,23]. The -lactalbumin protein is required for lactose synthesis, modifying the enzyme -1,4-galactosyltransferase (encoded by B4GALT1 on BTA8): in most tissues, B4GALT1 adds galactose moieties to N-acetylglucosamine (GlcNAc) residues. However, the binding of -lactalbumin to B4GALT1 changes the latter to instead add galactose to glucose, forming lactose: this binary enzyme is known as lactose synthase. Given the importance of these two genes for lactose synthesis, it is no surprise that a QTL for milk lactose concentration has also been identified at the LALBA locus [24], along with a milk yield at the B4GALT1 locus [25]. The importance of lactose synthesis for milk production was shown in -lactalbumin knockout mice, which produce highly viscous milk with otherwise normal fat and protein composition that cannot be extracted by the pups from the mammary gland [26]. It has been shown in vitro that multimeric -lactalbumin is cytotoxic to stem cells and transformed cell lines, though it does not harm healthy epithelial cells [27], suggesting that -lactalbumin may also have a protective function in either the mammary gland or the neotate digestive system. Another whey protein is the iron-binding antimicrobial protein lactoferrin, encoded by the lactotransferrin (LTF) gene on BTA22. In cattle, this protein exists in bovine milk at low but variable concentrations (average 115.4 μg/mL in [28] but ranging from 31.8 to 485.6), but reaches much higher concentrations in human milk—around 2 g/L in mature milk [29]. Interestingly, another antimicrobial protein, lysozyme, which is present at lower levels in human milk [4], is also present at relatively low concentrations in cattle [30]. Levels of lysozyme in Bovidae are reported to be as low as 1/1000th of those of other mammalian species [31]. Although lactoferrin expression can be induced by mastitic infection [32], it is also under partial genetic control [33], and genetic variants affecting expression have been identified at the LTF locus [34]. QTL at this locus have also been associated with casein number and lactose concentration [35]. A third antibacterial protein present in milk is lactoperoxidase, encoded by the LPO gene on BTA19. In contrast to lysozyme and lactoferrin, lactoperoxidase activity was found to be around higher in bovine milk compared to that of humans [36]. A trans-eQTL for LPO, overlapping a QTL for milk protein concentration, has been identified on BTA20 at the locus of the C6 and C7 genes [37]: these two genes encode proteins that comprise part of the complement pathway in the innate immune system, suggesting that lactoperoxidase may be co-regulated with this system. Fat is one of the major components of milk, forming membrane-bound droplets known as milk fat globules (MFG), mostly in the form of triglycerides. MFG membranes also contain a range of proteins, such as butyrophilin, adipophilin, mucin, lactadherin, lactoferrin, and xanthine oxidase [38]. Two of these, butyrophilin (encoded by butyrophilin subfamily 1 member A1; BTN1A1) and xanthine oxidase (XOR; encoded by xanthine dehydrogenase XDH), are required for enveloping the MFGs with the apical cell membrane, and therefore, for secretion of the MFG into milk [39]. Heterozygous knockout mice for XOR are unable to maintain lactation [40]. Many of the QTL for fat yield or milk concentration in cattle map to genes encoding fat synthesis or metabolic enzymes (see Figure 2). One of the most prominent QTL detected in cattle [41] for milk volume, fat, and protein phenotypes in cattle maps to the DGAT1 locus on BTA14 [42]. This gene encodes the enzyme diacylglycerol O-acyltransferase 1, which is responsible for the final stage triglyceride fat synthesis [43]. The causative variant for this QTL is a non-conservative lysine-to-alanine substitution at position 232 [42]. More recently, this same variant has been shown to cause an expression QTL for the DGAT1 gene by disrupting an exon splice enhancer, which in turn alters the splicing efficiency of several introns in the transcript [44]. Another enzyme sitting earlier in the same trigyceride synthesis pathway is glycerol-3-phosphate acyltransferase 4, encoded by the GPAT4 gene (formerly known as AGPAT6) on BTA27. Like DGAT1, a highly pleiotropic QTL has been detected at this locus for many milk phenotypes, including volume, fat, protein, and lactose traits [45]. The enzyme lipin 1 sits in a related pathway, catalysing the conversion of phosphatidate into diacylglycerol and functioning as a transcriptional coactivator for genes involved in fatty acid oxidation [46]. QTL for milk protein and casein concentrations [35] and milk yield [47] have been detected at the LPIN1 locus, which encodes this enzyme, in BTA11, in cattle. Before they can be assembled into triglycerides, fatty acids first need to be obtained either from the diet or from de novo synthesis. Several genes involved in this latter pathway have also shown QTL for milk-related phenotypes. The rate-limiting step in fatty acid synthesis is the carboxylation of acetyl-CoA to malonyl-CoA, catalysed by the enzyme acetyl-CoA carboxylase (ACC). The alpha form of this enzyme is encoded by the gene acetyl-CoA carboxylase alpha (ACACA) on BTA19, and QTL for fatty acid composition [48] and somatic cell score (a proxy phenotype for mastitis) [35] have been mapped to this locus. Another important enzyme in the fatty acid synthesis pathway is fatty acid synthase (FAS), encoded by the gene FASN on BTA19, dimers of which are responsible for synthesising the saturated C16 fatty acid palmitic acid from acetyl-CoA and malonyl-CoA [49]. QTL attributed to this gene have been identified for milk fat yield [8], fat concentration [9,50], and fatty acid composition [51] in cattle. The fatty acid synthase gene will produce only saturated fatty acids. To generate monounsaturated or polyunsaturated fatty acids, desaturase enzymes are required. One cluster of fatty acid desaturase genes on BTA29 includes the two genes fatty acid desaturases 1 and 2 (FADS1 and FADS2); these enzymes are responsible for the final, rate-limiting steps in omega-3 and -6 fatty acid syntheses [52,53]. Variants mapping in cattle to these two genes have been associated with concentrations of a range of polyunsaturated fatty acids in milk [54], and similar associations are also observed in human milk [55,56]. A third desaturase enzyme involved in fatty acid synthesis is stearoyl-CoA desaturase, which is encoded by the gene SCD on BTA26 and responsible for oxidising the C16 and C18 saturated fatty acid compounds palmitoyl- and stearoyl-CoA into the monounsaturated compounds palmitoleoyl- and oleoyl-CoA, respectively [57]. This enzyme is also important in regulating metabolism: knockout mice exhibited lower levels of tissue triglycerides and low-density lipoproteins [58], and showed increased insulin signalling and glucose uptake in muscle tissue [59]. In cattle, a QTL assigned to SCD has been identified for milk fat yield [8,50]. Hormones, and the receptors and signalling pathways they activate, are important in most if not all biological functions, and milk production is no exception. For example, knocking out the SCD gene discussed in the previous section leads to an increase in tyrosine phosphorylation of the insulin receptor, which has downstream effects on the PI3K-Akt signalling pathway, leading to increased levels of the glucose transporter GLUT4 in the plasma membrane and increased glucose uptake in muscle [59]. These knockout mice also showed lower levels of the hormone leptin, which is involved in regulating energy intake and partitioning. In cattle, leptin (encoded by the gene LEP on BTA4) has been associated with both milk yield and feed intake [60]. No milk phenotype QTL have been reported for the leptin receptor (LEPR on BTA3), though an association has been reported with body size [61]. In both humans [62] and mice [63], leptin resistance is associated with obesity. Another gene associated with obesity [64] is FTO, encoding the enzyme FTO alpha-ketoglutarate-dependent dioxygenase. This enzyme is involved in DNA repair; specifically, it demethylates 3-methylthymidine [65]. It can also demethylate bases in RNA, including 3-methyluracil and 6-methyladenosine [66]. Via this latter mechanism, FTO can inhibit adipogenesis by demethylating cyclin A2 and cyclin-dependent kinase 2 mRNA, reducing the expression of these genes and thereby prolonging the cell cycle [67]. In cattle, variants at the FTO locus on BTA18 have been associated with milk fat yield [68]. One hormone of particular importance for lactation is prolactin, a peptide hormone secreted by the anterior pituitary gland. In cattle, this peptide is encoded by the PRL gene on BTA23. The importance of this hormone in cattle was underlined by the discovery of a dominant missense mutation that caused, among other phenotypes, a failure to lactate [69]. Prolactin promotes development of the mammary gland during pregnancy, in conjunction with progesterone—generating ductal branching and alveolar buds [70]. Prolactin is detected by cells using prolactin receptor (PRLR; BTA20) and acts via the PRLR/JAK2/STAT5 signalling pathway (see Figure 3) to promote mammary gland development and milk protein expression [71], and it induces the expression of the enzyme UDP-glucose pyrophosphorylase 2 (UGP2) and the transporter UDP-galactose transporter 2 (SLC35A2), thereby promoting the synthesis of lactose [72]. In parallel, prolactin receptor also acts via the PI3K/Akt pathway to downregulate repressors of PRLR/JAK2/STAT5 signalling [73]. Akt1 also upregulates fat synthesis and glucose uptake into the cell for lactose production [74,75]. More recently, it has been shown that Akt signalling induces developing mammary epithelial cells to express prolactin, which in turn acts in an autocrine manner via STAT5 to cause terminal differentiation of the mammary epithelium [71,76]. Given the importance of these pathways, it is not surprising that QTL for milk yield have been widely identified at the PRLR locus [35,77,78] and for somatic cell score [78]. Likewise, many studies have reported milk-related QTL at the signal transducer and activator of transcription 5 (STAT5) locus on BTA19 (genes STAT5A and STAT5B) [8,23,24,51,79]. Beyond the leptin and prolactin pathways, other hormones have also been linked to lactation. One example is growth hormone (GH), also known as somatotropin, which comprises part of the same family of protein hormones as prolactin [80]. Growth hormone acts in the lactating animal to partition nutrients and energy towards the mammary gland [81], which is thought to be mediated by increased serum insulin-like growth factor (IGF-1) levels [82]. In the cow, growth hormone is encoded by the gene growth hormone 1 (GH1) on BTA19, and its receptor, encoded by growth hormone receptor (GHR), maps to BTA20. Both the GH1 locus [35], and especially the GHR locus [9,60,83,84,85], have been associated with milk, fat, and protein yield phenotypes in a range of cattle populations. Another important family of signalling molecules is the interleukin family of cytokines, a group of primarily immunomodulatory proteins that operate in a paracrine or autocrine manner to affect cell growth and differentiation during immune responses [86]. The gene colony stimulating factor 2 receptor subunit beta (CSF2RB) on BTA5 encodes the -chain of GM-CSF (also known as CSF2) receptor, and also forms a common subunit with the receptors for interleukin-3 (IL-3) and IL-5. QTL for milk, fat, and protein volume; and fat and protein concentrations, have been identified at this locus [8,21,87,88]. CSF2RB is suggested as the most likely candidate causative gene, although the neighbouring genes neutrophil cytosolic factor 4 (NCF4) [87,88] and thiosulfate sulfurtransferase (TST) [9] have also been put forward as candidates. The GM-CSF receptor operates via the JAK/STAT signalling pathway—specifically, JAK2 and STAT5 [86], the same pathway as used by prolactin signalling. In contrast to STAT5, which is linked to increased milk protein and lactose expression, STAT3 has been identified as a mediator of involution and apoptosis in the mammary gland [89], which is primarily activated by the cytokine leukaemia inhibitory factor (LIF) [90,91]. STAT3 phosphorylation is upregulated within one hour of physical distension of the mammary gland in cattle, ultimately leading to the cessation of milk production when the gland is not emptied [92]. Mice where either STAT3 or LIF is knocked out show delayed involution and reduced levels of apoptosis [90], raising the possibility of improving efficiency in dairy herds, especially those milking once a day, by breeding for animals lacking one or more of these genes. STAT3 is also involved in leptin signalling, interacting with the long form of the leptin receptor [93]. It is difficult to assign QTL unambiguously to the STAT3 or STAT5 loci, as STAT3 maps on BTA19 directly between the two STAT5 genes STAT5A and STAT5B. Yet another STAT protein, STAT1, is believed to be involved in the development of the mammary gland [94]. A QTL mapping to the STAT1 locus on BTA2 has been associated with milk, fat, and protein yield [94]. One group of proteins that is upregulated by the JAK/STAT pathway is the suppressors of the cytokine signalling (SOCS) gene family [95]. Proteins in this family downregulate JAK/STAT-mediated signalling. For example, SOCS1 (SOCS1 on BTA25) expression is induced by prolactin signalling, and in turn binds to JAK2, inhibiting its association with STAT5 and dampening signal transmission [70,96]. SOCS2 (SOCS2 on BTA5) negatively regulates GH signalling. SOCS2 knockout mice showed higher body weights and skeletal dimensions than control mice [97]. As SOCS proteins ultimately downregulate milk protein expression via prolactin and growth hormone signalling pathways, there is the potential that knocking out the genes encoding them could improve lactation phenotypes in dairy animals. For example, mice with homozygous Socs1 knockout genotypes showed enhanced alveolar development [96], and a point mutation in the ovine Socs2 gene has been associated with higher milk production in dairy sheep, albeit with increased susceptibility to mastitis [98]. In cattle, several different SOCS genes have been associated with milk volume, fat, and protein phenotypes [95]. Another important mechanism affecting milk production involves trans-membrane transport and ion channels. All major milk components need to either be produced within the mammary epithelial cells or transported across them from the blood, and in both cases need to cross the apical membrane into the lumen. While some small molecules, such as urea, can simply diffuse across the membrane, in most cases either passive or active transport channels are required. The volume of water excreted into the milk is driven by osmotic pressure, which is in turn created by exporting lactose and ions across the cell membrane against their concentration gradients. This means that QTL for milk phenotypes frequently map to genes encoding transporters. One important group is the sugar transporters. The gene solute carrier family 37 member 1 (SLC37A1) (on BTA1) encodes a phosphate-linked, glucose-6-phosphate antiporter [99], which is responsible for importing glucose into the cell, and QTL for milk volume have been detected at this locus in several populations [21,87,100]. Another glucose transporter, SWEET1, is encoded by the gene SLC50A1 on BTA3. SWEET1 has been observed in the Golgi in mammary cells and is possibly responsible for importing glucose into the Golgi for lactose synthesis [101]. QTL for lactose concentration [24] and protein concentration [87] have been identified near this gene, though the latter QTL has been assigned to GBA1, which encodes the lysosomal protein glucosylceramidase beta 1. Other glucose transporters have also been implicated in milk production, such as GLUT1 (SLC2A1 on BTA3) [102], the /glucose co-transporter SGLT1 (SLC5A1 on BTA17) [103], and GLUT12 (SLC2A12 on BTA9) [103]. While the mammary epithelial cells use osmotic pressure to secrete milk, it is important that they maintain their own cell volumes correctly. One mechanism by which they can do this is using voltage-regulated anion channels (VRACs), which help regulate cell volume by exporting Cl ions, and small organic anions such as taurine [104,105]. VRACs are comprised of heteromers of leucine-rich repeat containing 8 (LRRC8) proteins A to E, encoded by the genes LRRC8A–LRRC8E. In cattle, the locus on BTA3 containing LRRC8B–LRRC8D has been associated with milk lactose concentration [24,79]. LRRC8C is suggested as the likely causative gene on the basis of gene-expression data. Interestingly, LRRC8C has been associated with adipocyte differentiation (under the name fad158) [106] and is also present in the membranes of MFGs [107], suggesting it may also have a role in the storage or export of fat. As stated above, it is important that water be able to move across the cell membrane to balance osmotic pressure as milk is synthesised. However, the lipid bilayer of the cell membrane is impermeable to water, requiring channels to facilitate the crossing. These channels are provided by aquaporin proteins (AQPs). At least seven aquaporins are expressed in the mammary gland [108,109], where they are believed to play a role in gland development and in transporting water to the lactating gland for milk synthesis and secretion. For example, AQP1 is expressed in the capillary endothelial cells, and may be involved in oestrogen-mediated angiogenesis in the developing mammary gland [110], and AQP5 is expressed in mammary epithelial cells, and the protein pores are moved from the cytoplasm to the apical cell membrane under the regulation of prolactin [111], suggesting a role in milk production. Genetic effects mapping to aquaporin genes have been reported, such as a study in sheep [112] that mapped QTL for milk fat and protein concentrations to a window on OAR3 that contains the genes AQP2, AQP5, and AQP6, albeit alongside LALBA, which is also a strong candidate causal gene for milk-related traits, as described in the milk protein section above. Another important class of transporter is the potassium channel, a type of widely expressed ion channel found in the majority of cell types. The majority of these transporters are encoded by genes named KCN for the potassium channel, followed by a letter representing the subfamily. A number of different families of potassium channel have been identified. The largest family, the voltage-gated potassium channels (, reviewed in [113]), responds to voltage changes in the cell’s membrane potential. This family includes the subfamilies KCNA, KCNB, KCNC, KCND, KCNF, KCNG, KCNH, KCNQ, KCNS, and KCNV [113]. One channel, , which is encoded by the gene KCNC3 on BTA18, has been associated with milk yield in cattle [23]. Another QTL for both milk volume and fat yield maps to the gene KCNS2, encoding , on BTA14 [85]. A third gene in the same family, KCNH4, encodes the channel , and is a potential candidate for a lactose concentration QTL on BTA19 [24], although the QTL also encompass the genes STAT3, STAT5A, and STAT5B, which are also candidates. A second large family is the potassium inwardly rectifying channel family (, reviewed in [114]), comprising lipid-gated channels that are activated by phosphatidylinositol 4,5-bisphosphate (). These transporters correspond to the gene family KCNJ [114]. In cattle, a locus on BTA19 has been associated with QTL for fat and protein concentrations [23], lactose concentration [24,79], and milk yield [8]. This locus contains two genes encoding channels: KCNJ2 () and KCNJ16 (), and both genes have been proposed as candidate causatives underlying the QTL. A third, smaller family of potassium channels is the two-pore-domain potassium channels (), known as leak channels [115], corresponding to the KCNK family. A QTL for milk fat, protein, and volume has been identified on BTA26 at the locus of the KCNK18 gene, encoding the potassium channel [85]. The fourth family is the calcium- and sodium-activated potassium channels [116], of which the most well-known member is , also known as BK. The activity of is modulated by auxiliary and subunits [117,118], including 1, encoded by the gene LRRC26 on BTA11, where a QTL for fat concentration has been detected [23]. The channels discussed so far are limited to moving solutes along an existing concentration gradient. Another class of transporter requires energy in the form of ATP to concentrate solutes to establish a gradient or membrane potential. Many of these belong to the ATP-binding cassette family (ABC). One important example from this family is ATP binding cassette subfamily G member 2 (ABCG2). Initially identified as a xenobiotic drug transporter [119], ABCG2 also functions as a transporter of riboflavin into the milk [120] and a urate transporter in the kidneys [121]. QTL for several milk phenotypes have been mapped to the ABCG2 locus on BTA6, including milk yield [8,21,85,122], fat and protein concentration [9,79,122], lactose concentration [24,79], and -CN concentration [100]. The causative variant at this QTL is generally believed to be a tyrosine-to-serine substitution (Y581S), identified by Cohen-Zinder et al. [122]; however, the adjacent gene SPP1, encoding the protein osteopontin (involved in bone remodelling), has also been proposed as a candidate [123,124]. A second member of the ABC family is SUR2 (encoded by ABCC9 on BTA5), which forms a component of the ATP-sensitive potassium channel , alongside SUR1 (ABCC8) and the inward rectifying channels (KCNJ8) and (KCNJ11) [125]. The exact composition varies by tissue [126]. The channel is inhibited by ATP and activated by MgADP. The transporter is important for glucose-level sensing to control insulin release in pancreatic beta cells [127]: at low glucose levels, ATP levels are low and ADP levels are elevated, so the channel is open; and at high glucose levels, ATP closes the channel. This polarises the plasma membrane, a change that is detected by voltage-gated calcium channels, which then open, causing calcium to enter the cell and trigger the release of insulin. In cattle, a QTL for milk fat yield has been identified at the ABCC9 locus [23,84]. A third ATP-binding transporter is the calcium transporter SERCA2, encoded by the gene ATPase sarcoplasmic/endoplasmic reticulum transporting 2 (ATP2A2) on BTA17. This transporter pumps from the cytosol into the endoplasmic reticulum, whence it can be exported into milk [128]. A QTL at this locus has been detected for milk and protein yield, and for milk calcium [37]. Another ion transporter, showing widely reported associations with milk phenotypes, is inorganic pyrophosphate transport regulator, encoded by the gene ANKH on BTA20. The ANKH transporter controls extracellular mineralisation by regulating the levels of inorganic pyrophosphate in the extracellular matrix. In humans and mice, mutations in this transporter have been associated with arthritis and bone growth disorders linked to tissue calcification [129,130]. This transporter has previously been assumed to be a pyrophosphate transporter; however, recent work has shown that the channel in fact transports ATP, and that the production of extracellular pyrophosphate from ATP requires the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) [131]. In cattle, QTL mapping to the ANKH locus have been identified for milk yield [8,21], milk lactose concentration [24,79], and -lactalbumin concentration [100]. Another recent study [132] discovered that the ANKH protein cycles between the plasma membrane and trans-Golgi network using clathrin-coated vesicles mediated by clathrin adaptors AP1 and AP2. The phosphatidylinositol binding clathrin assembly protein also interacts with AP2 [133], binding to the signalling molecule phosphatidylinositol and recruiting the AP2 complex to form clathrin-coated pits. This protein is encoded by the gene PICALM on BTA29 and is associated with QTL for -CN [100] and lactose concentration [24,79]. The previous section may have given the impression that identifying the causative genes, or even variants underlying QTL, is easy. However, in many cases, there will be no obvious candidate genes in the QTL, possibly because the causative variant sits in a long-range regulatory element for a distant gene. In other cases, there may be many potential candidate genes, and different selection methods may highlight different candidates. For example, the window from 42.2 to 42.5 Mbp on BTA19 envelopes several candidate genes for milk phenotypes. A 2015 study by Raven et al. [87] highlighted the genes GH3-domain containing (GHDC), STAT5A, and STAT5B on the basis of differential expression and enrichment of significant variants, and proposed STAT5A as causative on the basis of knockout studies in mice. In contrast, later work by our group [24,79] used eQTL data and missense variants in strong LD with the top QTL variant to highlight the genes DExH-box helicase 58 (DHX58), GHDC, lysine acetyltransferase 2A (KAT2A), KCNH4, STAT5A, and STAT5B. The two STAT5 genes were again proposed as the most likely candidates. As discussed above, potassium channels, such as that encoded by KCNH4, have been associated with milk at a number of loci, and the histone deacetylase and transcription activator KAT2A2 is also a possible candidate, based on the high levels of gene expression required in the lactating mammary gland. It is possible that multiple QTL are segregated at this locus, and therefore, that more than one gene is causative. Another region with two strong candidate causal genes maps to between 15.3 and 15.6 Mbp on BTA3. At this locus, Raven et al. [87] identified a QTL for milk protein concentration and proposed the epithelial mucin gene MUC1 as the best candidate. The mucin 1 protein coded by this gene is considered a “metabolic master regulator” [134], regulating tyrosine-kinase signalling and the expression of metabolic genes. Work by our group [24,79] has identified a QTL for milk lactose concentration at the same locus, and proposed the gene SLC50A1, encoding a sugar transporter. Again, it is possible that these are two separate QTL, and that both genes are causative for the corresponding phenotypes; however, another possibility is that the QTL is pleiotropic, and only one QTL is present controlling both phenotypes. Distinguishing between these two possibilities is difficult or impossible using purely statistical or bioinformatic means, and additional experiments are likely to be required. Widely used phenotypes, such as fat and protein, effectively aggregate signals from a large number of milk components: individual fatty acids and other lipids for fat, and a number of casein and whey proteins for protein, for example. It is likely that these different components are not all under the same genetic regulation, and some correlations may even be negative. For example, Stoop et al. [135] observed genetic correlations of as low as between milk fatty acid measures (C14:0 and C16:0), and correlations over . This suggests that using finer composition measures, such as individual fatty acids or proteins, should give cleaner, and possibly a larger number of genetic signals compared to complex phenotypes such as fat or protein. This approach has been successfully applied to use milk minerals and individual protein measurements to identify novel QTL and to highlight SLC37A1 as involved in lactation [37]. These phenotypes can be considered “molecular”, as measurements for a single molecule, for example, using gas or liquid chromatography with mass spectroscopy, provide the phenotype. Molecular phenotypes in milk are often measured using Fourier-transform mid-infrared spectroscopy, as this method is commonly used for commercial herd testing and is cheaper to perform on a large scale than spectroscopy. This method uses the absorbance of around 900 different frequencies of infrared light (known as wavenumbers), then uses these values in a model to predict the phenotypes of interest. In commercial herd testing, the predicted phenotypes are typically fat, protein, and lactose concentrations; however, models have been developed for a range of other phenotypes, such as individual fatty acids [136] and proteins [137], and phenotypes more remote from milk, such as methane production [138] and fertility [139]. However, because FT-MIR phenotypes are predicted rather than measured, their utility for QTL discovery can be variable. For example, our recent work [140] showed that both HPLC measured and FT-MIR predicted phenotypes could detect significant cis-QTL for -casein, -casein, and -lactoglobulin. However, FT-MIR was unable to identify the highly significant cis-QTL for lactoferrin, which was identified using HPLC, and identified a QTL at the locus of the fat synthesis gene DGAT1 for -casein. The latter may be due to signal crossover from fat, which is highly correlated with protein in milk. This form of crossover, if present, would complicate the task of identifying pleiotropy. In addition to predicting concentrations of milk components, it is also possible to use the individual wavenumber data themselves as phenotypes [79]: different wavenumbers represent different chemical bonds that absorb MIR light at the corresponding frequencies, so wavenumber phenotypes effectively measure the concentrations in milk of compounds that contain specific chemical bonds. Wavenumber phenotypes have been shown to detect stronger and more numerous genetic signals compared to the predicted phenotypes such as fat and protein [79]. Examining the expression level of each gene in a relevant tissue sample provides an alternative to measuring or estimating protein concentrations. In milk, for example, expression levels of the genes encoding milk proteins in the lactating mammary epithelial tissue can give proxy measurements for milk proteins. Traditionally, this could be done using techniques such as qPCR or expression microarrays. More recently, RNA sequencing (RNA-seq) being used to sequence and count (relatively) mRNA molecules has become common. As RNA-seq captures data for every expressed gene, it is possible to search for expression QTL (eQTL) and allele-specific expression (ASE) for every gene, including those not expressed in the milk, such as fat synthesis enzymes and hormone receptors. The presence of a QTL for a given gene can provide evidence on the causality (or otherwise) of the gene at an overlapping QTL: when the QTL is caused by an underlying eQTL, we expect that the causal variant or variants will be strongly associated with both the QTL and eQTL, and that associations for other variants will decay away in proportion to linkage disequilibrium with the causal variants. This pattern can be identified by examining the correlations between variants regarding the QTL and eQTL variants’ effects [141], or p values (on a logarithmic scale) [24,45], or correlations between local genomic estimated breeding values (GEBVs) and gene expression [142]. Other methods used to associate eQTL with GWAS results include transcriptome-wide association scans (TWAS) [143], Mendelian randomisation [144,145], and Bayesian colocalisation methods [142,146]. Molecular phenotypes and their QTL have assisted in highlighting candidate causative genes in cases where no obvious candidates existed. For example, the gene MGST1 on BTA5 encodes microsomal glutathione S-transferase 1, which belongs to a family of detoxification enzymes [147]. It has no obvious role in milk production. Nevertheless, QTL for milk traits mapping to this locus have been reported in many different studies [21,87,100,148,149]. The neighbouring gene EPS8 (epidermal growth factor receptor kinase substrate 8) is sometimes proposed as a candidate. However, gene-expression data from lactating bovine mammary tissue have shown that the milk QTL at this locus co-segregate with an eQTL for MGST1, whereas EPS8 is barely expressed in this tissue. Similarly, a QTL for milk-fat percentage on the distal end of BTA11, has been linked to the gene encoding the ABO blood group, which is also named ABO (alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase), using RNA-seq data to show that a splice donor site mutation causes aberrant splicing of the ABO transcript, in turn causing an eQTL that co-segregates with the fat percentage QTL [79]. Chromatin is the name given to the compound comprising DNA wound around nucleosomes, which are themselves composed of histone proteins. The three-dimensional folded structure of chromatin can have an important impact on gene expression. On a proximal scale, the structure of the chromatin region surrounding a gene can be in open or closed configurations. The former provides access for transcription factors and RNA polymerase components to reach the promoter region and trigger gene expression, and the latter, closed, configuration blocks expression. Variants sitting within an open chromatin region in the appropriate cell type are more likely to have a regulatory impact on gene expression [141], and therefore, are more likely to present good candidate causal variants for a given trait, compared to other non-coding variants. One method commonly used to study chromatin state is chromatin immunoprecipitation (ChIP) [150], which can be used to identify transcription-factor binding sites or histone modifications. Histone proteins feature long tails, and a wide variety of post-translational modifications can be made to these tails to alter the chromatin state. For example, the modifications H3K4me2 (histone 3, lysine 4 dimethylation), H3K4Me3, and K3K27ac (lysine 27 acetylation) are associated with open chromatin in active promoters and enhancers; and closed, repressed regions can be marked by H3K9me2 or H3K27me3 [151]. More recent versions of ChIP, using DNA sequencing techniques to target the whole genome in a single experiment, include ChIP-seq [152,153] and CUT&RUN-seq [154]. Recent work [141] has identified cis-QTL for ChIP-seq phenotypes; and demonstrated that these QTL frequently exhibit strong correlation with nearby eQTL, and that eQTL tag variants are significantly enriched within ChIP-seq-identified open chromatin windows; this illustrates the utility of this type of data for understanding regulatory variation in the genome. Open chromatin is accessible to transcription factors and other proteins required for gene expression. However, it is also more accessible than other regions to nuclease enzymes, and this fact is used in another approach to identifying open chromatin regions. One commonly used nuclease is DNase 1, which can be used in conjunction with high-throughput sequencing via DNase-seq [155]. The hypersensitivity sites identified using this technique highlight the positions of regulatory elements such as promoters, silencers, and enhancers. A similar technique, using the enzyme micrococcal nuclease, is called MNase-seq [156,157], and identifies the positions of nucleosomes. As nucleosomes are scarcer in open chromatin, these data give an inverse signal to hypersensitivity sites identified by DNase-seq. A more recent technique called the assay for transposase accessible chromatin (ATAC-seq) [158] uses a modified hyperactive transposase enzyme Tn5 to fragment and load sequencing adaptors to open chromatin regions. ATAC-seq typically requires fewer cells and less time to perform compared to DNase-seq, although single-cell protocols have now been developed for both methods [159,160]. On a larger scale, the 3D folded structure allows distal enhancer and silencer elements to enter close contact with the gene to perform their respective functions on gene expression [161]. Identifying this structure allows linkages between genes and distal regulatory elements to be identified. These sorts of long-range interactions can be studied using chromosome conformation capture (3C) [162], and related methods, such as 3C-on-chip (4C) [163] and 3C carbon copy (5C) [164]. These older techniques can study only small parts of the genome. A more recent technique, Hi-C [165], could originally study the structure of the whole genome only at low resolution (), but later refinements of the method [166,167] have allowed for improved resolution and signal-to-noise ratios. The results of GWAS and similar experiments are typically genomic intervals containing several associated variants that are in strong LD with one another. Although candidate causal genes may be selected using known pathways, gene-expression data, or chromatin structure, as discussed above, for some applications it is useful or necessary to know the causal variant underlying the QTL. These applications include improving the accuracy of genomic selection, performing genetic testing, or creating gene-edited animals [168]. However, identifying the causal variant using purely statistical or bioinformatics approaches is typically not possible, as variants in strong LD cannot be distinguished from one other statistically. In some cases, variant annotation (using tools such as SnpEff [169] or Ensembl’s Variant Effect Predictor [170]) can highlight missense or nonsense mutations that will make stronger candidates [41], but many QTL are driven by regulatory effects rather than coding ones. This means that other tools will be needed to resolve QTL with underlying regulatory effects. One commonly used technique for discovering cis-regulatory elements (CREs) is transcription factor binding site (TFBS) prediction. Several tools have been developed for this, generally using information from databases containing binding site motifs for large numbers of transcription factors, such as TRANSFAC [171] and JASPAR [172]. A second approach to investigating regulatory effects is to use a reporter assay, where the effects of putative promoter variants can be tested against the expression of a reporter gene such as GFP. This method has recently been scaled up to test thousands of variants simultaneously using a massively parallel reporter assay (MPRA) [173]. A third method is to use CRISPR-Cas9 or other gene-editing technologies to test putative regulatory sequences, either by deleting them using NHEJ, or by editing in alleles of interest using HDR and then observing the resulting effect on gene expression, often using single-cell RNA-seq. Methods incorporating CRISPR include Perturb-seq [174], CROP-seq [175], and HCR-Flowfish [176]. Over the last twenty years since the seminal work of Grisart et al. [42] showed that the DGAT1 gene underlies the large milk QTL on BTA14, a large number of milk-related QTL have been identified in cattle, and candidate causative genes have been proposed for many of them. In the coming years, we can expect that sequence-resolution data sets will continue both to grow and diversify to include additional breeds from around the world. These larger, more diverse data sets will likely empower the discovery of many novel QTL. Although proving the causality of genes and variants underlying these QTL will likely remain difficult, the use of molecular phenotypes, massively parallel reporter assays, and CRISPR will extend the list of proven causatives. This new information should provide greater insights into the genes and pathways underlying the initiation and maintenance of lactation in mammals. Knowledge of causative genes and variants may also improve the accuracy of genomic selection in animal breeding, which would accelerate genetic gain and improve on-farm productivity. Causal variants will also provide for gene editing to rapidly spread beneficial variants through the population, should future regulatory environments allow it.

PMC10000087

Yan Wang,Wencan Ke,Qiang Lu,Guijie Zhang

Effects of Bacillus coagulans and Lactobacillus plantarum on the Fermentation Characteristics, Microbial Community, and Functional Shifts during Alfalfa Silage Fermentation

04-03-2023

alfalfa,Bacillus coagulans,fermentation composition,microbial diversity

Simple Summary Dry matter loss (DM loss) occurs during silage production, with the potential to reach up to 4–20% due to the aerobic respiration. Lower water-soluble carbohydrates (WSC) and a higher buffering capacity can contribute to this; however, the presence of Bacillus can help create an anaerobic environment, which is more conducive to the growth of anaerobic bacteria. The objective of this study was to evaluate the potential of Bacillus coagulans (BC) as an inoculant for silage fermentation. The results showed that adding BC increased the fermentation quality of alfalfa silage, especially when applied together with Lactobacillus plantarum (LP). This was evidenced by a reduction in the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, as well as an increase in Lactobacillus abundance and a decrease in Enterococcus abundance after 60 d of fermentation. Additionally, the application of LP, BC, and their combination stimulated cofactor and vitamin metabolism abundance, whilst suppressing drug resistance: antimicrobial pathway abundance. Thus, BC could be considered a viable bioresource for improving fermentation quality. Abstract This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 × 106 CFU/g FW), Lactobacillus plantarum (LP, 1 × 106 CFU/g FW), and their combinations (LP+BC, 1 × 106 CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH3-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman’s rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality.

Effects of Bacillus coagulans and Lactobacillus plantarum on the Fermentation Characteristics, Microbial Community, and Functional Shifts during Alfalfa Silage Fermentation Dry matter loss (DM loss) occurs during silage production, with the potential to reach up to 4–20% due to the aerobic respiration. Lower water-soluble carbohydrates (WSC) and a higher buffering capacity can contribute to this; however, the presence of Bacillus can help create an anaerobic environment, which is more conducive to the growth of anaerobic bacteria. The objective of this study was to evaluate the potential of Bacillus coagulans (BC) as an inoculant for silage fermentation. The results showed that adding BC increased the fermentation quality of alfalfa silage, especially when applied together with Lactobacillus plantarum (LP). This was evidenced by a reduction in the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, as well as an increase in Lactobacillus abundance and a decrease in Enterococcus abundance after 60 d of fermentation. Additionally, the application of LP, BC, and their combination stimulated cofactor and vitamin metabolism abundance, whilst suppressing drug resistance: antimicrobial pathway abundance. Thus, BC could be considered a viable bioresource for improving fermentation quality. This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 × 106 CFU/g FW), Lactobacillus plantarum (LP, 1 × 106 CFU/g FW), and their combinations (LP+BC, 1 × 106 CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH3-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman’s rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality. During each stage of silage production, there is a certain amount of dry matter loss (DM loss), which reduces the silage quality. Notably, the DM loss of aerobic respiration could be up to 4–20% [1], and even 2% higher in alfalfa silage than in grass silage due to the lower water-soluble carbohydrates (WSC) and higher buffering capacity [2]. Although a number of lactic acid bacteria (LAB) have been developed to reduce DM loss in silage fermentation, traditional LAB inoculants primarily target the anaerobic fermentation phase and have little effect on DM loss in the aerobic stage [3,4]. The development of silage inoculants is still ongoing, shifting from homofermentative LABs such as Lactobacillus plantarum and heterofermentative Lactobacillus buchneri to LAB strains with prebiotic functions [5]. Therefore, the strains that could consume oxygen, shorten the aerobic stage, and reduce DM loss also have the potential to be silage inoculants. Previous studies have demonstrated that Bacillus could consume free oxygen and generate an anaerobic environment, which favors the growth of anaerobic beneficial intestinal bacteria, such as Bifidobacteria and Lactobacilli, and improves animal performance when administered orally [6,7]. Currently, Bacillus have been used as a silage additive to enhance the fermentation quality and aerobic stability of alfalfa and corn silage [8,9,10]. Of the Bacillus species, Bacillus coagulans (BC) has become a focus of research due to its lactic acid-producing capability, degradation of lignin model compounds [11], and strong resistance to acid, heat, and pressure [12,13]. Additionally, BC has also been used as a feed supplement for monogastric animals, with satisfactory growth performances observed in pigs, fishes, and chickens [14,15,16]. However, few studies have assessed its effects on silage fermentation characteristics. We hypothesized that BC could shorten the aerobic phase of silage by consuming free oxygen during the initial stage, finally improving silage quality. Silage fermentation is a process involving the interactions of microbes, and a better understanding could help to regulate silage fermentation. With the progress of science and technology, microbiomics technology has been used to provide specific microbial information in the silage ecosystem [17,18]. It has only been in recent years that this new method has been widely applied to the study of silage microbial communities [19]. To the best of our knowledge, few studies have confirmed the potential of microbes to be inoculants by using microbiomics technology. Therefore, this study investigated the effects of BC on silage fermentation characteristics and microbial community, confirming the potential of BC as an inoculant in alfalfa silage fermentation. Alfalfa (cultivar “WL 440”) was harvested at the early bloom stage from three randomly selected plots of Ningxia University located in Yinchuan city, Ningxia province, China. Fresh alfalfa was wilted naturally to the dry matter (DM) content of 329.60 g/kg (within 8 h) and chopped into 1–2 cm pieces immediately using a hay cutter. The chopped forages from each plot were divided into 21 equal piles (each weighing approximately 500 g). One pile was immediately frozen at −20 °C for further analysis. The remaining 60 piles (5 ensiling durations × 4 treatments × 3 replications) were randomly assigned to one of the following treatment groups: (1) distilled water control (CON), (2) Lactobacillus Plantarum (LP, provided by Ningxia University, Yinchuan, China), (3) Bacillus coagulans (BC, provided by China center of industrial culture collection, identification number: CICC21735, Beijing, China), and (4) L. Plantarum plus B. Coagulans (LP+BC). The inoculants were applied at a rate of 1 × 106 CFU/g FW, and an equal volume of distilled water was used for the CON. All silage were vacuum packaged in polyethylene plastic bags using a vacuum packaging machine (Zhucheng Yizhong machinery Ltd., Shandong, China). The alfalfa silage bags were stored at room temperature (25 ± 2 °C) and sampled at 3, 7, 14, 30, and 60 d. The DM content was determined by drying the fresh and ensiled forages at 65 °C in an oven for 72 h and then ground with a mill (1 mm screen). Crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were analyzed according to the methods of the Association of Official Analytical Chemists (AOAC) [20], while ammonia nitrogen (NH3-N) and water-soluble carbohydrate (WSC) were measured according to Cai [21]. A 20 g of sample was placed in a juice extractor (BA-828, Mannengda Plasthetics Co., Ltd., Guangdong, China) and squeezed for 30 s with 180 mL of distilled water. The filtrate was then filtered through four layers of cheesecloth, and the pH was measured using a glass electrochemical pH meter. Lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were measured by high-performance liquid chromatography (KC-811, column, Shodex, Shimadzu, Japan; oven temperature, 50 °C; flow rate, 1 mL/min; SPD, 210 nm) [22]. Samples (5 g) of fresh forage and silage were blended with 45 mL of sterile water and diluted (10−1 to 10−5) after being shaken for 0.5 h at room temperature. To analyze the microbial composition, LAB was cultured on MRS medium in an anaerobic box (ANX-1; Hirosawa Ltd., Tokyo, Japan) at 37 °C for 48 h, whereas yeasts and molds were cultured on potato Dsandy AGAR at 28 °C for 72 h [21]. The number of viable microorganisms per gram of fresh matter (FM) was calculated in log10 CFU. For molecular analysis of microbial communities, genomic DNA was extracted using a FastDNA® SPIN Kit (Tiangen, DP302-02, Tiangen, China). Th e DNA quality was verified by an ultramicro spectrophotometer (Thermo, NanoDrop 2000, Waltham, MA, USA). The DNA used for high-throughput sequencing was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), targeting the V3-V4 regions of 16S rRNA. The PCR was conducted in a 20 μL mixture containing 1.6 μL primer mix (5 μM), 10 ng template DNA, 4 μL 5 × FastPfu Buffer, 2 μL dNTPs (2.5 mM each), 0.4 μL FastPfu Polymerase and 0.2 μL BSA, with dd H2O added to reach 20 μL. The PCR products were evaluated with 1% agarose gel and purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Amplified fragments were sequenced on an Illumina MiSeq PE300 platform (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China). All raw reads were cleaned by discarding those <50 bp and those not matching standard barcodes. The OTUs were clustered, and chimeras were removed based on 97% similarity. Each sequence was annotated for species classification using the RDP classifier, and compared to the Silva database, setting a comparison threshold of 70%, and counting each sample at the taxonomic level of community species composition. Principal coordinates analysis (PCoA) was drawn using the Vegan and Python packages. The linear discriminant analysis effect size (LEfSe) was used to calculate the communities or species with significant differences among the four groups. To investigate the relationship between silage quality and the bacterial community, Spearman’s rank correlation coefficients were generated using R language (version 3.0.2). Using the relative abundances of microbes at the species level, the top 50 independent variables were calculated, and the dependent variables were calculated based on pH, NH3-N, LA, and AA. The bacterial function was predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database Phylogenetic Survey of Communities in Unobserved States Reconstruction (PICRUSt2, v2.3.0_b, https://github.com/picrust/picrust2, accessed on 21 August 2022), which predicts the functional abundance of a sample based on the abundance of tagged gene sequences in the samples [23]. Data was analyzed using One-Way ANOVA of SPSS 21.0 (SPSS Inc., Chicago, IL, USA) for chemical composition and fermentation quality. Tukey’s test was used for multiple comparisons and p < 0.05 was considered significant. Comparisons of CON vs. LP, CON vs. BC, and LP vs. LP+BC were conducted to evaluate the effects of LP, BC, and their combination on silage fermentation quality and chemical composition using the Kruskal–Wallis test. Comparisons of inoculant treatments were analyzed using t-tests. Differences were considered significant when p < 0.05. The chemical composition and microbial populations of fresh materials are presented in Table 1. The pH, WSC, CP, NDF, and ADF contents in wilted alfalfa were 6.49, 19.61 g/kg DM, 201.29 g/kg DM, 476.73 g/kg DM, 311.48 g/kg DM, respectively. Table 2 shows the fermentation properties of silages. The pH values decreased with prolonged ensiling time. After 3 d of ensiling, the application of LP and BC alone resulted in lower pH values in treated silages compared with the CON silage. However, further application of BC to LP treatment did not lead to any improvement in pH value, with comparable pH values observed in LP and LP+BC-treated silages. On the contrary, LA concentrations increased with prolonged ensiling time, and greater LA concentrations were observed in LP and LP+BC-treated silages. Application of BC did not show significant difference on LA concentration from the CON group regards 3 to 30 days, but increased it after 60 d of fermentation. Compared with LP-treated silages, LP+BC-treated silage had moderately greater LA concentrations at the same ensiling time, significant after 60 d of fermentation. Additionally, the application of LP and BC resulted in decreased AA in treated silages, particularly in LP+BC-treated silages, which being significant lower AA than the CON silage after 14 d of fermentation. The chemical composition and microbial population of silages are shown in Table 3. Application of LPor BC had no effects on the contents of DM and DM loss. The treated silages had lower WSC concentration when only LP was applied, while greater WSC concentration was observed when BC was added alone. Compared with LP-treated silage, further application of BC increased WSC concentration in LP+BC-treated silage. Neither LP nor BC affected CP contents but limited proteolysis in treated silages, with lower NH3-N concentrations found. In comparison to LP-treated silage, BC-treated silages had comparable NH3-N concentrations, while lower NH3-N concentration was observed in LP+BC-treated silage when further BC was applied. Both LP and BC decreased NDF and ADF concentrations in the treated silage compared to the CON silage, especially when LP was applied. Moreover, further application resulted in lower ADF in LP+BC-treated silage relative to LP-treated. The application of LP and BC promoted the growth of LAB in treated silages, especially when their combination was applied. Applying BC had no effects on the population of the yeasts, whereas decreased yeasts were observed in LP-treated silage in comparison to the CON silage. Additionally, molds were not detected in this study. The principal coordinates analysis (PCoA) of the beta diversity revealed that the bacterial communities of fresh alfalfa and each treatment group were distinct after fermentation (Figure 1). The first two principal coordinates (PC1 and PC2) accounted for 42.05% and 25.89% of the total variance, respectively. The FM was grouped into a single category, whereas the CON- and BC-treated silages were clustered together and separated from the LP- and LP+BC-treated silages. The bacterial communities of fresh and silage samples were mainly composed of four phyla (Figure 2A). Before ensiling, Proteobacteria was the most abundant phylum (75.97%), followed by Actinobacteriota (15.53%), Firmicutes (4.98%), and Bacteroidota (2.44%). After fermentation, Firmicutes became the dominant phylum, reaching up to 80.34%, 87.41%, 92.87%, and 88.69% in CON silage, LP-, BC-, and LP+BC-treated silages, respectively. Proteobacteria were more abundant in CON- and LP-treated silages than in BC- and LP+BC-treated silages. At genus level, the predominant genera in FM were Pseudomonas (30.50%), Methylobacterium (11.48%), Sphingomonas (7.91%), Enterobacter (7.00%) (Figure 2B). The relative abundance of Lactobacillus increased in the treatment group, whereas Pseudomonas, Sphingomonas, and Enterobacter decreased. Weissella (41.59%) and Enterococcus (27.99%) were the leading genera in CON silage, whereas Lactobacillus dominated in LP- and LP+BC-treated silages, reaching 80.73% and 74.98%, respectively. Conversely, BC-treated silages had lower concentrations of Lactobacillus (15.28%), but greater concentrations of Weissella (42.88%). Linear discriminant analysis (LDA) effect size (LEfSe) was used to compare different treatments (LDA > 2.0) (Figure 3). There were 13 microbes that differed among the treatments. Thermoleophilia, Stappiaceae and Solirubrobacterales were more abundant in CON silages (LDA > 4.5). The LP-treated silage had higher relative abundances of Lactobacillus and Lactobacillaceae (LDA > 5.5), whereas the BC-treated silage had greater concentrations of Enterococcaceae and Enterococcus (LDA > 5.0). The Spearman’s rank correlation between fermentation parameters and microbial strains at genus level is visualized in the form of a heatmap in Figure 4. There was a positive correlation between pH and Lactococcus, Enterococcus, and Aerococcus, but a negative correlation with Lactobacillus. The NH3-N concentration was positively correlated with the abundance of norank-f-Solirubrobacteraceae. Additionally, the LA concentration was positively correlated with Lactobacillus, but negatively correlated with Lactococcus, Enterococcus, Aerococcus, Weissella, and Methylobacterium-Methylorubrum. Conversely, Lactococcus, Brachybacterium, Rubellimicrobium, Brevundimonas, unclassified-f-Rhizobiaceae, Leuvobacter, and unclassified-f-Microbacteriaceae were negatively correlated with AA concentrations. The PICRUSt2 was used to predict the potential functions and enzymes of bacterial communities of the four groups (Figure 5 and Figure 6). The predominant metabolism was the carbohydrate metabolism in silages, followed by amino acid metabolism. Compared to the CON silage, LP and LP+BC- treated silages showed a greater abundance of carbohydrate metabolism and amino acid metabolism, respectively (Figure 5A). The majority of predicted functions explained by KEGG pathways in FM and silages (Figure 5B) were classified into organismal systems (8 pathways), human diseases (12 pathways), environmental information processing (2 pathways), genetic information processing (4 pathways), and metabolism (11 pathways). Compared with FM, alfalfa silages had a higher proportion of amino acid metabolism and lower proportions of carbohydrate metabolism. The carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism abundances were lower in treated silages when compared with the CON silage (Figure 5C). The LP+BC-treated silage had the lowest abundances of amino acid metabolism and drug resistance: antimicrobial, the LP-treated and BC-treated silages had comparable abundances. Compared with CON-treated silages, LP-and LP+BC-treated silages had a higher abundance of peptidase, whereas no significant difference was observed between CON- and BC-treated silages (Figure 6). The LP- and LP+BC-treated silages had a lower abundance of cellulose than the control group, and the LP+BC-treated silages had the lowest abundance. It is usually difficult to ensile alfalfa due to its high buffering capacity and low WSC concentration [24], resulting in more significant DM loss in the aerobic phase compared to corn and grass silages [1]. Therefore, reducing the aerobic phase is essential for preserving more nutrients. This study found applying BC resulted in a reduced pH value in treated silage at each sampling time, particularly at 3 d, which is likely due to the acceleration of LA fermentation caused by oxygen consumption. Bai et al. [9,10] also observed similar results when alfalfa silage was treated with Bacillus and Lactobacillus buchneri. At 60 d of silage, compared to CON silage, further application of BC resulted in lower pH levels and greater concentrations of LA in treated silage. This result confirms the hypothesis and suggests that BC could be an excellent supplement to the current inoculants. After 14 d of ensiling, when LP and BC were used in combination decreased AA concentrations in treated silages. After 60 d of ensiling, a greater LAB population was observed in treated silages than that in the CON silages, which was correlated with the results of lower pH and greater LA concentration. Moreover, lower levels of yeast were found in silages when BC and LP were applied, particularly in LP+BC silage. It appears that both BC and LP used in the study had advantages in nutrition competition over undesirable microbes. To produce high-quality silage, a WSC is an essential component [25]. The study showed that silages inoculated with LP and its combination with BC had lower WSC concentrations after 60 d of storage; however, greater amounts of WSC were observed in BC-treated silage. It seems that the application of BC could shorten the aerobic phase and preserve more fermentation substrates. For legumes, proteolysis has been identified as a crucial problem in silage fermentation with a greater proportion of non-protein nitrogen, ranging from 440–880 g/kg total nitrogen [26,27]. The proteolysis could result in lower protein utilization efficiency. Therefore, there have been numerous studies evaluating the effects of inoculants on proteolysis, with positive results in legume silage fermentation [28,29]. The concentration of NH3-N, which is an indicator of proteolysis [30,31], was found to be lower in LP and BC-treated silages when compared to the CON silage, particularly when both BC and LP were applied. This could be attributed to the lower pH levels in treated silages. Additionally, lower NDF and ADF concentrations were found in treated silages when LP and BC were applied. These results could be explained by the acid degradation of hemicellulose. Furthermore, Bacillus could produce enzymes such as cellulase and feruloyl esterase, and it has been demonstrated that BC could degrade the lignin-related compounds ferulic acid and vanillin to vanillic acid [11]. This could be the reason for lower ADF concentrations in LP+BC-treated silage when compared with silage inoculated with LP alone. Silage fermentation involves the interactions of microbes, and a better understanding of the dynamic of microbial communities could be beneficial for regulating the fermentation process [32]. In this study, we investigated the effects of LP and BC on the bacterial communities of ensiled alfalfa. The results showed that fresh alfalfa was dominated by Proteobacteria and Firmicutes, which is in agreement with previous findings [33]. Li et al. [34] reported similar results for Cassava foliage after 60 days of ensiling with an increase in the relative abundance of Firmicutes to 88.69%. To further evaluate the effects of BC on the bacterial community during fermentation, we also analyzed the bacterial structures of alfalfa at the genus level. The dominant bacteria in the fresh alfalfa were Gram-negative bacteria, including Pseudomonas, Methylobacterium-Methylorubrum, and Sphingomonas. According to a previous report, the microbial flora of the alfalfa phyllosphere was predominately composed of Erwinia, Escherichia, Pseudomonas, and Pantoea [35]. This might be because the settling of bacteria on the plant surface is affected by plant species, climate, geographical location, and fertilizer type [32]. Lactobacillus and Weissella are the most common bacteria involved in the LA fermentation of silage [36,37]. The researchers discovered that, as compared to the CON silage, treated silages showed greater relative abundances of Lactobacillus after 60 d, especially in LP and LP+BC-treated silages. This could be attributed to the lower pH and greater lactic acid production. Similar results were reported by Bai et al. [9,10], who found that the abundance of Lactobacillus was higher in Bacillus subtilis and Bacillus amyloliquefaciens-treated silages than in the CON silage. Weissella, which was considered an early colonizer, had a lower acid tolerance, and was unable to survive when the pH was below 4.5, yet the relative abundance of Weissella in BC-treated silage increased from 41.59% to 42.88% after 60 d of ensiling [38,39]. This suggested that BC was not as effective in competing for nutrients with Weissella. A correlation analysis was conducted between the composition of microorganisms and end-products after ensiling. It was observed that Methylobacterium-Methylorubrum, an aerobic bacterium utilizing the serine pathway to consume methanol and other reduced one-carbon compounds [18], was gradually replaced by Lactobacillus in this study as the pH of the silage decreased, resulting in a positive correlation between pH and Methylobacterium-Methylorubrum and a negative correlation between pH and Lactobacillus. Lactobacillus is essential in inhibiting harmful microorganisms by rapidly acidifying silage in the late stages of ensiling [40]. In this study, a positive correlation was observed between NH3-N concentration and norank-f-Solirubrobacteraceae. This is likely due to the ability of certain species of Solirubrobacteraceae to thrive in anaerobic conditions and competes with Lactobacillus for nutrients [41]. These results also agreed with the greater NH3-N concentration and relative abundance of norank-f-Solirubrobacteraceae in CON silage. Additionally, LA and AA concentrations were shown to be positively connected with Lactobacillus and negatively correlated with Enterobacter, which is likely a result of the production of LA and AA by Lactobacillus. These findings were further supported by the higher NH3-N concentration and relative abundance of norank-f-Solirubrobacteraceae in CON silage. Alfalfa silage bacterial community functions were predicted using the KEGG pathways database and PICRUSt2. All samples contained a high proportion of the KEGG metabolism pathways, including the carbohydrate metabolism and amino acid metabolism. It was observed that four metabolic categories had a significant impact on microbial metabolisms. These categories were secondary metabolite biosynthesis, microbial metabolism in various environments, carbon metabolism, 2-oxocarboxylic acid metabolism, fatty acid metabolism, amino acid biosynthesis, and aromatic compound degradation [42]. After ensiling, microbial metabolism was inhibited in the anaerobic environment, resulting in weaker amino acid metabolism. However, LP-treated silage had a greater abundance of Lactobacillus than LP+BC-treated silages, which contributed to a higher microbial metabolism capacity. We concentrated our efforts on the metabolic pathways of carbohydrates, amino acids, energy, cofactors, vitamins, and drug resistance, as these are all associated with changes in fermentation, chemical composition, and human health [43,44]. Du et al. [18] determined that the relative abundance of total LAB in the microbial community affected the abundance of carbohydrates metabolism pathways. However, this study found that entire LAB was lower in silage with more carbohydrate metabolic pathways. Because carbohydrate metabolism is primarily glycolysis and gluconeogenesis, Enterococcus are more capable of metabolizing WSC in CON silage and LP+BC-treated silages than beneficial species of LAB [45]. Amino acids are integral to proteins and peptides, and play a substantial role in the energy metabolism and environmental tolerance of lactic acid bacteria [46]. The application of LP, BC, and their combination resulted in lower amino acid metabolism abundances, which correlates with the reduced NH3-N concentrations observed in the treated silages compared to the CON silage. The relative abundance of cofactor and vitamin metabolism was higher in treated silages, suggesting that BC could accelerate vitamin production or produce vitamins directly during ensiling. Similar results were reported by Bai et al. [47] who found that inoculating alfalfa silage with Enterococcus faecalis enhanced the relative abundance of cofactor and vitamin metabolism. Abuse of antibiotics leads to the development of multidrug-resistant bacteria, one of the greatest health threats [48,49]. The antimicrobial drug resistance relative abundance was found to be the greatest in the CON silage, whereas the LP+BC-treated silage had the lowest relative abundance, followed by LP-treated silages. Antimicrobial drug resistance is primarily found in undesirable bacteria, such as Salmonella and Escherichia coli [44]. The inhibition of undesirable and hazardous bacteria may be related to the lower pH in LP+BC-treated silages, which reduces resistance genes. The addition of BC increased the fermentation quality of alfalfa silages, indicated by lower pH values and greater LA concentrations, especially when BC was applied together with LP. Furthermore, the application of BC decreased DM loss, NDF, ADF and NH3-N contents, while increasing WSC. The microbial analysis demonstrated that the application of BC increasing Lactobacillus abundance and decreasing Enterococcus abundance in treated silages after 60 d of fermentation. Additionally, the application of LP, BC, and their combinations increased cofactor and vitamin metabolism abundance while decreasing drug resistance: antimicrobial pathway abundance. Thus, BC could be considered a viable bioresource for improving fermentation quality.

PMC10000092

Brigkita Venardou,John V. O’Doherty,Marco Garcia-Vaquero,Claire Kiely,Gaurav Rajauria,Mary J. McDonnell,Marion T. Ryan,Torres Sweeney

In Vitro Evaluation of Brown Seaweed Laminaria spp. as a Source of Antibacterial and Prebiotic Extracts That Could Modulate the Gastrointestinal Microbiota of Weaned Pigs

24-02-2023

brown macroalgae,bifidogenic,antibacterial,seaweed polysaccharides,weaned pig,gastrointestinal microbiota,batch fermentation

Simple Summary The transition from milk to solid feed in commercial pig-production systems negatively affects gut health, particularly the composition of the residing microbial community. This can subsequently impair pig growth and long-term health. Natural dietary supplements including seaweed extracts have the capacity to reduce pathogen load (antibacterial activity) and/or increase beneficial microbes (prebiotic activity). This study evaluated the antibacterial and prebiotic potential of two seaweed species, Laminaria hyperborea and Laminaria digitata, and their extracts using laboratory-based simulations of the gut microbial community of weaned pigs. Our investigation identified seaweed extracts that could decrease the numbers of pig- and food-related pathogens or increase the number of beneficial microbes, albeit to a different extent. These findings indicate that seaweeds are a promising source of antibacterial and prebiotic dietary supplements for use in pigs during the weaning period. Abstract Laminaria spp. and their extracts have preventative potential as dietary supplements during weaning in pigs. The first objective of this study was to evaluate increasing concentrations of four whole seaweed biomass samples from two different Laminaria species harvested in two different months in a weaned pig faecal batch fermentation assay. Particularly, February and November whole seaweed biomass samples of L. hyperborea (LHWB-F and LHWB-N) and L. digitata (LDWB-F and LDWB-N) were used. In the next part of the study, the increasing concentrations of four extracts produced from L. hyperborea (LHE1–4) and L. digitata (LDE1–4) were evaluated in individual pure-culture growth assays using a panel of beneficial and pathogenic bacterial strains (second objective). The LHE1–4 and LDE1–4 were obtained using different combinations of temperature, incubation time and volume of solvent within a hydrothermal-assisted extraction methodology (E1–4). In the batch fermentation assay, the L. hyperborea biomass samples, LHWB-F and LHWB-N, lowered Bifidobacterium spp. counts compared to the L. digitata biomass samples, LDWB-F and LDWB-N (p < 0.05). LHWB-F and LDWB-N reduced Enterobacteriaceae counts (p < 0.05). LHWB-F and LDWB-F were selected as the most and least promising sources of antibacterial extracts from which to produce LHE1–4 and LDE1–4. In the pure-culture growth assays, E1- and E4-produced extracts were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 reduced both Salmonella Typhimurium and Enterotoxigenic Escherichia coli with LDE1 having a similar effect on both of these pathogenic strains, albeit to a lesser extent (p < 0.05). Both LHE1 and LDE1 reduced B. thermophilum counts (p < 0.05). LDE4 exhibited strong bifidogenic activity (p < 0.05), whereas LHE4 increased Bifidobacterium thermophilum and Lactiplantibacillus plantarum counts (p < 0.05). In conclusion, antibacterial and bifidogenic extracts of Laminaria spp. were identified in vitro with the potential to alleviate gastrointestinal dysbiosis in newly weaned pigs.

In Vitro Evaluation of Brown Seaweed Laminaria spp. as a Source of Antibacterial and Prebiotic Extracts That Could Modulate the Gastrointestinal Microbiota of Weaned Pigs The transition from milk to solid feed in commercial pig-production systems negatively affects gut health, particularly the composition of the residing microbial community. This can subsequently impair pig growth and long-term health. Natural dietary supplements including seaweed extracts have the capacity to reduce pathogen load (antibacterial activity) and/or increase beneficial microbes (prebiotic activity). This study evaluated the antibacterial and prebiotic potential of two seaweed species, Laminaria hyperborea and Laminaria digitata, and their extracts using laboratory-based simulations of the gut microbial community of weaned pigs. Our investigation identified seaweed extracts that could decrease the numbers of pig- and food-related pathogens or increase the number of beneficial microbes, albeit to a different extent. These findings indicate that seaweeds are a promising source of antibacterial and prebiotic dietary supplements for use in pigs during the weaning period. Laminaria spp. and their extracts have preventative potential as dietary supplements during weaning in pigs. The first objective of this study was to evaluate increasing concentrations of four whole seaweed biomass samples from two different Laminaria species harvested in two different months in a weaned pig faecal batch fermentation assay. Particularly, February and November whole seaweed biomass samples of L. hyperborea (LHWB-F and LHWB-N) and L. digitata (LDWB-F and LDWB-N) were used. In the next part of the study, the increasing concentrations of four extracts produced from L. hyperborea (LHE1–4) and L. digitata (LDE1–4) were evaluated in individual pure-culture growth assays using a panel of beneficial and pathogenic bacterial strains (second objective). The LHE1–4 and LDE1–4 were obtained using different combinations of temperature, incubation time and volume of solvent within a hydrothermal-assisted extraction methodology (E1–4). In the batch fermentation assay, the L. hyperborea biomass samples, LHWB-F and LHWB-N, lowered Bifidobacterium spp. counts compared to the L. digitata biomass samples, LDWB-F and LDWB-N (p < 0.05). LHWB-F and LDWB-N reduced Enterobacteriaceae counts (p < 0.05). LHWB-F and LDWB-F were selected as the most and least promising sources of antibacterial extracts from which to produce LHE1–4 and LDE1–4. In the pure-culture growth assays, E1- and E4-produced extracts were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 reduced both Salmonella Typhimurium and Enterotoxigenic Escherichia coli with LDE1 having a similar effect on both of these pathogenic strains, albeit to a lesser extent (p < 0.05). Both LHE1 and LDE1 reduced B. thermophilum counts (p < 0.05). LDE4 exhibited strong bifidogenic activity (p < 0.05), whereas LHE4 increased Bifidobacterium thermophilum and Lactiplantibacillus plantarum counts (p < 0.05). In conclusion, antibacterial and bifidogenic extracts of Laminaria spp. were identified in vitro with the potential to alleviate gastrointestinal dysbiosis in newly weaned pigs. A healthy gut microbiota which is compositionally and functionally diverse and stable is essential to support host health and growth [1,2,3,4]. Contrarily, dysbiosis represents a state of imbalance in the composition and function of this microbial community, characterised by decreases in beneficial microorganisms and/or overgrowth of pathogens and/or a loss of overall diversity, with a subsequent negative impact on gastrointestinal health [5]. Commercial weaning in pigs is a typical example of dysbiosis, whereby the transition from milk to solid feed, coupled with emotional, social and environmental stressors leads to gastrointestinal dysfunction characterised by dysbiosis that predisposes them to intestinal infection and disease [6,7]. In a recent review, the potential of marine macroalgae or seaweeds were considered as natural dietary supplements with which to promote gastrointestinal health and subsequently growth in weaned pigs [8]. Brown seaweeds are rich in nondigestible polysaccharides, minerals, polyphenols and vitamins [9,10]. Wide-ranging biological activities [11] have been attributed to seaweed components, particularly fucoidan and laminarin, including prebiotic [12,13] and antibacterial [14,15] potential. However, various factors influence the concentration, structure and biological activity of seaweed-derived polysaccharides, such as seaweed species, harvest season, environmental conditions, and extraction methodologies [14,16]. Recently, a multivariate statistic technique, response surface methodology, has been utilised to improve the extraction efficiency by optimising the extraction conditions for a selected seaweed polysaccharide and/or bioactivity [17]. In that study, a novel hydrothermal-assisted extraction (HAE) methodology with combinations of temperature, time and solvent to seaweed ratio optimised for the best concentration of laminarin and/or fucoidan and/or antioxidant activity was developed using the response surface methodology. Seaweed extracts of Ascophyllum nodosum produced using this HAE methodology exhibited enhanced antibacterial and prebiotic activity compared to the conventional extraction methods [18]. The brown seaweed Laminaria spp. is a rich source of biologically active nondigestible polysaccharides. Previous in vitro investigation has associated this seaweed species with various biological activities including anti-inflammatory, immunomodulatory, antioxidant, antitumor and antihypertensive [9,11] effects, several of which have also been observed in in vivo studies with pigs [19,20,21,22]. Concerning its effect on the gastrointestinal microbiota, dietary supplementation of pigs with crude Laminaria spp. extracts consistently led to a reduction in the numbers of the Enterobacteriaceae family [22,23,24,25,26,27] which include several animal and human pathogens such as Salmonella enterica subsp. enterica serotype Typhimurium and pathogenic Escherichia coli [28,29]. Furthermore, an increase in Enterobacteriaceae family is considered to be an indication of dysbiosis and a risk factor for post-weaning diarrhoea in pigs [6,30]. Dietary supplementation of pigs with crude Laminaria spp. extracts led to a more variable response in the intestinal lactobacilli and Bifidobacterium spp. populations, as both increases [22] and decreases [23,24] in their counts have been reported. Lactobacilli are dominant members of the gastrointestinal microbiota in pigs and have an important role in growth and health due to their contributions to nutrient bioavailability, inhibition of pathogen colonisation and immunomodulation [31,32]. Bifidobacterium spp. are considered a beneficial bacterial population due to their probiotic status [33], but are present in low abundance in the gastrointestinal tract of pigs [34]. The current study focused on the antibacterial and prebiotic potential of two polysaccharide-rich members of the Laminaria spp., L. digitata and L. hyperborea, with an average total carbohydrate content of 70.7% and 65.5% of dry weight, respectively, as described in previous reports [35]. Batch fermentation and pure-culture growth assays were useful screening tools when assessing the direct effects of whole biomass seaweed samples and their extracts on key bacterial populations and species in the porcine gastrointestinal tract [18]. Thus, the first objective of this study was to assess the influence of seaweed species and harvest season on the effect of whole biomass samples of L. digitata and L. hyperborea with respect to selected faecal bacterial populations in a batch fermentation assay inoculated with pig faeces. The second objective was to investigate whether the different extraction conditions of the HAE methodology led to L. digitata and L. hyperborea extracts with improved antibacterial and prebiotic activities using a panel of pure-culture growth assays. The whole biomass samples (WB) of L. digitata (LD) and L. hyperborea (LH) were harvested in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N) by Quality Sea Veg Ltd., Co. (Donegal, Ireland). For each seaweed species, whole biomass samples were collected at a single time point and from the same collection site. The preparation (oven-dying, milling) and compositional analysis (dry matter, ash, protein, crude lipids, polysaccharide content, total phenols) of the dried whole seaweed biomass samples was performed as previously described [36]. LDWB-F, LDWB-N, LHWB-F and LHWB-N were stored at room temperature until their evaluation in the batch fermentation assay. A HAE methodology with optimised extraction conditions (temperature, incubation time and volume of solvent) was used to produce the extracts of LDWB-F and LHWB-F, as described previously by Garcia-Vaquero et al. [17] and presented in Table 1. The parameters for each extraction condition were optimised towards the concentration of fucoidan for E1, laminarin for E2, antioxidant activity for E3 and all the above for E4. The produced extracts of L. digitata (LDE1–4) and L. hyperborea (LHE1–4) were freeze-dried and their laminarin and fucoidan content was determined as previously described [18]. All extracts were analysed on two independent occasions (two biological replicates) with three readings each time. LDE1–4 and LHE1–4 were stored at −20 °C until their evaluation in the pure-culture growth assays. The preparation of the faecal inoculum and the batch fermentation assay were carried out as described previously [37]. Briefly, faeces from 29 healthy newly weaned crossbred pigs (Large White × Landrace) fed a cereal- and milk-based diet were pooled, aliquoted and stored at −20 °C. One day prior to the batch fermentation assay, the pooled faeces were diluted (1:5 w/v) in phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) after oxygen removal using oxyrase (Sigma-Aldrich, St. Louis, MO, USA) to prepare the faecal inoculum (FI) that was stored at 4 °C anaerobically. The FI was added to the fermentation medium at a 1:10 v/v ratio (21 mL final volume). The inclusion levels of LDWB-F, LHWB-F, LDWB-N and LHWB-N in the FI/fermentation medium were 0 (control tubes), 1, 2.5 and 5 mg/mL. The batch fermentation was carried out under anaerobic conditions using oxyrase and CO2 flushing at 39 °C for 24 h with gentle stirring (100 rpm). Sampling (5 mL fermentation broth) was performed at 0 and 24 h in duplicate. After centrifuging at 12,000× g for 5 min, the resultant pellets were stored in −20 °C until further analysis. All experiments were repeated on three independent occasions (biological replicates n = 3). DNA extraction: Bacterial DNA was extracted using QIAamp Fast DNA stool mini kit (Qiagen, West Sussex, UK) according to the manufacturer’s instructions, and its quantity and quality was evaluated spectrophotometrically (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA). Bacterial primers: The primers targeting the 16S rRNA gene of selected bacterial groups (total bacteria, lactobacilli and Bifidobacterium spp.) or the rplP gene (Enterobacteriaceae) are provided in Table 2. Primer design software, Primer3 (https://primer3.org/ (accessed on 26 June 2018)) and Primer Express™ (Applied Biosystems, Foster City, CA, USA) were used for larger amplicons (>150 bp) and smaller amplicons (<125 bp), respectively. Primer specificity was verified using Primer Basic Local Alignment Search Tool (Primer-BLAST), https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi, accessed on 26 June 2018. Bacterial enumeration by QPCR: The quantification of the above-mentioned bacterial groups was carried out using QPCR with plasmid-based standard curves as described previously [37]. Briefly, Competent E. coli was transformed with a pCR4-TOPO™ TA vector containing each fragment of the targeted 16S rRNA genes for total bacteria, lactobacilli and Bifidobacterium spp. or the rplP gene for Enterobacteriaceae and the resistance to ampicillin gene using a TOPO™ TA Cloning™ Kit for Sequencing (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and stored in cryoprotective beads (TS/71-MX, Protect Multi-purpose, Technical Service Consultants Ltd., Lancashire, UK). Transformed E. coli was recultured in 200 mL LB Broth Base (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) containing ampicillin (100 µg/mL) at 37 °C for 18 h at 150 rpm. Plasmids were extracted on a large scale using the GenElute™ HP Plasmid Maxiprep kit, (Sigma-Aldrich, St. Louis, MO, USA), linearised using APA1 restriction enzyme (Promega, Madison, WI, USA) and purified using GenElute™ PCR Clean-Up kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturers’ instructions. Following quantification, the plasmid copy number/μL was determined using the URI Genomics & Sequencing Centre online tool (http://cels.uri.edu/gsc/cndna.htmL, accessed on 14 May 2019). For the QPCR, the final reaction volume (20 μL) included 3 μL template DNA, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 5 μL nuclease-free water and 10 μL of Fast SYBR® Green Master Mix (Applied Biosystems, Foster City, CA, USA) for the lactobacilli or GoTaq® qPCR Master Mix (Promega, Madison, WI, USA) for the remaining bacterial groups. All QPCR reactions were performed in duplicate on the ABI 7500 Fast PCR System (Applied Biosystems, Foster City, CA, USA) using the following cycling conditions: a denaturation step (95 °C/10 min), 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Dissociation curve analysis and visualisation on a 2% agarose gel stained with ethidium bromide were used to confirm the production of single and specific PCR products. All PCR reactions used in this study exhibited 90–110% efficiency established by plotting the threshold cycles (Ct) derived from the 5-fold serial dilutions of each plasmid against their arbitrary quantities. Bacterial counts were determined using the standard curve derived from the mean Ct value and the log-transformed gene copy number of the respective plasmid and expressed as log-transformed gene copy number per gram of digesta (logGCN/g digesta). Pure-culture growth assays using a panel of commensal strains Lactiplantibacillus plantarum subsp. plantarum (formerly Lactobacillus plantarum, DSMZ 20174), Limosilactobacillus reuteri (formerly Lactobacillus reuteri, DSMZ 20016) and Bifidobacterium thermophilum (DSMZ 20210) and pathogens S. typhimurium PT12 and enterotoxigenic E. coli (ETEC) O149A+ selected for their beneficial roles and negative impacts on pig and human health, respectively, were carried out as described in our previous work [37]. Briefly, 24 h cultures of all bacterial strains were prepared using standard procedures and diluted in 10% medium: 10% de Man, Rogosa and Sharpe broth (MRS, Oxoid Ltd., Hampshire, UK) for L. plantarum, L. reuteri and B. thermophilum cultures; or 10% Tryptone Soya Broth (TSB, Oxoid Ltd., Hampshire, UK) for S. typhimurium and ETEC cultures, to obtain an inoculum of 106–107 CFU (colony-forming unit)/mL (verified for each assay). Two-fold dilutions (2–0.125 mg/mL) of LDE1–4 and LHE1–4 were performed in 10% MRS and 10% TSB prior to each assay from a working concentration of 4 mg/mL. 100 μL of each extract and each dilution and 100 μL of inoculum were added to duplicate wells of 96-well microtiter plates (CELLSTAR, Greiner Bio-One, Kremsmünster, Austria). Control wells were also included (bacterial inoculum only). Assay sterility was assessed using blank wells (no bacterial inoculum) for each dilution of each extract. After gentle agitation to ensure thorough mixing, plates were incubated aerobically at 37 °C for 18 h, apart from B. thermophilum, which was incubated anaerobically. Afterwards, bacterial enumeration was carried out by 10-fold serial dilution (10−1–10−8), spread plating onto MRS agar (Oxoid Ltd., Hampshire, UK) for L. plantarum, L. reuteri and B. thermophilum, and Tryptone Soya Agar (Oxoid Ltd., Hampshire, UK) for ETEC and S. typhimurium, and incubation aerobically at 37 °C for 24 h or anaerobically at 37 °C for 48 h for B. thermophilum. The dilution resulting in 5–50 colonies was selected for the calculation of CFU/mL using the formula, CFU/mL = average colony number × 50 × dilution factor. The bacterial counts were logarithmically transformed (logCFU/mL) for the subsequent statistical analysis. All experiments were carried out with technical replicates on three independent occasions (3 biological replicates n). All data were statistically analysed using Statistical Analysis Software (SAS) 9.4 (SAS Institute, Cary, NC, USA). Normality tests were initially carried out using PROC UNIVARIATE procedure for each data set. Batch fermentation assay: For each bacterial group and tested compound, the bacterial counts (n = 12, 3 flasks/each compound concentration) were analysed using PROC GLM procedure (Tukey’s test). The statistical model included the fixed effects of seaweed species (L. digitata, L. hyperborea), the season of collection (February, November), the concentration of whole biomass (0, 1, 2.5 and 5 mg/mL) and assay replicates (3 biological replicates) and their associated two- and three-way interactions with the bacterial counts at 0 h as a covariate. Pure-culture growth assay: To control for the natural variability in bacterial growth in the pure-culture assays, bacterial counts were expressed as the difference between the counts of each bacterial strain for each extract concentration and their respective control (0 mg/mL). The resulting positive or negative values representing the difference in bacterial counts were analysed using PROC GLM procedure (Tukey’s test). The statistical model assessed the effects of seaweed species (L. digitata, L. hyperborea), extraction conditions (E1–4) and concentration of extracts (0.125, 0.25, 0.5, 1 and 2 mg/mL) and their associated two- and three-way interactions. The biological replicate was the experimental unit. Probability values of < 0.05 denote statistical significance. Results are presented as least-square mean values ± standard error of the means (SEM). The proximate composition of the whole seaweed biomass samples LDWB-F, LHWB-F, LDWB-N and LHWB-N is presented in Table 3, as reported previously [36]. The laminarin and fucoidan contents in the L. digitata (LDE1–4) and L. hyperborea (LHE1–4) extracts are presented in Table 4. The effects of the whole biomass samples of L. digitata and L. hyperborea collected in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N) were evaluated on selected faecal bacterial populations in a batch fermentation assay. The effects of species, season and concentration and their interactions are presented in Table 5 and Table 6 and are described below. The species × concentration interaction and the season × concentration interaction were only significant for Bifidobacterium spp. (p < 0.05) and, as a result, were excluded from the statistical analysis of the other bacterial groups. Enterobacteriaceae: There was a species × season interaction whereby LHWB-F led to lower Enterobacteriaceae counts compared to LHWB-N, while the opposite was true for L. digitata (p < 0.05, Table 5). There was also a concentration effect on Enterobacteriaceae counts, whereby the 5 mg/mL reduced Enterobacteriaceae counts compared to the control (7.74 logGCN/g digesta (5 mg/mL) vs. 7.99 logGCN/g digesta (0 mg/mL) ± 0.056, p < 0.05). Bifidobacterium spp.: There was a species × season interaction, whereby LHWB-N led to lower Bifidobacterium spp. counts compared to LHWB-F, while harvest season had no effect on Bifidobacterium spp. counts with regard to L. digitata (p > 0.05, Table 5). There was a species × concentration interaction, whereby the concentrations of 2.5 and 5 mg/mL of L. hyperborea led to lower Bifidobacterium spp. counts compared to the control and 1 mg/mL (2.73 logGCN/g digesta (2.5 mg/mL) and below the limits of detection (5 mg/mL) vs. 6.72 (0 mg/mL) and 6.60 (1 mg/mL) logGCN/g digesta ± 0.056, p < 0.05)), while this effect was not as potent with the corresponding L. digitata concentrations (5.95 logGCN/g digesta (5 mg/mL) vs. 6.46 (0 mg/mL), 6.55 (1 mg/mL) and 6.37 (2.5 mg/mL) logGCN/g digesta ± 0.056, p < 0.05)). There was a season × concentration interaction, whereby the 5 mg/mL of both February and November suppressed Bifidobacterium spp. counts, while at 2.5 mg/mL, there was a greater reduction in the counts in November relative to February (p < 0.05, Table 6). Total bacteria: There was a species effect and a concentration effect on total bacterial counts. L. hyperborea increased total bacteria compared to L. digitata (9.83 logGCN/g digesta (L. hyperborea) vs. 9.72 logGCN/g digesta (L. digitata) ± 0.034, p < 0.05). The 1 and 2.5 mg/mL gave higher counts compared to the control (9.87 (1 mg/mL) and 9.87 (2.5 mg/mL) logGCN/g digesta vs. 9.64 logGCN/g digesta (0 mg/ mL) ± 0.048, p < 0.05). Lactobacilli: There was a species effect and a concentration effect on lactobacilli counts. L. hyperborea increased lactobacilli counts compared to L. digitata (8.79 logGCN/g digesta (L. hyperborea) vs. 8.45 logGCN/g digesta (L. digitata) ± 0.030, p < 0.05). The concentrations of 1 and 2.5 mg/mL were associated with higher counts compared to the control (8.66 logGCN/g digesta (1 mg/mL) and 8.69 logGCN/g digesta (2.5 mg/mL) vs. 8.56 logGCN/g digesta (0 mg/mL) ± 0.035, p < 0.05). In summary, whole seaweed biomass samples from L. hyperborea and L. digitata collected in February had the least negative impact on Bifidobacterium spp. counts. Furthermore, LHWB-F reduced Enterobacteriaceae counts to the greatest degree, while LDWB-F had no effect. Both LHWB-F and LDWB-F were selected to generate the extracts evaluated in the next part of the screening process to determine whether the extraction methodology could improve the bioactivity of two whole seaweed biomass samples with varying effects. The HAE methodology with four different extraction conditions (E1–4) was employed for producing the extracts from the L. digitata (LD) and L. hyperborea (LH) samples, collected in February, to investigate whether the extraction method could improve their biological properties. LDE1–4 and LHE1–4 were evaluated for their antibacterial and prebiotic activities in pure-culture growth assays with selected beneficial (L. plantarum, L. reuteri, B. thermophilum) and pathogenic (ETEC, S. typhimurium) bacterial strains. Bacterial counts were expressed as the difference between the counts of each bacterial strain for each extract concentration and their respective control (0 mg/mL). The effects of species, extraction condition and concentration and their interactions are presented in Table 7 and Table 8 and are described below. The species × concentration interaction was significant only for S. typhimurium (p < 0.05) and was excluded from the statistical analysis of the other bacterial species. ETEC and S. typhimurium: There was a species × extraction condition interaction, whereby LHE1 had more potent antibacterial activity than LHE2, LHE3 and LHE4, whereas the effect of the E1 extraction condition was not as potent with L. digitata, despite being significant (p < 0.05, Table 7). B. thermophilum: There was a species × extraction condition interaction, whereby LDE4 was more bifidogenic than LDE1, LDE2 and LDE3, whereas the effect of E4 extraction condition was not as evident with L. hyperborea, despite being significant compared with E1 and E2 (p < 0.05, Table 7). L. plantarum: There was a species × extraction condition interaction, whereby LHE4 was more stimulating on L. plantarum growth than LHE1, LHE2 and LHE3 (p < 0.05) and there was no effect of the extraction condition on L. digitata (p > 0.05, Table 7). L. reuteri: There was a species effect and an extraction condition effect on L. reuteri counts. LH extracts led to higher L. reuteri counts compared to LD extracts (0.24 logCFU/mL (LH extracts) vs. 0.16 logCFU/mL (LD extracts) ± 0.029, p < 0.05). The extraction conditions E1 and E2 increased L. reuteri counts compared to E3 (0.30 logCFU/mL (E1) and 0.27 logCFU/mL (E2) vs. 0.07 logCFU/mL (E3) ± 0.041, p < 0.05). ETEC and S. typhimurium: There was a concentration × extraction condition interaction, whereby 2 mg/mL was more potent than 1, 0.5, 0.25 and 0.125 mg/mL for the E1 extraction condition (p < 0.05), whereas the effect of concentration was not as evident with E2, E3 and E4 extraction conditions (p > 0.05, Table 8). There was also a species × concentration interaction for S. typhimurium (p < 0.05), whereby the 2 mg/mL LH extracts led to lower counts compared to the 2 mg/mL LD extracts (−1.56 logCFU/mL (LH extracts) vs. −0.66 logCFU/mL (LD extracts) ± 0.058, p < 0.05), however, species had no effect at any of the other concentrations. B. thermophilum: There was a concentration × extraction condition interaction, whereby all concentrations of E4 were more bifidogenic than the equivalent concentrations in E1, E2 and E3, where some of the concentrations had no effect, while some were antibacterial (p < 0.05, Table 8). L. plantarum: There was a concentration effect on L. plantarum counts. The concentration of 1 and 2 mg/mL of all extracts increased L. plantarum counts compared to the 0.125 mg/mL (0.15 (1 mg/mL) and 0.15 (2 mg/mL) logCFU/mL vs. 0.02 logCFU/mL (0.125 mg/mL) ± 0.032, p < 0.05). L. reuteri: There was a concentration effect on L. reuteri counts. The concentration of 2 mg/mL of all extracts increased L. reuteri counts compared to 0.125 mg/mL (0.34 logCFU/mL (2 mg/mL) vs. 0.10 logCFU/mL (0.125 mg/mL) ± 0.045, p < 0.05). The influence of seaweed species and harvest season on the effects of the whole biomass samples of L. hyperborea and L. digitata on selected bacterial markers of the porcine faecal microbiota were evaluated in a batch fermentation assay. In this study, seaweed species was the predominant factor affecting the growth of Bifidobacterium spp., Enterobacteriaceae, lactobacilli and total bacteria. Bifidobacterium spp. counts were also influenced by the harvest season. The February-harvested L. hyperborea biomass sample, LHWB-F, led to the lowest Enterobacteriaceae counts among all tested samples whilst also having a reduced negative impact on Bifidobacterium spp. compared to the November-harvested counterpart, LHWB-N. Contrarily, the February-harvested L. digitata biomass sample, LDWB-F, was the least promising in terms of its antibacterial properties, having no major effects on the tested bacterial groups. These two whole biomass seaweed samples were used to produce LHE1–4 and LDE1–4 using four extraction conditions (E1–4) of the HAE methodology. The extracts were assessed in a panel of pure-culture growth assays with selected beneficial and pathogenic bacterial strains, to evaluate whether the optimised extraction conditions could enhance their antibacterial and prebiotic activities. Regardless of the seaweed species, the extraction condition E1 was predominantly associated with improved antibacterial activity against S. typhimurium, ETEC and to a lesser extent B. thermophilum, while the E4 extraction condition was predominantly associated with bifidogenic activity. Total bacteria, lactobacilli, Bifidobacterium spp. and Enterobacteriaceae were monitored in the batch fermentation assay as part of the evaluation of the whole biomass of L. digitata and L. hyperborea collected in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N). The whole biomass of L. hyperborea, LHWB-N and LHWB-F, reduced the Bifidobacterium spp. counts in a concentration-dependent manner with LHWB-F having a lesser impact. The whole biomass of L. digitata, LDWB-F and LDWB-N, also showed evidence of minor reductions in this bacterial population. In addition, LHWB-F and LDWB-N were associated with reduced Enterobacteriaceae counts. Reductions in Bifidobacterium spp. and Enterobacteriaceae counts have been previously observed in the faeces and colonic and caecal digesta in pigs supplemented with crude extracts of L. hyperborea, L. digitata or Laminaria spp. [23,24,25,26]. In this study, whole biomass samples of L. hyperborea were associated with minor increases in lactobacilli and total bacterial counts compared to whole biomass samples of L. digitata. Thus, bacterial growth was predominantly influenced by the seaweed species rather than harvest season, which only had a significant effect on the Bifidobacterium spp. population. It is interesting to hypothesise what the bioactive components within the whole seaweed biomass samples could be based on their proximate composition analysis and the results of the batch fermentation assay. The whole biomass samples of L. hyperborea had higher total polysaccharide content compared to L. digitata for both months. The main polysaccharides present in L. digitata and L. hyperborea are laminarin, mannitol, alginate and cellulose, of which laminarin and mannitol have been reported to exhibit significant seasonal variation in their concentration [35]. This, along with the increase in total glucans (laminarin and cellulose combined) observed in November for both seaweed species in the current study suggests that the variation in the total carbohydrate content was due to laminarin. Fucoidan was confirmed to be a relatively minor polysaccharide in the whole biomass samples of L. digitata and L. hyperborea as expected for the Laminaria spp. [42] and increased in November in both seaweed species. Previous research has reported that laminarin reduced the Enterobacteriaceae counts in the caecum and increased lactobacilli counts in the faeces and colon of weaned pigs [22,27,43], while fucoidan from whole A. nodosum biomass samples was considered to be the bioactive reducing Bifidobacterium spp. and Enterobacteriaceae counts in a batch fermentation assay inoculated with faeces from weaned pigs [18]. The reduction in Bifidobacterium spp. counts could additionally be attributed to inhibitory effects due to the wide-ranging components within the extracts, including phenols, alginate, cellulose and fucoidan, on the activity of bacterial carbohydrate-degrading enzymes [44,45,46]. As whole seaweed biomass samples are inherently complex, it is not possible to attribute the observed effects on the faecal microbiota to specific bioactive components within the whole biomass samples of L. hyperborea and L. digitata with certainty. For the second part of the study, LHE1–4 and LDE1–4 were produced from LHWB-F and LDWB-F, respectively, using the HAE methodology with four extraction conditions (E1–4). Of these, LHWB-F was identified as the most promising antibacterial sample in the batch fermentation assay and was selected for further analysis. In parallel, LDWB-F was included to investigate whether the extraction protocol could improve its limited bioactivity, an effect that was demonstrated in a previous study [18]. LHE1–4 and LDE1–4 were evaluated for their antibacterial and prebiotic potential in a panel of pure-culture growth assays. The pathogens S. typhimurium and ETEC were selected as representatives of the Enterobacteriaceae family. While S. typhimurium infection in pigs is mostly asymptomatic, it is associated with intestinal inflammation and compositional changes in the gastrointestinal microbiota that can have a negative impact on animal health and performance [47,48,49]. Furthermore, pigs and their meat products can become a reservoir for S. typhimurium, which can impact on human health [50]. ETEC infection in newly weaned pigs contributes to the development of post-weaning diarrhoea, an economically significant disease characterised by diarrhoea, dehydration, stunted growth and significant mortality [51]. The effects of the L. hyperborea and L. digitata extracts on representative beneficial bacterial strains, B. thermophilum, L. plantarum and L. reuteri, were also evaluated. These bacterial species commonly colonise the porcine gastrointestinal tract and exert a range of beneficial roles such as inhibition of intestinal pathogens, immunomodulation, improved composition in the gastrointestinal microbiota and enhanced health and growth [52,53,54,55,56,57,58]. In the pure-culture growth assays, the E1 and E4 extraction conditions were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 was the most potent extract in reducing S. typhimurium and ETEC counts. LDE1 also inhibited the growth of both pathogenic strains to a lesser extent. Additionally, LHE1 and LDE1 reduced B. thermophilum counts, whereas both extracts were also associated with a slight increase in L. reuteri counts. Interestingly, LDE4 followed by LHE4 increased B. thermophilum counts in a concentration-dependent manner, with LHE4 additionally stimulating the growth of L. plantarum. Based on the above, the use of the E1 and E4 extraction conditions of the HAE methodology produced antibacterial and bifidogenic extracts with the potential to promote a healthy composition in the gastrointestinal microbiota of pigs. The laminarin and fucoidan contents of LHE1–4 and LDE1–4 were determined to establish the concentrations of these polysaccharides achieved by each combination of extraction conditions of the HAE methodology [17]. LHE1–4 extracts had higher concentrations of laminarin and fucoidan compared to LDE1–4, an expected outcome based on the proximate composition of the respective whole seaweed biomass. Interestingly, both sets of extracts had higher fucoidan content (12.76–14.68% for LHE1–4 and 3.84–5.80% for LDE1–4) than laminarin content (4.94–7.59% for LHE1–4 and ≤0.70% for LDE1–4). The presence of laminarin is reported to be at lower concentrations during the winter months in these seaweed species, in agreement with our observation [35,59]. Apart from laminarin and fucoidan, alginate is a polysaccharide which is present in high and relative stable concentrations throughout the year in both L. hyperborea and L. digitata [35], and could also be a significant component of the LHE1–4 and LDE1–4. While the alginate content of the tested extracts was not determined in the current study, this assumption is supported by the findings of a recent study evaluating an L. hyperborea extract produced using the E2 extraction conditions of HAE methodology [60]. Furthermore, the different extraction conditions (Table 1) could affect not only the content but also the structure of these seaweed polysaccharides in the produced extracts, and hence, their bioactivity. For instance, the use of HCl and increasing temperatures in the extraction protocol was previously associated with changes in the chemical composition (monosaccharide content, sulphation level) and lower molecular weight due to partial hydrolysis of fucoidan and partial depolymerisation of alginate [61,62,63]. Although we did not determine the antibacterial and bifidogenic components of the L. hyperborea and L. digitata E1 and E4 extracts, we hypothesise that fucoidan was likely the main bioactive, with the variation in bioactivities attributed to structural alterations due to the different extraction conditions (Table 1). Regarding the antibacterial activity, this assumption is supported by the following three facts: (1) LHE1 had both higher fucoidan content and stronger antibacterial activity against S. typhimurium and ETEC compared to LDE1, suggesting a connection between this bioactivity and fucoidan; (2) The fucoidan-rich A. nodosum extracts produced using the same E1 extraction protocol also led to significant reductions in S. typhimurium and ETEC counts in our previous studies [18,64]; (3) Depolymerised fucoidans from Laminaria spp., Sargassum spp. and Undaria spp. were reported to have improved antibacterial activity against various pathogenic strains including E. coli and S. typhimurium compared to the parent polysaccharide [65,66,67]. The antibacterial activity of LHE1 and LDE1 against B. thermophilum indicate that bioactives other than fucoidan are involved. The bifidogenic effect of LHE4 and LDE4 may also be attributed to the depolymerised fucoidan fraction due to the similar effects on Bifidobacterium spp. growth of the fucoidan-rich A. nodosum extract produced using the same E4 extraction protocol and depolymerised fucoidans of Laminaria spp. and Sargassum spp. in previous in vitro studies [18,68,69]. Alginate oligosaccharides have also exhibited a bifidogenic effect in pure-culture growth assays [70,71]. Therefore, depolymerised alginate may have contributed to the increases in B. thermophilum, particularly in the case of LDE4. The slight increases in L. plantarum and L. reuteri counts with LHE4 counts and E1-produced extracts, respectively, indicate limited ability of these bacterial strains to utilise seaweed polysaccharides, most likely laminarin [72] and alginate oligosaccharides [71]. Taken together, all of the above results suggest a strong indication that fucoidan is the candidate bioactive responsible for the antibacterial and bifidogenic activities, although other seaweed constituents such as alginate may also contribute to the latter in the E4-produced extracts, particularly LDE4. In future studies, investigation into the chemical composition of LHE1, LDE1, LHE4 and LDE4 would provide better insight into the prebiotic and antibacterial bioactive components of these extracts, which was not possible at the laboratory-scale production of the extracts during the development of the HAE methodology. The species of seaweed was the main determinant of the growth of Bifidobacterium spp., Enterobacteriaceae and lactobacilli when whole seaweed biomass samples were tested in a porcine batch fermentation assay. Whole biomass samples of L. hyperborea (LHWB-F) and L. digitata (LDWB-F) harvested in February were then selected as the most and least promising sources, respectively, for the generation of antibacterial extracts, based on their effects on the Enterobacteriaceae counts in the batch fermentation assay. E1- and E4-produced extracts from both seaweed species were associated with antibacterial and bifidogenic activities, respectively, indicating that the extraction conditions were a more important determinant of bioactivity than seaweed species. Of these extracts, LHE1 was the most potent extract against S. typhimurium and ETEC, whereas LDE4 stimulated the growth of B. thermophilum to the greatest extent. Further compositional characterisation of these extracts is required to facilitate the identification and purification of the bioactive components involved in the observed bioactivities. Nevertheless. these crude extracts, particularly LHE1, merit further exploration in terms of their ability to promote a more beneficial microbiota and, thus, overall health and growth in weaned pigs, as a means of minimising the costs associated with the purification of the responsible bioactives from these extracts.

PMC10000098

Bruno Stefanon,Michela Cintio,Sandy Sgorlon,Elisa Scarsella,Danilo Licastro,Alfonso Zecconi,Monica Colitti

Regulatory Role of microRNA of Milk Exosomes in Mastitis of Dairy Cows

24-02-2023

milk,exosomes,miRNA,mastitis,cow

Simple Summary The microRNA (miRNA) cargos of exosomes isolated from milk were investigated in relation to the healthy conditions of the mammary gland. Samples were collected from a group of cows without mastitis (H), a group at risk of mastitis (ARM), and a group with subclinical mastitis (SCM). The differential expression analysis identified 38, 18, and 12 miRNAs (p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only bta-mir-221 was shared between the three groups. The miRNA cargos of milk exosomes can be considered to study the complex molecular machinery set in motion in response to mastitis in dairy cows and the quality of milk. Abstract The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows.

Regulatory Role of microRNA of Milk Exosomes in Mastitis of Dairy Cows The microRNA (miRNA) cargos of exosomes isolated from milk were investigated in relation to the healthy conditions of the mammary gland. Samples were collected from a group of cows without mastitis (H), a group at risk of mastitis (ARM), and a group with subclinical mastitis (SCM). The differential expression analysis identified 38, 18, and 12 miRNAs (p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only bta-mir-221 was shared between the three groups. The miRNA cargos of milk exosomes can be considered to study the complex molecular machinery set in motion in response to mastitis in dairy cows and the quality of milk. The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows. The milk of cows is a source of nutrients and other compounds [1,2,3]. Moreover, breastfeeding modulates the development of the immune system [4], and this activity is also regulated by microRNAs (miRNAs) [5]. Milk is a rich source of extracellular vesicles (EVs), which are involved in cell communication [6]. Milk-derived EVs contain mRNA, miRNA, ribosomal RNA (rRNA), long noncoding RNA (lncRNA), transfer RNA (tRNA), and DNA in addition to lipids, metabolites, and proteins [7]. MiRNA in milk [8,9] is a class of single-stranded noncoding RNAs about 22 nucleotides in length that induce post-transcriptional silencing of genes by binding to the 3’ untranslated region of target genes [10]. In this way, miRNAs control multiple cellular functions, from cell differentiation to tissue development and immune regulation, and consequently many aspects of health and disease [11,12]. Mastitis is a common disease of dairy cows that causes high economic losses and often requires the administration of antimicrobial drugs [13,14], with potential implications for antimicrobial resistance [15,16,17], a topic under the scrutiny of the World Health Organization (WHO, https://www.who.int/, accessed on 14 December 2022). Somatic cell count (SCC) in milk is the most common routine tool used at the farm level to screen cows for mastitis, and 200,000/mL SCC is the European cutoff to identify an inflammatory response to infection [18], while the cutoff is >150,000/mL SCC for New Zealand and >250,000/mL for Australia [19]. Cows with high SCC levels can be later subjected to microbial culture or molecular analysis to confirm and diagnose the type of infection and stage of disease. Recently, it has become possible to routinely calculate the percentage of polymorphonuclear neutrophils (PMN) and lymphocytes (LYM) within the SCC [20,21,22], also defined as differential somatic cell count (DSCC), which is an estimate of the percentage of polymorphonuclear cells and allows a more accurate classification of intramammary infection in dairy cows [20]. Interestingly, analysis of SCC and DSCC in milk is also related to milk quality, in particular to coagulation characteristics for cheese production [23,24]. The identification of early biomarkers of mastitis is important and forms the basis for defining and applying farm protocols to reduce the spread of infectious agents, implement hygiene measures, and thus limit the use of antimicrobial drugs. Moreover, signaling pathways and cellular functions activated in response to an inflammatory response or disease can be assessed by the study of miRNA contained in milk exosomes, which have gained attention as markers of cow health [25,26,27,28,29] and mastitis [30,31,32,33]. In fact, the availability of next-generation sequencing technology has stimulated studies that use genomics as a diagnostic tool for mastitis while enabling the molecular response of the host to microbial invasion [33]. These findings may also provide new insights for understanding the variable prevalence of mastitis among dairy cow breeds and the high individual susceptibility [34]. It has been reported that miRNAs contained in milk exosomes are conserved among mammalian species, at least among humans, pigs, cows, and pandas [35]. The miRNAs in milk exosomes are acid-resistant [36,37] and protected from degradation in the intestine [38]. Their transfer from mother to infant has been documented [39]; notably, miRNAs in milk exosomes of cow can be incorporated into human cells such as intestinal cells and macrophages [40,41,42]. Bovine exosomes isolated from milk have been used as vehicles for drug delivery for therapeutic purposes [43], confirming their uptake in the gut. These findings open the perspective of considering milk exosome loading as a newer aspect of milk safety and quality [44]. The aim of this study was to measure miRNA cargoes in exosomes isolated from milk samples of lactating cows classified as healthy or at risk or affected by mastitis based on the European cutoff of 200,000/mL SSC and the proportion of PMN. The regulatory network of differentially expressed miRNA between these groups of cows was also investigated to reveal possible signaling pathways and cellular functions activated in response to an inflammatory response or disease. Moreover, to evaluate whether the presence of an inflammatory process can influence the expression of miRNAs and milk quality, in the present study milk was collected from the all the four quarters of each cow. Sixty cows were recruited for the study from a herd on a commercial farm (46.1134059, 13.2817455; N 46°6′48.261″, E 13°16′54.283″; Italy). Cows were housed in loose housing with cubicles, fed the same total mixed ration, and not treated with antibiotics during the previous 20 days. The milking parlor (parallel 12 + 12) was located next to the bedding area. All protocols, procedures, and care of the animals were in accordance with the Italian legislation on animal care (DL n.116, 27/1/1992), and the study was approved by the Ethics Committee of the University of Udine (OBPA Prot. N. 9/2020). Before starting the sampling, milk from these cows was analyzed for SCC and DSCC values obtained from the official register of the breeders’ association (Associazione Allevatori del Friuli Venezia Giulia, Codroipo, Italy; www.aafvg.it, accessed on 14 December 2022). SCC and DSCC were measured in milk samples using a Fossomatic 7DC (FOSS Electric A/S, Hillerød, Denmark; according to ISO 13366-2/IDF 148-2:2006). The 60 cows in the herd were divided into 3 groups of 20 cows each according to the classification proposed by Zecconi et al. [24]: healthy (group H, SCC/mL < 200,000 and DSCC ≤ 69.3%); at risk (group ARM, SCC/mL < 200,000 and DSCC > 69.3%); with subclinical mastitis (group SCM, SCC/mL > 200,000 and DSCC > 69.3%). Milk samples were collected again during the subsequent monthly official record of the breeders’ association. Based on the analysis of SCC and DSCC of the second sampling, the 60 cows were reclassified as follows: 34 cows were in group H (11 primiparous and 23 multiparous; 10 Days in Milk (DIM) < 70 and 24 DIM > 70), 13 cows were in group ARM (6 primiparous and 7 multiparous; 2 DIM < 70 and 11 DIM > 70), and 13 cows were in group SCM (2 primiparous and 11 multiparous; 3 DIM < 70 and 10 DIM > 70). For EVs isolation, the teats were cleaned with disposable wipes and disinfected (ethanol, 70%) before milking; then, the first flow of milk from each quarter was discarded, and a pooled sample of about 100 mL of milk for each cow was collected in sterile Falcon tubes, which were kept on ice during the collecting period and stored at −80 °C until analysis. EVs were isolated using a modified isoelectrical precipitation method [45]. Briefly, frozen milk samples were thawed at room temperature and centrifuged at 2000× g at 4 °C for 10 min to separate fat. After elimination of the supernatant fat, the samples were centrifuged at 12,000× g at 4 °C for 40 min to remove cell debris. The supernatant was diluted 1:1 with distilled water and heated to 37 °C for 10 min in a water bath after adding HCl 6N to reach pH 4.6 for casein precipitation. Samples were centrifuged at 5000× g at room temperature for 20 min, and the supernatant was transferred into clean tubes and frozen at −80 °C overnight. After thawing, the samples were filtered with Millipore membrane filters of 1.00, 0.45, 0.20 μm (Merck Life Science, Milan, Italy), and the filtrate was centrifuged at 100,000× g at 4 °C for 1 h. An aliquot of the pellet was resuspended in 0.1 M PBS pH 7.4 for the exosome characterization analysis. The Nanosight (Malvern Panalytical, Malvern, UK) under light scatter mode was used to visualize size distribution of the isolated milk exosomes in the 50–250 nm range. Transmission electron microscopy (TEM) with the immunogold labeling method was used to directly detect exosomes based on their size and specific surface proteins. Nickel TEM grids (Electron Microscopy Sciences, Hatfield, PA, USA), 400 mesh, with a formvar/carbon film, were floated on a drop of the pellet suspension fixed in 4% paraformaldehyde. Immunogold labeling was performed as previously described [26] using anti-HSC70 (ab19136, Abcam, Cambridge, MA, USA) and CD63 (ab193349, Abcam, Cambridge, MA, USA) primary antibodies coupled with a 10 nm gold particle (EY Lab. Inc., San Mateo, CA, USA). The grids were then washed with several drops of water and stained with methyl cellulose-uranyl acetate (4% uranyl acetate and 2% methyl cellulose in a ratio of 1:9) before being subjected to microscopic analysis. Grids were analyzed on Philips CM10 and images recorded at 80 kV. The results of TEM confirmed the presence of round-shaped exosomes with diameters of about 80 nm. Another aliquot of the pellet was suspended in 300 μL of mirVana™ miRNA Isolation Kit lysis buffer (Ambion, ThermoFisher Scientific, Milan, Italy) and frozen at −80 °C. Extraction of miRNAs from the isolated EVs was performed with the commercial kit mirVana™ (ThermoFisher Scientific, Milan, Italy). The lysate was then mixed with 30 µL of homogenate additive and incubated on ice for 10 min and extracted once with 300 µL of acid phenol:chloroform. Samples were then centrifuged, and the aqueous (upper) phase was collected in a new tube. After washing in 100% absolute ethanol at room temperature, the lysate–ethanol mixture was placed in a filter cartridge and centrifuged until all the lysate solution had passed through the filter. The filter cartridge was then washed repeatedly until 40 µL of RNase-free water preheated to 95 °C was added. MiRNAs were collected by spinning for 25 s at 10,000× g. Ten samples of the H group, 11 samples of the ARM group, and 11 samples of the SCM group were sequenced. RNA purity, integrity, and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US). FASTQ raw sequence files were subsequently quality-checked with FASTQC software [46]. Then, sequences with a low quality score Q < 20 or including adaptor dimers were trimmed using Cutadapt 4.2 software [47]. Samples were selected on the basis of an RNA Integrity Number (RIN) of >7 and an rRNA 28 s/18 s ratio of >1.8. Libraries were prepared with Illumina® TruSeq® Small RNA Library Prep protocol (Illumina Inc., San Diego, CA, USA); 1 μg of total RNA for each library (minimum concentration of 200 ng/μL) was sequenced to 50 bp long single reads (average of 64 million reads per sample) using 8 lanes in 6-plex on an Illumina HiScanSQ (Illumina Inc., San Diego, CA, USA). Sequences were mapped against Btau_5.0.1 using Bioconductor Rsubread [48]. The raw data in FASTQ format were uploaded to NCBI Sequence Read Archive (Bioproject ID PRJNA902552). The resulting miRNA sequences were firstly divided into those annotated in Bos taurus (no. 184) and those in other vertebrate taxa (no. 622), whilst sequences annotated to other phyla were not included in the downstream analysis. To discover which miRNAs annotated in various vertebrate organisms overlapped in Bos taurus, sequences were searched in the miRbase [49]. The final number of miRNAs considered for Bos taurus was 225. These miRNAs were uploaded on the miRNet suite [50] and normalized with the DESeq2 method [51] for the comparisons of H vs. SCM, ARM vs. SCM, and H vs. ARM. The target genes of these mRNAs for Bos taurus were found based on miRTarBase version 8.0 [52] and miRanda database [53]. The list of differentially expressed (DE) target genes (p < 0.05 after Benjamini and Hochberg correction for multiple testing) resulting from each comparison were enriched to the Function Explorer on the Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/, accessed on 14 December 2022) database to find the statistically relevant pathways linked to the DE miRNAs and genes. Only enrichments with a p-value of <0.05 after Benjamini and Hochberg correction are reported. The characterization of the isolated milk exosomes was obtained by Nanoparticle Tracking Analysis (NTA), and the results are displayed as averaged finite track-length adjusted (FTLA) concentrations (Supplementary Figure S1A). Under electron microscopy, the exosomes were round with a diameter of approximately 80 nm (Supplementary Figure S1B,C). Moreover, the exosomes were positive for known exosomal markers CD63 and HSC70 via immunogold labeling. The total number of reads from RNAseq obtained from 32 samples was 63,219,487, but after the quality check, 28,199,543 were annotated as RNAfam, and 18,507,870 were detected as miRNAs. A value of 656,318 counts per million (CPM) was calculated for miRNA followed by 196,430 CPM for RNA (Figure 1), and the contribution of other RNA types was minimal. In the miRNet workflow, DESeq2 was applied to assess the DE of miRNAs in the different health states (H_ARM, ARM_SCM, and H_SCM). A total of 38 (16 upregulated and 22 downregulated), 18 (5 upregulated and 13 downregulated), and 12 (3 upregulated and 9 downregulated) miRNAs were significantly DE in the H_ARM, ARM_SCM, and H_SCM comparisons (Supplemental Table S1). The Venn diagram (Figure 2) showed that 1 DE miRNA was shared between the three groups (bta-mir-221), 1 DE miRNA in the H_SCM comparison (bta-mir-1247-5p), 9 DE miRNAs in the ARM_SCM comparison (bta-mir-142-5p; bta-mir-128; bta-mir-671; bta-mir-19a; bta-mir-146b; bta-mir-222; bta-mir-142-3p; bta-mir-15b; and bta-mir-205), and 21 DE miRNAs in the H_ARM comparison (bta-mir-2892; bta-mir-96; bta-mir-1343-3p; bta-mir-194; bta-mir-1281; bta-mir-2885; bta-mir-30c; bta-mir-1307; bta-mir-2415-3p; bta-mir-365-3p; bta-mir-542-5p; bta-mir-502°; bta-mir-7857-3p; bta-mir-30b-5p; bta-mir-2431-3p; bta-mir-328; bta-mir-210; bta-mir-186; bta-mir-31; bta-mir-1839; and bta-mir-425-3p). The target genes of the DE miRNA were 2195 for the H_ARM comparison, 713 for the H_SCM comparison, and 1313 for the ARM_SCM comparison (Supplementary Table S2). Based on these genes, analysis of pathways revealed 57, 47, and 56 pathways significantly affected in the H_ARM, ARM_SCM, and H_SCM comparisons, respectively (p < 0.05 after HB correction for multiple testing) (Supplementary Table S3). A comparison of these enriched pathways from healthy, subclinical mastitic, and mastitis-prone cows showed that 19 pathways were differentially expressed in the three groups (Figure 3), while 16 were expressed only in the H_SCM group, 12 in the H_ARM comparison, and 18 in the ARM_SCM comparison. Based on the degree and betweenness results, the top miRNAs were highlighted for each network (Supplementary Table S4). In the H_ARM network, bta-mir-2415-3p, bta-mir-2431-3p and bta-mir-6517, bta-mir-339a, bta-mir-1343-3p, bta-mir-2407, bta-mir-328, and bta-mir-1306 are reported, and these miRNAs were downregulated in the healthy cows compared with the at-risk cows. In the H_SCM pairwise comparison, the miRNAs bta-mir-339a, bta-mir-1247-5p and bta-mir-1306, bta-mir-345-5p, bta-mir-320a, and bta-mir-2388-3p were downregulated in the healthy cows compared to the cows with subclinical mastitis. The significantly enriched KEGG signaling pathways are also shown in Figure 4, highlighting the NF-KB signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, adherens junction, and leukocyte transendothelial migration for H_ARM. For H_SCM, the NF-KB signaling pathway was not enriched and was replaced by the NOD-like receptor signaling pathway. Figure 4A,B depict the connections between the miRNAs and target genes according to the selected KEGG pathways. They show the relevance of one miRNA in relation to the other miRNAs in regulating the relevant genes and pathways for immune functions. The analysis of miRNAs was performed in milk samples collected from a single milking and revealed 225 unique miRNAs associated with Bos taurus or showing sequence homology. The total number of miRNAs in the isolated milk exosomes was higher than that reported by Saenz-de-Juano et al. [31] and lower than that reported by Sun et al. [33], Cai et al. [30], and Ma et al. [54], and is likely dependent on library construction, reference Bos taurus genome and sequence depth, and isolation techniques. In the present study, the mammary glands health status of the lactating cows was assessed by total (SCC) and differential counts (DSCC) of somatic cells in milk [18]. This method is routinely used by the Italian Breeders Association to classify lactating cows as healthy, affected by subclinical or chronic mastitis, or at risk of mastitis, based on validated cutoffs [22]. The leukocyte counts and miRNA cargos of exosomes in milk were measured in milk samples from healthy and mastitis-affected cows collected during a single milking. In this context, Saenz-de-Juano et al. [31] reported that the miRNA in exosomes and SCC did not vary on consecutive days and thus could reflect the condition of the individual cow at the level of the mammary gland. The results in Figure 2 and in the Supplementary Table S1 clearly show that the number of DE miRNAs from healthy cows (H) was higher compared with the ARM groups (21 miRNAs) than with the SCM groups (1 miRNA). Among the different studies on the cargos of isolated milk EVs, only four investigated the effects of mastitis on the presence and expression of miRNAs, three of them referred to Staphylococcus aureus infection, and only one compared subclinical with clinical mastitis. In particular, Ma et al. [54] collected milk samples from the four quarters of cows infected with this bacterial species, and Cai et al. [30] collected milk from the udder after infusion of Staphylococcus aureus colonies. Sun et al. [33] also infected the mammary gland with this pathogen, but only in two quarters, and used the corresponding quarters of individual cows as negative controls. In the fourth study [31], milk from cows with subclinical mastitis was collected from the infected quarters and compared with milk from the quarters with low SCC. The DE miRNAs in healthy and diseased cows were not the same in all four studies, except bta-miR-142-5p, which was always upregulated, not only in the cows infected with Staphylococcus aureus but also in the milk exosomes isolated from the milk of cows with subclinical mastitis. Our results showed that this miRNA was not DE compared with healthy cows (H_ARM, H_SCM), whereas it was downregulated in cows in the early phase of inflammation (ARM) compared with cows with subclinical mastitis (SCM) (p < 0.01). The upregulation of bta-miR-142-5p in cows with subclinical mastitis (SCC > 200,000/mL) compared with at-risk cows (SCC < 200,000/mL) is consistent with the results of Saenz-de-Juano et al. [31] and with the results of other studies in which cows were at the onset of mastitis after infection with Staphylococcus aureus. However, only one target gene for bta-miR-142-5p was found in cattle in the miRbase database. Interestingly, bta-miR-1247, the only upregulated miRNA in the H_SCM comparison, has been related to inflammation in bovine endometrial stromal cells treated with lipopolysaccharide affecting the MAPK pathway, which was significantly found in an H_ARM comparison [55]. Among the downregulated miRNAs in H_SCM and H_ARM comparisons, bta-mir-339a and bta-mir-320 were negatively associated with lactogenic differentiation in bovine mammary epithelial cell culture [56]. Bta-mir-22 and mir-345 microRNAs have been recently identified as differentially expressed in bovine macrophages in response to M. bovis expression [57]. Their differential expression is suggested to regulate host gene expression to enhance pathogen survival. Mir-345 is also a methylation-sensitive microRNA involved in cell proliferation. In addition to being downregulated in macrophages in response to M. tuberculosis, it is hypermethylated in T cells from TB-infected cattle [57]. A few other miRNAs found in the present study agreed with previous results, namely, bta-miR-142-3p, bta-miR-221, and bta-miR-103 with Cai et al. [30] and bta-miR-22-3p with Saenz-de-Juano et al. [31]. In addition, bta-miR-146a, bta-miR-146b, bta-miR-223, bta-miR-2285b, bta-miR-378-b, and bta-miR1246 were common between the studies, but the overlap did not extend to all of these studies. However, bta-miR-146a was found to alleviate intestinal colitis in a mouse colitis model by activating NF-κB [58], and it was found to be differentially expressed in bovine mammary glands affected by mastitis [59,60]. The experimental conditions in these investigations differed from those in the present study, suggesting that there is a great deal of individual variability in terms of infection-specific change, as previously suggested by Saenz-de-Juano et al. [31]. Indeed, the immunological response and milk concentrations of biomarkers also differed according to the type of mastitis [61]. Nevertheless, genetic variants [62] and factors other than infection influence the cargos of EVs in the milk of lactating cows. Stressors such as group relocation and ambient temperature have been reported to modulate miRNA levels in milk EVs from dairy cows [26,28]. Since miRNAs are also involved in mammary gland plasticity and the synthesis of milk components, genetic background is another factor to be considered [63]. Notably, bta-mir-320 and bta-mir-345 show 100% homology with human sequences, whereas bta-mir-146a is 93.8%. Since it is known that miRNAs can be absorbed in the human intestine, the study of miRNAs in milk exosomes may be a new area to investigate in relation to human health. Significantly enriched pathways (Supplementary Material Table S3) underscored a different pattern between subclinical and at-risk cows compared with healthy cows. In the H_SCM comparison, at least nine pathways were directly or indirectly involved in the cellular immune response (leukocyte transendothelial migration; prolactin signaling pathway; Rap1 signaling pathway; signaling pathways regulating pluripotency of stem cells; NOD-like receptor signaling pathway; regulation of actin cytoskeleton; VEGF signaling pathway; thyroid hormone signaling pathway; and thyroid hormone synthesis). From a biochemical point of view, these data support the evidence for the increase in leukocyte count in subclinical mastitis. In the H_ARM group, however, the pattern was different. In the ARM samples, an increase in the percentage of PMN and lymphocytes (DSCC) was observed, but the total leukocyte count (SCC) was not yet above the cutoff generally considered as inflammation (200,000 cells/mL). Among the signaling pathways of this comparison, at least 11 related to immune cell function and modulation of inflammation (phosphatidylinositol signaling system; lysosome; Fc epsilon RI signaling pathway; Jak-STAT signaling pathway; chemokine signaling pathway; platelet activation; NF-kappa B signaling pathway; inflammatory mediator regulation of TRP channels; inositol phosphate metabolism; sphingolipid signaling pathway; and HIF-1 signaling pathway). Moreover, the target genes of bta-mir-339a were enriched for 10 significant pathways, including the MAPK signaling pathway [64], as for bta-mir-1247 in the present study. The different patterns observed in the two comparisons can be explained by the different health status of the mammary gland. In the H_SCM comparison, we compared healthy with subclinical mastitis conditions, and in SCM there were clear signs of inflammation with an increase in leukocyte count above 200,000 cells/mL [18,22]. Therefore, one can expect a prevalence of signaling pathways involved in cell immune responses. The H_ARM comparison includes samples in the initial stages of inflammation (ARM). In these animals, overt inflammation had not yet started, and SCC was <200,000 cells/mL, while PMN content was increased. ARM status can develop in two ways: spontaneous healing or subclinical mastitis. The regulation of the immune and inflammatory response is crucial for the final outcome. Therefore, we can assume that at this stage genes regulating the activity of cells, including epithelial cells that are also able to secrete inflammatory mediators and antibacterial substances [65,66], may play a role leading to one of the two possible outcomes in terms of the success of the defense response and the virulence of the invading pathogens. These results are supported by assessing the degree (number of connections of nodes) and the betweenness (a measure of the number of shortest paths through a node; this filter retains genes connecting clusters) from the miRNet analysis, which allows us to highlight the most relevant miRNAs for the H_ARM and H_SCM networks (Figure 4; Supplementary Table S4 for the full list). The interconnections of these miRNAs with selected significantly enriched KEGG pathways highlighted in Figure 4 (T cell receptor signaling pathway, TNF signaling pathway, leukocyte transendothelial migration, adherens junction, and NF-KB signaling pathway) were based only on the target sequences of the genes. The H_ARM comparison was the most interconnected and showed that a complex network exists between the miRNAs and the target genes within the selected signaling pathways. In contrast, in the H_SCM comparison, the DE miRNAs formed separate gene networks, indicating a more focused regulatory role of miRNAs within each pathway. However, these miRNAs do not correspond to those reported in the limited studies of exosomes in bovine mastitis [30,31,33,54]. Searches of the miRbase database and the literature revealed limited information on the regulatory activity of these miRNAs in the inflammatory process and immune response. The expression of bta-miR-2431-3p, bta-miR-2415-3p, bta-mir-2407, bta-mir-345-5p, and bta-mir-328 was reported in an in vitro study of viral infection of a bovine kidney cell line, but these miRNAs were not significantly affected by treatment [67]. In another study of DE miRNAs in bovine testicular and ovarian tissues [68], bta-miR-6517, bta-miR-1343-3p, and bta-mir-2388-3p were highlighted, but their biological significance was not elucidated. A sequencing and annotation study reported the expression of bta-mir-671 and bta-miR-1306 [69], but their regulatory roles were not investigated. The limitations of the present study are that the extracellular vesicles were isolated from whole milk and were not related to a specific pathogen but were referred to an inflammatory process. Moreover, potential confounding factors, such as parity and stage of lactation, were not evaluated. Nevertheless, the origin of the extracellular vesicles is another aspect that deserves attention, and further studies are needed to determine the role of miRNAs in the inflammatory response of the mammary gland and their potential effects on human health to reach more comprehensive conclusions. Extracellular vesicles and their miRNA cargos in milk are considered a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows and could influence milk quality. However, the question of whether miRNAs are influenced only by mastitis or also depend on various experimental conditions and environmental factors deserves further investigation.

PMC10000099

Larissa Cuta,Christoph Georg Baums,Kerstin Cramer,Maxi Harzer,Jutta Hauptmann,Kristin Heenemann,Maria-Elisabeth Krautwald-Junghanns,Ines Stegmaier,Thomas W. Vahlenkamp,Volker Schmidt

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors

03-03-2023

Turkey Osteomyelitis Complex,green liver,hemorrhagic enteritis virus,aseptic bone necrosis

Simple Summary Organically raised turkeys investigated in a recent study showed a high prevalence of green liver discoloration. The condition is commonly associated with bone alterations and is potentially caused by opportunistic bacteria. This study was performed to determine possible infectious risk factors and reduce disease prevalence. The prevalence of green livers has decreased between both studies, and there was no explicable significant correlation with bacterial or parasitological findings. However, there was a significant correlation between green livers and the immunosuppressive turkey hemorrhagic enteritis virus at the early fattening stage. Hens with virus detection had impaired physical health. At the late fattening stage, there was a significant correlation between green livers and joint/bone lesions, as described in previous literature. It can be assumed that the hemorrhagic enteritis virus affects the pathogenesis of green liver discoloration at the early fattening stage. Therefore, an adequate vaccination schedule should be implemented in poultry farms to reduce the prevalence of discoloration and improve animal health. However, further standardized investigations to determine and evaluate possible infectious risks regarding green liver discoloration are necessary. Abstract A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health.

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors Organically raised turkeys investigated in a recent study showed a high prevalence of green liver discoloration. The condition is commonly associated with bone alterations and is potentially caused by opportunistic bacteria. This study was performed to determine possible infectious risk factors and reduce disease prevalence. The prevalence of green livers has decreased between both studies, and there was no explicable significant correlation with bacterial or parasitological findings. However, there was a significant correlation between green livers and the immunosuppressive turkey hemorrhagic enteritis virus at the early fattening stage. Hens with virus detection had impaired physical health. At the late fattening stage, there was a significant correlation between green livers and joint/bone lesions, as described in previous literature. It can be assumed that the hemorrhagic enteritis virus affects the pathogenesis of green liver discoloration at the early fattening stage. Therefore, an adequate vaccination schedule should be implemented in poultry farms to reduce the prevalence of discoloration and improve animal health. However, further standardized investigations to determine and evaluate possible infectious risks regarding green liver discoloration are necessary. A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health. The organic farming associations and Council Regulation (EU) No 2018/848 of 20 May 2018 on organic production and labeling of organic products place special requirements on the organic rearing of turkeys in Germany [1]. This serves to improve animal welfare as well as climate and environmental protection. During extensive statistical surveys from 2007 to 2017, the health status of conventionally and organically raised turkeys in Germany was recorded [2,3,4]. The latest study revealed that organically raised turkeys (Kelly Broad Breasted Bronze [BBB]) showed an approximately nine times higher prevalence of green livers (GL) at processing compared to conventionally raised B.U.T. 6 (British United Turkeys). The prevalences of GL for organically raised Bronze toms and hens were 34.8% and 33.2%, versus 3.8% and 4.4% for conventionally raised B.U.T. 6 toms and hens, respectively [2,4,5]. The pathogenesis of GL is mainly attributable to the accumulation of inter- and intrahepatic endogenous biliverdin [6]. This bile pigment represents a degradation product of erythrocytes. It is particularly relevant in birds due to the absence of the enzyme biliverdin reductase, which converts biliverdin to bilirubin [7]. Besides bacterial infection, other possible causes for the accumulation of biliverdin are increased hemolysis due to mechanical trauma, intoxication, hepatitis, necrosis, or obstructive outflow disorders [8]. Turkey osteomyelitis complex (TOC) is a disease predominantly described in conventionally fattened turkeys. It is commonly associated with GL and bone alterations, leading to economic losses due to the discarding of complete or partial carcasses [9]. The descriptions of this disease complex have many similarities with the findings from the predecessor study. A statistically significant correlation (p ≤ 0.05, r = 0.49) between prevalences of GL and joint pathologies was found at processing [4]. However, previous studies indicate that the alterations of TOC are associated with opportunistic bacteria like Escherichia (E.) coli or Staphylococcus (S.) aureus [10,11,12]. A further immunosuppressive effect of infection with the turkey hemorrhagic enteritis virus (HEV), leading to a higher prevalence of pathological findings associated with coliform bacteria, has been suggested [12]. Transient immunosuppression and related hemorrhagic enteritis are mediated by the release of proinflammatory cytokines as well as necrosis and apoptosis of primary target cells, B lymphocytes, and macrophages [13,14,15]. HEV belongs to the family Adenoviridae, genus Siadenovirus, and species Turkey siadenovirus A. It includes two strains, the avirulent turkey HEV and the virulent turkey adenovirus 3 [16]. It is a non-enveloped, linear, double-stranded DNA virus with an approximate genome length of 26.3 kb and is characterized by an open reading frame (ORF). The ORF encodes for the glycoside hydrolyzing enzyme sialidase [17,18,19]. Besides different bacteria, several other viruses and diseases in turkeys are known to affect the liver. These include, among others, avian hepatitis E virus, turkey viral hepatitis, aviadenoviruses, highly pathogenic avian influenza A viruses, and reoviruses [17,20,21,22,23,24,25,26,27]. This study was designed to identify associations between GL and infectious risk factors in organically raised turkeys. It was carried out to gain knowledge about the potentially infectious causative agents of GL disease in organic turkey farming. Further, it was conducted to implement strategies to avoid circumstances leading to discoloration and the associated TOC at the time of processing. The results may help to assess the importance of preventing HEV infection during an early stage of fattening to avoid economic losses and improve health and welfare. The examinations did not require notification or approval, as in accordance with the German Animal Welfare Act (§ 7, paragraph 2, sentence 3). A total of five organic turkey farms fattening Bronze turkey hens of a Cartier genetic (including rearing farms) were selected for this investigation. In part, the selection was based on the selection criteria of high prevalence of GL at processing from the previous study [4,5]. This study included two consecutive trials, with the first examination trial extending over six months, starting in September 2020 and ending in March 2021. The second trial started in February 2021 and finished with the final examination in August 2021. As a consequence of avian influenza control measures in one farm’s area (flock No. 41, second examination) and quarantine measures in the scope of the SARS-CoV-2 pandemic (flock No. 31, second examination), two examinations were canceled. Due to the avian influenza epidemic in Germany in 2020/2021, flocks No. 21 (second examination trial) and No. 22 (first examination trial) were not granted any access to the outdoor enclosure. In Flock No. 11 (second examination trial), access to the outdoor enclosure became available during the end of the fattening phase. The turkeys of flock No. 41 were kept outside during the most part of the fattening period with variable access to woodland and a small stable (in the second trial, no access was allowed from week 11 onward). Recorded data of the farm workers of each farm considered in this investigation included the vaccination program, flock diseases and treatments, and daily mortality (Table S1). With respect to health protection measures during the SARS-CoV-2 pandemic, no personal attendance at the slaughterhouses was possible. Clinical and detailed post-mortem examinations were performed on 20 turkeys per flock between the 70th and 75th days of fattening (the first or early examination time) and the 120th to 127th days of fattening (the second or late examination time), respectively. In addition, an inspection of the turkeys was performed on the first days of rearing. The sample size was calculated based on an average prevalence of GL of 27.7% (Bronze- and B.U.T. 6/TP7/TP9-hens) in organically raised hens, with the lower limits of the 95% confidence intervals of 18.5% [4]. Using this lower limit with a maximum herd size of 2.500 turkeys [the maximum permissible number of fattening turkeys in one housing unit according to Regulation (EU) No. 2020/464 of 26 March 2020] and calculation according to Cannon and Roe, a sample size of n = 16 was required to demonstrate the presence of GL in organically raised hens [28]. Sample size was limited to 20 randomly drawn individuals per investigation time and flock. This can be explained by the fact that the sample, on the one hand, had to comprise a sufficient number of carriers, while on the other hand, all animals had to be purchased and thus removed from the food chain. This study includes only female turkeys to maximize validity, as the prevalence of GL showed a notably greater variance in the Bronze hen flocks (1.7% to 75.0%) than in the tom flocks (1.7% to 55.0%). Therefore, influences on GL development could potentially be demonstrated [4]. Hens were then taken to the Clinic for Birds and Reptiles at the University of Leipzig and provided with food and water overnight. The next day, all turkeys were euthanized after being stunned (Large Poultry Stunner, Friedr. Dick GmbH & Co., KG, Deizisau, Germany) by subsequent exsanguination through a unilateral neck cut severing the carotid artery and jugular vein. All turkey hens were euthanized for routine diagnostic necropsy. Necropsy was conducted following the protocol by Schmidt et al. including the 10-cut procedure protocol implemented by the Food Safety and Inspection Service [29,30]. Pathoanatomical examinations were performed on 360 turkeys. Depending on the occurrence of GL, six hens without and additionally up to six further hens with discoloration were chosen for histopathological, bacteriological, parasitological, and virological examinations. Further examinations were performed on 67 hens at the early and 63 hens at the late fattening stages (in total, 130 hens) (Table S1). Swabs for microbiological examination were taken from the liver, heart, kidney, spleen, lungs, small intestine, shoulder joint, hip joint, knee joint, ankle joint, and the epiphysis of the proximal tibiotarsus. In the case of an ambiguous parasitological result, the diagnosis was confirmed by PCR of the 18S rDNA fragment specific for Eimeria of turkeys and/or for Tetratrichomonas (T.) gallinarum and Histomonas (H.) meleagridis [31,32]. The chymus of the jejunoileum was sequentially diluted 1:10 using phosphate-buffered saline (PBS) for quantifying coliform bacteria as colony-forming units (CFU) (starting with 0.5 g in 4.5 mL PBS). Different volumes of the first two dilutions were cultivated for 24 h at 37 °C on a selective Gassner agar (Sifin, Berlin, Germany), which allows differentiation of lactose fermentation. A Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometer (Bruker, microflex LT mass spectrometer, Bruker Daltonik GmbH Leipzig, Germany) was used to identify bacteria forming colonies on Gassner agar. Analyses for Brachyspira (B.) sp. were performed randomly at six time points, including at least one examination of each flock. The chymus of the cecum was cultured anaerobically on selective horse blood agar plates designed to detect avian brachyspira as described by Harms et al. [33]. Samples of liver, kidney, spleen, lungs, heart, duodenum, ileum, cecum, and bursa cloacalis were fixed in 4.5% neutral buffered formalin (Formaldehyde-Solution 37%, Merck, Darmstadt, Germany) for at least 24 h. Bone fragments from the femoral head, proximal tibiotarsus, distal tibiotarsus, and proximal tarsometatarsus were decalcified (OSTEOMOLL, Merck KGaA, Darmstadt, Germany) for at least 24 h prior to further processing. Formalin-fixed samples were dehydrated, routinely embedded in paraffin wax, and sectioned at 4 µm. All sections were stained with hematoxylin and eosin for histopathological examination. Samples for molecular biological examinations were pooled separately for hens with and (if applicable) without GL, despite the assumption that a whole flock would be affected by virus infection. In each case, DNA was extracted from 10 to 15 mg of each pooled sample using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) or IndiSpin Pathogen Kit (INDICAL BIOSCIENCE GmbH, Leipzig, Germany). RNA was isolated from 15 to 30 mg of each pooled sample using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Samples included the trachea and a joint swab for detection of mycoplasma [34]. Samples of liver and duodenal tissue as well as a joint swab were collected for amplification of avian orthoreoviruses [35]. Liver tissue samples were taken to detect the avian hepatitis E virus [36]. For the detection of adenoviruses, spleen and duodenum homogenates were examined [37]. In the case of HEV detection, positive samples were further characterized to determine the virulence and, thus, relevance of the virus. For this purpose, the partial ORF1, E3, and fib knob domains were amplified [38,39]. For amplification of the partial ORF1, E3, and fib knob domains, 2 µL of DNA was mixed with 5 µL of 5X Q5 Reaction Buffer (New England BioLabs, Ipswich, MA, USA), 0.5 µL of dNTPs (final concentration 200 µM; Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1.25 µL of each oligonucleotide primer (final concentration 0.5 µM), and 0.25 µL of Q5 Hot Start High-Fidelity DNA Polymerase (final concentration 0.02 U/µL) (New England BioLabs, Ipswich, MA, USA). The PCR protocol started with activation of the polymerase for 30 s at 98 °C, followed by 40 cycles of denaturation for 10 s at 98 °C, annealing for 15 s at 60 °C, and elongation for 60 s at 72 °C. The reaction ended with a final elongation step lasting 2 min at 72 °C. Subsequently, the PCR product was analyzed by agarose gel electrophoresis. Positive PCR products were purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and subsequently sent to Microsynth Seqlab (Göttingen, Germany) for Sanger sequencing. Nucleotide sequences were analyzed and edited using the GENtle program (Magnus Manske, University of Cologne, Germany), and comparison was performed using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/, accessed on 16 May 2022). Phylogenetic analyses and the construction of phylogenetic trees were carried out using the software MEGA X [40]. The evolutionary history was illustrated using the maximum likelihood method and the JTT (Jones-Taylor-Thornton) matrix-based model [41]. For this analysis, the Neighbor-Join and BioNJ algorithms were applied to a matrix of pairwise distances estimated using the JTT model, and then the topology with superior log likelihood value was selected. All sequences of HEV were deposited in NCBI GenBank, with NCBI accession numbers (acc. No.) OM994418, OM994422 to OM994426 for the E3 and fib knob domain genes, and OP171872, OP171876 to OP171880 representing the partial ORF1 gene (Tables S2 and S3). Statistical analysis was performed with IBM SPSS Statistics for Windows Version 28.0 (IBM SPSS, Armonk, NY, USA) [42]. Tests were implemented to examine infectious agents based on the appearance of GL. Calculations were performed separately for both examination dates to differentiate between the different ages of the hens. A Chi-square test was used to investigate the relationship between two nominal or categorical variables. Fisher’s exact test was utilized instead of the Chi-square test when cell frequencies were below five for 2 × 2 tables. The means of two independent groups were compared using Student’s t-tests for independent samples with a 95% confidence level. The model assumptions of normality and homogeneity of variance were examined by Shapiro-Wilk and Levene’s test. If the data did not follow a normal distribution, the non-parametric Mann-Whitney U test was performed to compare the median values of two samples. A one-way analysis of variance, including a Tukey post-hoc test, was implemented to compare the means of multiple samples. The assumption of normal distribution and homogeneity of variances was determined using the tests described above. A Games-Howell post-hoc test was performed if the data failed to meet the assumption of homogeneity of variances. The non-parametric alternative for the one-way analysis of variance was the Kruskal–Wallis test followed by a post-hoc test. A 95% confidence interval was calculated for the prevalence rates of clinical and pathological findings. For all calculations, results were considered significant if the double-sided p-value was equal to or less than 0.05. The p-value is supplemented by the specific effect size for the statistical test (d = Cohend’s d, φ = phi coefficient, r = Pearson correlation coefficient). In total, 31 of 343 (9.04%; 95% CI [0.06, 0.12]) Bronze hens (of which 17 turkeys were toms and excluded from further examination) showed GL discoloration. Divided by age, 16 of 184 (8.76%; 95% CI [0.05–0.13]) were affected at the early fattening stage, and 15 of 159 (9.43%; 95% CI: [0.06–0.15]) at the late fattening stage. The prevalence at the individual examination times varied between 0.0 and 68.4%. The most common finding was a focal GL discoloration limited to the caudal margin of the liver. Multifocal or diffuse green coloration appeared rarely. In a previous study, the mean prevalence of GL in organically farmed turkeys was 33.15% (processing batches from nine flocks with 60 Bronze hens each) [4]. Therefore, the mean prevalence for GL is significantly lower in the current study. In addition, examinations in the current study were performed on Bronze hens of a Cartier genetic. This is due to the decreased proportion of organically fattened Kelly BBB turkeys in Germany, which were investigated in the previous study in favor of other turkey genetics. Because both genetics were kept under the same fattening conditions, a comparison of the two studies seems legitimate. The marked discrepancy in GL prevalence between the two studies, however, may be in part due to the different genetics of the turkeys examined. The histopathological examination of the livers revealed the accumulation of bile pigment in 4.62% (95% CI [0.02, 0.09]) of the examined livers and solely at the late stage of fattening. Contrarily, inflammatory reactions consisting of slightly multifocal infiltrates consisting of heterophils, lymphocytes, and plasma cells were seen at both examination times, with a prevalence of 9.23% (95% CI [0.05, 0.15]). There was a significant correlation between GL and the presence of inflammatory cells at both fattening stages (early fattening stage: p ≤ 0.05, φ = 0.34; late fattening stage: p ≤ 0.05, φ = 0.30), as well as with the accumulation of bile pigment (p ≤ 0.001, φ = 0.58) at the late fattening stage (Table 1). Therefore, damage to the hepatic tissue concerning GL can be assumed. The bodyweight (BW) of hens between their 70th and 75th days of fattening varied from 1.83 kg to 5.41 kg (mean = 3.53 kg, SD = 0.57). Between 120 and 127 days, it ranged from 4.27 kg to 11.91 kg (median = 8.94, variance = 1.60). At the late examination date, differences in BW of more than 2.0 kg between individuals were found in all flocks. Hens with GL had a significantly (p ≤ 0.05, d = 0.56) lower mean BW compared to hens without GL in the early fattening period. The discrepancy increased up to 1.45 kg (95% CI [0.36, 2.54]) at the later stage of fattening for hens with GL as opposed to those without (p ≤ 0.001, d = 1.19) (Table 1). The reduced body weight in the presence of GL indicates impaired physical development and emphasizes that GL is an indicator of a superordinate pathologic condition. Over both examination dates, the mean of the relative liver weight was statistically significantly (early fattening stage: p ≤ 0.05, d = 0.36; late fattening stage: p ≤ 0.01, d = 0.28) higher in hens with GL (Table 2). High relative liver weights as a consequence of liver pathology in cases of GL were already described in the literature [11]. Splenomegaly, defined as follicular hyperplasia, was documented in 5.54% (95% CI [0.035, 0.083]) of hens. Most were seen at the early examination date and predominantly belonged to flock No. 31 in the second trial. However, there was a significant correlation between GL and splenomegaly at the late fattening stage (p ≤ 0.001, φ = 0.44). The hens showed a statistically significant (p ≤ 0.01, r = 0.35) higher median spleen weight at this stage (Table 1 and Table 2). Gastrointestinal lesions were seen in 81.54% (95% CI [0.74, 0.88]) of the turkey hens. The most common finding was a slight to mild mucosal and submucosal inflammation consisting of heterophils and lymphocytes. They were occasionally accompanied by intestinal villi thickening and merging. Inflammatory reactions frequently affect more than one intestinal segment. Concerning the targeted organ of this study, a significant relation was proven between the occurrence of GL and catarrhal duodenitis at the early fattening period (p ≤ 0.001, φ = 0.47) (Table 2). A total of 34.69% (95% CI [0.29, 0.40]) of the hens showed skin injuries predominantly located at the snood, and there was no significant correlation for GL. Bilateral footpad dermatitis (FPD) of differing severity was present in every hen. The evaluation method for the footpads is based on a scheme implemented by Mayne and Hocking et al. [43,44]. All lesions were restricted to the upper skin layers, and no alterations of the subcutaneous tissue were found. There was no statistically significant correlation between the severity of FPD and GL (Table 1). Macroscopic alterations of the joints were solely seen in the late fattening phase, with a prevalence of 6.92% (95% CI [0.04, 0.12]). There was a statistically significant correlation between macroscopic bone alterations and GL in the late fattening phase (p ≤ 0.001, φ = 0.42) (Table 1). Aseptic bone necrosis was characterized by cell debris and heterophilic and plasmacellular infiltrates accompanied by proliferation of connective tissue and fibrin. It was localized within the epiphyseal growth plate at the proximal tibiotarsus in three cases (2.31%, 95% CI [0.007, 0.06]) and at the distal tibiotarsus in another case (0.77%, 95% CI [0.001, 0.035]). Of those findings, three were seen at the later stage of fattening. Therefore, a significant (p ≤ 0.05, φ = 0.40) correlation with GL is given at this stage (Table 2). TOC is defined by green livers associated with lesions of the musculoskeletal system [10,11,45]. Although this study found a correlation between GL and joint/bone lesions, swollen joints did not necessarily indicate GL discoloration. Regarding histological examinations, the relation between aseptic bone necrosis and GL discoloration corresponds to the literature describing TOC as an immune-associated process possibly leading to further lesions [9,11,46]. However, a bacterial infection is not necessarily involved or no longer detectable. This could explain the finding of a single hen with systemic S. aureus infection and simultaneous GL discoloration [10,11]. Due to the small sample size (five flocks within two trials), no statistical calculations regarding husbandry and management-associated factors were performed. However, an impact of the housing situation in addition to climatic conditions on several health parameters is likely. Regarding the overall fattening period, total losses ranged from 1.38% to 10.32% (mean = 5.32%, SD = 2.69). A higher median mortality existed in flocks where GL appeared during necropsy. Vaccination against hemorrhagic enteritis virus with DINDORAL SPF (Merial GmbH, Hallbergmoos, Germany) was implemented in flocks No. 21 and 22 in the first examination trial and flock No. 41 in the second examination trial. The time point of vaccination varied between the 21st and the 34th day of fattening (Table S1). The prevalences of cecal parasites were 45.49% (95% CI [0.37, 0.54]) for T. gallinarum, 26.92% (95% CI [0.20, 0.35]) for Eimeria (E.) meleagridis, 23.08% (95% CI [0.17, 0.31]) for H. meleagridis, and 3.08% (95% CI [0.01, 0.07]) for Heterakis (H.) gallinarum. No significant relationship between GL and the detection of E. meleagridis or H. gallinarum could be proven. In contrast, hens with GL significantly more often harbored T. gallinarum (p ≤ 0.05, φ = 0.27) at the late fattening stage and H. meleagridis at the early fattening stage (p ≤ 0.001, φ = 0.52) (Table 2). The parasite T. gallinarum usually induces latent infections of the ceca without clinically manifest outbreaks. Similar to H. meleagridis, the documented association with GL may indicate a general weakening of the affected hens and a higher susceptibility for secondary infections [47,48]. Extraintestinal bacterial growth was rarely seen in the hens examined. This included one hen at 72 days of fattening with detection of E. coli in the spleen, shoulder joint, and knee joint. Despite the presence of osteomyelitis in the distal tibiotarsus, this hen showed no GL. Another hen at 120 days of fattening had a systemic infection from the cultivation of S. aureus in her liver, spleen, knee joint, and duodenum. Concurrently, this hen had GL discoloration and inflammatory responses in the liver and spleen on histology. The detection of Mycoplasma sp. succeeded with a prevalence of 9.23% (95% CI [0.05, 0.15]) and predominantly at the late fattening stage. Concerning GL, there was no significant relationship to the detection of mycoplasma. Even though joint/bone lesions predominantly occurred at a later age as well, there was no significant correlation between macroscopic and/or histologic bone lesions and the presence of mycoplasma (Table 2). Most isolates showed high sequence identities with uncultured Mycoplasma sp. from geese of ambiguous relevance for turkeys. Swabs taken from the duodenum revealed E. coli in 29.23% (95% CI [0.22, 0.37]) and Candida (C.) albicans in 24.62% (95% CI [0.18, 0.33]) of hens. There was no significant correlation between the detection of C. albicans or E. coli and GL. The median of the results for quantitative analyses of the commensal E. coli in the jejunoileum from each examination time varied between 180 and 110.000 CFU/mL. There was no statistically significant correlation between E. coli counts and GL. The most diverse bacterial culture was present in the ceca. Slow-growing thermophilic and microaerobic Campylobacter sp. were detected with a total prevalence of 84.62% (95% CI [0.78, 0.90]). Furthermore, Clostridium (C.) perfringens was commonly isolated, with a prevalence of 52.31% (95% CI [0.44, 0.61]). Brachyspira were found with a prevalence of 13.04% (n = 46; 95% CI [0.03, 0.23]). This includes B. innocens in 50.00%, B. pilosicoli in 16.67%, and brachyspira, which could not be further typified in the remaining positive samples. There was no significant correlation between the detection of Campylobacter sp., C. perfringens, or Brachyspira spp. and GL discoloration (Table 2). It must be noted that four examination trials had problems with clostridial infection during the fattening phase, followed by antimicrobial treatment. Samples were taken at specific time points in which the infection may have resolved despite the persistence of inflammation. Therefore, limited detection of bacterial agents may be explicable. In six out of 27 pooled samples from three flocks, HEV was detected irrespective of vaccination status. In addition, turkey adenovirus 4 (TAdV-4) from two flocks in the late fattening period and turkey adenovirus 5 (TAdV-5) from two flocks in the early fattening period were detected. Avian orthoreoviruses or avian hepatitis E virus RNA were not detected in any pooled sample. Hens from flocks with a positive finding of HEV in the pooled sample revealed GL significantly more often (p ≤ 0.001, φ = 0.35) than those without virus detection. Furthermore, hens from positive tested flocks more frequently showed a predominantly slight or moderate catarrhal duodenitis (p ≤ 0.001, φ = 0.47) at the early fattening stage (Table 1 and Table 2). In contrast, there was no significant association between the finding of GL and the detection of TAdV-4 or TAdV-5 in the pooled sample at this stage. At the late examination date, hens with HEV-positive samples (7.91 kg, variance = 1.03) showed a significantly (p ≤ 0.001, r = 0.59) lower median BW compared to hens without virus detection (9.20 kg, variance = 1.14). Intranuclear inclusion bodies and splenic lymphocytic depletion are characteristics of a pathologically manifest infection with HEV [49]. These alterations were found in five hens in the early fattening period in the second examination trial from flock No. 31. These hens were not vaccinated against HEV but tested virus positive, and one of these hens concurrently had a GL. One key finding of HEV is the eponymous hemorrhagic enteritis, which is primarily localized in the duodenum [49]. In this study, hens with HEV detection only exhibited slight catarrhal duodenitis, indicating a certain effect on intestinal health, nonetheless. Immunization against HEV is usually part of the routine vaccination regimen for turkeys in Germany. Therefore, we compared the obtained HEV samples with the avirulent turkey HEV strain Virginia (Dindoral® SPF, Merial GmbH, Hallbergmoos, Germany; NCBI acc. No.: AY849321) used for vaccination in Germany [50]. The HEV samples showed high sequence identity (98.98–100.00%) at nucleotide levels at partial ORF1, E3, and fiber knob compared to the listed sequence of the avirulent turkey HEV strain Virginia. This is emphasized by the high sequence identity (98.62–100.00%) at amino acid levels (Tables S2 and S3). The phylogenetic analysis revealed the presence of two main clusters. Isolates from flock 31 cluster close to the Virginia avirulent vaccine strain (Dindoral® SPF) as well as the Virginia virulent strain isolated from turkeys with clinical HE. Isolates from flocks 21 and 22 cluster next to the commercial Oralvax HE vaccine strain listed at NCBI (Figure 1, Table S4). Based on the present phylogenetic analysis, it can be assumed that all isolates cluster close to other field and vaccine strains described in the literature [39,51]. Turkey adenoviruses are known to be widespread in Germany, with particularly avirulent vaccine strains circulating between different turkey flocks. However, knowledge of types and disease conditions is lacking [38,52]. Possible reasons for the contact with the virus could be insufficient cleaning between herds during the rearing or fattening phase or indirect transmission of the virus through employees, insects, rodents, or equipment. Clinical manifestations of HEV infection in flock 31 can be explained by the genetic drift of the circulating HE field and the vaccine strains through which strains gain virulence. However, infection with a HE-isolate does not necessarily lead to a clinical outbreak but can still increase susceptibility to opportunistic pathogens [53]. Therefore, it is necessary to use vaccines that are adapted to the currently circulating strains [15]. Examined turkey flocks were assigned to five subgroups to clarify the potential impact of HEV-vaccination and HEV- or TAdV-5 infection in the early fattening period (flock virus-positive at the first examination time point). The groups were as follows: (a) hens unvaccinated against HEV with detection of TAdV-5; (b) hens unvaccinated against HEV with detection of HEV; (c) HEV-vaccinated hens with detection of HEV; (d) HEV-vaccinated hens without AdV-detection; and (e) hens unvaccinated against HEV without AdV-detection (Table 3). The highest mean mortality rate was seen in the subgroup of HEV-unvaccinated, positive hens. This group also showed the highest prevalence of GL in the early and late fattening periods. Most of the histological bone lesions occurred in groups with either HEV- or TAdV-5-detection for both examination times. At the early fattening period, unvaccinated hens with HEV detection showed the highest prevalence of catarrhal duodenitis. Most of the histological bone lesions were also found during the late fattening period in this group. The mean BW of the hens chosen for further examination did not differ significantly between the five subgroups at the early fattening stage. The same calculations were performed for the relative spleen and liver weights. Although there were no statistically significant differences regarding the relative liver weight, the highest BW proportion at the early fattening stage was found within the group of HEV-unvaccinated and HEV-positive hens. In addition, the mean relative spleen weight at this stage differed significantly between the five groups (p ≤ 0.001, η2 = 0.32). A Tukey post-hoc analysis revealed a significantly higher relative spleen weight (p ≤ 0.05) for unvaccinated hens with HEV detection compared to the other groups. At the early fattening stage, hens of the HEV-unvaccinated but infected subgroup most often harbored C. perfringens (100.00% of the hens) and H. meleagridis (58.82% of the hens, 95% CI: [0.36–0.79]). Regarding the detection of HEV, a virally mediated long-term adverse impact can be implied based on the reduced fattening performance at the end of the fattening stage. Overall development of flocks with HEV detection that had not been vaccinated against HEV was worse compared to those that were either HEV-infected but vaccinated or unvaccinated against HEV and AdV-negative. Furthermore, a poor health condition can be presumed due to high prevalences of GL in HEV-unvaccinated but HEV-positive flocks, indicating liver damage; high relative spleen weights, indicating an immunogenic reaction; the presence of inclusion bodies and splenic lymphatic depletion as a pathological side effect of natural HEV infection; and finally, high mortalities. Confirmation is given when assessing the development of HEV-positive and HEV-unvaccinated hens with a high prevalence of GL in addition to joint/bone lesions at the late fattening period. Since the isolates originate from unvaccinated hens infected with a presumably mutated vaccine or field strain, this emphasizes the role of HEV as a predisposing factor for GL and the importance of an adequate vaccination. The causative etiology of GL cannot be restricted to one single pathogen. Statistical calculations partially need to be interpreted carefully due to the comparatively low prevalence of GL. Despite the fact that only one hen had a simultaneous S. aureus infection, an initial bacterial etiology for the other hens could not be excluded in this study. Furthermore, antimicrobial treatment during the fattening phase could be an explanation for the limited detection of bacterial agents. Bacterial or viral infections determine the general health condition and should be managed adequately to avoid potentially higher GL prevalences. Discoloration should be considered an indicator of a negative impact on individual health status and animal welfare that also entails economic damage. This can be assumed due to decreased weight gain, high relative liver weights, the presence of inflammatory cells in the liver, increased prevalence for catarrhal duodenitis and joint/bone lesions, and a tendency for higher flock mortality in the presence of GL. Furthermore, HEV as an immunosuppressive agent correlated significantly with GL, facilitated secondary infections, increased flock mortality, and led to impaired physical development. This is particularly evident when the hens were not vaccinated against this viral pathogen, which emphasizes the need for proper management of this virus. This study indicates two different pathogeneses for GL with a correlation with immunosuppressive HEV at the early and bone/joint lesions at the late fattening stage. However, the results of this study may be coincidental because other important factors, such as husbandry practices or climate, were not evaluated. Further standardized investigations to determine and evaluate possible infectious risk factors are necessary. This study expanded the knowledge about green liver discoloration in fattening turkeys, nevertheless.

PMC10000107

Alessandra Iannuzzi,Leopoldo Iannuzzi,Pietro Parma

Molecular Cytogenetics in Domestic Bovids: A Review

06-03-2023

animal cytogenetics,cattle,river buffalo,sheep,goat,FISH mapping,PCR

Simple Summary Molecular cytogenetics, and particularly the use of fluorescence in situ hybridization (FISH), has allowed deeper investigation of the chromosomes of domestic animals in order to: (a) create physical maps of specific DNA sequences on chromosome regions; (b) use specific chromosome markers to confirm the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchor radiation hybrid and genetic maps to specific chromosome regions; (d) better compare related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) study meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better show conserved or lost DNA sequences in chromosome abnormalities; (g) use informatic and genomic reconstructions, in addition to CGH arrays in related species, to predict conserved or lost chromosome regions; and (h) study some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications. Abstract The discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility focused the interest of many scientific groups on using chromosome banding techniques to reveal chromosome abnormalities and verify their effects on fertility in domestic animals. At the same time, comparative banding studies among various species of domestic or wild animals were found useful for delineating chromosome evolution among species. The advent of molecular cytogenetics, particularly the use of fluorescence in situ hybridization (FISH), has allowed a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of the chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, to predict conserved or lost chromosome regions in related species; and (h) the study of some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications.

Molecular Cytogenetics in Domestic Bovids: A Review Molecular cytogenetics, and particularly the use of fluorescence in situ hybridization (FISH), has allowed deeper investigation of the chromosomes of domestic animals in order to: (a) create physical maps of specific DNA sequences on chromosome regions; (b) use specific chromosome markers to confirm the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchor radiation hybrid and genetic maps to specific chromosome regions; (d) better compare related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) study meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better show conserved or lost DNA sequences in chromosome abnormalities; (g) use informatic and genomic reconstructions, in addition to CGH arrays in related species, to predict conserved or lost chromosome regions; and (h) study some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications. The discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility focused the interest of many scientific groups on using chromosome banding techniques to reveal chromosome abnormalities and verify their effects on fertility in domestic animals. At the same time, comparative banding studies among various species of domestic or wild animals were found useful for delineating chromosome evolution among species. The advent of molecular cytogenetics, particularly the use of fluorescence in situ hybridization (FISH), has allowed a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of the chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, to predict conserved or lost chromosome regions in related species; and (h) the study of some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications. The application of cytogenetics to domestic animals emerged about 60 years ago with the study of normal stained chromosome preparations from some cases of domestic animals with reproductive defects [1,2,3]. However, the discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 [4,5] and the demonstration of its deleterious effects on fertility [6,7] was what piqued the interest of many scientific groups and focused their attention on studying the chromosomes of domestic animals. This approach was particularly useful for selecting bulls to be used for artificial insemination, as it could avoid the transmission of chromosome abnormalities (i.e., rob1;29) from bull carriers to their progeny. Evolutionary studies also benefitted from advancements beyond normal chromosome staining. Among the various studies, the most important was the study of the Bovidae family by Wurster and Benirske [8], who looked at the diploid number and shape of chromosomes. They concluded that while the diploid number varies from 38 to 60 among all bovid species, the number of chromosome arms (Fundamental Number = NF) varies only between 58 and 62, with three exceptions; therefore, they hypothesized a high degree of autosome arm conservation among all bovid species. This hypothesis was later confirmed with the application of chromosome banding techniques [9], which ushered in a new era of chromosome studies in various domestic animal species, allowing (a) the establishment of standard karyotypes of the most important domestic species as a point of reference for various applications; (b) better characterization and identification of the chromosomes involved in chromosome abnormalities of domestic animals [10], particularly domestic bovids [11], pigs [12], horses [13], and dogs [14]; (c) the study of the chromosome homologies between related and unrelated species [15,16,17]; and (d) the study of chromosome fragility in animals exposed in vivo or in vitro to particular mutagens [18,19]. The molecular cytogenetics, particularly the introduction of fluorescence in situ hybridization (FISH), offered a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid (RH) and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities or aneuploidies in both oocytes and embryos; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities by CGH (comparative genomic hybridization) or SNP (single-nucleotide polymorphism) arrays; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, for the prediction of conserved or lost chromosome regions in related species; and (h) the study of chromosome abnormalities and genomic stability using PCR (polymerase chain reaction). This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with particular emphasis on FISH mapping applications. The FISH mapping technique is based on two main principles: the target and the probe. The target can be a whole chromosome (or chromosome arms) or a specific chromosome region. The probe is prepared according to the size of the target and is typically: (a) cDNA (generally applied when the target gene is a multi-copy); (b) cosmids with DNA insert sizes of 20–40 kb; (c) bacterial artificial chromosomes (BACs) with DNA insert sizes of 100–300 kb; (d) yeast artificial chromosome (YAC) clones (these are actually not used because they have a low cloning efficiency and show a high level of chimerism); (e) chromosome painting probes (obtained by cell sorter or chromosome microdissection techniques) that can visualize parts of or entire chromosomes; and (f) CGH arrays to check for genomic gains or losses. The probes are labeled directly with fluorochromes or indirectly with molecules that bind to the probe via fluorochrome-conjugated antibodies. The probe is specific for the target, based on complementary DNA base pairing, which allows the fluorescence-labeled probes to hybridize and form specific fluorescent signals on specific chromosome regions. The advent of the fluorescence in situ hybridization (FISH) technique, initially applied to human chromosomes [20,21], noticeably expanded cytogenetics research and investigations applied to domestic animals due to the possibility of revealing specific chromosome regions, entire chromosomes, or chromosome arms according to the choice of probe. One of the great advantages of the FISH technique is that it can be applied to interphase cell nuclei, meiotic preparations (sperm and oocytes), embryos, and elongated chromatin fibers, in addition to metaphase chromosomes, thereby allowing more complete cytogenetic investigations of animal cells. The following sections describe the main uses of FISH in domestic bovids. The first study to apply FISH for the precise identification of the chromosomes involved in a chromosome abnormality was published by Gallagher et al. [22], who discovered an X-autosome translocation (X;23) using both Q-banding and a BoLA Class I cDNA probe. The probe shows hybridization signals to the normal chromosome 23 and to the translocated autosomal material present on the X chromosome, allowing a more precise localization of MHC (major histocompatibility complex) in cattle than was achieved earlier by genetic mapping. Several subsequent studies also applied FISH to obtain better confirmation of the chromosome(s) involved in abnormalities (especially when banding was poor) and identification of the break points, especially in reciprocal translocations. Table 1 shows the main studies that applied FISH mapping, either alone or in combination with other classical cytogenetic techniques (e.g., C-banding, G-banding, R-banding, and Ag-NORs), to study the chromosome abnormalities of domestic bovids in somatic cells at the metaphase (Figure 1) or interphase nuclei of germinal cells, such as sperm and oocytes, or embryos at different cell stages. A more complete classification of all chromosome abnormalities studied by classical cytogenetic techniques alone or (in some cases) with other molecular cytogenetic techniques is provided by Iannuzzi et al. [11]. Two examples of the importance of the use of FISH for the correct identification of the chromosomes involved in chromosome abnormalities of cattle were a case of autosome trisomy and two types of Robertsonian translocations. A case of autosome trisomy 28 in an abnormal calf, revealed by both R-banding and FISH mapping with a specific molecular marker [33], was identified, and the same abnormality was reported earlier as trisomy 22 using only the banding technique [86]. Two robs earlier reported as rob (4;8) [87] and rob (25;27) [88] in cattle were later corrected as rob (6;8) and rob (26;29), respectively, using C-, G-, and R-banding and FISH mapping with specific molecular markers and the use of HSA painting probes [28]. Table 1 shows that FISH mapping applications were used for the diagnosis of chromosome abnormalities in both metaphase (the majority) and interphase cells, the latter applied to lymphocyte nuclei (Figure 2), sperm (Figure 3), oocytes, and embryos. Concerning the studies on meiotic preparations, those performed on the synaptonemal complexes (SCs), especially in spermatocytes, were particularly important for establishing the regularity of the pairing processes during the pachytene substage of meiotic prophase in animals carrying chromosome abnormalities (reviewed in [89]). Recent analyses of meiotic preparations have been performed using immune fluorescence approaches and have provided more detailed information on SCs [90,91,92]. Other studies have addressed the fragile sites in the chromosomes of domestic animals (reviewed by [93]), and limited studies have used CGH and SNP arrays to establish possible genomic losses occurring during chromosome rearrangements (Table 1). FISH mapping was also very important for the definitive establishment of the agreement between various chromosome nomenclatures due to some discrepancies found during the Reading conference [94] and the subsequent ISCNDA1989 [95] (the inverted position between BTA4 and BTA6, as well as the correct position of BTA25, BTA27, and BTA29). This aspect was vital for the clinical cytogenetics of domestic bovids, as it allowed a correct identification of the chromosomes involved in chromosome abnormalities. During the Texas conference [96], specific molecular markers (only type I loci) were selected for each bovine syntenic group and each cattle chromosome based on previous standard chromosome nomenclatures. The next advance was the application of FISH mapping by two labs that used 31 selected BAC clones (from the Texas Conference) on RBG- and QBH-banded cattle preparations [97]. The chromosome-banding homologies among bovids (cattle, sheep, goats, and river buffalo) were then used to establish a definitive standard chromosome nomenclature for the main domestic bovid species [98]. Subsequent studies using FISH mapping and the same Texas markers on river buffalo, sheep, and goat R-banded chromosomes [99,100] definitively confirmed the chromosome homologies among domestic bovids, as established at the ISCNDB2000 [98]. The identification of the DNA structure [101] paved the way for the development of in situ hybridization technology. In the early stages of its development, this technology allowed the localization of genes using radioactive probes [102]. It was also used in studies of domestic animals [103,104], but the greatest diffusion of the physical mapping of genes awaited the development of fluorescent probes [105]. At that moment, we entered the golden years of gene mapping, and domestic animals were not excluded. One of the first examples was the localization of bovine alpha and beta interferon genes [106], and this localization was rapidly replicated in buffalos, goats, and sheep [107,108]. Subsequently, many other localizations were obtained using this technology (Figure 4). Considering the practical impossibility of compiling a complete list of all gene localizations obtained using this technology, some significant examples are listed in Table 2. Localization sometimes involved a single gene [124,129] or a family of genes [132]. Other reports, however, mapped many genomic markers [100,141]. A point to remember is that FISH technology has significantly benefited from the availability of BAC genomic libraries—elements that represent the ideal source for the construction of the probes. Among these, the INRA library [144] and the CHORI-240 have played relevant roles. The publication of genomes [145,146,147,148] has since inevitably diminished interest in using this technology for mapping genetic factors, although genetic factor mapping continued for species whose genomes were sequenced later, such as the water buffalo [149]. However, this technology has proved useful in several aspects, including: a) the identification of errors in genomic assembly [150]; b) the refinement of genome assembly [151]; and c) the mapping of sequences not included in genomic assemblages [152]. Clearly, the interest today is very limited in locating a genetic factor in a species whose genomic sequence is available, but this does not mean that FISH technology is no longer indispensable for solving other problems related to the organization of genomes. The mapping of genomic elements by FISH has also been used successfully for the physical mapping of data obtained by other technologies. The first examples concerned the physical anchoring of a genetic map to a chromosome [153,154,155] and the mapping of a synteny group to a specific chromosome [114]. Subsequent examples of the combined use of FISH and genetic maps followed [127,156]. Two main methods have been applied thus far to obtain a FISH mapping comparison between related and unrelated species: Zoo-FISH, which uses chromosome painting probes, and FISH mapping, which uses specific molecular markers of both type I and type II. Zoo-FISH is a molecular technique that provides an easier comparison between related and unrelated species from a macro point of view. The term was first reported by [157], based on earlier studies that used genomic chromosome painting probes, obtained by cell sorter chromosomes, to compare related species [158,159,160]. Zoo-FISH was first applied in domestic animals when human chromosome painting probes became commercially available. This approach demonstrated the conservation of several human chromosome segments in both domestic bovids (Table 3) and other domestic species (reviewed in [161]). The use of human-chromosome painting probes allowed the identification of a substantial number of human chromosome segments (around 50) in bovid chromosomes [175,176,217,218,219]. Zoo-FISH has also been applied to correctly identify some chromosomes involved in the chromosome abnormalities shown in Table 1. The availability of specific painting probes obtained by both cell sorting and/or by the microdissection of specific chromosomes (or chromosome arms) from domestic animals extended these studies to investigations between related species (Table 3). For example, in cattle, Zoo-FISH was applied to study X-Y aneuploidy in sperm [55] and in oocytes [58] (Table 1). An interesting approach was demonstrated in two studies characterizing two cases of goat/sheep [220] and donkey/zebra [221] hybrids using multicolor FISH (M-FISH), starting from painting probes obtained from microdissected river buffalo chromosomes (or chromosome arms) and from flow-sorted donkey chromosomes, respectively. Chromosome painting probes allow the delineation of large, conserved chromosome regions between related and unrelated species, as reported above. The use of comparative FISH mapping using several chromosome markers to map a single type I or type II locus along the chromosomes allows a more accurate establishment of the gene order within chromosome regions, thereby confirming that chromosome rearrangements occurred to differentiate related or unrelated species in key evolutionary studies (Table 3). These detailed comparisons have confirmed a high degree of autosome (or chromosome arm) conservation among all bovid species. The main autosome difference found thus far in bovids was a chromosome translocation of a proximal chromosome region from Bovinae chromosome 9 to Caprinae chromosome 14, as demonstrated by both chromosome banding and, in particular, by a molecular marker (COL9A1) mapping proximal to Bovinae chromosome 9 and proximal to Caprinae chromosome 14 (reviewed in [9]). This translocation involved a genome region of about 13 MB and was followed by an inversion in Caprinae chromosome 14, as demonstrated earlier [213]. This chromosome event was common to all remaining Bovidae subfamilies, leading to the conclusion that the Bovinae subfamily is an ancestor to the remaining Bovidae subfamilies (reviewed in [9]). In contrast to autosomes, sex chromosomes are differentiated by more complex chromosome rearrangements. Indeed, the Caprinae X chromosome (as for all remaining X chromosomes of the other Bovidae subfamilies) is differentiated from the ancestor Bovinae X (very probably a large acrocentric chromosome, such as that of the water buffalo) by at least three chromosome transpositions and one inversion (reviewed in [9]). Detailed FISH mapping data are also useful for better anchoring of both genetic and RH maps [203,222,223,224]. The availability of detailed cytogenetic maps in bovid species allowed a better comparison of the bovid and human chromosomes, especially using type I loci. These comparisons facilitated the translation of genomic information from the human genome to the genomes of domestic animals, especially in those with no genome sequencing available. These comparisons also revealed a very high number of chromosome rearrangements that differentiate bovid species from humans. Indeed, the conservation of entire chromosomes or large regions of them between bovid and human chromosomes, as revealed by Zoo-FISH, was the result of complex chromosome rearrangements that differentiated human and bovid species according to their gene order. An example is presented in Figure 5 which illustrates the comparison of FISH mapping between HSA2q and BTA2. As seen, when utilizing the Zoo-FISH technique with the HSA2q painting probe, almost all BTA2 is painted [217], indicating a high degree of chromosome conservation between the chromosomes of the two species. By conducting the same comparison using comparative FISH mapping and examining the gene order along the chromosomes of the two species, we observe a distinct gene order between the two species, thus revealing complex chromosome rearrangements that differentiated the chromosomes of the two species during their evolution. The various FISH mapping techniques developed for human cytogenetics (reviewed by [225]) include SKY-FISH (spectral karyotyping FISH), Q-FISH (quantitative FISH), M-FISH (multicolor FISH), heterochromatin-M-FISH, COBRA-FISH (combined binary ratio labeling FISH), cenM-FISH (centromere-specific M-FISH), and fiber-FISH. Among these techniques, only fiber-FISH and M-FISH have been applied to domestic bovids. The use of fiber-FISH yields high-resolution maps of chromosomal regions and related genes on a single DNA fiber. This approach establishes the physical location of DNA probes with a resolution of 1000 bp. It is particularly useful for detecting gene duplications, gaps, and variations in the nuclear genome. The DNA fibers are obtained from nucleated cells by releasing the DNA fibers from the nucleus, stretching them mechanically, and then fixing them on slides [226] (Figure 6). Table 4 summarizes the studies that have used this technique in domestic bovids. The CGH array technology, an evolution of in situ comparative genomic hybridization (CGH), is a method of cytogenetic investigation that emerged in the 1990s to overcome the limitations of common banding cytogenetic analyses, especially those involving the presence of genomic imbalances, such as duplications or deletions [231,232]. In situ CGH technology has many similarities to FISH: the support used is the same, i.e., denatured metaphases fixed on slides and the approaches to label the probes are identical. However, in this case, the probes are produced using complete genomic DNA deriving from two subjects: typically, one healthy and one relating to the subject being investigated. The two DNAs are labeled with two different fluorochromes and then hybridized simultaneously on the slide. In the hybridization phase, a competition is therefore created between the probes, and in the presence of a normal chromosomal segment, an intermediate color is obtained, while in the presence of chromosomal alterations, a fluorescence closer to one of the two colors used is obtained. Although this technology has been widely used and has provided important results, its major limitation lies in the resolution. CGH array technology follows the same principle, but the support is no longer represented by slides but by synthetic DNA fixed on slides. Initially, the chips for CGH array analyses contained DNA extracted from BAC to provide as uniform a representation of the genome as possible [233]. Current CGH array analyses are performed using devices containing oligonucleotides chosen that uniformly cover the whole genome and achieve resolutions of 5–10 kb [234,235]. More information about this technology and its use is provided by [236]. In species of zootechnical interest, CGH array analyses (Figure 7) became common following the appearance of the first commercial arrays, and these analyses are conducted essentially for two purposes: the identification of copy number variation (CNV) polymorphisms and the characterization of chromosome anomalies. CNVs are polymorphic variations present very frequently in the genomes of higher organisms [237,238,239]. In humans, approximately 4.8–9.7% of the genome contains CNVs [240]. The introduction of commercial arrays has allowed the use of this technology to obtain a great amount of information about the distribution of CNVs in species differences and how these variations are related to phenotypic traits. The transfer of this technology to the animal field and the availability of commercial arrays has led to the publication of several reports (Table 5). The publication of animal genomes [145,146,147,148,149,250] has made available a very large series of data that required the development of sophisticated analysis techniques and often required the use of computers with large processing capacities. The first bio-informatic analyses were used to assemble thousands of short genomic sequences, produced by modern high-throughput sequencing technologies, into genomes. Today, most of these programs are available free of charge through web pages that function as interfaces between the user and calculation tools [251]. Currently, dozens of bio-informatics programs are available to analyze the data contained in genomic assemblies, and many of these are accessible through various web platforms. Making a complete list is very complicated, in part because this is a rapidly evolving discipline that introduces, almost daily, new analytical tools. The genomic sequences produced by the various assemblies can be visualized using one of the available websites available, including Genome Data viewer [252], UCSC Genome Browser [253], and Ensembl [254]. Currently, these websites provide the ability to view and process data relating to several genome assemblies (Table 6). These genome viewers are constantly evolving and contain several tools within them that allow the user to obtain highly relevant genetic data and information. This includes, but is not limited to, the possibility of: (a) identifying the structure of genetic factors (in terms of exon–intron boundaries); (b) identifying SNP polymorphisms in a particular region of the genome; (c) identifying the position of BACs by mapping the BES (Bac Ends Sequences, particularly useful when the user wants to choose the BACs to use in FISH analysis); (d) observing the genomic regions expressed in particular types of tissues; (e) analyzing the relationships between different assemblies of the same species; (f) visualizing the relationships between similar regions in different species (comparative genomics); and (g) viewing the repeating regions. In this review, we do not specify a best genome viewer, as this will often depend on personal needs and experience. However, as each genome viewer has its own specific analysis tools, sometimes the best solution is to use all three to obtain more complete information. The availability of genomic assemblages has, on the one hand, limited the interest in the physical mapping of genomic elements, but has, on the other hand, allowed the evolution of a very large number of genetic and genomic analyses. Probably one of the most common uses (even if not directly related to cytogenetics) is to design primers for use in PCR amplifications. This operation can be performed using different software, both available for free and for a fee. Among those available free of charge, the most frequently used is Primer3 [255]. The availability of genomic assemblages also makes rapid evolutionary investigation possible (i.e., visualizing, in a simple and rapid way, the similarities that exist between the various genomic regions of different species). The publication of genomes has certainly had a great impact on cytogenetics (both negatively and positively). If the golden era of gene mapping has ended, the possibility of rapidly identifying BACs for use as probes in FISH experiments has certainly provided great benefits to cytogenetics, as it avoids long and tedious testing of BAC libraries. This aspect has allowed the rapid characterization of some chromosomal anomalies, such as a centromere repositioning event in cattle [66], a reciprocal translocation, also in cattle [62], and cryptic evolutionary rearrangements between cattle and sheep [213]. Finally, the rapid localization of BACs on genomes has allowed the development of complex approaches for the identification of chromosomal abnormalities, which are also difficult to identify [71]. Obviously, these are not all the possible uses of genomic assemblies, but they represent the best examples in relation to cytogenetics. Each genomic assembly contains substantial information that can be used for very specific purposes and avoids the need for probes that would be complex to synthesize. The continuous evolution of these data analysis tools creates difficulty in any attempt to compile their possible uses. Simultaneously with the publication of the genomes, bio-informatics tools were developed for the analysis of the vast amount of data generated—data that are characterized by both their great variety and their large quantity. One of the main repositories of tools for analyzing genomic data is Galaxy [251]. This repository provides access to bio-informatic analysis tools, which are constantly updated. SNP variations represent the major source of variation in genomes, and the genomes of the species covered in this review are no exception. Currently, identifying these sources of variation is quite simple (through modern high-throughput sequencing techniques at ever-lower cost), but this does not characterize the effect that these variations can cause. For this scenario, the variant effect predictor (VEP available on the Ensembl website) software is helpful [256]. Without a doubt, BACs represent one of the most useful tools for molecular cytogenetics, and, as previously mentioned, their identification in genomes is currently greatly facilitated. However, the current situation would not be possible without the existence of two important institutions that have dedicated part of their activities to the construction, maintenance, and distribution of BAC libraries: the BACPAC Resources Center (BPRC, https://bacpacresources.org/ (accessed on 2 March 2023)) and INRA (http://abridge.inra.fr/index.php?option=com_flexicontent&view=item&cid=17&id=61&Itemid=202&lang=fr (accessed on 2 March 2023)). Through these two institutes, BACs belonging to different libraries can be obtained. In recent years, the decreasing costs of sequencing have made it possible to analyze many subjects. The purposes of these sequencings are different; in many cases, the aim is the identification of signatures of selection [257,258,259], but other purposes are represented, such as: (a) the identification of genetic variants in specific genes [260]; (b) the verification of data obtained regarding the identification of SNPs with chip arrays [261]; (c) the identification of the run of homozygosity in breeds intended for different productions [262]; (d) prediction and QTL mapping [263]; and (e) the identification of copy number variants [264] and transcriptome characterization [265]. Similar analyses were performed on sheep [266,267] and goats [268,269]. Additionally, in this case, the water buffalo seems to be slightly behind, as there are very few papers available on it [265]. The polymerase chain reaction (PCR) [270] is a method largely used to make millions of copies of a specific DNA sample in a fast and economical way for the detection, quantification, and typing of infectious diseases and genetic changes. Current PCR-based methods are distinguished as: (a) first-generation PCR, (b) second-generation quantitative PCR (qPCR), and (c) third-generation droplet-based digital PCR (dPCR). PCR detects endpoint, qualitative, or semi-quantitative assays by gel electrophoresis, separating DNA fragments according to size. The qPCR measures DNA/RNA in real time using PCR methods, fluorescent dyes, and fluorometry for relative quantification and quantitative assays with standard curves. The dPCR splits a PCR sample labeled with fluorescent dye into millions of microsamples to digitize the pool of DNA molecules with a single or no copy in each droplet. It quantifies the DNA/RNA copy number faster than qPCR based on standard curves [271]. In recent years, PCR-based methods have replaced the classic cytogenetic techniques for detecting chromosome abnormalities and aneuploidy due to greater precision, lower cost, and faster data than are possible with cytogenetic methods, because of the small quantities of DNA (30 ng) required from any stored or fresh biological samples. PCR-based approaches are most commonly used in bovid studies to examine sex chromosomes in early-sex-determination assays to detect aberrations (Table 7). Telomere assessment is another critical goal of cytogenetics research due to the central roles of telomeres in chromosome stability, aging, cancer development, apoptosis, and senescence. The telomeres consist of thousands of noncoding repetitive sequences of DNA composed of six nucleotide motifs (TTAGGG)n localized at the ends of chromosomes and are responsible for maintaining DNA integrity during each cell division. They are associated with several proteins, with the most abundant being the shelterin complex, which is made up of six different polypeptides. Telomeres also contain other genomic structures, such as T-loops, D-loops, G-quadruplexes (G4), R-loops, and long noncoding RNA (TERRA) [286]. In farm animals, telomere length (TL) did not receive much interest initially due to the difficulty in determining the natural limits of their lifespans. However, a recent study related TL to health, genome stability, and aging in cattle aged between 2 and 13 years and transformed TL into a sensitive biomarker for longevity and wellness (critical traits of selective breeding), responding to the “One Health” approach (improving animal welfare) [287]. TL is not often used as a unique marker of aging in humans because of its poor predictive accuracy due to increased telomere shortening in elderly humans as a consequence of age-related diseases (e.g., cancer, atherosclerosis, autoimmune disorders, obesity, chronic obstructive pulmonary disease, diabetes, hematological disorders, and neurodegenerative diseases) [288]. By contrast, TL proved to be a relevant biomarker of the general state of farm animals due to their lack of age-related pathologies [289,290]. Approaches for measuring TL include: (a) telomere restriction fragment (TRF) length [291]; (b) length analysis by Southern blotting; (c) fluorescent in situ hybridization (FISH) by flow cytometry (flow-FISH) or in metaphase cells (Q-FISH) [292,293]; and (d) PCR-based methods. Most of these methods have several limitations. For example, TRF and flow-FISH are labor-intensive and expensive; Southern blot analysis requires large amounts of genomic DNA, and Q-FISH works only on chromosomes (metaphase stage). Of the available methods, the PCR-based ones are the fastest, most recent, and least costly and require only small quantities of DNA (30 ng) from stored or fresh biological samples [294]. The qPCR method amplifies telomere repeats relative to a single-copy gene (reference gene) according to a method described by Cawthon et al. [295] and follows the MIQE guidelines [296]. One limitation of qPCR is the inconsistent repeatability and reproducibility of different TL measurement methods, producing a high variation in results [297]. Several studies on humans and animals indicated that the DNA extraction method might affect TL measurements using q-PCR, as DNA yields were higher using the non-silica membrane kit (salting-out method), and DNA integrity on electrophoresis gels varied [298,299]. A recent study showed comparable results for DNA quality and purity (tested using a NanoDrop instrument and electrophoresis gels) in cattle blood and milk samples using two different extraction kits (a salting-out kit for blood and a silica membrane kit for milk samples) due to the difficulty of extracting DNA from milk matrices. The DNA quality results were similar in both matrices, demonstrating a synchronous trend between them for the first time [287]. Molecular cytogenetics is approaching its first 30 years of history and during this period, it performed important functions that evolved over time. It therefore seems normal that in the coming years, we will witness further developments; however, some approaches will always be current and irreplaceable. The FISH technology represents, and will represent, the main methodology for the verification of chromosomal anomalies eventually identified with other approaches, just as the CGH array technology that will be increasingly used for the identification of genomic variants linked to a particular phenotype. Molecular cytogenetics could be very useful for the study of those species which have not yet benefited from the genomic revolution, or which are still in its early stages: in this sense, the water buffalo (Bubalus bubalis) is the main example. Despite possessing a great economic importance, its genome has been decrypted and made available only recently, and the application of other technologies is very late. A further gap that can be filled is the development of a technological approach that can allow the identification of all chromosomal types identifiable by cytogenetic analyses. A similar approach has already been published [71], but only the transfer of SKY-FISH technologies [300] from humans to bovids will bridge this gap. Finally, the certain decrease in costs will mean that even the species considered in this review will be able to benefit from long-read genomic sequencing, such as PacBio [301] and Oxford Nanopore [302]. The study of the chromosomes of domestic bovids is about to enter its seventh decade, and, as expected, it has undergone a notable evolution along the way. This evolutionary process for this discipline is mainly a result of the appearance of technologies that have significantly increased the potential of applied cytogenetics. Banding techniques, FISH, CGH arrays, and PCR have radically changed animal cytogenetics, making them irreplaceable tools for understanding the genetics of bred animals. Therefore, considering the history of cytogenetics, a quite easy prediction is that even the next evolutions will be dictated by technological advances. Predicting the next technological leap is difficult, but if we were to make a prediction, it would be that long-read genomic sequencing technologies will have important impacts on cytogenetics. Cytogenetics will likely retain its functionality, particularly in the confirmation of genomic results and the characterization of cytogenetic anomalies, as well as in evolutionary studies. This is because the most significant genetic mutations have accumulated at the chromosome level during the evolution of species. Finally, the implication and progresses from animal cytogenetics can be summarized as follows: In the pre-genomic era, FISH technology represented the almost exclusive technology available for the localization of genes in genomes. Prior to the availability of low-cost genomic sequencing, molecular cytogenetics was the only approach for identifying similarities between karyotypes of different species. The technologies of molecular cytogenetics represent the best approach for the characterization of chromosomal abnormalities. Despite scientific progress in similar disciplines, molecular cytogenetics will always find its place and represent an inescapable investigation methodology.

PMC10000111

Katarzyna Perz,Sebastian Andrzej Kaczmarek,Sebastian Nowaczewski,Aaron Joell Cowieson,Marcin Hejdysz

The Effect of Exogenous Amylase Supplementation on the Nutritional Value of Pea (Pisum sativum L.) for Broiler Chickens

23-02-2023

pea,amylase,nutritional value,digestibility,poultry

Simple Summary Pea (Pisum sativum L.) can be successfully grown and harvested in almost all the climatic zones across the world. Currently, the main protein source that is used in animal nutrition is soybean meal (SBM). Pea seeds are characterized by a relatively high content of crude protein and starch; therefore, they can serve as a potential energy source when included in feed. However, a portion of starch present in pea seeds is defined as resistant starch (RS). Previous studies have shown that the extrusion process significantly reduced the content of RS; therefore, enzyme supplementation may be more economically advantageous because it is known as an efficient method to improve the digestibility of nutrients. Amylase can be highly beneficial for pea seeds due to the presence of a relatively high starch concentration. However, reports regarding the use of amylase alone in diets based on pea seeds are limited. The present study hypothesized that exogenous amylase can exhibit a positive influence on the nutritional value of pea seeds when included in the feed of broiler chickens. Abstract The present study aimed to investigate whether the exogenous addition of amylase enhances the nutritional value of pea seeds for broiler chickens. In total, 84 1-day-old male broiler chickens (Ross 308) were used for the experimental study. During the first phase of the experiment (1–16 d), all birds in each treatment were fed with a corn–soybean meal reference diet. After this time, the first treatment (control) was still fed the reference diet. In the second and third treatment, 50% of the reference diet was replaced with 50% pea seeds. In addition, the third treatment was supplemented with exogenous amylase. Animal excreta were collected on 21 d and 22 d of the experiment. The birds were sacrificed at the end of the experiment (23 d), and samples of ileum content were collected. The experimental results showed that the exogenous addition of amylase significantly improved (p < 0.05) the apparent ileal digestibility (AID) of the crude protein (CP), starch, and dry matter (DM) of pea. In addition, an improvement in the AID of essential amino acids in pea seeds (except Phe) was observed. The trend in the AMEN values was also noted (p = 0.076). It can be concluded that supplementation with exogenous amylase improves the nutritional value of pea seeds in broiler chicken nutrition.

The Effect of Exogenous Amylase Supplementation on the Nutritional Value of Pea (Pisum sativum L.) for Broiler Chickens Pea (Pisum sativum L.) can be successfully grown and harvested in almost all the climatic zones across the world. Currently, the main protein source that is used in animal nutrition is soybean meal (SBM). Pea seeds are characterized by a relatively high content of crude protein and starch; therefore, they can serve as a potential energy source when included in feed. However, a portion of starch present in pea seeds is defined as resistant starch (RS). Previous studies have shown that the extrusion process significantly reduced the content of RS; therefore, enzyme supplementation may be more economically advantageous because it is known as an efficient method to improve the digestibility of nutrients. Amylase can be highly beneficial for pea seeds due to the presence of a relatively high starch concentration. However, reports regarding the use of amylase alone in diets based on pea seeds are limited. The present study hypothesized that exogenous amylase can exhibit a positive influence on the nutritional value of pea seeds when included in the feed of broiler chickens. The present study aimed to investigate whether the exogenous addition of amylase enhances the nutritional value of pea seeds for broiler chickens. In total, 84 1-day-old male broiler chickens (Ross 308) were used for the experimental study. During the first phase of the experiment (1–16 d), all birds in each treatment were fed with a corn–soybean meal reference diet. After this time, the first treatment (control) was still fed the reference diet. In the second and third treatment, 50% of the reference diet was replaced with 50% pea seeds. In addition, the third treatment was supplemented with exogenous amylase. Animal excreta were collected on 21 d and 22 d of the experiment. The birds were sacrificed at the end of the experiment (23 d), and samples of ileum content were collected. The experimental results showed that the exogenous addition of amylase significantly improved (p < 0.05) the apparent ileal digestibility (AID) of the crude protein (CP), starch, and dry matter (DM) of pea. In addition, an improvement in the AID of essential amino acids in pea seeds (except Phe) was observed. The trend in the AMEN values was also noted (p = 0.076). It can be concluded that supplementation with exogenous amylase improves the nutritional value of pea seeds in broiler chicken nutrition. Pea (Pisum sativum L.) can be successfully grown and harvested in almost all the climatic zones across the world. Currently, the main protein source that is used in animal nutrition is soybean meal (SBM). However, alternative protein sources obtained particularly from non-GMO plants have been the subject of interest for many authors conducting studies in poultry nutrition [1,2,3]. Pea seeds are characterized by a relatively high content of crude protein and starch, and they can serve as a potential energy source when included in feed. It has been reported that starch is the most important energy source for poultry [4], and starch widely determines the apparent metabolizable energy (AMEN) value. Starch is usually digested by the endogenous enzymes, but the efficiency of digestion depends on many factors, such as growth phase [5], composition of diet, and the chemical structure of starch [6]. A small portion of starch present in pea seeds is defined as resistant starch (RS), which, depending on the subtype, is harder to digest or not digested at all [7]. Recently, several methods have been explored to enhance the nutritional value of pea seed meals. Technological processes are very effective because they induce changes in the molecular structure of starch. Previous studies have shown that the extrusion process significantly reduced the content of RS in pea seeds [8], improving the digestibility of starch, CP, and, crude fat in broiler chickens [2]. However, these techniques are expensive and led to increases in the cost of production. Compared with this process, enzyme supplementation may be more economically advantageous. Amylase can be highly beneficial for pea seeds because of the presence of a relatively high starch concentration. However, reports regarding the use of amylase alone in diets based on pea seeds are limited. Earlier studies demonstrated the positive impact of amylase when used in soybean–corn diets. Some authors showed that the addition of amylase improves growth performance [9], the digestibility of starch and CP [9,10], and AMEN value [5,11] in broiler chickens. In contrast, Kaczmarek et al. (2014) [12] showed that amylase supplementation in a corn–soybean meal diet did not cause any impact on starch and CP digestibility, or on AMEN values. Based on the above-mentioned results and due to the lack of information about the effects of amylase supplementation when added to a diet based on pea, the present study hypothesized that exogenous amylase could exhibit a positive influence on the nutritional value of pea seeds when included in the feed of broiler chicken. Moreover, the amount of excreted sialic acid will allow for the estimation of endogenous losses [13] after amylase supplementation. The aim of the present study was to determine the influence of amylase on nutrient digestibility, energy value, and the excretion of sialic acid in pea-seed-based diets for broiler chickens. All animal procedures were conducted in accordance with the guidelines proposed by the Polish Council of Animal Care [14]. Pea seeds The samples of pea seeds (Pisum sativum L.) cv. Mentor were obtained from the Polish Plant Breeding Station located in Tulce, and harvested in 2019 in Poland. The seeds were initially ground by using a hammer mill (RG11 model; Zuptor, Gostyn, Poland) with a screen size of 2.0 mm. The chemical composition and the antinutritional factors regarding the concentration of pea are shown in Table 1. This experiment was conducted in a completely randomized design with a control group and two experimental groups, which differed by the addition of exogenous α-amylase. The study was conducted on 84 1-day-old male broiler chickens (Ross 308) with an initial individual weight of 42 ± 2 g, which were obtained from a Polish hatchery (Dan Hutch, Wolsztyn, Poland). Each group was composed of 14 replicates, with 2 birds in each replicate. All the environmental conditions were controlled and kept constant and were in agreement with the recommendations of AVIAGEN [15]. During the first 7 days of the experiment, the temperature was maintained at about 33 °C and then gradually reduced to 23 °C. The chickens were exposed to light for 24 h during the first 7 days, and then the time of light exposure was gradually reduced to 16 h. Birds were kept in metabolic cages in one common room, and collection trays were installed in every cage. Access to the feed and water was ad libitum during the entire experiment. All diets were prepared according to AVIAGEN recommendations [15], such that they meet the nutritional needs of the chickens. The chemical composition and the nutritional value of the reference diet (control) are presented in Table 2. Birds in treatment 1 (control) were fed with a reference diet from 1 d to 16 d of the experiment. Birds in treatment 2 and treatment 3 were fed with the same reference diet from 1 d to 16 d of the experiment, and after this period, 50% of the reference diet was replaced with pea seed. The third treatment was supplemented with exogenous amylase, according to the dosage recommended by the manufacturer of the enzyme (0.14 g/kg; 80 KNU/kg as fed). The enzyme was obtained from a commercial source (Rononzyme HiStarch; DSM Nutrition, Heerlen, The Netherlands). Titanium dioxide (3 g/kg as fed) was added to each diet in each treatment, which serves as a nonabsorbable marker to determine the digestibility coefficients and AMEN value. For excreta collection, collection trays were installed under each cage, and excreta were collected thrice a day at 21 d and 22 d of experiment. One sample was collected from each replicate group from each treatment. Hence, the total number of samples collected was 14. At the end of the experiment (23 d), all the birds were slaughtered by cervical dislocation. The ileum was removed and the ileal digesta (15 cm, adjacent to the ileocecal junction) was acquired as samples for further analysis. To ensure that a sufficient amount of material was available for chemical analysis, digesta samples of two birds were combined to form a single sample, and thus the total number of samples obtained was 14. Immediately after sample collection, each sample was frozen and then lyophilized. All samples were ground and passed through a 0.5 mm sieve. Chemical analysis of pea seeds (ran in duplicate) was carried out to determine the amounts of DM, CP, ether extract, crude ash, neutral detergent fiber (NDF), aNDF (NDF with heat-stable amylase and expressed inclusive of residual ash), and acid detergent fiber (ADF; expressed inclusive of residual ash), which was performed according to AOAC standard methods 934.01, 976.05, 920.39, 984.27, 973.18, and, 942.05, respectively [16]. These methods were used to determine the content of DM and CP in digesta and excreta. The assay kit (Megazyme International, Dublin, Ireland) was prepared using thermostable α-amylase and amyloglucosidase according to the standard guidelines of AOAC [16] using method 996.11. This method was used to determine the starch content in pea seeds, diet formulations, and digesta. RS assay kit (Megazyme International, Wicklow, Ireland) was prepared according to the method 2002.02 [16], with a slight modification in the time of incubation, as presented by Weurding et al. (2001) [17], and was used for the determination of the content of RS in pea seeds. Gross energy was calculated in pea seeds, feed, and excreta by using an adiabatic bomb calorimeter (KL 12 Mn; Precyzja-Bit PPHU, Bydgoszcz, Poland) standardized with benzoic acid. The tannins were evaluated according to the method described by Kuhla and Ebmeier (1981) [18]. Phytates were determined according to the method presented by Haug and Lantzsch (1983) [19]. RFOs were determined using a high-resolution gas chromatography technique according to the method suggested by Zalewski et al. (2001) [20]. Phosphorus was determined using the colorimetric method with ammonium molybdate, and calcium by the spectrophotometric method using a split AES Agilent instrument. Non-starch polysaccharides (NSP) in pea seeds were determined by using gas–liquid chromatography-neutral sugars, according to method presented by Englyst and Cummings (1988) [21], with modifications presented by Slominski and Campbell (1990) [22]. Colorimetry method was used for uronic acids, according to method presented by Scott (1979) [23]. Postcolumn derivatization using ninhydrin reagent (according to method 994.12) [16] was performed on an AAA-400 Automatic Amino Acid Analyzer (INGOS s.r.o., Prague, Czech Republic) to determine the amino acid content in pea seeds, experimental diets, and digesta samples. The concentrations of titanium dioxide in diet formulations, digesta, and excreta samples were determined by using the method described by Short et al. (1996) [24], and each sample was prepared according to the procedure presented by Myers et al. (2004) [25]. The excreted free and total sialic acid content was evaluated according to the method presented by Jourdian et al. (1971) [26] after extracting crude mucins from excreta [27]. The determination of WEV was carried out as follows: 1 g of sample was mixed with 5 mL distilled water for 1 h at 40 °C and was centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was withdrawn and viscosity was determined in a Brookfield Digital DV-II+ cone/plate viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA, USA) maintained at 40 °C at a shear rate of 12 × s−1. WEV units are mPas·s = cP = 1 × 100 dyne s cm2. Apparent ileal digestibility (AID) values in reference diet (treatment 1) for DM, CP, starch, and amino acids were calculated from the ratio of titanium dioxide concentration in the diet to its concentration in digesta (indigestible marker method) by using the following formula (with starch (S) as an example): where S represents the content of starch and TiO2 is the dietary marker. The AID values of DM, CP, starch, and amino acids in pea seeds (treatment 2 and 3) were determined (difference method) using the following formula: where AIDStarch—digestibility of starch; AIDStarch diet—digestibility coefficient of starch in diet; CStarch diet—concentration of starch in diet; 0.50—amount of investigated pea seeds in diet; AIDStarch reference—digestibility coefficient of starch in reference diet; and CStarch reference—concentration of starch in reference diet. The difference method approach was used to calculate the AMEN and apparent ileal digestibility coefficient of various dietary components contained in pea. The AMEN value was corrected to zero nitrogen balance using 34.4 kJ/g of N retained [28] and was calculated by using the following equation (treatment 1): where GE represents the gross energy (kcal/kg), N represents nitrogen, and TiO2 is the dietary marker. The AMEN value for treatment 2 and 3 was estimated using the formula: where 0.50 is the proportion of the investigated pea seeds in the pea diet and 0.50 is the proportion of the reference diet in the pea diet. The obtained data were explored to discard any possible outliers. The experimental results were analyzed by using statistical software SAS, (2012). Pooled standard error of mean and group mean values were calculated. Significant differences between means values of the factors investigated in this study were estimated by performing a t-test at a significance level of p ≤ 0.05. The following formula was used: where t-Student’s t-test value is the difference in the means between the two groups being compared; are variance estimates from each independent group; and are the respective sample sizes for each independent group. In this study, the standard error of the mean (SEM) was established as a measure of error. The overall mortality was found to be low (<2%) and was not attributed to any specific dietary treatment (data not shown). All research procedures were conducted according to EU directives, and the protocol for this study was approved by the Local Ethics and Animal Experimentation Committee at Poznan University of Life Science. The effect of enzyme addition on the AID values in pea seeds of DM, CP, and starch is presented in Table 3. The addition of exogenous amylase significantly increased the AID value of DM (from 0.691 to 0.730; p < 0.05). A similar result was observed for the AID value of CP, from 0.800 to 0.864. Additionally, the AID value of starch was significantly higher from 0.852 to 0.886 (p < 0.05). No significant differences in the AMEN values were observed between supplemented and non-supplemented diets. However, a tendency was observed (p = 0.076). No significant differences in the amount of excreted total and free sialic acids were observed as a result of amylase addition (Table 3). The effect of amylase supplementation on the AID of amino acids is presented in Table 3. The addition of amylase significantly increased the AID of almost all essential amino acids (except Phe; p < 0.05). A maximum improvement in AID (8% increase) was observed for Thr and Ile. Additionally, a high (7.00%) increase was noted for Lys and Val. In the case of the non-essential amino acids of pea seeds, a significant difference was observed only for Pro, an almost 12.00 % improvement (p < 0.05). Pea seeds are considered to be an important source of protein. The CP concentration was found to be in the reported range of about 208.0–276.0 g/kg as fed depending on the cultivar or type of pea (white or colored flower) [29]. The concentration of starch was found to be higher than the value presented by another study [29]. The content of resistant starch was low (136.6 g/kg as fed) when compared with that observed in a previous study [2]. The pea seeds are a good source of essential amino acids, such as Leu, Lys, and Arg [30,31]. The main RFO found in the pea seeds was stachyose, which was also found by Hejdysz et al. (2015) [29]. The total concentration of NSP was much lower than in data presented by others authors [32]. Indeed, the influence of the cultivar should be distinguished among the factors influencing the concentration of nutrients, as well as antinutritional factors. Amylase is an enzyme that specifically degrades starch, and supplementation with amylase can have a positive impact on the growth performance of broiler chickens [11], which can be attributed to higher AMEN value. No significant differences were noted between non-supplemented and supplemented pea seeds for AMEN value in the present study. However, there was a tendency (p = 0.076), which is likely related to enhanced starch digestibility. The lack of significant difference in AMEN, and at the same time an important difference in the AID of starch, was interesting. However, we can speculate that RS in the group without the enzyme was fermented in the caeca and, due to this fact, the energy value level was close between groups. It has been earlier confirmed that fiber and CP also contribute to energy value measurements, although the apparent digestibility of energy alone does not provide a complete response regarding the efficacy of enzyme use [10]. In this study, the addition of amylase significantly increased the AID of starch by 8.00% (p < 0.05), similar to the results of Gracia et al. (2003) [9] and Amerah et al. (2017) [10]. The crucial factor that determines the level of digestion is the ratio of amylose to amylopectin, and increasing the ratio of these two compounds reduced starch digestibility. Gunawardena et al. (2010) [33] reported that the ratio of amylose to amylopectin in pea seeds was 31:69, due to which pea native starch is classified as type C. C-type starches do not undergo hydrolysis easily unlike A-type starch, which is found in corn. Since type-A starch is more susceptible to degradation, greater digestibility of this compound is observed in corn-based diets. In addition, a previous study by Amerah et al. (2017) [10] reported that exogenous amylase increased starch digestibility by about 1.12%, while an improvement of 8.00% was noted in this study. However, the level of starch digestibility was found to be much higher in the above-mentioned study. Therefore, it may be concluded that the overall starch digestibility will always be higher with type-A starch. However, it should be noted that the addition of exogenous amylase could also provide good results with type-C starch. Additionally, since exogenous amylase was able to degrade RS effectively, a higher digestibility was observed. On the other hand, Stefanello et al. (2015) [11] found the positive effect of amylase supplementation on the AMEN value in conventional diets. However, Kaczmarek et al. (2014) [12] and Amerah et al. (2017) [10] did not observe any increase in the AMEN value when amylase was supplemented in conventional diets. It can be assumed that many factors could affect the AMEN value, such as the source and the type of starch present in the feed, the different components used in the diet, and the techniques used for the processing of raw materials. However, the crucial factors that determine the AMEN value could be the concentration of ANF and RS. The addition of exogenous amylase significantly increased (p < 0.05) the AID of DM and CP by about 5.64% and 3.99% in pea seeds, respectively. An improvement in the AID of DM is the result of an overall improvement in the AID of starch and CP. Gracia et al. (2003) [9] and Amerah et al. (2017) [10] also reported an improvement in the digestibility of CP in studies where conventional diets based on corn and SBM were used. Starch granules contain almost 3 g of protein per 1 kg of starch [7]. As a result of the hydrolysis of starch glycosidic bonds, the portion of protein molecules that were involved in the formation of complexes with starch are now be available for further digestion processes. Another factor that may contribute to improvements in the AID of CP is the fact that the addition of amylase could, by increasing the digestibility of starch and RS, have an effect on the viscosity of the digesta. This could have resulted in changes in the passage time of digesta through the gastrointestinal tract, thus prolonging the time for the action of endogenous proteases. An increase in the concentration of exogenous amylase in diets used for chickens resulted in the lower production and activity of endogenous amylase [34]. In addition, Jiang et al. (2008) [35] showed that amylase supplementation reduces the mRNA expression of amylase. The results of the present study indicate an increasing in the AID of almost all essential amino acids (except Phe). We can assume that a reduction in the production of endogenous amylase can be attributed to the enhanced AID of amino acids. The probable mechanism can be due to the reduced utilization of amino acids for the production of amylase by the pancreas. However, this finding should be confirmed in future studies. As mentioned earlier, starch binds to specific domains of the amylase enzyme. The cleavage of glycosidic bonds in the presence of amylase increases the availability of the substrate to enzymatic hydrolysis by proteases, which further leads to an increase in the digestibility of amino acids. Liu et al. (2020) [36] reported that increasing the concentration of RS decreases the digestibility of CP. Amylase supplementation could effectively decrease the levels of RS in the feed. Schramm et al. (2021) [37] reported that amylase addition increased the digestibility of RS. Therefore, we can conclude that a reduction in the levels of RS resulted in an increase in the digestibility of CP and amino acids. A previous study indicated that sialic acid excretion was lower after the extrusion process, which can be associated with lower levels of RS [8]. Additionally, sialic acid can be considered to be an indicator of endogenous losses [13]. However, no effect of amylase supplementation on sialic acid secretion was observed in the present study, which indicates that ANF concentration, rather than RS concentration, is a strong determiner of this parameter; however, this requires confirmation. In conclusion, the addition of exogenous amylase enhances the apparent ileal digestibility of DM, starch, and CP in pea seeds. In addition, the significantly higher apparent ileal digestibility of almost all essential amino acids was observed. The tendency to obtain a higher AMEN value was also observed. Therefore, exogenous amylase can improve the nutritional value of pea and affects the nutrients and amino acid utilization in broiler chicken nutrition.

PMC10000112

Rafał Osiecki,Mieszko Kozikowski,Beata Sarecka-Hujar,Michał Pyzlak,Jakub Dobruch

Prostate Cancer Morphologies: Cribriform Pattern and Intraductal Carcinoma Relations to Adverse Pathological and Clinical Outcomes—Systematic Review and Meta-Analysis

21-02-2023

prostate cancer,cribriform pattern,intraductal carcinoma,meta-analysis,radical prostatectomy

Simple Summary Prostate cancer is one of the most common male cancers. A more accurate disease assessment is needed to better stratify patients’ risks and guide treatment decisions. It has already been studied that some prostate cancer submorphologies are associated with worse outcomes. We performed a systematic review and meta-analysis on the impact of distinct prostate cancer morphologies: the cribriform pattern and intraductal carcinoma on adverse pathological and clinical outcomes after radical prostatectomy. Our results showed that the cribriform pattern together with intraductal carcinoma are negative prognostic factors associated with both adverse clinical and pathological outcomes in the radical prostatectomy cohort, and the presence of those patterns should be implemented in the surgical planning and postoperative treatment guidance. Abstract The present study aimed to assess the association between the cribriform pattern (CP)/intraductal carcinoma (IDC) and the adverse pathological and clinical outcomes in the radical prostatectomy (RP) cohort. A systematic search was performed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis statement (PRISMA). The protocol from this review was registered on the PROSPERO platform. We searched PubMed®, the Cochrane Library and EM-BASE® up to the 30th of April 2022. The outcomes of interest were the extraprostatic extension (EPE), seminal vesicle invasion (SVI), lymph node metastasis (LNS met), risk of biochemical recurrence (BCR), distant metastasis (MET) and disease-specific death (DSD). As a result, we identified 16 studies with 164 296 patients. A total of 13 studies containing 3254 RP patients were eligible for the meta-analysis. The CP/IDC was associated with adverse outcomes, including EPE (pooled OR = 2.55, 95%CI 1.23–5.26), SVI (pooled OR = 4.27, 95%CI 1.90–9.64), LNs met (pooled OR = 6.47, 95%CI 3.76–11.14), BCR (pooled OR = 5.09, 95%CI 2.23–11.62) and MET/DSD (pooled OR = 9.84, 95%CI 2.75–35.20, p < 0.001). In conclusion, the CP/IDC belong to highly malignant prostate cancer patterns which have a negative impact on both the pathological and clinical outcomes. The presence of the CP/IDC should be included in the surgical planning and postoperative treatment guidance.

Prostate Cancer Morphologies: Cribriform Pattern and Intraductal Carcinoma Relations to Adverse Pathological and Clinical Outcomes—Systematic Review and Meta-Analysis Prostate cancer is one of the most common male cancers. A more accurate disease assessment is needed to better stratify patients’ risks and guide treatment decisions. It has already been studied that some prostate cancer submorphologies are associated with worse outcomes. We performed a systematic review and meta-analysis on the impact of distinct prostate cancer morphologies: the cribriform pattern and intraductal carcinoma on adverse pathological and clinical outcomes after radical prostatectomy. Our results showed that the cribriform pattern together with intraductal carcinoma are negative prognostic factors associated with both adverse clinical and pathological outcomes in the radical prostatectomy cohort, and the presence of those patterns should be implemented in the surgical planning and postoperative treatment guidance. The present study aimed to assess the association between the cribriform pattern (CP)/intraductal carcinoma (IDC) and the adverse pathological and clinical outcomes in the radical prostatectomy (RP) cohort. A systematic search was performed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis statement (PRISMA). The protocol from this review was registered on the PROSPERO platform. We searched PubMed®, the Cochrane Library and EM-BASE® up to the 30th of April 2022. The outcomes of interest were the extraprostatic extension (EPE), seminal vesicle invasion (SVI), lymph node metastasis (LNS met), risk of biochemical recurrence (BCR), distant metastasis (MET) and disease-specific death (DSD). As a result, we identified 16 studies with 164 296 patients. A total of 13 studies containing 3254 RP patients were eligible for the meta-analysis. The CP/IDC was associated with adverse outcomes, including EPE (pooled OR = 2.55, 95%CI 1.23–5.26), SVI (pooled OR = 4.27, 95%CI 1.90–9.64), LNs met (pooled OR = 6.47, 95%CI 3.76–11.14), BCR (pooled OR = 5.09, 95%CI 2.23–11.62) and MET/DSD (pooled OR = 9.84, 95%CI 2.75–35.20, p < 0.001). In conclusion, the CP/IDC belong to highly malignant prostate cancer patterns which have a negative impact on both the pathological and clinical outcomes. The presence of the CP/IDC should be included in the surgical planning and postoperative treatment guidance. Prostate cancer (PCa) is the second most common male malignancy worldwide [1]. The introduction of the prostate-specific antigen (PSA) at the end of the 1980s has revolutionized both the diagnosis and the management of the disease. The subsequent years brought a constant decline in prostate cancer-specific mortality in the US [2]. At the same time, numbers of indolent PCa soared and resembled overdiagnosis. It was followed by overtreatment. The mainstay therapy for localized PCa remains either a radical prostatectomy (RP) or radiotherapy. Unfortunately, the two modalities are widely known to be associated with significant morbidity. Therefore, the protocols of active surveillance (AS) aiming at postponing the treatment were eagerly implemented across the world [3]. AS oncological safety has been shown in many studies, and it has become the pivotal therapy of low-risk disease. The success has encouraged a number of investigators to explore the role of AS in intermediate-risk PCa and expand the indications above the lowest risk category. To avoid harm, potential predictive markers of the AS outcome in this specific group of PCa patients are currently under scrutiny. The major advantage of RP is the postoperative histopathological assessment that provides the highest possible accuracy of the staging and grading of PCa. A detailed report embraces valuable information regarding a patient’s prognosis which aids in postoperative clinical decision making. The pathological features in the RP specimen that have a confirmed impact on the treatment outcome include the cancer grade and stage, positive surgical margins (PSM), and lymph node metastasis (LNs met) [4,5,6]. In addition to the prognosticators, there are other emerging pathological features that appear to compromise the oncological outcomes. The 2014 International Society of Urological Pathology Consensus Conference on the Gleason Grading of Prostatic Carcinoma stated that the cribriform pattern (CP) should be considered part of the spectrum of the Gleason Grade 4 pattern along with glomeruloid, fused and poorly formed glands [7]. Previous studies indicate its association with the extraprostatic extension (EPE), PSM, biochemical recurrence (BCR), distant metastasis (MET) and disease-specific death (DSD). Another pathological PCa entity that may have a dismal prognosis is intraductal carcinoma (IDC). In 2006, Guo and Epstein published the most commonly used morphological description of IDC [8]. The WHO Classification of Prostatic Tumors in 2016, for the first time, recognized IDC as a new entity and provided a detailed histopathological description of it. [9]. The incidence of IDC in an RP specimen ranges from 20–40%, depending on the tumor grade and stage [10,11]. The studies conducted so far associate the presence of IDC with the Gleason pattern (GP) 4 and 5, a more advanced tumor stage, an increased risk of BCR and shorter MET-free survival [12,13,14,15,16,17]. From a pathological point of view, IDC and the CP show architectural similarities and, as a result, can sometimes be misinterpreted. It has been confirmed that they might coexist in the same tumor [18,19]. Therefore, in equivocal cases, additional immunohistochemical staining for basal cells is recommended. Despite that, sometimes it is impossible to distinguish between the two patterns. In the present study, we aimed to investigate the correlation of both the CP and IDC with pathological as well as clinical outcomes by performing a systematic review and meta-analysis of the published data. In the present study, only articles with pathological assessment based on the 2005 or 2014 ISUP guidelines were analyzed. The 2005 ISUP guidelines recommended CP to be graded as GP4 but allowed some cribriform glands to be included as GP3 [20]. Among various changes that the 2014 ISUP conference brought to the pathological assessment of the RP specimen, one of the most important was the recommendation to include all cribriform glands as one of the GP4 spectrum morphologies [7]. However, until 2021, there was no uniform definition of CP. Nonetheless, a study by Kweldam et al. [21] proved that CP had good interobserver reproducibility among pathologists, unlike other Gleason 4 patterns, such as ill-formed or fused glands. To further improve the pathological assessment of CP, in 2021, ISUP provided consensus definition of CP. Currently, CP is defined as a confluent sheet of contiguous malignant epithelial cells with multiple glandular lumina that are easily visible at low power. There should be no intervening stroma or mucin separating individual or fused glandular structures [22]. In 2016, WHO recognized IDC as a new entity, not included in the Gleason classification, and defined it as an intra-acinar and/or intraductal neoplastic epithelial proliferation that has some features of high-grade prostatic intraepithelial neoplasia (HGPIN), exhibiting much greater architectural and/or cytological atypia, typically associated with high-grade and/or high-stage PCa [9]. The 2022 EAU Prostate Cancer Guidelines require reporting of both patterns in prostate biopsy and RP specimens. These guidelines, however, do not specify whether immunohistochemical staining is demanded to distinguish those entities [3]. Even the absence of basal cells cannot be considered pathognomonic for invasive CP as they may not be visible on a particular slide [18]. CP and IDC can morphologically mimic each other and even belong to a pathological and biological continuum [23,24]. Kryvenko et al. [25] found that the frequency of IDC was related to the proportion of CP. Kweldam et al. [19] reported that CP and IDC tend to coexist in both biopsy and RP specimens. Therefore, we decided to assess both entities CP and IDC together. This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines [26]. The protocol was registered in PROSPERO (CRD42020183346). The research question for this systematic review was established in the PICO (population, intervention, comparison and outcomes) framework, which was as follows: What is the prognostic value of PCa patterns: CP/IDC for clinical and pathological outcomes in men who underwent radical prostatectomy? A thorough literature search was conducted, PubMed®, the Cochrane Library and EMBASE® databases, with the following keywords: (“prostatic neoplasms” OR “prostate cancer” OR “pca” OR “prostate” OR “prostatic”) AND (“intraductal” OR “intraductal cancer” OR “intraductal carcinoma” OR “IDC” OR “cribriform” OR “cribriforming”). The search was restricted to English language publications and included every article fulfilling the inclusion criteria from the inception of the databases until 30 April 2022. The Mendeley Desktop version 1.19.4 citation manager was used to store records and remove duplicates. For completeness, references to incorporated articles and review articles were also extensively searched. Two investigators (R.O. and M.K.) independently screened and assessed article eligibility based on their titles and abstracts, adopting the PICO approach. Any disagreements were resolved by discussion with a senior author (J.D.). Eligible were original studies with a cohort of patients with PCa, whose initial treatment modality was RP (surgical approach was not considered). Additional inclusion criteria were a comparison of adverse pathological/clinical outcomes between PCa with and without CP/IDC. Both investigators extracted data from full-text publications and cross-checked values. In the case of multiple reports of the same cohort, the most complete data were selected. We excluded articles not in English, reviews, editorials, case reports and animal or in vitro studies. Additionally, references where CP/IDC was evaluated in a prostate biopsy, or where data were not stratified, or the authors were not able to extract data by the presence of CP/IDC, or the record did not provide data on prognosis/adverse outcome were also excluded. The selection process is shown in Figure 1. Data extracted from eligible studies and study characteristics are presented in Supplementary Table S1. Despite direct e-mail contact with authors, it was not possible to obtain more precise data from 3 studies [17,27,28]. The adverse outcomes reported in the analyzed studies included EPE, SVI, LNs met, BCR, local recurrence (LR), clinical recurrence (CR), MET, DSD, cancer-specific survival (CSS), overall survival (OS). The PSM as an outcome measure was excluded from this systematic review and meta-analysis because it does not necessarily confer non-organ-confined disease and may result from surgical flaws, postsurgical tissue handling or misinterpretation. Quantitative analysis was performed wherever data for calculations were available. The assessment of the risk of bias in the studies included in the meta-analysis was conducted with the Newcastle–Ottawa Quality Assessment Scale (NOS), which was developed to assess the risk of bias in observational studies [29]. We decided to dismiss the response rate in the exposure domain in case-control studies as this subdomain was not applicable to this article. Each study could receive a maximum of 8 stars for case-control studies and 9 stars for cohort studies. Two investigators (R.O. and M.K.) independently evaluated each article, any disagreement was discussed with and resolved by the third senior investigator (J.D.). Observational comparative studies with scores of <4, 4–6 and >6 were considered, having a high, intermediate and low risk of bias, respectively. According to the NOS, 11 studies were considered high quality and 2 were evaluated as intermediate quality (Supplementary Tables S2 and S3). Statistical analyses were performed with the use of Review Manager software (RevMan version 5.4 Cochrane, UK) and StatsDirect 3 link software (version 3.3.5; StatsDirect Ltd. Wirral, UK). The strength of associations between CP/IDC and pathological as well as clinical outcomes were analyzed by a calculation of the pooled odds ratios (OR) with 95% confidence interval (CI). The degree of heterogeneity between included studies was estimated with the I2 test which describes the proportion of variance (from 0% to 100%). The random effects method (DerSimonian–Laird; REM) was used to calculate the pooled OR with the 95% CI. The obtained results were summarized in tables as well as illustrated using forest plots. Potential publication bias was analyzed with Egger’s regression and Begg’s rank correlation tests for all comparisons. In addition, sensitivity analyses were made by sequential exclusion of each study to evaluate the stability and in turn reliability of the results. The article selection process was conducted according to the PRISMA statement, and a total of 1798 references were identified. Of 16 studies included in the systematic review, 13 articles provided sufficient data for a quantitative evaluation. The studies included in this systematic review were published between 2011 and 2020. The systematic review encompassed 164,296 patients. The sample sizes varied significantly among the studies, ranging from 28 to 159,777 participants. It is noteworthy that cohorts from two studies partially overlapped, but the endpoint measures were different; therefore, both studies were included and assessed separately [30,31]. The statistical analysis was based on 3254 patients; of those, 1405 had the CP/IDC morphology (43%). All the included studies were retrospective (16/16). Most of them were case-control studies (14/16), two of which were designed as paired and nested paired case-control studies [25,32]. Additionally, there was one population-based study and one cohort study [13,33]. Eight studies focused on the CP [28,30,31,32,33,34,35,36], whereas five evaluated IDC [10,13,14,17,25]. Three studies allowed for a combined assessment of both patterns [27,37,38]. The pathological evaluation of resected specimens was conducted according to the ISUP 2005 criteria in six studies [25,27,30,31,32,39]. The ISUP 2014 Consensus Conference Working Group criteria were adopted by the authors of six studies [14,17,28,34,37,38]. In the study by Kweldam et al. [36], the adopted histopathological criteria differed depending on the time of the RP. Two articles did not include detailed information regarding a pathological assessment [10,33]. In the study by Dinerman et al. [13], the data on the histopathological evaluation were not provided. Two studies investigated the large (LC)/expansile CP (EC) [27,33]. According to the 2021 WHO recommendation, the CP should be reported without subtypes. Therefore, we considered the LC as the CP. Luo et al. [28] focused on invasive cribriform lesions (ICLs), which we also included as the CP. In the studies which focused on IDC, this pattern was defined using the Guo and Epstein criteria in four studies [14,25,27,38]. Kato et al. [17] adopted the McNeal and Yemoto definition of IDC. Miyai et al. [10] defined IDC as one or both of the following patterns: (1) solid or dense cribriform (less than 50% lumen in a duct) intraductal lesions or (2) loose cribriform (50% and more than 50% lumen in a duct) or micropapillary intraductal lesions with prominent nuclear pleomorphism (nuclear size greater than 6× normal) and/or non-focal comedonecrosis. In the study by Hollemans et al. [37], IDC was identified if the cribriform structures were clearly continuous with the pre-existing glands lined by normal basal epithelium or containing corpora amylacea. The presence or absence of the CP/IDC in the evaluated specimen was reassessed by at least two specialized genitourinary pathologists in 62.5% of the studies (10/16). Luo et al. [28] stated only that the slides were reviewed for the purpose of the study. In two other studies, the diagnosis of the CP/IDC was extracted from pathology reports [10,33]. In the remaining articles, this information was either not disclosed or there was a single reviewer [17,30]. Dinerman et al. [13] extracted data on the presence of IDC from the SEER database. In equivocal cases, the authors of nine papers used immunohistochemical staining (IHC) to distinguish IDC from the CP or HGPIN [10,14,27,28,30,31,36,37,38], whereas the authors of three papers relied solely on the cancer cell morphology [32,33,34]. In four articles, the information regarding the use of immunohistochemical staining was not disclosed [13,17,25,39]. The median follow-up period in the studies that assessed the BCR varied significantly from 360.5 days to 130.6 months. For the MET or DSD, the median follow-up duration also differed across the studies and ranged from 20 to 120 months. The BCR definitions varied among the studies with a serum PSA level ≥ 0.2 ng/mL being the most commonly used threshold (6/9) [10,32,33,34,37,38]. In two studies, the BCR was defined as a PSA ≥ 0.1 ng/mL (2/9) [28,31], and in one case, the definition differed depending on the period of the sample and data collection [39]. Although Kweldam et al. [36] in their study investigated the BCR, the presented data were insufficient for a quantitative analysis. The LR was investigated by two authors [14,28]. Both authors assessed either the CP or IDC in RP specimens. Trinh et al. [14] defined the LR as a peri-prostatic involvement after surgery as confirmed by imaging, biopsy and/or suspect results from a digital rectal examination (DRE), followed by exclusive radiotherapy to the prostatic bed leading to a serum PSA-level reduction without concurrent hormone therapy. Luo et al. [28] did not elaborate on the LR definition. MET was also diagnosed differently. In the studies by Kweldam et al. and Trinh et al., the diagnosis of MET required either radiological or pathological confirmation [14,36]. Dong et al. relied only on imaging and Hollemans et al. either on a biopsy or multidisciplinary consensus [37,39]. In one study, the diagnostic criteria for MET were not defined [28]. None of the authors included information regarding the imaging modality used for the confirmation of metastatic spread. Two studies provided additional value for this systematic review. Luo et al. [28] conducted a systematic review and meta-analysis on the impact of the CP in prostate biopsy/RP specimens on adverse outcomes. The studies included in the analysis by Luo and the current systematic review had some overlap, but the addition of articles evaluating IDC in the RP cohort extended the scope of the current study (see Discussion). Greenland et al. [33] assessed the DECIPHER score in case it was either positive for the CP or the glomerulation pattern. The DECIPHER assay was developed to predict the risk of metastases within 5 years after a prostatectomy and measures the expression levels of the RNA of 22 genes in the radical prostatectomy specimen. A DECIPHER score < 0.45 was interpreted as low risk, 0.45–0.6 corresponded to average risk and > 0.6 was assessed as high risk. The authors of that study emphasize that the assay was performed at the discretion of the treating physician, and patients with negative pathological risk factors were more likely to undergo genetic testing. Nonetheless, CP-positive patients were characterized by higher mean DECIPHER scores than patients with glomerulation patterns (0.61 vs. 0.47; p = 0.02; SD = 0.18, 0.17). Studies by Dinerman et al., Kato et al. and Trudel et al. were qualitatively assessed in the systematic review, but because of either different study concepts or a lack of detailed statistical data, they were disqualified from the meta-analysis (see Discussion) [13,17,27]. The correlation between the EPE and CP/IDC was analyzed in eight studies, with a total number of 903 patients with CP/IDC and 1285 without CP/IDC. Significant heterogeneity was observed between the studies (I2 89%). The random effect models analysis revealed that the EPE was significantly more common in patients with than without CP/IDC (41.3% vs. 17.7%, respectively: pooled OR = 2.55 95%CI 1.23–5.26, p = 0.01) (Figure 2). The authors of seven studies presented information regarding the relationship between SVI and the CP/IDC. In this meta-analysis, there were 788 patients with CP/IDC and 1215 without CP/IDC, respectively. SVI was significantly more common in patients with CP/IDC than without (19.9% vs. 4.9%), respectively, which resulted in a pooled OR = 4.27 (95%CI 1.90–9.64, p < 0.001) (Figure 3). Seven studies provided data for the analysis of the LNs met in correlation with the CP/ICD, with a total of 639 patients with CP/IDC and 1217 patients without CP/IDC. The LNs met were significantly more prevalent in patients positive for CP/IDC (21.6% vs. 5.8%), with a pooled OR = 6.47 (95%CI 3.76–11.14, p < 0.001) (Figure 4). Nine studies analyzed the association between the BCR and CP/IDC. The total number of patients with CP/IDC was 1023 and there were 1498 cases without CP/IDC. The random effect models analysis showed that the BCR was significantly more frequent in individuals positive for CP/IDC (32.2% vs. 8.0%), with a pooled OR = 5.09 (95%CI 2.23–11.62, p < 0.001) (Figure 5). MET/DSD as an adverse outcome was analyzed in five studies, with a total number of 581 positive for CP/IDC and 411 without CP/IDC. The random effect models analysis revealed that MET/DSD was also significantly more common in patients with CP/IDC (23.4% vs. 2.9%), which resulted in a pooled OR = 9.84 (95%CI 2.75–35.20, p < 0.001) (Figure 6). In the sensitivity analysis, no changes in the OR value were demonstrated in all the performed comparisons after excluding the subsequent studies. Therefore, the above-described analyses were considered stable and reliable. The publication bias tests indicated that there is no publication bias in the studies on the relationship between the EPE, SVI, LNs met, BCR, MET/DSD and CP/IDC. The exact results of Egger’s regression and Begg’s rank correlation tests, i.e., the intercept with a 95%CI and Kendall’s tau are shown in Supplementary Table S4. In addition, the publication bias was evaluated visually with the inspection of funnel plots, which were eventually found to be symmetric (Figure 7). We performed this systematic review and meta-analysis to investigate the impact of the CP/IDC on the adverse pathological and clinical outcomes after an RP. To the best of our knowledge, this is the first study to systematically and quantitively assess the prognostic factors of CP/IDC in the RP cohort. The results of this study indicate a relationship between the CP/IDC and unfavorable prognostic factors, such as the advanced stage of the disease, BCR, MET and DSD. The aim of a radical prostatectomy is to remove the whole prostatic gland, seminal vesicles and distal part of the vas deferens with the adjacent periprostatic tissue in select cases to ensure the best oncological outcome. Proper surgical planning includes neurovascular bundle (NVB)-sparing techniques in order to restore continence and erectile function after surgery [40]. To date, no recommendations have been released regarding the extent of the surgery in cases with CP/IDC morphology. The results of the meta-analysis clearly indicate a close relationship between the EPE/SVI and CP/IDC (OR = 2.55 and OR = 4.27, respectively). Although we decided to exclude the PSM from this systematic review and meta-analysis, as explained earlier, the PSM has an added value with regard to an oncological prognosis. The articles included in the systematic review reported inconclusive results on the association between the CP/IDC and PSM. According to Sarbay et al. [30], GG1 cases with a CP have been found to have a much higher incidence of PSM (23.1% vs. 5.1%, p < 0.011), but no such correlation was observed in patients with GG2 and 3 PCa (p < 0.331). Dong et al. [39] and Kweldam et al. [36] also did not report an association between the CP and PSM. However, in two other studies, PSMs were more commonly found after an RP in the presence of the CP [34,37]. PSMs in the presence of IDC were also investigated. In a retrospective population-based analysis, the PSM was found to be more commonly encountered in the presence of IDC (25.6% vs. 19.5%, p = 0.02) [13]. According to Trinh et al. [14], there was not any significant correlation between IDC and the PSM. Contrary to our results, Flammia et al. [41] found no correlation between the CP and the local disease stage, the SM in GG 2–5 RP patients. The EAU recommends offering NVB-sparing surgery to patients with a low risk of EPE, which is, among other factors, based on the GG [3]. GP 4 itself does not exclude the NVB-sparing approach, but those guidelines do not take into consideration the GP4 submorphologies or IDC. The results of our meta-analysis indicate a higher prevalence of both the EPE and SVI in CP/IDC cases. Therefore, in the presence of the CP/IDC, intrafascial NVB preservation may pose a risk of a PSM. We also identified the CP/IDC in the radical RP as being associated with LNs met. These results are consistent with our previous study, which investigated the clinical importance of the CP in a prostate biopsy [42]. Nodal metastases, especially in greater numbers, are a known negative prognostic factor associated with a worse BCR-free survival, MET-free and OS [43,44,45,46,47]. Unfortunately, the included studies did not provide detailed data on the extent of the LN dissection (LND) and the number of dissected LNs, which made it impossible to draw meaningful conclusions regarding the true significance of the LNs met in the presence of the CP/IDC. At the same time, especially extending the LND during an RP provides more tissue for a histopathological analysis. Detailed information on the disease stage may help guide the adjuvant treatment. The extent of the LND and the role of adjuvant therapy in the presence of the CP/IDC and other postprostatectomy negative prognostic factors have yet not been fully understood. The results of our meta-analysis clearly indicate the negative impact of the CP/IDC on the long-term oncological outcomes such as the BCR, MET and DSD and are consistent with the results presented in the literature [12,35,36]. Moreover, a meta-analysis by Luo et al. [28] also revealed the negative impact of the CP on the BCR, MET and DSD, which is understandable as the articles included by Luo et al. partially overlapped with those evaluated in our study. However, a study by Flammia et al. [41] showed that no association between the CP and BCR was found in the GG 2–5 subjected to an RP with a median follow-up of 22 months. A comprehensive systematic review and meta-analysis conducted by Miura et al. [12] on the clinical importance of IDC in localized and advanced PCa showed that in the case of localized PCa, the presence of IDC in either a biopsy or radical prostatectomy specimen was associated with worse BCR-free survival (pooled HR 2.09) and CSS ((pooled HR = 2.93). Additionally, there was a correlation between IDC and shorter OS in advanced PCa (pooled HR = 2.93). Dinerman et al. [13] found a 3-fold higher risk for DSD in the presence of IDC but reported no association between IDC and OS. BCR-free survival in more than 1000 RP patients was significantly worse in any ISUP with IDC than without in a study by Kato et al. Moreover, GG2 men with IDC had a prognosis similar to GG4 or GG5 patients without this morphology [17]. Trudel et al. [27], who investigated the BCR-free rate, concluded that in the presence of LC/IDC, the BCR occurred more commonly, and again, any GG without LC/IDC had a better prognosis. In a subgroup analysis, Kaplan–Mayer curves for the BCR-free survival were similar in men with LC and in men with LC/IDC. IDC as an isolated morphology was, on the other hand, associated with worse outcomes. Nonetheless, the authors highlighted that isolated IDC was present in only 10 men and this cohort was too small to draw meaningful conclusions [27]. Trinh et al. [48] investigated the effect of adjuvant radiotherapy (ART) on the BCR in patients with IDC and found that the worst BCR-free survival was in men positive for IDC, who did not undergo ART (log-rank p = 0.023). AS oncological safety has been shown in many studies. As a result, it has become the pivotal therapy of low-risk disease, acknowledged by many international and national urological associations [3,49]. The success has encouraged a number of investigators to explore the role of AS in intermediate-risk PCa and expand the indications above the lowest risk category. Decisions regarding AS are made based on the results of a prostate biopsy. This systematic review and meta-analysis refers to the RP histopathological assessment which is more detailed and provides the most accurate evaluation of the disease stage and grade. In our study, CP/IDC emerge as negative prognosticators in terms of both pathological and clinical outcomes after an RP. Considering our results, the negative impact of the CP/IDC should be discussed with every patient qualified for AS. This statement is supported by international urological associations. The current EAU guidelines allow AS in highly selected GG2 cases (<10% GP4, PSA< 10 ng/mL, cT2a); the identification of CP/IDC in a biopsy sample is considered as an absolute contraindication for this type of deferred treatment [3]. Similarly, the PCa guidelines provided by the America Urological Association (AUA) recognize CP/IDC in intermediate-risk patients as unfavorable characteristics and therefore do not recommend AS in those cases [49] Several factors could not be included in the meta-analysis due to insufficient data. In the systematic review by Luo et al. [28], we found that the risk of LR is more than twice as high in patients with the CP (OR 2.32). Trinh et al. [14] showed that IDC was linked to distant metastasis (mainly bone metastases) as the initial site of the CR on both univariate and multivariate analyses (OR = 6.27), but surprisingly, the time to the CR and CSS was not significantly different between men with and without IDC. A subgroup analysis revealed that radiotherapy (adjuvant or salvage) was superior to pre-RP ADT in respect to the CSS in men with IDC (170 months, 95%CI (124–215) vs. 159 months, 95%CI (122–196)). That was one of the first studies to highlight the detailed evaluation of a pathological specimen and IDC detection in terms of post-RP management. A single study included in this meta-analysis evaluating the impact of the CP on the OS found the OS in GS7 (includes both GG 2 and 3) men with CP to be shorter (log-rank p = 0.001) [36]. The malignant potential and basis for the joint assessment of CP/IDC are reflected in the genomic and epigenetic alternation observed in these histopathological patterns. Elevated SChLAP1 expression and well as genomic instability were found in PCa with CP/IDC [50]. Mehra et al. [51,52] indicated that increased SChLAP1 expression was associated with a higher risk of BCR, DSD and MET. Based on the whole slide images of the TGGA database and dataset from CPCGN, Böttcher et al. [53] found that IDC and the CP were associated with an increased genomic instability that affected specific regions: the chromosomal deletions of 3p13, 6q15, 8p21–23, 10q23, 13q14 16q21–24 and 18q21–23, and the amplification of chromosome 8q24. Those regions are known to be involved in aggressive PCa, for example, the BRCA 2 gene is located on the long arm of chromosome 13. In CP/IDC-positive cases, genomic alternations also affect the epigenetic profile. Olkhow-Mitsel et al. [54] reported that CP/IDC cases were characterized by DNA hypermethylation in the APC, RASSF1 and TBX15 genes. Hypermethylation in those genes has already been linked to more aggressive PCa [55]. The authors also suggested the utilization of methylation profiles in those genes as a marker of CP/IDC [54]. In a study by Taylor et al. [56], who investigated the correlation between the CP and IDC with the DECIPHER genomic classifier, found that the predominance of CP in RP specimens significantly increased the DECIPHER score. IDC was non-significantly associated with an increased score (OR 1.92 p = 0.24). On the other hand, the presence of CP/IDC did not reach statistical significance in terms of high-risk categorization (OR = 4.53, p = 0.08), which could be due to the small sample size (11 men) [56]. In a study by Risbridger et al. [57], which investigated patients-derived xenografts, BRCA 2 germline mutations were more common in IDC-positive men with localized PCa. In contrast to those results, Lozano et al. [58] found no association between the germline BRCA 2 mutations and CP/IDC morphology. In the same study, however, the bi-allelic BRCA2 loss in primary PCa was significantly correlated with the CP and IDC. The authors, therefore, advised against the routine germline BRCA2 testing in the presence of CP/IDC but felt that further research was needed to investigate the relevance of CP/IDC as a marker of the bi-allelic BRCA2 loss in primary PCa. The present study has several limitations. The key one is the different diagnostic criteria applied by authors for the identification of CP/IDC. Various definitions of IDC used by authors and high interobserver variation in the identification of IDC might have contributed to its under- or overdiagnosis in the selected articles. Additionally, the histopathological evaluation of the RP specimens was conducted according to either the ISUP 2005 or 2014 diagnostic criteria. Therefore, the true prevalence of CP/IDC might differ from what was reported. Moreover, the definitions of BCR and MET varied across the studies, which in turn might have had an influence on the presented results. Our conclusion to exclude patients with CP/IDC from the AS is based on studies in which the RP specimen was assessed and not the prostate biopsy. Nonetheless, this attitude is supported by international urological associations. Although a statistical analysis revealed that CP/IDC compromise the long-term oncological outcome in the RP cohort, an analysis of the impact of radiotherapy on patients with CP/IDC might contribute meaningful insight into the prognosis in the setting of different radical treatment modalities. The CP and IDC should be considered as highly aggressive histopathological morphologies of the prostatic adenocarcinoma, which have a negative impact on both the pathological and oncological outcomes. The presence of CP/IDC should be taken into account in the planning of surgery and proper postoperative counselling. In intermediate-risk patients with either of the two entities, a detailed explanation of their higher malignant potential is required and AS should be discouraged, in line with international urological guidelines.

PMC10000116

Alexandra Teixeira,Luís Carreira,Sara Abalde-Cela,Belém Sampaio-Marques,Anabela C. Areias,Paula Ludovico,Lorena Diéguez

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review

21-02-2023

acute myeloid leukemia (AML),measurable residual disease (MRD),microfluidics,surface-enhanced Raman scattering (SERS)

Simple Summary Acute myeloid leukemia is the most common type of leukemia in adults. It is frequently associated with a limited response to conventional therapies that have a high recurrence and mortality rate in the elderly population. Even patients with clinical remission after initial treatment eventually relapse due to measurable residual disease. An earlier and more accurate diagnosis of this condition, using tools with higher sensitivity and specificity, would allow for a more reliable prognosis of patients, leading to more favorable outcomes. Abstract Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient’s prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review Acute myeloid leukemia is the most common type of leukemia in adults. It is frequently associated with a limited response to conventional therapies that have a high recurrence and mortality rate in the elderly population. Even patients with clinical remission after initial treatment eventually relapse due to measurable residual disease. An earlier and more accurate diagnosis of this condition, using tools with higher sensitivity and specificity, would allow for a more reliable prognosis of patients, leading to more favorable outcomes. Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient’s prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD. Hematopoiesis is a process that promotes the formation, development, and maturation of all blood constituents from hematopoietic stem cells (HSCs) [1]. HSCs are multipotent cells that can self-renew and have the capacity to regenerate all the different cell types of the hematopoietic system, including the formation of erythroid cells, platelets, neutrophils, monocytes/macrophages, basophils, eosinophils, and lymphocytes [2,3]. These cells have a key role in the continuous replenishment of blood cells throughout life [2]. However, several intrinsic and extrinsic factors might have an impact on hematopoiesis [3,4]. Disturbance in the ability of the HSCs to proliferate and differentiate in the different blood cells can cause an accumulation of immature blasts and hematopoiesis failure, consequently leading to the development of distinct hematopoietic disorders, such as leukemia. Leukemia can be grouped according to the affected cell lineage and the proliferation rate of immature cells. As such, leukemia can affect myeloblasts (myeloid) or lymphocytic precursors (lymphoid), and can be classified as acute (fast proliferation) or chronic (slow proliferation) [5]. Consequently, leukemia is mainly divided into four types: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL), and chronic lymphoid leukemia (CLL) [6]. Depending on the type of leukemia, patients undergo tailored chemotherapy until they clinically reach complete remission (CR). However, after achieving CR and despite having no symptoms or signs of the disease, some patients may present small undetectable numbers of leukemic stem cells (LSCs) in the bone marrow [7]. This condition is named minimal, or, more recently, measurable residual disease (MRD), and it constitutes the major cause of leukemia relapse. Until very recently, none of the conventional diagnostic tests used was sensitive enough to assess MRD in the early stages. In the following sections, we provide an overview on AML and the current tools used in the clinical setting for their diagnosis, monitoring, and therapeutics, and then focus on the new technologies and their potential to assess MRD. AML is a heterogeneous group of hematopoietic neoplasms and the most common type of acute leukemia in older individuals [8,9]. AML incidence rises with age, increasing from ~1.3 per 100,000 of the population in those less than 65 years old to 12.2 cases per 100,000 of the population in those over 65 years of age, and with a median age of diagnosis of 68 years old [10]. Prognosis also changes with age, younger individuals having a more favorable prognosis. In the majority of cases, the disease appears as a de novo malignancy in healthy individuals, but patients with underlying hematological disorders or prior chemotherapy are at higher risk of developing the disease [10]. The estimated 5-year overall survival (OS) for older patients, those with secondary AML, or those with relapsed disease is an alarming 5–10%. The appearance and even the accumulation of mutations in HSCs and normal dividing cells are often associated with aging [11]. A common age-related condition characterized by the acquisition of somatic mutations in leukemia-associated myeloid malignancy driver genes is clonal hematopoiesis of undetermined potential (CHIP) [12,13]. However, these mutations must be present at a variable allele frequency ≥2% in healthy individuals non-diagnosed with myeloid neoplasm or other obvious hematologic conditions [14]. Taking this into account, CHIP is considered a potential pre-stage of leukemia, associated with a 0.5–1.0% risk (per year) for developing the hematologic disease [14,15,16]. AML disease arises from continuous genetic and epigenetic alterations in HSCs and progenitor cells [17,18]. It is known that somatic mutations in genes codifying epigenetic modifiers as NPM1, DNMT3A, IDH1/2, and TET2 are accumulated with age and/or during AML progression in HSCs [17,19,20,21]. Shlush et al. found that there is a potential order for a mutation to happen, therefore mutations in DNMT3A and IDH2 occur in pre-leukemic HSCs, and mutations on NPM1c and FLT3 [21,22,23] and NRAS and RUNX1 [24] appear later in a leukemic state. In addition, even in cases of absence of any large chromosomal abnormality, genetic mutations which are identified in more than 97% of the cases are involved in the development of AML [10]. Furthermore, emerging evidence suggests that LSCs induce molecular changes in the distinct hematopoietic and non-hematopoietic cell populations in the bone marrow (BM) niche [25]. The latter happens via exosome secretion, facilitating the development of a malignant microenvironment while disrupting normal hematopoiesis [26]. However, the conundrum of ‘the chicken or the egg’ of whether the niche favors some mutations or whether the mutations precede alterations in the niche remains an open question. It is possible that these two events are concurrent, where either niche alterations are genotoxic and oncogenic and/or that malignant cells might transform the normal niche to promote their survival and expansion. As a result of the dysregulated differentiation of normal blood cells, an accumulation of immature myeloid progenitor cells is observed not only in the BM (Figure 1), but also in the peripheral blood (PB). Consequently, the ability of the BM to produce normal white and red blood cells and platelets is reduced [25,27,28]. Occasionally, clonal blasts can be found in other organs, such as the liver, spleen, and lymph nodes, resulting in anemia, bleeding, or organ infiltration and, ultimately, in hematopoietic failure [29,30,31]. Finally, due to the highly heterogeneous genetic landscape of LSCs, early detection and personalized treatment constitute the biggest challenges in the field of AML. Furthermore, expanding the knowledge of the genetic landscape might promote a better understanding of therapeutic fragilities in AML, contributing to the definition of new potential targets, the development of new therapies, and the improvement of current strategies [32]. At the initial stages of the disease, PB analysis showing a high number of abnormal white blood cells (WBC), or even a very low blood count, can indicate the presence of AML. Hence, the confirmation of the AML diagnosis is performed by further morphologic and cytogenetic assessment in BM aspirates [6,33]. The immunophenotyping by multiparameter flow cytometry (MFC) is conventionally used to accurately diagnose AML based on the identification of intracellular and extracellular markers (e.g., CD13, CD33 and CD34). On the other hand, the conventional cytogenetic analysis is mandatory in the assessment of AML disease. An alternative to detect specific abnormalities (RUNX1::RUNX1T1, CBFB::MYH11, KMT2A (MLL) and MECOM (EVI1) gene fusions or myelodysplasia-related chromosome abnormalities) is fluorescence in situ hybridization. Beyond these technical approaches, different molecular genetic tests have been used to screen all AML characteristic genetic abnormalities, and the tests are needed for differential diagnosis and targeted treatments. The final diagnosis and monitoring of AML always involves a BM tissue biopsy (BM trephine biopsy) or a BM biopsy. Those are invasive, painful, and create discomfort and harm for leukemia patients [34]. Therefore, less invasive procedures are needed while ensuring an accurate diagnosis revealing more detailed information about the disease progression. One of the less invasive procedures that have been extensively studied and applied as a complementary method is the PB biopsy, also known as liquid biopsy [6]. This is currently a complementary technique to the gold standard method for the detection of leukemia or MRD because of a smaller number of LSCs in the PB when compared with BM aspirates [34]. The classification of hematological malignancies has advanced tremendously since the 1970s. The classification of AML is based on two main systems: the French-American-British (FAB) and the World Health Organization (WHO) (Table 1) [24,35,36,37,38,39]. The FAB classification was introduced in 1976 and it is based on the morphology and cytochemistry of blasts, the degree of myeloid or monocytic differentiation, and their degree of maturation [35,36,40]. As such, this classification recognizes eight distinct subtypes of AML, from M0 to M7 [35,40]. This system has been widely used for the classification of hematological disorders in the last decade since characterization of the blasts is easily accessible and low cost. However, with recent advances in the genetic and molecular characteristics of AML, this classification is now becoming incomplete and outdated. Consequently, the WHO system has been increasingly used to classify AML. This system was firstly introduced in 1999 [17,41] and reviewed in 2016 [42], combining the previous features from the FAB classification with genetic data (chromosomal and molecular), biological data, and clinical data [42]. More recently, in September 2022, new updates about AML classification were published [39]. This updated version of the classification provides a more genetically defined classification as it retains most of the previously defined AML types (with recurrent genetic abnormalities) and includes other genetically-related entities to define AML and changes in the blast thresholds. In summary, this revision of the WHO classification (completed by leading experts), maintains the blast threshold at 20% for the diagnosis of the majority of AML subtypes, but decreases it to ≥10% blasts in BM or blood in the presence of recurrent genetic abnormalities (except for AML with t(9;22)(q34.1;q11.2)/BCR::ABL1) (Table 1) [24,39]. The case of AML with BCR::ABL1 still requires ≥20% blasts, due to avoiding overlap with chronic myeloid leukemia in the accelerated phase [24,39]. In addition, some predisposing features (therapy-related, prior myelodysplastic syndrome (MDS) or MDS/myeloproliferative neoplasm (MPN), and germline predisposition) designed as qualifiers of the primary diagnosis have also been added. The WHO system provides a better characterization of AML patients, offering clear advantages for treatment personalization [17,41]. In the new diagnosis of AML, a high percentage (50–60%) of cytogenetic abnormalities are found [17]. These abnormalities are usually associated with non-random chromosomal translocations that result in gene arrangements implicated in a modification in the locus encoding for a transcriptional activator, leading to the expression of a fusion protein [43]. These fusion proteins promote changes in the expression of target genes responsible for myeloid development maturation and proliferation, an abnormality that potentiates AML transformation [44]. As a result, AML is constituted by a heterogeneous population of malignant cells. The different AML clones have distinct cytogenetic alterations and mutations that produce considerable genetic complexity. Taking this into account, a risk classification system in AML patients at diagnosis was also defined. This system was also updated this year (September, 2022), but maintained the division into three risk categories: favorable, intermediate, and adverse [24]. However, it is important to note that after the recent revision and refinement, in addition to the baseline genetic abnormalities, the response to initial therapy and early MRD testing are now considered factors that contribute to the patient’s risk classification [24]. Thus, AML patients with a favorable prognosis present translocations of t (8; 21) (q22;q22.1)/RUNX1::RUNX1T1 with an occurrence of 10%; inversions of inv. (16) (p13.1q22) with an occurrence of 5% [17,42,45]; and mutated NPM1 without FLT3-internal tandem duplication (ITD) and in-frame mutations affecting the bZIP region of CEBPA [24]. The intermediate risk group includes all AML cases with FLT3-ITD; mutated NPM1 with FLT3-ITD; wild-type NPM1 with FLT3-ITD; with translocations: t(9;11)(p21.3;q23.3) involving the MLLT3::KMT2A genes, and all the cytogenetic and/or molecular abnormalities not classified as favorable or adverse [24]. Finally, the adverse-risk group now includes recurring cytogenetic abnormalities, such as the translocations: t(3q26.2;v) involving the MECOM gene; t(8;16)(p11;p13) associated with the KAT6A::CREBBP gene fusion; t(6;9)(p23;q34.1)/DEK::NUP214; t(v;11q23.3)/KMT2A-rearranged; t(9;22)(q34.1;q11.2) involving BCR::ABL1 genes; t(3;3)(q21.3;q26.2) involving the GATA2, MECOM(EVI1) genes or inversions: inv(3)(q21.3q26.2); −5 or del(5q); −7; −17/abn(17p). AML patients that present hyperdiploid karyotypes with multiple trisomies and complex karyotypes or monosomal karyotypes are also included in this group. Additionally, mutated ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 and mutated TP53 are also classified as adverse risk [24]; and if adverse-risk cytogenetic abnormalities are present in NPM1-mutated AML, they are also classified as adverse risk [24]. Patients included in the adverse risk group (~40%) have reduced rates of CR being prone to develop recurrence [23,46]. The current AML intensive treatment protocols are the same that were established more than 50 years ago. The remission-inducing chemotherapy usually consists of a combination of high doses of two cytotoxic drugs, cytarabine and anthracycline, with or without a purine analog [24,47,48,49,50]. After the induction therapy to clear LSCs and consequently induce CR, patients follow consolidation chemotherapy regimens (consisting of an intermediate dose of cytarabine) to deepen remission and maximize response [24,51]. In some cases, maintenance therapy is also applied to patients that have achieved remission, with the main goal of decreasing the risk of relapse [24]. In other cases, such as patients classified with adverse risk, allogeneic hematopoietic stem cell transplantation (alloSCT) is also recommended, but it is dependent on the risk of relapse (>35% to 40%), age (<60 years), and other comorbidities [24,52]. Although conventional therapy has better results in younger patients, this approach presents poor results in elderly individuals or in patients that present some comorbidities [53]. This is due to the combination of the high toxicity of the drugs and the heterogeneity of the disease [9,54,55,56]. Thus, some alternative treatment options are idarubicin (FLAG-IDA), fludarabine, cytarabine, azacitidine, decitabine, granulocyte colony-stimulating factor, and also mitoxantrone-based cytarabine regimens. Current research has been devoted to the identification of molecular targets allowing the implementation of personalized treatments. In 2017 and 2018, a handful of new drug formulations were approved by the FDA: new liposomal formulation of cytarabine and daunorubicin (CPX-351), a BCL-2 inhibitor (venetoclax), the IDH inhibitors (ivosidenib and enasidenib), the FLT3 inhibitors (gilteritinib and midostaurin) the anti-CD33 monoclonal antibody gemtuzumab ozogamicin, the hedgehog signaling pathway inhibitor (glasdegib), and the oral HMA CC-486 [17,50,57,58,59,60,61]. Some of these therapeutic options have already demonstrated very good results in clinical trials. For example, CPX-351 treatment improved the 5-year OS by 10% in patients aged 60–75 years, and with newly diagnosed high-risk or secondary AML when compared with conventional chemotherapy (cytarabine for 7 days plus daunorubicin on days 1, 2, and 3 [7 + 3 regimen] and cytarabine for 5 days and daunorubicin on days 1 and 2 for the second induction [5 + 2 regimen]) [62]. The use of a kinase inhibitor, midostaurin, as an adjunct to conventional therapy, daunorubicin and cytarabine (induction therapy), and then to high doses of cytarabine (consolidation therapy) also promoted an increase in overall and event-free survival in AML patients with FLT3 mutation, and, more precisely, increased the 4-year OS from 44.3% to 51.4% [63]. Currently, midostaurin is often used as first-line therapy in AML patients with FLT3 mutation [63]. Despite these advances, the choice of the therapeutic regime (combination of drugs on doublet and triplet regimens) has always been based on the patient’s eligibility or ineligibility to undergo intensive chemotherapy [8,17]. It is important to note that there are still no generally accepted or validated criteria for considering a patient ineligible for intensive therapy. However, in the context of clinical trials, there are some criteria to consider a patient not suitable, which can be used as a guideline in routine practice [24]. According to the 2022 revision completed by Dohner et al., patients not suitable for intense therapy are (1) patients >75 years old (cannot be an absolute criterion, because AML patients with a more favorable disease and without relevant comorbidities can be submitted to intensive chemotherapy); or (2) Eastern Cooperative Oncology Group (ECOG) performance status >2 and/or age-related comorbidities, such as severe cardiac disorder, severe pulmonary disorder, creatinine clearance <45 mL/min, a hepatic disorder with total bilirubin >1.5 times, or any other comorbidity that the physician assesses to be incompatible with intensive chemotherapy [24]. Thus, for patients who are not candidates for intensive remission-induction therapy, venetoclax plus either hypomethylating agents or low doses of cytarabine have emerged as an effective treatment option based on the achievement of a high and rapid rate of response [24,64,65]. Finally, for patients that do not want any treatment or cannot tolerate any type of anti-leukemic therapy, the best support care is proposed. More recently, immune therapies based on antibody therapies have revolutionized the treatment of leukemia. This includes chimeric antigen receptor T cells (CAR T cells), which are T cells taken from the patient and educated in the laboratory to express a specific antigen receptor target or CAR designed against a specific cell-surface antigen. These immunotherapeutic strategies have shown good results in other types of leukemia, particularly CLL and ALL [66,67], showing superior remission rates when compared with conventional therapy regimens. Despite several efforts to achieve similar success of antibody-based or cellular immunotherapies in AML [68], it is still a challenge to identify a good target antigen for this hematologic malignancy. In conclusion, due to the heterogeneous nature of AML, there is a pressing need for more fundamental research and biomarker identification to optimize and personalize AML therapies. Advances in this field will have the potential to improve quality of life, increase the time of at-home care, and reduce early organ damage and mortality while reducing the clinical, emotional, psychosocial, and economic burden associated with current intensive therapies. After the first induction of chemotherapy, CR rates reach approximately 80% and the disease burden is reduced by 99.9%. However, some residual LSCs (up to 1010 to 1012 cells) can remain, causing almost every patient to relapse if no consolidation therapy is provided [69,70]. Even when consolidation therapy is performed to eradicate any persistent LSCs, above 50% of patients will still relapse or develop refractory disease [71,72,73] (Figure 2). This is caused by the persistence of LSCc not detected by conventional technologies, and constitutes the termed minimal residual disease. However, more recently (in 2018), this nomenclature has been replaced by measurable disease (MRD) [74]. The term “measurable” has been proposed to indicate contexts where levels of LSCs are detectable by modern technologies that already have a higher sensitivity [70,75,76]. MRD monitoring is performed routinely in clinical practice since it is known that the presence of MRD is a strong prognostic indicator of relapse risk. Besides, MRD can also have implications in the planning and personalization of treatment, when assessed in conjunction with other well-established clinical, cytogenetic, and molecular data [77,78]. Furthermore, it has been shown that when MRD detection is successfully performed at an early stage, it results in improved prognosis, disease management, and patient outcome [79]. Given the importance of efficient and sensitive detection of MRD, which to date remains challenging, a large body of research has concentrated on developing new precise and sensitive detection strategies. Due to AML and the short period between CR and relapse, finding particular genetic mutations or aberrant protein expression patterns constitutes a major challenge [80,81,82,83]. Additionally, the challenge of detecting a MRD condition is due to the fact that the number of leukemia cells is very low compared to other blood cells; i.e., in ratios of 1:104 to 1:106 LSCs to WBCs, which makes this type of evaluation an extremely difficult task [75,84,85,86,87]. Thus, powerful and highly sensitive techniques are required to perform the molecular MRD assessment, reaching a limit of detection of 10−3 or lower, and PB and BM may be used [74,88]. Recently, the research community has demonstrated the relevance of MRD assessment towards guiding and personalizing therapy in leukemia patients [89]. The results of studies and clinical trials carried out in this field point towards two main MRD-driven patient management strategies: (i) the use of MRD in the pre-transplant scenario (induction and before transplantation), helping to select the most appropriate consolidation therapy and risk stratification; and (ii) the evaluation of MRD in the post-transplant setting, such as taper off maintenance therapy [89,90,91,92,93]. Thus, the stronger added value of MRD is based on its use as a (a) monitoring tool to detect imminent disease relapse, (b) a prognostic biomarker to allow the refinement of post-remission relapse risk assessment, and also (c) as a potential surrogate endpoint (still under exploration) [70,74,94]. In agreement, the recent ‘Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN’, recommends MRD assessment only after two cycles of induction/consolidation treatment, at the end of treatment, and during follow-up (every 3 months for up to 24 months) [24]. This recommendation validates not only the more frequent assessment of MRD at different and specific time points during treatment, but also the application of MRD as an independent biomarker to predict outcome, and an asset for detailed patient monitoring [24,95,96]. Although MRD negativity has been best stablished as a potential surrogate for clinical benefit in ALL, APL, and chronic lymphocytic leukemia, for AML this is still unclear due to the heterogeneity of the disease and the complexity of the MRD assessment [89,95]. In AML, MRD assessment is relevant for understanding the efficiency of induction chemotherapy and the intensity of the conditioning regimen; determination of the optimal type of post-remission therapy, such as hematopoietic stem cell transplantation in patients at high risk of relapse or maintenance therapy with hypomethylating agents; and choice of immunosuppressive agents, among others [94,96]. Currently, the use of MRD analysis to guide treatment decisions in AML patients is still not widely accepted, despite being a promising strategy. Nevertheless, many clinical centers have already started to guide treatment based on MRD, especially in the early stages of disease, given its strong clinical relevance and its potential to aid therapeutic decisions [95]. However, it is important to mention that most of the information until now is derived from clinical studies in patients submitted to intensive treatment. In the context of less intensive treatments (hypomethylating agents treatment or targeted therapies), the investigation about the predictive value of MRD assessment is still less extensive [95]. For example, MRD detection on AML patients with CR guides the early decision to proceed with an alloSCT. Post-transplantation monitoring of MRD could also guide the prescription of immune-suppressive drugs or more intensified chemotherapy regimens or with hypomethylating agents plus venetoclax; targeted therapy combinations when indicated, usually taking into account specific molecules abnormalities (FLT3 or IDH inhibitors); and antibody therapies [97]. While having already demonstrated the high relevance of MRD detection, one of the challenges is still to be able to detect this condition, particularly in the early stages. Thus, it is crucial to use reliable tools, and with high sensitivity. The European LeukemiaNet MRD Working Party has released comprehensive, consensus recommendations for the use of appropriate techniques having taken into consideration their technical limitations [98]. Well-established techniques, such as reverse transcription polymerase chain reaction (RT-PCR) and multiparameter flow cytometry (MFC), are recommended by the European Leukemia Network AML Measurable Residual Disease Expert Committee to detect MRD in AML. Additionally, other strategies have also been applied as well as next-generation sequencing (NGS) [75]. All these techniques present advantages and disadvantages regarding levels of sensitivity, applicability to the various heterogeneous types of AML, and costs and level of technical expertise needed to process the data. It is worth noting that an ideal MRD detection method should discriminate different cellular populations that could or not cause relapse. Except for the detection of acute promyelocytic leukemia by PCR, current detection methods are not sensitive enough to detect MRD in a regime with very low residual LSCs [84,99]. MFC is one of the most common techniques used for the diagnosis, classification, and prognosis of AML disease. This technique identifies a signature of aberrant expression of antigens present on AML cells, through specific panels of fluorochrome-labeled monoclonal antibodies [100]. Recent advances, based on the development of multicolor panels (>8 colors), brought the levels of sensitivity of cytometric analysis closer to those obtained by molecular biology techniques [75,84]. Thus, MRD analysis based on MCF presents moderate sensitivity, with a LOD of 1:10−3 up to 1:10−4 (1 leukemic cell in 10,000 to 10,000 WBCs), which is dependent on the number of cells analyzed, the gating strategy, and the number of antibody markers used [24,70,98]. The main advantage of MCF over other techniques is the ability to characterize millions of cells in a short period of time, while distinguishing viable cells from debris and dead cells [101,102,103]. Furthermore, it has the advantage of being applicable to the vast majority of AML patients, unlike other techniques that focus on the genetic and/or molecular signature [98]. MRD condition can be assessed/monitored in AML patients by MFC, based on leukemia-associated immunophenotypes (LAIPs) [104,105]. Briefly, it is known as LAIPs, the abnormal expression of immunophenotypic markers that provide the distinction of the leukemic cells. Conventionally, for the detection of LAIPs, several markers can be used, including CD34, CD117, CD45, CD33, CD13, CD56, CD7, HLA-DR (for MRD), among others [24]. Much work has been completed to improve this strategy and its application in MRD. In most of the cases, the improvements consist of the combination of several antibodies with different fluorophores (at least eight) to detect more than one LAIP at a time, improving the diagnostic performance [106,107]. Several other studies were conducted to understand the ability and efficiency of cytometric analysis to detect MRD. For example, a study with 451 BM samples from adult AML patients assessed the clinical utility of MRD detection, demonstrating that MFC is useful for outcome prediction and guidance of post-remission in AML [108]. The biggest drawback of this technique is the requirement of significant technical expertise and the intervention of an interpreting pathologist. The future use of artificial intelligence to reduce potential bias and/or human interpretive errors can bring accurate interpretations of the results. The low sensitivity and the high change of the aberrant immunophenotype during disease progression makes the assessment of MRD more difficult, leading to a high rate of false negatives and limiting the detection of the different aberrant markers. Molecular techniques are used to assess the presence of MRD by analyzing well-known AML mutations [24,70]. The most common markers used to monitor MRD are NPM1 insertion, CFB-MYH11, AML1-ETO, and PML-RARA (chimeric fusion genes) [109]. NPM1 is one of the most prevalent mutations in AML (present in 25–35% of patients) and is reported as a definitive leukemia-founder mutation that has already been validated as an optimal and reliable MRD marker [110,111]. Recent studies are focused on introducing/validating new markers for MRD monitoring; for example, many studies are focused on the relevance of IDH mutations [24]. The IDH1 and IDH2 mutations area is also prevalent in AML (occur in ~20% of cases) but there are still conflicting data, whether they represent pre-leukemic mutations or dominant clonal mutations, hence their value in monitoring MRD is not yet well established [75,110,112,113]. Moreover, NPM1 is frequently one of the references for the studies involving IDH mutations [114,115]. Furthermore, the prognostic significance of combined mutations, such as NPM1, FLT3, DNMT3A, and IDH is still unclear. However, recent studies state that some combinations may have an impact on the risk of relapse and overall survival of AML patients compared with other combinations [116,117,118]. Thus, in AML disease, and even in the MRD condition, it is crucial to use reliable and sensitive molecular techniques to detect these mutations, even when present in small amounts, in order to have a better understanding of their prognostic value. In AML, the development of RT-PCR has been crucial to detect residual elements conventionally characterized by molecularly cloned chromosome translocations [119,120]. The use of RT-PCR enables the detection of the fusion genes RUNX1-RUNX1T1 and CBFB-MYH11 [121] as well as PML-RARA [122,123] and mutated-NPM1 [86,124], important for the sensitive detection and quantification of MRD in AML [75,125]. In fact, in the case of CBF fusion transcripts (RUNX1-RUNX1T1 and CBFB-MYH11), some studies in PB or BM have demonstrated the prognostic value of detection and quantification of MRD at specific time points allowing the identification of patients with a high risk of relapse [121,126]. In addition, several studies have investigated the clinical implications of monitoring NPM1 and its association with relapse and survival rates [127,128]. In the study performed by Ivey et al., using BM and PB of the AML patients, the presence of NPM1-mutated transcripts after the second chemotherapy cycle was associated with a significantly higher relapse risk and poorer survival rates, independent of other known prognostic factors [127]. Given the high specificity and sensitivity for LSCs, decreased risk of contamination and better evaluation of RNA quality, the technique of RT-PCR has been widely implemented for routine patient care [70]. However, the detection of MRD based on RT-PCR or PCR is currently limited to around 50% of patients, since not all patients carry the fusion transcript RUNX1-RUNX1T1 (characteristic of t[8;21]), CBFB-MYH11 [inv 16] or t[16;16]), or NPM1 mutations [91]. The optimized RT-PCR assays presents a limit of detection (LOD) of 10−4 to 10−6, and is therefore more sensitive or equal than other technologies used, such as MFC (range 1:10−3 up to 1:10−4 ) [75,129,130,131]. As is the case with any other technique, there are some limitations of RT-PCR-based MRD tests, namely their dependence on specific mutations, requiring individual standard reference curves based on serial dilutions of targets, intensive labor, expertise, cost, time-consuming, and computationally demanding work [84,132]. Next-generation sequencing (NGS) refers to the deep, high-throughput, in-parallel DNA sequencing technologies that were developed in the decades after 1977, when the Sanger DNA sequencing method was developed. Unlike Sanger sequencing, NGS procedures provide a massive parallel and extremely high-throughput analysis (millions to billions of nucleotides) of multiple samples, bringing much faster results (due to multiplexing), and at a lower cost than individual testing [133]. Therefore, NGS is a tool that easily assesses genomic, transcriptomic, and epigenomic features [134]. More specifically, in the AML context, NGS has extreme relevance for diagnosis, due to the high clonal heterogeneity characteristic of this disease [135]. It provides information about the different AML mutations present in a patient, providing more information and allowing the design of personalized therapies, which will ultimately result in a better prognosis of remission and less cases of relapse [84]. It was already described that for patients in CR, the estimated percentage of the MRD measured by NGS was much greater than of aberrant blasts detected by MFC [136]. This technology has also revealed a set of mutations in rare mutant cells and gene sequences, as well as the discovery of genetic alterations that occur between diagnosis and relapse times [137]. In a study with a cohort of 482 patients, at least one mutation was detected in 430 patients (89.2%) showing the broad clinical application of targeted NGS [138]. Additionally, NGS showed a significant additive prognostic value compared to flow cytometry for the detection of MRD [138]. Recently, Alonso et al. tested a 19-gene AML-targeted NGS in a small cohort of 162 patients and showed that well-defined NGS panels are reliable in guiding clinical decisions by the current standards. Results had a 100% correlation with conventional molecular biology techniques, and all patients were successfully classified according to 2016 WHO classification systems (2016 WHO diagnostic categories, 2017 European LeukemiaNet, and Genomic classification) [139]. NGS was also explored in the context of allogeneic hematopoietic cell transplantation (alloHCT). For example, Thol et al. published a clinical study with a cohort of more than one hundred patients, demonstrating that MRD detection utilizing NGS before alloHCT is highly predictive of relapse and survival, and can therefore improve patient management [140]. It is worth noting that in this study similar results were obtained from both BM and PB samples, demonstrating the usefulness of using less invasive body fluids [140]. Thus, through the molecular characterization of AML cells, NGS promotes a personalized and precise method for the assessment of MRD, since can reach a sensitivity of 10−4 to 10−5 [95, 132]. However, the widespread use of NGS has been limited due to its high cost, and the expertise required for the data analysis and interpretation [94,141]. Furthermore, other drawbacks of this technique are the non-standardization of the assay, the high variance of sensitivity across different platforms, and a long waiting time for the result analysis. In summary, MRD has been shown to be an important and independent prognostic factor and predictor of relapse, and also plays a crucial role in the design of personalized treatment strategies. However, when values of cells are lower than 10−5, it is generally more challenging to be detectable by the diagnostic procedures used in clinical settings. Thus, the development of the aforementioned techniques enables a more reliable and satisfactory detection of MRD and CR as well as the stratification of patients into different subtypes. Still, the sensitivity of some of these techniques is far from ideal, and their implementation in clinical routine is still pending. Despite the technological challenges, it remains crucial to develop new technologies for MRD detection and quantification that are more affordable, faster, and offer higher accuracy and sensitivity. The major limitation of current MRD detection techniques is the inability to detect very low amounts of LSCs, considered rare tumor cells and named circulating leukemic cells (CLCs). These type of cells are present in body fluids, such as BM or PB. Hence, the pressing need of developing new tools with high sensitivity that could be easily implemented in clinical practice. Microfluidic systems are able to process fluids at high throughputs in microchannels. Due to the channel dimensions, in the order of 10 s to 100 s of micrometers, and to the laminar flow conditions, these devices exhibit many advantages to manipulate cells from a complex sample. Microfluidic devices also display a very high surface-to-volume ratio, holding extremely low amounts of fluids (10−9 to 10−18 L) [142]. The substantial reduction of the scale of the experiments offers unique advantages compared to more conventional analytic approaches, such as reducing the necessary amounts of samples and reagents. Microfluidics is also compatible with multiplexing and automation, and allows quick and efficient cell separation and detection at high resolution, high sensitivity, and low costs [142,143]. Currently, there are reports of many microfluidic devices that offer the capacity to selectively isolate cells from a mixture using several methodologies based on different cell characteristics, such as size [144], deformability [145,146,147], density [148], electrical [149] and magnetic properties [150,151], and surface charge [152,153]. Microfluidic strategies for cell isolation also exploit immunoaffinity, either for negative [154,155] or positive [147,156,157] enrichment of cells in body fluids, or using a combination of both [158]. These assays are not only very efficient but also maintain cell integrity, which is crucial for downstream analyses. Negative enrichment consists of using immunoseparation methods to target specific antigens on the membrane of unwanted cells, for example targeting the CD45 surface marker for the depletion of WBCs [159]. This method allows an unbiased separation of target cells, but it results in lower purity when compared with positive enrichment approaches [160]. The CellSearch® system is an example of a positive enrichment technique that has obtained FDA clearance for the separation of Circulating Tumor Cells (CTCs) from the blood of metastatic patients. In this case, magnetic nanoparticles immunoconjugated to target EpCAM are used to separate the cells of interest from the whole blood sample. Although positive selection promotes greater purity of CTCs, this strategy fails to isolate cells with low expression or negative expression of the surface-marker used [161,162]. Similarly to CTCs, microfluidics can be used to isolate CLCs from body fluids. The application of microfluidics in AML has been explored in many other ways, including a very interesting work for single-cell DNA sequence [163]. In this review, we have focused on microfluidics as a tool to isolate CLCs in order to demonstrate the high relevance of this technique in the MRD detection as a less invasive tool for patient diagnosis and monitoring. Enriching and isolating CLCs would not only provide a way to better detect and monitor MRD (where the number of LSCs is very low), but it would also be invaluable for early diagnosis and accurate patient stratification. Recent studies have shown the capacity of microfluidics to isolate CLCs, which can be used as biomarkers to determine the occurrence of MRD in AML patients [79]. However, the isolation of CLCs can be more complicated than that of CTCs, since they do not have any ubiquitous characteristic that can aid their identification. It is important to emphasize that CLCs and CTCs differ in both, morphological and biological aspects. The most evident parameter is the size, while CTCs have bigger sizes (~14–25 μm), CLCs are a very similar size to WBCs (~7–15 μm) [164,165]. Therefore, in the case of CLCs, it is not useful to use filtration systems or other types of size-based assays. Another very common technique described to isolate CTCs is positive immunoisolation. The antibodies most commonly used for this purpose are the ones that recognize epithelial cell adhesion molecule (EpCAM). This approach renders high levels of purity because this marker is highly expressed in cancer cells from solid tumors, while WBCs do not express this glycoprotein. On the other hand, antibodies that are used in the context of AML to promote the enrichment of CLCs can also be expressed by blood cells, so the number of interfering cells would be higher, meaning low purity outcomes. Therefore, to overcome these obstacles, an additional step is necessary to distinguish cancer cells from healthy blood cells, using specific markers of aberrant leukemic cells. In this sense, and to detect MRD in patients with AML, Jackson et al., used a microfluidic assay to target several antigens, including CD33, CD34, and CD177 [79]. The antibodies used to recognize these antigens cover about 70% to 90% of the AML patient population. They also included CD7 and CD56 to detect aberrant markers and consequently identify the CLCs fraction of the total of cells isolated (Figure 3). The study also demonstrated that this microfluidic assay is able to detect relapse two months earlier when compared with the technologies currently used––PCR or flow cytometry analysis of a BM biopsy [79]. Recently, an inertial microfluidic chip based on cell stiffness (LSCs are stiffer than the non-target cells) and differential cell size to detect rare leukemic cells was developed by Khoo and collaborators (Figure 4) [34]. For the optimization and validation of the system to detect low amounts of cells in blood, the authors started using samples spiked with LSCs (cell count < 5%) showing a detection limit of 5 LSCs among 106 leukocytes. This efficiency exceeds the minimum detection rate obtained by the methods currently used. After preliminary validations with cell lines, the study used clinical blood samples (including ALL, MDS for various subtypes of AML patients) demonstrating that the system had the ability to promote rapid enrichment of blasts, although the blood samples had to be pre-processed before being run in the system to remove red blood cells [34]. After isolation, the morphology and expression of the recovered cells was identified by cytopathological and/or immunocytochemistry analysis [34]. This procedure allows the potential detection of low amounts of blasts and, subsequently, has added value in the detection of MRD. In a recent work, Lai et al. also developed a non-invasive strategy for LSCs capture designed LSC-Chip (Leukemic Stem Cell Specific Capture Chip) [166]. This strategy consists of microfluidic herringbone chips with a CD34-antibody-modified surface. To bind the antibody to the surface of the chips, they used a reversible disulfide strategy to promote further analysis, including scRNA-Seq [166]. The main objective of the herringbone structures is to ‘force’ cells to travel through streamlines, and to interact more frequently with the antibody-modified surface, with the primary aim of improving target cell capture. The capture efficiency of the chip was tested using different cell lines such as KG-1a (CD34+) and HCT116 (CD34-). The best results obtained reached a value of 84.55% using KG-1a cells in PBS and 77.75% using the same cells spiked in whole blood, and both used a flow rate of 1.5 mL/h. The LSCs chips were validated using PB samples (non-invasive sampling) from 36 AML patients diagnosed as CR and non-remission, and 10 healthy donors [166]. The results achieved demonstrated the potential of the application of the LSCs-Chip for remission status monitoring in AML. A novel approach aimed at the isolation and detection of AML cells, more precisely drug-resistant ones, has recently been implemented based on the combination of superparamagnetic nanomaterials and microfluidics [167]. The magnetic capture efficiency of the cells of interest using the microfluidic device, which in this case were HL-60 cells that overexpress CXC chemokine receptor 4 (CXCR4), was 82.92% [167]. All these studies have demonstrated the relevance and potential of using microfluidics to separate/isolate cells of interest more efficiently, with lower sample volume consumption and in a shorter time compared to traditional methods. The inclusion of new techniques with the ability to identify disease-related mutations present at very low frequencies is crucial for the assessment of the MRD evaluation in the clinical and research stages. Digital PCR (dPCR) is a high-performance technology that produces an absolute quantification. It is based on the amplification of target genes without a standard reference curve, unlike its conventional version, RT-PCR [168,169,170]. Due to its advantages when compared with conventional PCR, dPCR is starting to be explored as a replacement of the outdated version in numerous applications, and AML is no exception. Compared with NGS, it allows less multiplexing, but it is cheaper and presents a good sensitivity. In this context, Hindson et al. compared the microfluidic-based system (dPCR) with traditional Taqman probe (or similar) based quantitative PCR systems, which resulted in even greater sensitivity (up to 10 times), accuracy, and high reproducibility of the newly developed technology [171]. Due to the fact that this technology is considered an asset in MRD monitoring, it has recently been introduced as a molecular assay, and it has been applied in patients for the detection of mutations, such as IDH1/2, DNMT3A, and NPM1 [172,173]. Parkin and colleagues published a work in which they tried to optimize the dPCR to characterize MRD and to be used in AML prognosis. The authors essentially applied dPCR to measure a variety of mutated alleles of recurrently mutated genes in BM samples of CR AML patients. It was possible to detect variant alleles with a frequency as low as 0.002% [174]. Koizumi et al. used mRNA to detect the presence of Wilms’ tumor 1 (WT 1) in several hematological malignancies, including AML, by dPCR, and compared the results with quantitative real-time polymerase chain reaction (RQ-PCR). Both methods correlated strongly with each other, but dPCR was capable of detecting lower levels of WT1 [175]. Bussaglia et al. also applied a dPCR method for the detection of low levels of WT1, but, in parallel, also performed the MFC technique for immunophenotype analysis (using markers for MRD assessment) to establish some prognostic correlations [176]. The optimization of dPCR allowed the first quantitative measurement of frequent genetic mutations in AML and many other malignancies, using the detection of rare variants with high sensitivity, managing to detect 1 in 50,000 mutant cells. dPCR also allowed the provision of several conclusions on the biology of residual AML and pre-leukemia in early CR [174]. In a cohort of 99 patients the occurrence of molecular MRD (NPM1mut) by dPCR strongly correlated with RQ-PCR, showing prognostic relevance [177]. Beyond WT1 and NMP1, dPCR was also performed to IDH1/2 [115], the PML-RARA [178,179], BAALC [180], MN1 [181]. We may conclude that dPCR is a promising tool for MRD detection since it presents good levels of sensitivity, able to achieve from 10−4 down to 10−6 [24,131]. Besides, dPCR offers other advantages, such as being much faster and easier to interpret data, without requiring technical expertise, and with lower error rates. As such, dPCR is considered a powerful and feasible tool to improve the diagnosis and stratification of patients and to monitor MRD [182]. Another promising tool that has also been used is the incorporation of locked nucleic acids (LNA) nucleotides into PCR probes and primers. Briefly, LNA bases can be incorporated into any DNA or RNA oligonucleotide, inflicting a conformational modification in the local helix [183,184,185]. This alteration provides a higher hybridization affinity and stronger binding strength for complementary sequences [183,186,187] as well as better duplex formation [188]. Moreover, the incorporation of LNAs increases melting temperatures by several degrees (per LNA monomer introduced: +1–+8 °C against DNA and +2–+10 °C against RNA [189]), which permits probes and primers to be shortened [190]. All these positive aspects of the incorporation of LNAs in PCR increase the success of the amplification and, additionally, provide a higher specificity due to the elimination of undesired products [191], and, besides PCR, it has also been integrated into Molecular Beacon, TaqMan Beacon, microarray probes, and antisense oligonucleotides [189,192,193]. In the AML context, the LNAs strategy has been applied with the main goal of improving the detection of AML-specific mutations. For example, Laughlin et al. developed a method for the detection of NPM1 mutations that are frequently found in AML patients with a normal karyotype [194]. It consists in using an oligonucleotide that contains LNA nucleotides to act as a PCR clamp, thereby inhibiting amplification of the normal sequence and promoting preferential amplification of DNA with a mutation. This method provides increased analytical sensitivity of the assay [194]. In another work, the E-ice-COLD-PCR method (enhance improved and complete enrichment co-amplification at lower denaturation temperature polymerase chain reaction) was used for the detection of low levels of NPM1 mutations [195]. This method uses a non-extendable blocker probe that integrates LNA bases to block wild-type amplification, promoting the enrichment of the four different types of NPM1 mutations, such as A, B, D, and J [195]. Some studies have already been conducted using the LNAs for the assessment of specific mutations during MRD monitoring [114,196]. For example, the work developed by Abdelhamid and collaborators used LNA-RQ-PCR, which is a RQ-PCR-based technique incorporating LNA to modify the reverse primer in order to amplify only the mutant allele that is weakly present in the neoplastic DNA studied [196]. This strategy allowed a rapid and sensitive quantification of IDH1 and IDH2 gene mutations in MRD AML [196]. A similar strategy was previously used by Jeziskova et al. for the detection of IDH2 mutations [114]. In addition, a very recent work developed by Kao et al. also demonstrated the application of LNA-qPCR in detecting IDH1/2 mutations and monitoring MRD [197]. For this study, BM samples from 88 IDH1/2-mutated AML patients that received standard chemotherapy, azacitidine, or low-dose cytarabine as induction therapy were used. The authors then applied LNA-qPCR to quantify the IDH1/2 mutants MRD kinetics in the samples and compared the results with those obtained for NPM1 qPCR. The results demonstrated that IDH1/2 LNA-qPCR MRD was concordant with remission status or NPM1-MRD in 79.5% of patients [197]. Finally, authors demonstrated the potential of LNAs for detecting NPM1 mutations using both PB and BM samples [198]. The results demonstrated the potential of MRD measurement by mutant NPM1, allowing the identification of a group at high risk of recurrence and even detection of relapse. Thus, all these studies demonstrated the potential of LNAs integrated with quantitative PCR to provide an easier but more specific and sensitive way to detect low levels of specific and relevant mutations in AML and MRD conditions. At present, Raman spectroscopy and its variant surface-enhanced Raman scattering (SERS) spectroscopy are among the most powerful analytical techniques available for the analysis of biochemical markers. Its application has already been demonstrated in a wide number of fields, from environmental pollution [199], food industry [200], biomedical [201], medicine [202,203] to arts [204]. Raman spectroscopy is a scattering technique that studies the specific vibrations of molecules [205] by the analysis of their characteristic Raman spectrum (Raman intensity versus wavelength shift spectrum) [206]. This technique provides a vibrational fingerprint of the studied sample that can be directly correlated to its structure, constituents, symmetry, and environment [207]. Thus, Raman spectroscopy can be used to determine chemical composition, molecular structures/conformations, and interactions between different molecules, allowing qualitative and quantitative results [207]. Qualitative analysis can be performed by measuring the frequency of scattered radiation, while quantitative analysis is performed by measuring the intensity of scattered radiation [208]. However, the sensitivity of conventional Raman spectroscopy is intrinsically low, which limits its wide application in the biomedical field [207]. One way to overcome this limitation is to use metal nanoparticles or other plasmonic nanostructures to enhance the Raman signal of molecules that are in their vicinity, known as SERS [209]. SERS is an advanced analytical technique that can be used for the ultra-sensitive detection of various analytes, with astonishingly low detection limits, even up to a single molecular level [210]. This technology was already explored in rare cells detection in the distinction of different cell populations or even in DNA mutation detection. For example, Nima et al. demonstrated a strategy based on SERS to detect CTCs, where four different antibodies and Raman tags were used, showing the possibility of identifying a single cancer cell, with high accuracy/precision, in the middle of millions of blood cells [211]. Wu et al. also developed three types of active SERS nanoparticles to detect CTCs in blood samples with high sensitivity (detection limit of 1 cell/mL) and specificity, without prior enrichment [212]. Additionally, an efficient and non-invasive method was also developed based on an active SERS platform. Briefly, this method consists of the incorporation of the platform in the microfluidic device to isolate CTCs, promoting a label-free detection and its identification through structural and biochemical analysis [213]. This demonstrates the relevance and applicability of SERS in the difficult task of detecting rare cells. On the distinction of different cell populations, Teixeira et al. demonstrated the potential of the SERS technique to detect the profiles of different cell types (malignant and non-malignant) and to distinguish them based on spectral differences [214]. In a recent study, Oliveira et al. demonstrated the multiplex phenotyping of single cancer cells using SERS probes [215]. Furthermore, the possibility to detect DNA mutations using SERS was also demonstrated. More precisely, a SERS chip with the ability to detect specific KRAS mutations and differentiate between wild-type and mutated KRAS genes in different cancer cells was developed [216]. The same SERS chip was optimized and combined with supervised machine learning allowing the intelligent cancer classification according to its mutational landscape [217]. The application of this sensitive and precise technology, with added value in the detection of malignant cells, has already been explored in the context of leukemia or even to distinguish these cells from the healthy ones. In 2014, González-Solís and colleagues mentioned the relevance of SERS technology associated with leukemia. They analyzed the biochemistry of blood serum from leukemia patients and healthy donors, through Raman spectroscopy, and managed to distinguish between normal and leukemia cells, and also to identify different types of leukemia [218]. In CLL, a SERS technique was used to detect leukemic cells, using SERS probes composed by nanoparticles functionalized with PEG and antibodies (to recognize three surface proteins of interest in malignant B cells). To confirm the results of this method, flow cytometry was used [219]. In ALL, the differentiation between normal B-lymphocytes and B-ALL cells at different maturation stages using Raman spectroscopy and Principal component analysis (PCA) was demonstrated, with important implications for the clinical practice. The high sensitivity of Raman and feature-rich Raman spectra offers the possibility of multiplex detection, and it could represent a major step in blood analysis through the realization of a non-destructive, label-free Raman-based flow cytometer [220]. Furthermore, in the context of B-cell hematological malignancy, a SERS-based strategy was also used to simultaneously detect two MRD surface markers (CD19 and CD20) in patient blood samples. The results were compared with flow cytometry and demonstrated the great potential of the approach for MRD detection with high sensitivity [221]. Finally, in the context of AML, a preliminary study was carried out using a sensitive and robust platform, based on nanoparticles and hollow core photonic crystal fibers, to monitor and detect leukemic cells (using HL-60 cells) [222]. In addition, electroporation-based SERS spectroscopy using silver nanoparticles was used to detect AML cells (HL-60 and K562 cells) and also differentiate them from healthy cells (PBMCs, Peripheral blood mononuclear cells) [223]. It is important to note that some SERS methods have been also used in AML patient samples, more precisely in APL subtype, to discriminate subtypes or molecular tests. For example, Ye et al. used Ag nanoparticles-based SERS technique to discriminate acute monocytic leukemia and the AML subtype acute promyelocytic leukemia using plasma from patient samples [224]. Another study also demonstrated the possibility of using SERS (through Ag nanoparticles) to distinguish between AML subtypes and control samples, and also for the prognostic stratification of AML patients using characteristic SERS bands [225]. Interestingly, for the discrimination between AML patients and control samples, the relevant specific patterns referred to nucleic acids, and also amino acids, glucose (and products), and protein (and derivatives). On the other hand, to distinguish between different subtypes of AML, different nucleic acids patterns were used. This study demonstrated the potential of SERS technique to be translated to clinical practice, then, and to aid in the differential diagnostic and prognostic stratification of AML patients [225]. A different approach developed by Moisoiu et al. showed the application of SERS for the detection of cancer DNA, more precisely for the characteristic methylation pattern of cancer DNA without pre-amplification in AML patients. The results demonstrated that with SERS, it is possible to detect a unique methylation pattern of cancer DNA and, even more, to promote a discrimination between control samples and AML patients, with a specificity of 82% and a sensitivity of 82% [226]. The studies described above have evidenced the high sensitivity and precision of Raman spectroscopy, and its capacity to differentiate tumor cells from healthy blood cells in a non-destructive way, and even to discriminate patient subtypes. Despite the very promising potential of this technique in cancer and also specifically in AML context, more studies are needed. For example, no specific studies using SERS for the detection of CLCs from blood samples and for the evaluation of MRD or even for specific DNA mutations in AML have been described so far. The detection of MRD has a strong clinical relevance, but it is still the greatest challenge in AML disease management. New technological advances have been focused on increasing sensitivity and specificity to create essential tools for the early diagnosis of AML and its relapse. Some of the most promising tools to improve the current landscape include microfluidics and SERS or a combination of both. Indeed, several techniques may be needed, in order to maximize the availability of clinically useful information, with the ultimate goal of promoting more personalized treatment approaches.

PMC10000120

Davide Adriano Santeufemia,Giuseppe Palmieri,Gianmaria Miolo,Maria Colombino,Maria Grazia Doro,Laura Frogheri,Panagiotis Paliogiannis,Giampiero Capobianco,Massimo Madonia,Antonio Cossu,Giovanni Lo Re,Giuseppe Corona

Current Trends in Mucosal Melanomas: An Overview

21-02-2023

cancer,mucosal melanomas,diagnosis,prognosis,genetic,metabolism,immunotherapy

Simple Summary This review provides an updated overview about molecular features, clinical advancements and therapeutic directions of the primary mucosal melanoma which represents a rare and heterogeneous malignancy characterized by a poor prognosis. Abstract Primary mucosal melanomas (MMs) are uncommon tumors originating from melanocytes located in the mucous membranes at various anatomic sites within the body. MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies. Despite these differences, that have important implications for both disease diagnosis and prognosis, MMs are usually treated in the same way as CM but exhibit a lower response rate to immunotherapy leading to a poorer survival rate. Furthermore, a high inter-patient variability can be observed in relation to therapeutic response. Recently, novel “omics” techniques have evidenced that MM lesions have different genomic, molecular, and metabolic landscapes as compared with CM lesions, thus explaining the heterogeneity of the response. Such specific molecular aspects might be useful to identify new biomarkers aimed at improving the diagnosis and selection of MM patients who could benefit from immunotherapy or targeted therapy. In this review, we have focused on relevant molecular and clinical advancements for the different MM subtypes in order to describe the updated knowledge relating to main diagnostic, clinical, and therapeutic implications as well as to provide hints on likely future directions.

Current Trends in Mucosal Melanomas: An Overview This review provides an updated overview about molecular features, clinical advancements and therapeutic directions of the primary mucosal melanoma which represents a rare and heterogeneous malignancy characterized by a poor prognosis. Primary mucosal melanomas (MMs) are uncommon tumors originating from melanocytes located in the mucous membranes at various anatomic sites within the body. MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies. Despite these differences, that have important implications for both disease diagnosis and prognosis, MMs are usually treated in the same way as CM but exhibit a lower response rate to immunotherapy leading to a poorer survival rate. Furthermore, a high inter-patient variability can be observed in relation to therapeutic response. Recently, novel “omics” techniques have evidenced that MM lesions have different genomic, molecular, and metabolic landscapes as compared with CM lesions, thus explaining the heterogeneity of the response. Such specific molecular aspects might be useful to identify new biomarkers aimed at improving the diagnosis and selection of MM patients who could benefit from immunotherapy or targeted therapy. In this review, we have focused on relevant molecular and clinical advancements for the different MM subtypes in order to describe the updated knowledge relating to main diagnostic, clinical, and therapeutic implications as well as to provide hints on likely future directions. Primary Mucosal Melanoma (MM) is a rare malignancy originating from melanocytes located in the mucosae at various anatomic sites within the body [1]. Estimation of cancer incidence in United States for 2023 indicates that melanomas will represent 5% of new cancer cases, of which MM will constitute ≤2% diagnoses [2]. Melanocytes’ primary role is the synthesis of the melanin pigment that, in the skin, is transferred to keratinocytes and serves as a shield to protect the human body surface from sunlight UV-radiation. Despite the fact that these pigmented cells are markedly distributed into the skin and the vast majority of melanomas are of cutaneous origin [2,3], they can also be found in other tissues. Originating from pluripotent embryonic cells (namely, the neural crest cells), melanocytes have the property to migrate not only under the epidermis to populate all parts of the skin but also within the basal cell layer of the epithelium at different anatomic districts. Melanocytes are mainly present in the inner ear, nervous system, and heart; in addition, generally they can be found in nearly all tissues to a differing degree [4]. Melanin-containing melanocytes are present in the basal cell layer of the epithelium even at mucosal sites with no visible signs of melanin pigmentation [5,6,7]. At the oral level, melanocytes may or may not produce melanin and, analogously to what happens into the skin, the amount of synthetized melanin is constitutionally determined [3,7]. As is commonly known, there are substantial variations in rates of melanin pigmentation among people from different racial/ethnic groups as well as among those belonging to the same racial/ethnic group [3,8,9]. Furthermore, when considering the oral site, melanin pigmentation is common in black people [10], being even more frequent in darker skinned whites (Caucasians) than in lighter skinned whites [11]. Additionally, melanin distribution into the oral mucosae may be heterogeneous, with the pigmentation being patchy or uniform and more frequently affecting the gingiva [10]. Smaller amounts of melanin-containing melanocytes can be also found in other distinct mucosal membranes, determining that melanomas can arise in any site where such cells are present: the respiratory, gastrointestinal, and urogenital tracts as well as in the eye [3,11]. MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies [5,6]. Despite these differences that have important implications for disease management, the MMs are usually treated in the same way as CMs [6]. However, MM patients exhibit a lower response rate to immunotherapy than CM patients, with a poorer survival rate [5,6,7]. Furthermore, a high inter-patient variability can be observed in relation to treatment response. Recently, novel omics’ techniques have revealed that MMs have a different molecular landscape and genomic profiling as compared with CM that can explain their different response [8,12]. Such specific molecular aspects are useful for identifying new biomarkers for improving diagnosis as well as the selection of MM patient candidates to achieve the best benefit from immunotherapy or targeted therapy. Here, we will we provide an updated overview of the relevant molecular and clinical advancements on MM subtypes in order to describe the state of the art on major diagnostic, clinical, and therapeutic implications as well as likely future directions. MMs are very rare tumors representing less than 2% of all melanomas [8,9], with about 850 new cases reported in Europe in 2013 [6]. Unlike CM, whose incidence is increasing, the MM incidence is estimated to remain stable [2,10,11,13,14]. Usually, MMs are diagnosed in people aged over 65 years, with a rate of 6.3 per million, and they are more frequent in women than men [6]. MMs occur with about equal frequency in all races, therefore accounting of a substantial fraction of all melanomas diagnosed in black people, Asian people, and Hispanic people when compared with melanomas occurring amongst Caucasian people [15]. This reflects the incidence of CM in these populations [15]. Defined as melanomas occurring in mucosal membranes at various anatomic sites, these lesions are most frequently observed in the head and neck regions (approximately 50% of cases) followed by gastrointestinal (37%), female genital (6%), and urinary tracts (4%) rather than other locations such as pharynx, larynx, urinary tract, cervix, esophagus, and gallbladder [16]. Furthermore, lung site involvement is mostly secondary [17]. Unlike CM, where association with UV sunlight exposure represents a well-defined risk factor, genomic studies have suggested that UV light plays a nearly null role in MM carcinogenesis [8,12,18,19]. In fact, MMs develop on sun-shielded surfaces. Risk factors for this rare melanoma subtype remain poorly understood. Currently, there is no clear evidence to support the pathogenic role of chemical carcinogens or viruses despite their suggestion as etiologic factors [15]. Interestingly, some authors have reported that oral melanosis, which precede about one third of MMs in the oral cavity, may be associated with cigarette smoking [20,21]. Globally, MMs are characterized by the presence of distinct molecular features, frequently characterized by DNA structural changes and mutation signatures of unknown etiology, with respect to CM [22,23]. MMs are characterized by a relatively low tumor mutational burden (TMB) compared to CM [24]. In particular, MM has an average of about 8000 non-synonymous single nucleotide variants (nsSNVs) per tumor, a more than 10-fold lower mutation rate than CM (which, with an average of more than 80,000 nsSNVs, exhibits one of the highest somatic neoplastic mutation rates) [8,24,25]. This difference may explain the molecular reason for discordant clinical responses to immunotherapy that are observed in Asian people, where MM represents a substantial fraction of all diagnosed melanomas, versus white Caucasian people [15,26,27]. From the qualitative point of view, mutational profiling of MM as inferred by whole genome/exome sequencing approaches is different from that observed in CM [28]. According to recent NGS-based studies as well as meta-analysis and systematic review, MMs are characterized by a lower load of somatic mutations and lack of either UV-induced mutational signatures or oncogenic fusion gene transcripts frequently reported in CM [29,30,31]. In Figure 1, the main genes and their functional pathways involved in MM pathogenesis are represented. Overall, the genes mostly mutated (>10%) are neurofibromin 1 (NF1), KIT proto-oncogene receptor tyrosine kinase (KIT), and splicing factor 3b subunit 1 (SF3B1); the remaining genes commonly mutated but at lower rates (5% to 10%) are neuroblastoma RAS viral oncogene homolog (NRAS), sprout-related EVH1 domain containing protein 1 (SPRED1), ATP dependent helicase (ATRX), and B-Raf proto-oncogene (BRAF) [29,30,31]. The MITF gene, involved in melanocyte development and differentiation, has been found to either rarely carry the germline variant E318K—widely demonstrated to confer an increased CM risk—in vulvar melanoma either to be commonly amplified in several MMs [30,32]. Despite the fact that the BRAF and NRAS genes are preponderantly mutated in CM lesions (together accounting for about 75% of cases), their role in MM pathogenesis is not secondary. Activating mutations of the BRAF oncogene are detected in approximately 50% of CM [28], but at much lower frequency in MMs [13,14,19,33,34,35]. Considering NGS-based data, the frequency of mutations for BRAF and NRAS oncogenes has been reported at even higher rates (up to more than 20%) in different types of MM, with the most prevalent incidence in the mucosae of the head and neck district (Figure 2) [29,30,31]. In sinonasal MM, a high prevalence of NRAS mutations has been reported [36]; however, a lack of KIT alterations with a high prevalence of BRAF mutations has been reported in some populations, such as those from South Italy [13] or from Germany [37], but not in the majority of the others (as those from Japan, Poland, Spain, and the United States [38]), further suggesting that patients’ origin may account for different mutation rates in candidate cancer genes. In some MM cases with a lower rate of BRAF mutations, an increased activity of the Cyclin D1-Cyclin-dependent kinase 4/6 (CCDN1-CDK4/6) complex, mostly through gene amplification, has been demonstrated as contributing to MM pathogenesis [39]. Finally, in BRAF/NRAS-wild-type MMs, a higher rate of mutation into the Telomerase Reverse Transcriptase (TERT) promoter has been described, specifically for sinonasal melanomas [29,36]. Not having UV radiation as the most preponderant carcinogenic effect—usually occurring in the CM etiology—the pathogenic sequence variations found in the BRAF and NRAS genes appear to be more heterogeneous in MMs [33]. In fact, a markedly high prevalence of mutations at the V600 codon of BRAF (nearly all BRAF-mutated CM lesions present V600E/K variants, with particular reference to melanomas arising on skin areas intermittently exposed to UV) is lacking among MM cases; conversely, sequence variations mostly affect other regions of the BRAF gene than codon 600, though most of them in codons of the exons 11 and 15 are deputed to encode the protein domains where the kinase activity resides (among others, codons D594, G469, and K601 are frequently altered) [22,33,40,41]. This situation closely resembles what occurs in the adenocarcinoma subtype in the non-small cell lung cancer (NSCLC), in which the V600E mutations account for a fraction of BRAF-mutated NSCLC cases (about 40%, mainly females, and never-smoker patients), whereas the majority of them (about two thirds, regardless of the smoking habitus) carry non-V600 mutations [42]. Similarly, a poor prevalence of mutations into the Q61 codon of the NRAS exon 3, which is found in nearly all RAS-mutated CM cases, has been observed in MM; conversely, the G12 and G13 codons within the exon 2 of the gene are maximally involved in NRAS-mutated MM cases [29,30,31]. The latter codons are also prevalently mutated in the KRAS gene among lung and colorectal adenocarcinomas [43]. Such evidence strongly supports the hypothesis that causative factors promoting mutations in BRAF and NRAS genes in MM can be more similar to those involved in the onset and progression of lung and colorectal carcinomas than to those involved in cutaneous melanocytic transformation. Unfortunately, such causative factors remain to be determined. As already depicted above, the anatomical sites where MMs originate deeply affect the spectrum and distribution of the mutations in driver genes (Figure 2). Considering the main one, head and neck melanomas present a lower prevalence of KIT mutations as compared to the genitourinary tract and anorectal melanomas in which mutations and amplifications of this gene are common; among genital MMs, vaginal but not vulvar melanomas present very low rates of KIT mutations [40,41,44,45,46,47]. Furthermore, the frequency of different mutations may vary across different studies and reports on patients of different geographical origin and different genetically driven constitutional features. For other genes, a different anatomical distribution of alterations has also been reported: ATRX and SF3B1 mutations occurred more commonly in lower anatomy MMs and TERT or CTNNB1 in the upper anatomy [30]. Overall, MMs show specific genetic signatures depending on the site of origin. A higher percentage of mutations involving the SF3B1 gene was observed in patients with MM of anorectal origin. In patients with vulvovaginal melanoma, the involvement of the TP53 gene is common, while mutations involving the MYC gene are frequently detected in patients with sinus or nasopharyngeal melanoma [30,31,47]. In summary, the mitogen-activated protein kinase (MAPK) pathway—including the RAS-RAF-MEK-ERK signal transduction cascade—regulates the main processes of cell proliferation and cell survival in both MM and CM lesions. However, while in CM the MAPK cascade is directly activated by a constitutive alteration of one of its component (BRAF or NRAS), in MM the oncogenic MAPK activation is indirectly promoted by effectors external to the pathway (KIT and/or NF1) (Figure 1). In particular, KIT encodes a tyrosine kinase receptor of the cell membrane and its activation through mutation and/or amplification result in a continuous RAS-driven induction of cell proliferation, whereas NF1 encodes for a negative regulator of RAS signaling and its genetic or functional silencing results in RAS activation and enhancement of the malignant transformation and progression [48]. Inactivation or functional loss of NF1 may also cooperate with other so-called RASopathy genes—such as SPRED1, which is involved in melanoma genesis (Figure 1)—in sustaining constitutive RAS activation [48]. Tumors with mutations in NF1 and constitutive activation of RASopathy genes are expected to be associated with a higher mutational load and, consequently, a greater probability of generating neoantigens [49,50]. Unfortunately, MMs are poorly immunogenic, regardless of their intracellular mutational status, probably due to their lack of ability to induce a strong adaptive immune response in the tumor microenvironment; the reduced susceptibility to PD-1/PD-L1 and/or CTLA-4 blockade is highlighted by the extremely poor clinical outcomes of patients with advanced MM using immune checkpoint inhibitors [51]. Globally, MMs are characterized by an aggressive clinical behavior and a prognosis worse than CM [40]. Early diagnosis of the disease can be hindered by both its rarity and its mostly occult anatomical origin site; a possible multifocality; and/or the fact that about half of MMs are amelanotic may contribute to delaying diagnosis [41,52]. MMs patients are most likely to be diagnosed with regional or distant metastases, with a reported 5-year overall survival rate of approximately 25% [9,40]. In the large series reported by Lian et al. [40], predominant metastatic sites included regional lymph nodes (21.5%), lung (21%), liver (18.5%), and distant nodes (9%). Interestingly, when matched for staging, prognostic, and molecular factors, no significant survival difference among MMs arising at different sites was found by Cui et al. in a series of 706 patients [53]. MM clinical presentation is extremely variable. The site of origin and the size of the tumor, along with the possible metastatic involvement of other locations within the human body, can clearly determine different clinical scenarios. Although almost all malignant melanomas of the head and neck district are constituted by cutaneous lesions, mostly arising on the face skin, this region represents the most common site of MM origin and it accounts for more than half of all diagnosed MMs [10,54]. MMs may involve the nose and paranasal sinuses, oral cavity, pharynx, larynx, and esophagus [1]. The nose and paranasal sinuses followed by the oral cavity are the most common sites of origin [54,55], whereas it is uncommon to find it in the primitive mucosa of the larynx, pharynx, esophagus, or tracheobronchial tree [1,17,54,56,57,58]. MMs arising from the respiratory mucosa (such as the nasal cavity) have different clinicopathologic characteristics than those involving the oral mucosa, though they are characterized by similar adverse prognosis and outcome [54]. Within the nasal cavity, the origin of the disease from either the lateral nasal wall or nasal septum is preferential, whereas, considering the sinuses, the maxillary sinus, followed by the ethmoid, the frontal, and the sphenoid sinuses, these represent the anatomic privileged sites of its origin [54,59]. Typically, most of patients developing a MM in the nasal cavity presents a clinically localized disease and tend to exhibit a more favorable prognosis than those affected by MMs of paranasal sinuses or of other head and neck sites, which are often diagnosed at a more advanced disease stage with subsequent poorer outcome [54,59]. Sinus melanomas are asymptomatic in this anatomically silent area until the neoplastic growth invasion of adjacent structures is evident. In a pooled data analysis of approximately 200 patients from five series, the five-year survival for patients with nasal melanoma was 31% compared to approximately 0% for those with sinus melanoma [1,54,60]. Sinonasal MM commonly metastasizes to the lung and liver. At the time of the diagnosis, nodal secondary involvement by the tumor is present in about 20% of patients [54,60]. Clinical symptoms—such as epistaxis, unilateral nasal obstruction, or persistent rhinorrhea, loss of smell, and facial pain—are nonspecific, often indistinguishable from those of benign sinonasal disease; therefore, early diagnosis can be delayed in several cases [59]. Moreover, proptosis, diplopia, or neurological symptoms are symptomatic of a more advanced disease stage [60]. Most of such MMs is likely to show features endoscopically as polypoid, fleshy lesions with strict unilateral involvement with different degrees of pigmentation while they are rarely amelanotic [6]. Oral cavity MMs represent almost 30% of MMs of the head and neck, mostly involving the palate and maxillary gingiva [60,61]. Again, they are asymptomatic in the early stages, usually presenting with a pigmented patch or a mass characterized by a rapid growth rate [62]. Diagnosis may be easier due to greater accessibility of the oral cavity for clinical examination. Those lesions can be amelanocitic or characterized by a wide range of colors varying from black, brown, grey, to reddish or white [6,63]. At the time of the diagnosis, the probability of detecting distant metastases is low (5–10%) [6] with no significant difference between oral and sinonasal MMs; however, the incidence of nodal metastases at this level is highest than in sinonasal lesions, especially in presence of a depth of infiltration > 5 mm [6,61]. Approximately, 37% of MMs arise from the gastrointestinal tract, which represents the second most privileged site of origin for those malignancies [16]. Their preferred location is the anorectal tract (>50%), followed by the stomach, small intestine, and colon [64]. However, anorectal MMs are very rare accounting for approximately 0.5% of all colorectal and anal cancers [65]. MM rectal origin is more frequent than anal primary involvement and notably, given their infrequent occurrence, those tumors are studied together as a unique entity [64]. They are most frequent in women aged over 50 years and are characterized by a very poor prognosis [66]. It is also interesting to report that there is an unclear association between HIV infection and anorectal MM, though we think it noteworthy to take this information into account in the management of such patients [66]. Because of the hidden location and the lack of specific symptoms, patients are likely to present with disseminated disease [66]. Typical, early symptoms such as itching or rectal pain are often mistakenly attributed to a benign pathology, such as hemorrhoids, polyps, or anal fissures, and so the diagnosis is delayed many times. Moreover, changes in bowel habits, bowel obstruction, rectal bleeding, anal pain, and/or rectal tenesmus occur in a more advanced disease stage. MM arising from the genitourinary (GU) tract is an extremely rare disease, characterized by a clinically aggressive behavior, that comprises 0.2–1% of all melanoma cases [64]. GU MMs include tumors arising from the female GU tract (vulva, vagina, and cervix), male GU tract (penis and scrotum), and urinary tract (urethral and bladder) [64]. In a series of 817 primary GU melanoma cases from the Surveillance, Epidemiology, and End Results (SEER) database, the female GU tract was the most commonly involved site (89.4%), followed by the male GU tract (6.6%), and the urinary tract (4.3%) [64,67]. Most cases of GU MMs occurred in the vulva, with the highest age-specific incidence rates in patients aged 85 years and older for both women and men [67]. The labia minora followed by the labia majora and clitoris represent the most common sites of disease occurrence, whereas urethra and cervix locations are less frequently involved [68]. Symptoms of vulvovaginal melanoma include itching, vaginal discharge, bleeding, dyspareunia, mass as well as occasional dysuria and voiding dysfunction if urethral involvement occurs [69]. Vulvovaginal melanomas present frequently with regional lymph node or distant metastatic disease, probably due to the richly innervated lymphatic system that facilitates nodal spreading; again, their prognosis is very poor [64]. GU melanoma usually presents in males as a pigmented macule, papule, plaque, or an irregularly demarcated, ulcerated lesion on the glans penis at an early disease stage. Moreover, patients may present obstructive symptoms, hematuria, urethral discharge, and urinary fistula in more advanced disease stages [64]. Prognosis is also very poor. Hematuria, complaints of a protruding mass, obstructive urinary symptoms, including a weak urinary stream, urethral discharge, flank pain, and hydronephrosis are symptoms related to urinary tract MMs [64]. MMs presenting ‘‘ABCDE’’ features can be clinically recognized in visible regions such as the vulva, the penis, and the oral cavity [15]. However, it is important to take into account that amelanocitic lesions may be difficult to diagnose. A careful clinical examination as well as palpation for the detection of suspicious regional or distant lymphadenopathies along with a visual inspection for detection of occult mass must be carried out in such patients. Particular attention should be paid to cutaneous examination and careful exploration of visible mucosae due to the possibility of metastatic CM. Specialist consultations by an otolaryngologist, urologist, gynecologist, and gastroenterologist are often necessary for MM diagnosis because of the necessity to perform biopsies of suspicious lesions. Pathology examination of biopsy specimen represents the gold standard for diagnosis. Macroscopically pigmented lesions are highly suspect for melanoma. If pigment is absent, then diagnosis may be less simple. Immunohistochemical staining positive for protein S-100, HMB-45, Melan-A, Mart-1, and tyrosinase support the diagnosis of melanoma [1]. Once diagnosis of malignancy is performed an accurate staging of the disease is mandatory in order to obtain prognostic information and to determine the further management of the patient. Usually, laboratory analyses along with instrumental tests including endoscopic studies of the head and neck region or gastrointestinal tracts, a total body computed tomography (CT) scan, positron emission tomography (PET), ultrasonography of various areas within the human body, as well as magnetic resonance imaging (MRI) are useful to complete the diagnostic path and to detect putative loco-regional and systemic extension of the disease. However, no consensus on staging MMs from various anatomic sites exists [41]. Due to the rarity of disease, the adequacy of staging criteria for providing prognostic information is not clear and many oncologists use different systems to stage MM [41]. Globally, MMs of the aero-digestive tract are staged in Italy according to AJCC staging (8° ed) and vulvar melanomas are staged following the AJCC staging criteria for CM; for the urethral, vaginal, rectal, and anal MMs, there is no consensus for staging [70]. Some clinicians prefer to utilize a simple and practice system for staging MM patients, which was proposed by Ballantine for Head and Neck MMs in 1970 [71]. This system is characterized by three stages, depending on disease extension: (I) local, (II) regional (nodal metastases), or (III) disseminated. Surgery represents the mainstay for treatment of localized disease. Complete tumor removal with the pathological finding of clear margins seems to be associated with better outcomes [72,73], though with no clear benefit on overall survival (OS) [41]. Despite aggressive surgery, the occurrence of local or distant recurrences are very common with the majority of patients ultimately dying of disseminated disease. Factors associated with local recurrence include tumor size, vascular invasion, and not radical tumor resections. Furthermore, the surgical strategy may be challenging and it should be tailored individually taking into account the tumor stage or its anatomic site, because of its potential morbidity. Overall, the poor prognosis related to the disease induces the consideration of the residual patient quality of life in any clinical decision-making in order to avoid unnecessary and harmful surgical efforts. For instance, endoscopic resections can be offered to selected patients with lower morbidity and similar local control [74]. Radiotherapy may be helpful for increasing local control and reducing the rates of local failure, especially when surgery achieves no negative margins or is aimed at obtaining conservative surgical management of the disease such as in anorectal MMs [75]. It is also a treatment worth considering for bulky mass or symptomatic lesions with palliative scope. Before 2010, the prognosis of patients affected by advanced CM was very poor irrespective of melanoma subtype, as few effective systemic therapies were available. In their series, Kuck et al. [7] reported a median survival ranging from 10 to 13 months in patients affected by metastatic melanoma from cutaneous, acral, uveal and unknown origin [27]. In the same report, patients affected by MM exhibited a poorer outcome with a median survival of approximately 9 months [7]. Whilst the introduction of new systemic treatment options for the management of patients affected by CM, such as immunotherapies (anti-CTLA-4 and anti-PD-1 antibodies [76]) or targeted therapies (BRAF, MEK inhibitors [77]) either alone or in combination, represented a significant breakthrough in clinical practice, the development of systemic therapies for MMs has been much slower [7]. Actually, 3 immune checkpoint inhibitors (ICIs) have been approved for treatment advanced melanoma patients: ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4); the anti-programmed cell death receptor 1 (PD-1) agents pembrolizumab and nivolumab; as well as the combination of ipilimumab plus nivolumab [78]. Recently, based on results of the phase 2/3 Relativity-047 randomized trial [79], a new drug that combines nivolumab with relatlimab, opdualag, has been approved in an attempt to block both the immune checkpoint protein PD-1 and LAG-3. Across different trials, PD-1 blockade with either nivolumab or pembrolizumab resulted in response rates ranging from 26% to 44% when used as single agents and significantly improved OS when compared with ipilimumab and dacarbazine [78,80,81,82,83,84,85]. Furthermore, a durable, sustained survival benefit can be achieved with first-line nivolumab plus ipilimumab or nivolumab alone [86]. However, CM and MMs are molecularly different tumors and most of these studies have been performed on patients affected by CM, limiting data extrapolation to MMs because of their rarity. It was also suggested that the efficacy outcome of ICIs was poorer in MM than CM patients, but no formal perspective comparisons, to the best of our knowledge, were made between such melanoma subtypes [51,87]. Moreover, due to the rarity of MMs, knowledge about disease treatment with ICIs are limited, mostly deriving from small population studies, retrospective analyses, and case reports [26,88,89,90,91,92]. A multicenter retrospective study comparing immunotherapy (anti-PD-1 and anti-CTLA-4) versus chemotherapy (mostly dacarbazine) showed a longer median OS for patients affected by advanced MM who received ICIs (approximately 16 months) respect to those treated with chemotherapy (8.8 months) [19,93]. Furthermore, Hamid et al. reported an overall response rate (ORR) of 22% and 15% with a median progression-free survival (PFS) of approximately 2.8 months (in ipilimumab-naive or ipilimumab pre-treated patients, respectively) in a post-hoc analysis of pembrolizumab of KEYNOTE-001, -002, and -006 trials [19,94]. A large report of the efficacy and safety of an ICI in MM was reported by D’Angelo et al. in their pooled analysis, where the combination regimen nivolumab plus ipilimumab was compared with either nivolumab or ipilimumab alone [89]. This combo immunotherapy achieved a better ORR (37.1%) as compared with both single agents (23.3% and 8.3%, respectively), but showed a higher toxicity. Despite this positive result, ORR by this combination was found to be markedly inferior as compared with the ORR of 60% observed in CM patients. Furthermore, the same combo achieved 43% of ORR with a PFS of 5.8 months in a naive subgroup of MM patients from Check-Mate 067 as compared with nivolumab (ORR 30% and PFS 3 months) and ipilimumab (ORR 7% and PFS 2.6 months) [19,95]. The combo ipilimumab–nivolumab regimen appears to be a rational systemic approach for treating fit patients affected by advanced MM, though this type of treatment is burdened by important grade 3–4 toxicities (approximately 55%) [76]. Alternatively, a less toxic monotherapy with anti PDl-1 agents, such as nivolumab or pembrolizumab, may be considered for unfit patients in order to implement their treatment compliance. A retrospective cohort study evaluated treatment efficacy of anti-PD-1 ± ipilimumab in 545 MM patients. Three hundred and forty-eight (64%) patients received anti-PD-1 whereas the remaining 197 (36%) received the anti-PD-1/ipilimumab combination. The ORR, PFS, and OS did not significantly differ by primary site (naso-oral, urogenital, anorectal, other), ethnicity/race, and treatment. Only the ORR for naso-oral primary site was numerically higher for the anti-PD-1/ipilimumab combination compared with anti-PD-1 without survival benefit. These data suggest that in MMs, the addition of ipilimumab has a poor clinical benefit over anti-PD-1 [96]. In phase 2/3 Relativity-047, 51 (7.1%) of the 714 melanoma patients were MM patients. The subgroup analysis comparing 23 cases undergoing the relatlimab–nivolumab combo toward the 28 cases undergoing nivolumab alone showed no significant differences in PFS. However, the small sample size and the rarity of MMs show that additional studies are needed to understand the efficacy of the relatlimab–nivolumab combo in patient populations that are often excluded from clinical trials [79]. Overall, it is not clear why the response rates observed to ICIs are lower in MMs with respect to CM. It has been hypothesized that this may be a result of the lower proportion of MM patients carrying high levels of PD-L1 expression (>5%), which was found in the pooled analyses, although no evidence fully supports the role of PD-L1 in predicting response to ICIs in such patients [41]. On the other hand, the lower MM mutation burden as compared to that observed in CM lesions may justify this clinical observation, whereas the presence of tumor-infiltrating lymphocytes (TILs) could be a predictor of anti PD-L1 response [41]. Recently, a phase II trial aimed to compare toripalimab versus high-dose interferon-α2b (HDI) as an adjuvant therapy for resected MM. A total of 145 patients with resected MM were randomized (1:1) to receive HDI (n = 72) or toripalimab (n = 73) for 1 year. Toripalimab showed a similar relapse-free survival and a more favorable safety profile than HDI, suggesting that toripalimab might become the better treatment option [97]. To date, limited data regarding the efficacy of neoadjuvant checkpoint inhibition for resectable MMs are available. However, the Ho et al. study, despite the small sample size (n = 36) demonstrated that the neoadjuvant anti- PD1 +/− anti-CTLA4 immunotherapy can be a feasible approach. Indeed, median event-free survival (EFS) was 9.2 months, ORR was 47%, PRR was 35%, whereas the 3-year OS rate was 55%. Interestingly, the pathologic response achievement was associated with a significant improvement in OS and EFS suggesting the need for further investigation [98]. Unlike CM, BRAF mutations are infrequent in MMs and therefore treatment with BRAF kinase inhibitors (i.e., dabrafenib or vemurafenib), mostly in combination with a MEK inhibitor (i.e., trametinib), rarely represents a therapeutic option for these patients [41]. However, all MM patients should be tested for BRAF mutations and the treatment with the combination of a BRAF inhibitor and a MEK inhibitor may be considered when activating BRAF mutations are detected [41]. Treatment with BRAF inhibitors achieved a 20% of ORR and 70% disease control rate (DCR) in a small cohort of 12 MM patients with metastatic or unresectable BRAF V600E-mutant melanoma [99]. MMs may present activating mutations in KIT. These mutations, mainly detected in exons 11 and 13, are most common in vulvovaginal and anorectal disease sites [18,19,22,46]. Tyrosine kinase inhibitors, such as imatinib or nilotinib, targeting the aberrant protein gene product were found to be able to induce a clinical response. In a multicenter phase 2 trial involving 17 MM patients harboring mutationally activated or amplified KIT, Hody et al. [100] reported a 64% of ORR among those with KIT mutations (in exon 11, 13, and 17) treated with imatinib mesylate, whereas the drug was ineffective in patients only presenting KIT amplifications. Furthermore, nilotinib exhibited similar responses as imatinib, demonstrating a clinical effect in circumstances of disease progression after imatinib [101,102,103]. Interestingly, new strategies for the systemic treatment of MMs have been recently reported [26,104,105]. These systemic approaches were developed considering the important role played by the vascular endothelial growth factor (VEGF), which is strongly expressed in MMs, on either disease progression or immunosuppression [106,107,108,109]. Sheng et al. [26,104] investigated a combination of a VEGF inhibition (axitinib) with PD-1 blockade (toripalimab) in a phase Ib trial of 33 patients suffering from metastatic MM. Among 29 treatment naïve patients, an ORR of 48.3% with a disease control rate (DCR) of 86.2% was reported. Median PFS was 7.5 months while median OS was 20.7 months. In their report, PD-L1 expression or TMB value presented no significant differences in responder versus non-responder patients, whereas gene expression profile (GEP) scores of eight selected immune-related and four angiogenesis-related genes, showed a strong correlation with clinical response [26,104]. Notably, 39.4% of patients experienced Grade 3–4 toxicities but only one patient discontinued treatment. Most common side effects were diarrhea, proteinuria, hand–foot syndrome, and hypothyroidism [104]. On the other hand, Yan et al. [105] conducted a multicenter 2:1 randomized, open-label, phase II study involving 114 naïve advanced MM patients receiving chemotherapy (carboplatin AUC 5 plus paclitaxel 175 mg/m2 every 4 weeks) with or without VEGF inhibition (bevacizumab 5 mg/kg every 2 weeks). The primary end point of the trial was PFS while OS was a key secondary end point [110]. The combination of carboplatin plus paclitaxel and bevacizumab was found to be more active than carboplatin plus paclitaxel. A combination with the antiangiogenic drug bevacizumab significantly increased median PFS from 3.0 to 4.8 months and median OS from 9.0 to 13.6 months [103]. Treatment appeared to be well tolerated and no significant safety signals (including no gastrointestinal perforation or hemorrhage) were reported [105,110]. This regimen could represent an alternative for the treatment of advanced MMs in an immunotherapy-ineligible or immunotherapy-failure population and further studies are warranted as suggested by the authors [105]. The different sites of origin associated with a remarkable genetic heterogeneity make MM a straightforward model to evaluate the cell biochemical changes that enhance tumor spreading. Indeed, mucosal melanoma cell lines capable of maintaining their heterogeneity following long-term cell culture and allowed to establish as two different metabolic profiles, the first characterized by the production and excretion of lactate in the extracellular microenvironment while the second, oxidative type was characterized by the uptake of lactate from the microenvironment and its use as primary carbon source, showed different phenotypic profiles in promoting cancer metastasis spreading [111]. Thus, a deep knowledge of metabolic reprogramming processes may highlight potential targets useful in interfering with the dynamic cancer progression. Another targetable metabolic pathway for MM is represented by the degradation of tryptophan to kynurenine, which plays a key role in regulating the immune response. In particular, it has been found that in tumor tissue an increase in the enzyme indoleamine 2,3-dioxygenase (IDO) that regulates the degradation of tryptophan is able to promote the development of an immunosuppressive microenvironment that can inhibit effective antitumor immunological responses [112,113]. To date, it is unknown whether in MM the IDO activity changes reflect or are able to modify the metabolism of the host, leading to serum variations in the concentration of tryptophan and kynurenine. Interestingly, it has been observed that the surgical treatment of non-cutaneous melanoma is able to profoundly change the metabolic profile of the host. The removal of a primary melanoma of the lung re-established the level of 5-S-Cysteinyldopa within the normal range; thus, indicating how melanoma is able to condition not only the microenvironment surrounding the tumor but also to create important metabolic changes in different body matrices [114,115,116]. Considering that the metabolic profile tended to change according to the recurrence of the disease and to the treatment, the achievement of a homeostatic balance similar to normal range or its further imbalance could be considered as two different trajectories associated to different prognoses. The cell-free microRNas (miRNAs) may play a significant role in the development and progression of MM since in MM cells it has been observed that overexpression of some miRNa, such as miR-let-7b or miR-let-7c, is able to inhibit cell growth, migration, invasion, and metastatization whereas, conversely, it seems to induce apoptosis and cell cycle shutdown after chemotherapy treatment [117]. The overexpression of these miRNAs was therefore able to reduce the recurrence of MM increasing the sensitivity to chemotherapy treatment thus leading to a lengthening DFS. In agreement with such evidence, a low expression of microRNA-23a-3p has been significantly associated with poor outcomes. Indeed, the Kaplan–Meier survival analysis of 117 MM patients showed that low mir-23a-3p expression was significantly associated with poor outcomes, with both the DFS and OS being markedly reduced. In this study, even after a multivariate analysis, the low expression of miR-23a-3p was confirmed as an independent prognostic indicator of reduced OS [118]. Finally, the recent SARS-CoV-2 pandemic allowed for the introduction of new technologies based on mRNA vaccines. This methodology has proven to be safe and effective and could be translated into the oncology field. The building of mRNAs that go inside the immune cells favoring the production of individual, specific tumor proteins able to enhance immunological responses would definitively pave the way for highly personalized therapies. Briefly, mRNA vaccines represent an attractive vector to deliver tumor antigens into dendritic cells (DCs) that are the antigen-presenting cells of our immune system [119]. The encoded proteins can be translated and processed into peptides, which can be subsequently exhibited on the DCs’ surface; thus, enhancing the immunological effects mainly through adaptive immune responses. In this context, the peptides sharing with the CD8+ T cells trigger the cytotoxic reaction while exposure to CD4+ T cells determines antibody production against the tumor cells [119]. Underlying the immune response processes turns out to be the relevant modulation of the interferon type I (IFNs) capable of initiating or adjuvating the cytotoxic activity of CD8 cells [120]. However, to date it remains controversial whether IFNs have a stimulatory or inhibitory action on CD8+ T-cell immunity since the mechanisms behind this duality are unclear. Strategies to optimize the intracellular delivery of mRNA vaccines, allowing a more efficient antigen translation and providing potent and specific immune activations, are being investigated. Among others, lipid formulations seem to induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway and result in systemic cytokine expression and enhanced anti-tumor efficacy [121]. To further support such a therapeutic strategy to a wider extent, preliminary results from a study on adjuvant treatment with the personalized cancer vaccine mRNA-4157 and pembrolizumab in stage III/IV melanoma patients undergoing complete resection of cutaneous melanoma have just demonstrated a statistically significant and clinically meaningful reduction in the risk of disease recurrence or death compared to pembrolizumab monotherapy (KEYNOTE-942) [122]. The life expectancy of patients with MMs remains lower than that of patients with CMs since they are diagnosed at a more advanced stage and have a lower burden of point mutations and a wider number of structural chromosomal variants that make them less responsive to immunotherapy treatments. These biochemical features impose the need to identify new specific and selective druggable transcriptomics and metabolic pathways for future more efficient therapeutic applications. In this context, the application of omics’ technologies could represent an innovative impulse to better understand the dynamic cell metabolic reprogramming that leads to cancer development and progression explaining the phenotypic heterogeneity and its biological and biochemical behavior of MMs that can contribute to improve their diagnosis and treatment.

PMC10000128

Ajay Major,Pierluigi Porcu,Bradley M. Haverkos

Rational Targets of Therapy in Extranodal NK/T-Cell Lymphoma

21-02-2023

extranodal NK/T-cell lymphoma (ENKTL),biomarkers,novel therapies

Simple Summary Extranodal NK/T-cell lymphoma (ENKTL) is an aggressive blood cancer with poor survival rates, particularly for patients with advanced-stage and relapsed disease. Emerging research on the genetic and molecular causes of ENKTL have revealed new potential treatment strategies. In this review, we summarize how research on the biological causes of ENKTL has translated to new targets for the treatment of ENKTL, as well as the identification of new bi-omarkers which may predict prognosis and responses to specific anti-cancer therapies and enable a personalized medicine approach towards ENKTL therapy. Abstract Extranodal NK/T-cell lymphoma (ENKTL) is an aggressive extranodal non-Hodgkin lymphoma (NHL) with poor outcomes, particularly in advanced-stage and relapsed/refractory disease. Emerging research on molecular drivers of ENKTL lymphomagenesis by next-generation and whole genome sequencing has revealed diverse genomic mutations in multiple signaling pathways, with the identification of multiple putative targets for novel therapeutic agents. In this review, we summarize the biological underpinnings of newly-understood therapeutic targets in ENKTL with a focus on translational implications, including epigenetic and histone regulatory aberrations, activation of cell proliferation signaling pathways, suppression of apoptosis and tumor suppressor genes, changes in the tumor microenvironment, and EBV-mediated oncogenesis. In addition, we highlight prognostic and predictive biomarkers which may enable a personalized medicine approach toward ENKTL therapy.

Rational Targets of Therapy in Extranodal NK/T-Cell Lymphoma Extranodal NK/T-cell lymphoma (ENKTL) is an aggressive blood cancer with poor survival rates, particularly for patients with advanced-stage and relapsed disease. Emerging research on the genetic and molecular causes of ENKTL have revealed new potential treatment strategies. In this review, we summarize how research on the biological causes of ENKTL has translated to new targets for the treatment of ENKTL, as well as the identification of new bi-omarkers which may predict prognosis and responses to specific anti-cancer therapies and enable a personalized medicine approach towards ENKTL therapy. Extranodal NK/T-cell lymphoma (ENKTL) is an aggressive extranodal non-Hodgkin lymphoma (NHL) with poor outcomes, particularly in advanced-stage and relapsed/refractory disease. Emerging research on molecular drivers of ENKTL lymphomagenesis by next-generation and whole genome sequencing has revealed diverse genomic mutations in multiple signaling pathways, with the identification of multiple putative targets for novel therapeutic agents. In this review, we summarize the biological underpinnings of newly-understood therapeutic targets in ENKTL with a focus on translational implications, including epigenetic and histone regulatory aberrations, activation of cell proliferation signaling pathways, suppression of apoptosis and tumor suppressor genes, changes in the tumor microenvironment, and EBV-mediated oncogenesis. In addition, we highlight prognostic and predictive biomarkers which may enable a personalized medicine approach toward ENKTL therapy. Extranodal NK/T-cell lymphoma (ENKTL) is a rare extranodal non-Hodgkin lymphoma (NHL) that primarily occurs in the upper aerodigestive tract and has an aggressive presentation, with locoregional invasion in the nasopharynx causing necrosis, hemorrhage, and impingement on anatomic structures including the orbits [1,2,3]. The incidence of ENKTL is much higher in East Asia and Latin America than in Europe and North America, representing up to 15% of all NHL diagnoses, likely due to underlying geodemographic differences in human leukocyte antigen (HLA) genes and genetic susceptibility [4,5,6,7]. Outcomes for ENKTL are generally poor, with 5-year overall survival (OS) rates of approximately 50% when treated with asparaginase-based multi-agent combination chemotherapy regimens such as SMILE [8,9] but as low as 25–28% in high-risk patients as assessed by various prognostic indices of NK lymphoma [10,11]. The majority of patients with ENKTL present with limited-stage, localized disease, in which outcomes are better, with 5-year and 10-year OS rates of 64–89% and 57%, respectively, utilizing combination chemotherapy and radiation [12,13,14]. However, patients with relapsed or refractory disease have dismal outcomes, with median survival of less than 12 months [15,16]. Despite advances in the frontline management of ENKTL with combination chemoradiotherapy approaches [14], there is a pressing need for novel and rational therapies, informed by the molecular biology of ENKTL, particularly for patients with relapsed/refractory disease, for patients who cannot tolerate intensive frontline induction therapy, and for patients with high-risk disease features and advanced-stage disease. Historically, the pathobiology of ENKTL and its molecular drivers have been poorly understood. Gene rearrangement studies suggest that the majority of ENKTL cases are of NK cell rather than T cell lineage [17,18,19]. Although there are no apparent differences in clinical presentation or survival between NK or T cell lineages [20], there is emerging evidence that different signaling pathways are constitutively activated in each cell lineage, reflecting a complex and heterogeneous molecular driver landscape of ENKTL [19]. Classical descriptions of ENKTL oncogenesis by canonical T and NK cell signaling have focused on the ubiquitous presence of EBV in ENKTL tumor cells, and it is posited that EBV infection induces the overexpression of the transmembrane oncoprotein LMP1 in infected NK and T cells, resulting in ligand-independent activation of the NF-κB and MAPK signaling pathways [18,19,21]. These signaling pathways, with loss of tumor suppressor genes on chromosome 6q21, as is commonly observed in ENKTL, cause tumor cell proliferation and avoidance of apoptosis by regulation of c-MYC and survivin and also upregulate cell surface expression of PD-L1 to promote escape from immune surveillance [18,22]. However, ENKTL cells do not experience immortalization in response to EBV infection, as is seen in B-cell lymphomas, but rather become sensitized to proliferative cytokines such as IL-2, suggesting that other molecular alterations must occur in a multistep process for EBV-infected NK or T cells to undergo malignant transformation [23,24]. Recent advances in somatic next-generation and whole-genome sequencing have provided emerging evidence that ENKTL is characterized by genomic aberrations in multiple signaling pathways, including broad epigenetic and gene methylation changes [17,19,23,25]. In this review, we summarize newly-understood putative driver mutations in ENKTL with a focus on translational implications and novel therapies with rational biologic underpinnings (Table 1), as well as prognostic and predictive biomarkers which may enable personalized therapeutic approaches (Table 2). It is increasingly observed that epigenetic modifiers are the largest group of mutated genes in ENKTL, with one meta-analysis of nine next-generation sequencing (NGS) studies finding that 25% of identified mutations in ENKTL affected epigenetic regulators [19]. Epigenetic aberrations play a critical role in tumorigenesis by silencing tumor suppressor genes and altering the regulation of oncogenes. A cohesive understanding of how epigenetic changes drive the ENKTL clone is starting to emerge [32]. In many types of cancer, promoter regions of tumor suppressor genes are frequently hypermethylated during carcinogenesis, resulting in transcriptional silencing via the recruitment of histone deacetylases (HDACs) and repressive chromatin formation [32]. Comparison of ENKTL tumor cells and normal NK cells with methylation assays have demonstrated global promoter hypermethylation and gene silencing in ENKTL, with decreased mRNA transcription of 95 putative tumor suppressor genes, including BCL2L11 (BIM), DAPK1, PTPN6 (SHP1), TET2, SOCS6, and ASNS with known functions [26]. Specifically, BIM sensitizes NK cell lines to chemotherapy-induced apoptosis, DAPK1 mediates p53-dependent apoptosis, SHP1, and SOCS6 inactivate the JAK/STAT signaling pathway, and TET2 silencing may contribute to early global hypermethylation in ENKTL, so it is easily understood how hypermethylation of multiple tumor suppressor promoters may create additive effects leading to tumor growth and malignant transformation [26]. These findings have been confirmed in other studies, with the identification of additional tumor suppressor genes, including DLC1 involved in RAS signaling [27], PRDM1 involved in apoptosis and promotion of NK cell growth in cooperation with activating STAT3 mutations [28,29], PTPRK involved in JAK/STAT signaling and apoptosis [30], HACE1 involved in apoptosis [31], as well as several other genes (P73, hMLH1, CDKN2A, CDKN2B, RARβ, PCDH10, DLEC1, CADM, DAL1) with currently unknown functions [32]. A recent genome-wide DNA methylation and transcriptomic study by Mundy-Bosse et al. (2022) demonstrated that ENKTL cells likely represent a malignant transformation from NK cells normally present in mucosal tissues, with ubiquitous and profound DNA hypermethylation driven by EBV infection causing the arrest of normal NK cell differentiation [17]. In this study, patient-derived xenograft (PDX) mouse models of ENKTL were treated with the DNA hypomethylating agent 5-azacytidine, which resulted in a significant decrease in tumor burden and an increase in OS compared to control mice. In addition, 5-azacytidine treatment resulted in a global reduction in DNA methylation and the emergence of mature NK cell markers, suggesting that treatment with hypomethylating agents may induce terminal differentiation of ENKTL cells, as is seen in patients with acute promyelocytic leukemia treated with all-trans retinoic acid [17]. These findings confirm the results of other studies, which have found that exposure of ENKL cell lines to hypomethylating agents such as 5-azacytidine or decitabine restores the expression of these putative tumor suppressors [26,27,30,146]. In a phase I study, one patient with ENKTL responded to a combination of 5-azacytidine and romidepsin with profound demethylation of their tumor after treatment [147]. There is a strong pre-clinical rationale for the treatment of ENKTL with hypomethylating agents, although there may be discordant effects from other therapeutic agents. For example, treatment with 5-azacytidine upregulates genes associated with immunoregulatory functions, suggesting possible synergy with immunotherapy agents [17], while there is concern that hypomethylating agents may desensitize ENKTL cells to asparaginase by upregulating ASNS and also lead to lytic reactivation of EBV [26,148]. Previous studies of ENKTL have revealed mutations in genes that regulate epigenetic changes to the genome, including genes responsible for chromatin remodeling, histone acetyltransferases and methyltransferases, and DNA demethylases [32,149]. One of the most frequently mutated genes in 17–32% of ENKTL cases is BCOR [28,40,41,42], a BCL-6 interacting corepressor which is involved in the epigenetic modification of histones via HDACs and which is suggested to be a tumor suppressor given frequent loss-of-function mutations seen in ENKTL [19,41] and enhanced cell proliferation and IL-2 production when BCOR is silenced [42]. MLL genes are also commonly mutated in up to 19% of ENKTL cases, specifically MLL2 (KMT2D) and MLL3 (KMT2C), which are involved in histone methylation and may be tumor suppressors in B-cell lymphomas, although the role of these epigenetic regulators specifically in the tumorigenesis of ENKTL is unknown [19,28,32,43]. Other commonly mutated epigenetic regulatory genes in ENKTL include TET1/TET2, EP200, ASXL3, CREBBP, and ARID1A, with yet unknown functions in ENKTL biology [19,32,44]. An epigenetic regulator gene with documented oncogenic function in ENKTL is EZH2, a histone methyltransferase that is not mutated in ENKTL but rather is aberrantly overexpressed and promotes NK tumor cell growth independently of its histone methyltransferase activity [33]. This non-canonical activity of EZH2 is likely due to its direct phosphorylation by JAK3, which converts EZH2 from a gene repressor to a transcriptional activator of genes involved in cell proliferation [34]. The JAK/STAT pathway is likely an upstream regulator of EZH2, as evidenced by studies demonstrating that treatment of ENKTL cells with the JAK3 inhibitor tofacitinib reduced EZH2 expression [38] and decreased ENKTL cell growth [34]. EZH2 is a known druggable target, with an FDA-approved EZH2 inhibitor (tazemetostat) for EZH2-wild type and EZH2-mutated follicular lymphoma [35]. However, treatment of ENKTL PDX mice with tazemetostat alone as well as in combination with 5-azacytidine did not improve survival in one study [17] and was ineffective in ENKTL cell lines [34], suggesting that inhibition of the EZH2 methyltransferase catalytic site does not affect its non-canonical and enzyme-independent oncogenic functions. EZH2 inhibitors with novel mechanisms of action, such as 3-deazaneplanocin-A (DZNep), which disrupts the metabolism of methyl donors required for histone methylation by EZH2 and also accelerates proteasomal degradation of EZH2, have demonstrated enhanced lymphoma cell apoptosis compared to tazemetostat [36,37,38]. EZH2 inhibitors are a rational drug target in ENKTL, although there is pre-clinical evidence that combination with agents targeted at its upstream regulators, such as JAK/STAT inhibitors, may also cause ENKTL cell death in patients with high EZH2 tumor expression [34]. Further, targeting downstream target genes of EZH2, such as cyclin D1, may also be efficacious, with one study demonstrating that combined inhibition of upstream JAK with ruxolitinib and CDK4/6 with ribociclib produced synergistic inhibition of ENKTL cell growth [39]. Large-scale somatic sequencing of ENKTL has enabled the description of new biomarkers which are both prognostic and predictive, although large-scale studies will be required for clinical validation. Mutations in MLL2 (KMT2D) and TET2 were associated with inferior prognosis in ENKTL [43], and overexpression of EZH2 was associated with higher tumor cell proliferation, advanced stage, and inferior survival [38]. Further, the presence of KMT2D mutations in circulating tumor DNA (ctDNA) was prognostic and correlated with total metabolic tumor volume, suggesting that serial measurements of ctDNA could be used to monitor treatment response and the presence of residual disease [128]. MicroRNAs (miRNAs) are short noncoding RNA sequences that inhibit the expression of target genes by suppressing translation or promoting mRNA degradation, and many have been found to be overexpressed in ENKTL as putative oncogenes as well as prognostic biomarkers [22]. For example, elevated plasma levels of miR-221 are associated with inferior overall survival [129], and miR-155 is associated with disease response [130]. Predictive biomarkers are particularly important for ENKTL to promote maximal response to therapy for patients with an aggressive and difficult-to-treat malignancy. In a study of 17 patients with ENKTL, patients with methylated PTPRK tumors who were treated with the SMILE protocol had significantly worse overall survival and a trend towards inferior disease-free survival [30]. In addition, ASNS expression is strongly correlated with cell survival in response to asparaginase treatment, which may serve as a clinically useful biomarker to determine which patients will respond to asparaginase-containing chemotherapy or which patients with low ASNS expression may be treated with lower doses of asparaginase to avoid toxicity [26]. miRNAs have been identified as predictive biomarkers with putative therapeutic targets; for example, a novel inhibitor of miR-155 induced apoptosis in ENKTL cell lines as well as xenografts by downregulation of STAT3 and VEGF signaling pathways and upregulation of the pro-apoptotic BRG1 [45]. High expression of SNHG12, a long noncoding RNA (lncRNAs) sequence which is overexpressed in ENKTL, conferred cisplatin resistance in ENKTL cells [131] and also may be responsible for multidrug resistance in ENKTL owing to its contribution to P-glycoprotein overexpression, which is a known mechanism for anthracycline resistance in ENTKL [150]. Another lncRNA, brain cytoplasmic RNA 1 (BCYRN1), is also overexpressed in ENKTL and is associated with inferior PFS as well as resistance to asparaginase [132]. The second most frequently mutated genes in ENKTL NGS studies are those of the JAK/STAT signaling pathway, predominantly in ENKTL cells of NK cell lineage, while T cell lineage ENKTL more commonly exhibits mutations of the RAS/MAPK signaling pathway and aforementioned epigenetic regulators [19]. Aberrant regulation of both of these signaling pathways results in ENKTL survival and proliferation [19,149], driven by mutations in JAK3, STAT3, STAT5B, and DDX3X [19,25,149]. However, these mutations rarely occur together, suggesting that there are disparate molecular signaling pathways leading to ENKTL tumorigenesis [25] and confirmed in contemporary studies identifying three distinct subtypes of ENKTL by gene expression signatures: the TSIM (tumor suppressor-immune modulator) subtype with JAK/STAT activation and PD-L1 overexpression; the MB (MGA-BRDT) subtype with MYC overexpression and MAPK activation; and the HEA (HDAC9-EP300-ARID1A) subtype with changes in histone acetylation and NF-κB activation [63]. Each of these subtypes may be targeted by specific therapeutic agents based on activated signaling pathways, and putative targeted agents for each pathway are outlined as follows. Constitutive activation of the JAK/STAT pathway is responsible for ENKTL survival and proliferation, with activating mutations in STAT3 being the most common driver, followed by activating JAK3 mutations [28,46,47,48,49]. As previously discussed, activation of the JAK/STAT pathway in ENKTL converts the histone methyltransferase EZH2 into a non-canonical transcriptional activator of genes involved in cell proliferation, with inhibition of JAK3 decreasing EZH2 expression and decreasing ENKTL tumor growth [34,38]. There are multiple inhibitors of the JAK/STAT pathway which have been studied broadly in hematologic malignancies, including specifically in ENKTL [151]. The JAK1/3 inhibitor tofacitinib has demonstrated inhibition of cell growth in JAK3-mutant [47] and STAT-3 mutant ENKTL cells [50], as well as ENKTL cells with EZH2 overexpression [34]. Similar pre-clinical results have been demonstrated with novel pan-JAK inhibitors [23] and STAT inhibitors [47,50], although none of these have yet been studied in patients. Ruxolitinib, a JAK1/2 inhibitor that is already approved for use in hematologic malignancies, is a therapy of interest in ENKTL, given pre-clinical work that JAK1/2 inhibition can disrupt JAK/STAT signaling in STAT3-mutated or overexpressed ENKTL [46]. A phase II biomarker-driven study of ruxolitinib in patients with relapsed/refractory T-cell lymphomas revealed high response rates of 45–53% in patients with activating JAK/STAT mutations or STAT3 overexpression, compared to low response rates of 13% without those biomarkers [51]. Although no patients with ENKTL were included, this study provides a strong clinical rationale for the use of ruxolitinib and other JAK/STAT inhibitors in ENKTL cases with JAK/STAT aberrations and suggests that a biomarker-based therapeutic approach utilizing the aforementioned TSIM, MB, and HEA subtypes may be warranted. Further, in vitro studies suggest that ruxolitinib may be combined with novel TP53-MDM2 inhibitors in TP53-wild type disease, farnesyltransferase inhibitors in TP53-mutated disease, and with dexamethasone or novel MCL-1 inhibitors [52], or with the BCL2 inhibitor venetoclax or aurora kinase inhibitor alisertib in another study [53], to maximize activity and avoid long-term resistance. DDX3X is an RNA helicase gene that is one of the most commonly mutated genes in ENKTL [23,28,40,64], with significant upregulation of the NF-κB and MAPK pathways found in DDX3X-mutated ENKTL tumors, suggesting that DDX3X is a possible tumor suppressor [17,19,64]. The RAS/MAPK signaling pathway is broadly implicated in carcinogenesis by promotion of cell survival and proliferation [19] and is upregulated in the MB subtype of ENKTL [63], likely related to overexpression of the EBV-associated LMP1 oncoprotein [18]. Previous pre-clinical studies have found that inhibition of the MAPK pathway is not effective in vitro or in vivo in NHL cells, likely due to the regulatory complexity of the pathway [65]. However, pre-clinical studies have found that statins inhibit cell growth and enhance chemotherapy-induced cytotoxicity in NK leukemia cell lines due to inhibition of the MAPK pathway, suggesting an opportunity for future combination studies [66]. Of note, DDX3X mutations portend a poor prognosis in patients with ENKTL treated with CHOP-based chemotherapy [64], although not in patients treated with more contemporary asparaginase-based regimens [63]. There are multiple drivers of NF-κB signaling pathway upregulation in ENKTL, including DDX3X mutations as well as EBV-associated LMP1 oncoprotein expression [18,54]. The NF-κB pathway is considered a major driver of tumorigenesis in lymphomas and specifically in ENKTL, likely related to overexpression of a downstream anti-apoptotic protein called survivin which is elevated in nearly all cases of ENKTL and is prognostic [22,23,54,55,56,57]. There has been considerable study focused on the inhibition of the NF-κB pathway in ENKTL, particularly with the proteasome inhibitor bortezomib, which inhibits ENKTL in vitro [58]. Clinical use of bortezomib has been restricted to small case series [59], including a phase II study of bortezomib, gemcitabine, ifosfamide, and oxaliplatin in 7 patients with newly-diagnosed ENKTL which demonstrated an overall response rate of 43% but a median progression-free survival of only 4 months [60]. A phase II study of bortezomib in combination with CHOP chemotherapy included 10 patients with ENKTL, for which the complete response rate was much lower than other lymphoma subtypes at 30%, with ENKTL patients having rapid disease progression after the first two cycles, likely due to known anthracycline resistance in ENKTL [61]. There is a suggestion that bortezomib may be effective if combined with asparaginase-based chemotherapy, as inhibition of the NF-κB pathway would downregulate CD25, which induces asparaginase resistance in ENKTL cell lines [23], and effective combinations of proteasome inhibitors with other chemotherapeutics or targeted agents merits further study. For example, pre-clinical data has identified that terameprocol and mithramycin, inhibitors of survivin and its related transcription factors, induce apoptosis of ENKTL cells, with mithramycin also suppressing ENKTL growth in a mouse xenograft model, both of which may be appropriate partners for multipronged inhibition of the NK-κB pathway [55,62]. C-MYC is a well-known oncogene in lymphomas and is overexpressed in ENKTL, likely driven by LMP1 and EBV infection and causing upregulation of downstream targets EZH2 and RUNX3, the latter of which is a master transcriptional regulator and a putative oncogene in ENKTL [22,67,68]. C-MYC inhibitors are of particular interest in multiple aggressive lymphomas, and treatment of ENKTL cell lines with a small-molecule novel MYC inhibitor caused downregulation of both MYC and RUNX3 and, subsequently, apoptosis [67]. Platelet-derived growth factor receptor alpha (PDGFRa) is a tyrosine kinase pathway that is overexpressed in ENKTL, possibly driven by the PI3K/Akt/mTOR and JAK/STAT pathways, and mediates cell survival [69,70]. Previous pre-clinical work has demonstrated that imatinib, a tyrosine kinase inhibitor (TKI), inhibits ENKTL cell proliferation in vitro [71] as well as in mouse models [69], supporting further study of TKIs in ENKTL. In addition, high PDGFRa expression is a prognostic biomarker of survival [69,70]. The PI3K/Akt/mTOR signaling pathway, which is a master regulator of growth and survival in normal and malignant cells, is downstream of the JAK/STAT pathway and is upregulated in many types of lymphoma, including ENKTL, where it is driven by EBV infection and LMP1 [18,23,72]. Inhibitors of multiple targets along this signaling cascade have been explored in lymphomas, particularly PI3K inhibitors, which have already been approved by the FDA for various relapsed/refractory lymphoma subtypes. Several small phase I studies of duvelisib and tenalisib in peripheral T-cell lymphomas (PTCLs) have demonstrated an ORR of ~50% [73,74,75], and a phase I/II trial of copanlisib and gemcitabine in PTCL included 3 patients with ENKTL with 2 partial responses and 1 complete response [76]. Combination targeting of the PI3K/Akt/mTOR signaling pathway appears to be more effective due to the activation of alternative signaling pathways by negative feedback, with drug sensitivity profiling revealing that inhibition of both mTOR and JAK may be more effective than single agents in NK/T-cell lymphoma cell lines [77]. As an example, a phase I/II trial of the mTOR inhibitor temsirolimus in combination with the Akt inhibitor and immunomodulatory agent lenalidomide in relapsed/refractory lymphomas demonstrated an ORR of 67% in 9 patients with T-cell lymphomas, a superior ORR compared to single-agent mTOR inhibition or lenalidomide alone [78]. Avoiding apoptosis, either via overexpression of anti-apoptotic proteins or suppression of tumor suppressor genes, has been identified as a significant contributor to tumorigenesis in ENKTL. The tumor suppressor TP53 is one of the most frequently mutated genes in ENKTL and is present in up to 63% of cases [19,28,40,64], with a worse prognosis in TP53-mutated ENKTL [133]. Although genomic studies have identified numerous other putative tumor suppressor genes in ENKTL with functional suppression of cell growth in ENKTL cell lines, such as PRDM1 and FOXO3, there are not currently targeted agents to re-enable these tumor suppressors [152]. However, histone deacetylase inhibitors have been explored extensively in lymphomas to reactivate intrinsic apoptotic pathways, with several HDAC inhibitor agents under exploration in ENKTL [79]. As previously discussed, HDACs are histone deacetylases that frequently lead to the inactivation of tumor suppressor genes when aberrantly activated during tumorigenesis [32]. As such, HDAC inhibitors lead to the reactivation of downstream apoptotic pathways, including upregulation of TP53 and downregulation of the PI3K/Akt/mTOR pathway by the accumulation of autophagic factors [79]. Given the known activity of the HDAC inhibitor romidepsin in T-cell lymphomas, a prospective pilot study of romidepsin, specifically patients with relapsed/refractory ENKTL, was conducted in 2013 [80]. However, of the first 5 patients enrolled in the study, 3 patients developed treatment-emergent EBV reactivation manifesting as severe liver injury and 1 death, resulting in early termination of the trial. A correlative analysis confirmed that romidepsin caused a significantly greater increase in EBV reactivation in ENKTL cell lines compared to vorinostat [80]. This early experience decreased interest in HDAC inhibitors for ENKTL, although there was a suggestion that not all HDAC inhibitors would cause the same degree of EBV reactivation given different HDAC isoform specificity of different inhibitors. Subsequent clinical studies have explored various HDAC inhibitors in ENKTL, including chidamide, which is of particular interest given it also inhibits the PI3K/Akt/mTOR and RAS/MAPK signaling pathways, with an ORR of 15–50% in small studies of 15–19 patients [81,82,83]. There have also been responses in single patients to belinostat [84] and vorinostat [85]. Simultaneous targeting of the NF-κB signaling pathway with proteasome inhibitors was posited to reduce the risk of EBV reactivation in a phase II study of panobinostat and bortezomib, in which 1 of 2 patients with ENKTL had a partial response, and neither patient had EBV reactivation [153,154]. Several other HDAC inhibitor combinations have been explored, including chidamide in combination with chemotherapy as well as chidamide in combination with immunotherapy, the latter to be discussed later. In the former study of 53 patients with ENKTL, the ORR was significantly higher when chidamide was combined with chemotherapy (40%) compared to chidamide alone (15%), confirming pre-clinical data that HDAC inhibitors sensitize lymphoma cells to chemotherapy-induced DNA damage [82]. In a phase II randomized trial of 74 patients with newly-diagnosed, high-risk, early-stage ENKTL, patients were treated with radiation therapy followed by gemcitabine, dexamethasone, and cisplatin (GDP) either with or without chidamide [155]. Response rates, PFS, and OS were similar in the control group of GDP alone as compared to the experimental arm of GDP combined with chidamide, suggesting that chidamide may need to be paired with a different chemotherapy backbone or studied in patients with specific biomarkers such as the HEA subtype of ENKTL which is enriched with histone acetylation mutations [63]. In that vein, a phase II single-arm trial of 36 patients with ENKTL in first complete response were administered chidamide, cladribine, gemcitabine, and busulfan conditioning chemotherapy prior to autologous stem cell transplantation [156]. The 4-year PFS of ENKTL patients was 73%, which was significantly higher than historical PFS rates of 33–40% after autologous transplantation and suggested that the optimum pairing of HDAC inhibitors with chemotherapy partners merits further study. Novel HDAC inhibitors with different mechanisms of action have also been developed, including nanatinostat in combination with valganciclovir [122], citarinostat in combination with the JAK/STAT inhibitor momelotinib in cell lines [157], and the histone acetyltransferase KAT5 inhibitor NU9056 in cell lines [158]. Mobilizing the tumor microenvironment (TME) is a major therapeutic advancement in cancer, particularly with checkpoint inhibitors which augment the PD-1/PD-L1 signaling pathway to induce a cytotoxic T cell immune response against tumor cells. In lymphomas, immunotherapy has been most successful in Hodgkin lymphoma, in which anti-PD1 treatment alters the TME by downregulating pro-survival factors rather than inducing an immune response [159]. The role of the TME in ENKTL is under investigation, with previous studies identifying the upregulation of PD-L1 in ENKTL cells driven by EBV-mediated LMP1 and constitutive activation of the JAK/STAT pathway [134,160]. However, there are additional elements of the TME in ENKTL that may be amenable to therapeutic targeting. PD-L1 is very commonly expressed in ENTKL in 56–80% of cases [86,87], with the interplay of PD-1 and PD-L1 in the TME of particular interest in the therapeutic targeting of ENKTL, with a study finding that PD-L1 is expressed on both tumor cells as well as tumor-infiltrating macrophages in the ENKTL TME, suggesting that ENKTL cells may additionally induce immunosuppression by upregulation of PD-L1 in macrophages [88]. Commercial monoclonal antibodies against PD-1, such as pembrolizumab and nivolumab, have been studied in small cohorts of patients with relapsed/refractory ENKTL: ORR 100% in 7 patients using various radiographic and serologic response criteria [89], ORR 57% in 7 patients [90] and in 2 out of 3 patients of whom 2 quickly died due to infections and poor performance status [91], as well as in case reports [92,93]. However, the follow-up in these studies was very short at less than 6 months, with PFS and OS also measured in less than 6 months for most of the patients. Sintilimab, an anti-PD-1 antibody with a different binding site than pembrolizumab and nivolumab with greater affinity for PD-1, has been extensively studied in ENKTL. In an initial phase II study of sintilimab monotherapy in 28 patients with relapsed/refractory ENKTL, the ORR was 75% (CR 21%) with a 2-year OS of 79% with 30 months of median follow-up [94]. This trial generated significant interest in sintilimab, given high survival rates in patients with an otherwise dismal prognosis, and also demonstrated that patients with pseudo-progression due to immunotherapy, which developed in 18% of patients in the study, did not have an inferior survival and benefitted from ongoing continuous treatment with sintilimab. Given the low complete response rates with sintilimab monotherapy, there have been several efforts to combine immunotherapy with chemotherapy and other agents to induce deeper and prolonged responses, including in the frontline setting. The initial experience combining various anti-PD-1 agents with peg-asparaginase, gemcitabine, and oxaliplatin (P-GemOx) in 9 patients with newly-diagnosed, advanced-stage ENKTL revealed an ORR of 89% (78% CR) with 1-year PFS and OS of 67% and 100%, respectively, with follow-up of 10.6 months [95]. This led to a subsequent phase II study of sintilimab plus P-GemOx, with preliminary results of 6 patients revealing an ORR of 100% (33% CR) with only 7.8 months of follow-up [96]. The short follow-up of these studies does not confirm if combination therapy improves the promising survival observed with sintilimab monotherapy, although a stage-adapted study of sintilimab plus chidamide induction followed by P-GemOx (plus radiation for early-stage disease) demonstrated an ORR of 100% for 19 patients with early-stage disease and 100% in 7 out of 9 patients with advanced-stage disease who were able to complete therapy [97]. The 1-year PFS and OS rates were both 93% across the entire cohort, demonstrating that a non-chemotherapy pre-phase may be effective in patients with early-stage disease who present with poor performance status. However, given that the ORR was 100% after sintilimab and chidamide induction in the early-stage cohort and only 44% in the advanced-stage group, with 3 patients developing rapid progression during induction in the latter cohort, the appropriate choice and sequence of combination therapies need to be carefully selected based on stage as well as predictive biomarkers. The combination of sintilimab plus chidamide also has activity in the relapsed/refractory setting, with an ORR of 60% (CR 49%) and an 18-month OS of 76% in 38 patients [98]. Other combinations have been explored, including anti-PD-1 tislelizumab with chidamide, lenalidomide, and etoposide in 8 patients (ORR 88%, CR 63%) [99], anti-PD-1 toripalimab with chidamide, etoposide, and thalidomide in 3 patients (ORR 100%, CR 67%) [100], case reports of sintilimab [101] and atezolizumab [102] with chidamide, and ongoing study of the hypomethylating agent azacitidine with tislelizumab and peg-asparaginase [103]. A more recent 2022 abstract reported the results of 27 patients with relapsed and refractory ENKTL who were treated with various anti-PD-1 antibodies in conjunction with hypomethylating agents (either decitabine or azacytidine), finding an ORR of 63% (CR 59%), including high ORRs in patients who had previously received checkpoint inhibitors, with median PFS of 12.8 months [104]. Sintilimab is not currently available in the United States, although there are other novel immunotherapy agents which are amenable to study, such as the anti-PD-L1 agent sugemalimab which received breakthrough therapy designation by the FDA for relapsed/refractory ENKTL in 2020 based on the GEMSTONE-201 trial, which preliminarily demonstrated an ORR of 46% (CR 37%) with 2-year OS of 55% in 80 patients with relapsed/refractory ENKTL [105,106]. The anti-PD-L1 agent avelumab has activity in relapsed/refractory disease, with an ORR of 38% (CR 24%) among 21 patients, with expression of PD-L1 significantly associated with complete responses [107]. A novel recombinant anti-PD-1 agent, geptanolimab, was studied in 102 patients with relapsed/refractory PTCL, with the highest ORR of 63% among the 19 patients with ENKTL. There are also novel agents under investigation, including the anti-PD-1 IgG4 antibody camrelizumab [161], as well as monoclonal antibodies for newly-described immune checkpoint receptors in NK cell lymphomas such as killer cell lectin-like receptor G1 (KLRG1) [162]. The available data suggest that immunotherapy is a rational and efficacious treatment for both newly-diagnosed and relapsed/refractory ENKTL, although long-term follow-up is necessary to assess if responses as monotherapy or combination therapy are durable. There have been mixed results in ENKTL with regards to the prognostic value of PD-L1 in ENKTL, with one study finding that higher expression of PD-L1 expression was associated with inferior treatment response and survival in early-stage disease when defined as ≥38% on histology [134], but improved survival in advanced-stage disease when defined as ≥10% on histology [86]. Given emerging data about different molecular subtypes of ENKTL, it is possible that a biomarker-driven approach may be warranted, with immunotherapy targeted towards patients with the TSIM subtype characterized by PD-L1 overexpression, or patients with mutated PD-L1 who have better responses to pembrolizumab [135]. Recent whole-genome sequencing has further identified 4 TME subgroups of ENKTL: an ‘immune tolerance’ group with high numbers of Tregs more common in early-stage disease; two ‘immune evasion’ groups with frequent cytotoxic T cells and high PD-L1 expression; and an ‘immune silenced’ group with an exhausted immune response more common in advanced-stage disease [136]. The ‘immune silenced’ group had the worst prognosis and also had the worst response to pembrolizumab, with the best responses seen in the ‘immune tolerance’ group. These data suggest that immunotherapy may be targeted towards patients with the aforementioned predictive biomarkers or potentially to patients with early-stage disease who are more likely to have immunotherapy-responsive phenotypes. Several important molecular drivers of angiogenesis are upregulated in ENKTL, including vascular endothelial growth factor (VEGF) and its genes (VEGFA, VEGFC, and VEGFR2), as well as the oncogene MET and its related hepatocyte growth factor (HGF), all of which are regulated by the JAK/STAT pathway [71]. Anti-VEGF antibodies such as bevacizumab have been studied in lymphomas, including a study of bevacizumab plus CHOP in 39 patients with T and NK cell lymphomas with an ORR of 90% (CR 49%) which demonstrated significant treatment-limiting toxicities and poor durability [108]. However, a phase II trial of bevacizumab, gemcitabine, peg-asparaginase, oxaliplatin, and dexamethasone in 43 patients with newly-diagnosed ENKTL demonstrated an ORR of 100% (CR 100% for early-stage and 88% for advanced-stage disease) with a 2-year PFS and OS of 83% and 79%, respectively [109]. An oral tyrosine kinase inhibitor specific for the VEGF receptor VEGFR2, apatinib, is being studied in conjunction with the anti-PD-1 inhibitor camrelizumab in relapsed/refractory PTCL, with preliminary results demonstrating that 2 of the 3 patients with ENKTL achieved a partial response [110]. Pre-clinical data also support the use of MET inhibitors, which induce enhanced helper T cell recognition of ENKTL cell lines and may be combined with immunotherapy [111], and oral MET inhibitors such as crizotinib have already been studied in other types of PTCL with an ORR of 90% in ALK-positive lymphomas [112]. Serum VEGF levels are prognostic of PFS and OS in patients with ENKTL treated with non-anthracycline-based chemotherapy [56]. The inflammatory milieu of the ENKTL TME is thought to contribute to lymphomagenesis, as EBV infection causes NK/T cells to become sensitized to growth-promoting cytokines such as IL-2 [24]. Several serum cytokines are prognostic in ENKTL, including IL-18, which is associated with HLH, advanced-stage disease, and poor OS [137]; IL-10, which is associated with worse OS, low CR rate, and higher early relapse rate [138]; and IL-2Ra, IL-9, IL-15, and MIP-1a [139]. In addition, interferon, IL-6, and IL-10 are all associated with serum EBV DNA load, a known prognostic biomarker in ENKTL, suggesting that EBV-induced cytokine storm may contribute to poor prognosis in ENKTL [140,141]. Inhibition of proliferative cytokines has been proposed as a treatment strategy in ENKTL, although there is no clinical data. However, a recent study suggests that IL-10 contributes to gemcitabine resistance in ENKTL cell lines by regulation of drug resistance genes, suggesting that inhibition of IL-10 in select patients may be utilized to increase tumor sensitivity to chemotherapy [113]. Chemokines are chemoattractant cytokines that also mediate the TME in EBV-driven malignancies and promote metastasis and tumor angiogenesis, with chemokine receptor 4 (CCR4), the receptor for chemokine ligand 17 and 22 (CCL17 and CCL22), prominently identified in ENKTL tumors [114]. Mogamulizumab, a monoclonal antibody against CCR4, has demonstrated cytotoxicity in ENKTL cell lines and has clinical activity in other T cell malignancies, warranting further study [114]. Recent proteomic analysis has identified a novel biomarker in ENKTL, S100A9, an immunosuppressive molecule that is overexpressed in serum and tumor samples in ENKTL and likely mediates tumorigenesis by upregulation of PD-L1 expression [115]. High levels of S100A9 are associated with poor response to therapy, early relapse, and advanced stage, and it has been proposed as a possible therapeutic target. Given the abysmal prognosis of relapsed and refractory ENKTL, numerous targeted agents have been trialed in single patients or small cohorts with variable efficacy. However, there is pre-clinical rationale for the combination of these targeted agents with other active chemotherapeutics in ENKTL. Cell surface protein CD38 is expressed in the vast majority of ENKTL cases, and high expression of CD38 is correlated with inferior responses and survival [116]. The anti-CD38 monoclonal antibody daratumumab has been explored in relapsed/refractory ENKTL in a phase II study of 32 patients, which demonstrated an ORR of 25% (no CRs) with a very short response duration, with a median duration of response of 55 days and a 6-month OS of 43% [117]. Although daratumumab has low single-agent activity, it is frequently used in combination with other therapies in multiple myeloma, and there is emerging pre-clinical evidence that daratumumab enhances the cytotoxicity of asparaginase in a PDX mouse model; in that same study, pretreatment of ENKTL cell lines with all-trans retinoic acid (ATRA) increased expression of CD38 and increased daratumumab-induced cytotoxicity, setting the stage for future combination studies [118]. The cell surface protein CD30 is also expressed in the majority of ENKTL cases [119], with an unclear role as a prognostic marker [142,143]. Brentuximab vedotin (BV), a CD30-directed antibody-drug conjugate that is used extensively in lymphomas, has low single-agent activity in relapsed/refractory ENKTL, with a phase II study that included 7 patients with ENKTL demonstrating an ORR of 29% (1 patient with CR) [120]. P-glycoprotein (MDR1) may mediate resistance to BV in ENKTL, and cyclosporine, an MDR1 inhibitor, may increase BV response rates [121]. There are ongoing studies to combine BV with combination chemoimmunotherapy as is done in other lymphomas, as well as CD30-directed CAR T cell therapy which may provide therapeutic avenues [23,142]. Targeting EBV and its myriad viral proteins which drive ENKTL tumorigenesis, including LMP1 and LMP2, have been explored [123], particularly the use of antigen-specific cytotoxic T-lymphocytes (CTLs) as has been utilized in EBV-associated PTLD. In one study, LMP-targeted autologous CTLs were infused into patients with EBV-associated lymphomas, including 11 patients with ENKTL [124]. The ORR was 67% (all CRs) in the 6 patients with active disease, with durable remissions of 4 or more years in 3 of the patients and no treatment-emergent toxicities. In the 5 patients who were already in remission and felt to be at high risk of relapse, all 5 patients had ongoing sustained remission after CTL infusion with no CTL-related toxicities, suggesting that consolidation with CTLs may also be a therapeutic approach. This has been demonstrated in a cohort of 26 patients who had undergone allogeneic transplantation for EBV-associated lymphomas and who adjuvantly received allogeneic donor-derived LMP-specific T-cells, which demonstrated a 2-year EFS of 57% [125]. A similar study of adjuvant CTLs in 10 patients with ENKTL who were in a CR after induction therapy demonstrated a 4-year PFS and OS of 90% and 100%, respectively, suggesting that the post-remission timepoint may be a critical period to consolidate with CTL therapy [126]. A more recent study explored the activity of a novel T cell product, baltaleucel-T, in which autologous peripheral T cells are stimulated with antigen-presenting cells containing peptide libraries of multiple EBV-associated proteins, including LMP1, LMP2, and multiple cytokines [127]. Of the 54 patients with advanced-stage, relapsed ENKTL screened for the study, only 47 had adequate whole blood collection, and only 15 patients actually received baltaleucel-T treatment, with the other patients either having manufacturing failure and death or disease progression death before product administration. The ORR among 10 patients with measurable disease was 50% (CR 30%), with a median PFS of 12 months. Although there was activity of the product in relapsed ENKTL, manufacturing issues present a significant challenge in an aggressive and rapidly-progressive malignancy, and off-the-shelf allogeneic products are under investigation. Antiviral therapy against EBV requires the presence of lytic phase viral proteins, and HDAC inhibitors are able to activate the lytic cycle to sensitize EBV-infected cells to ganciclovir. A phase I/II study of nanatinostat with valganciclovir in 55 patients with relapsed/refractory EBV-positive lymphomas included 9 patients with ENKTL and demonstrated an ORR of 60% (27% CR) in all patients with T/NK cell lymphomas and an ORR of 63% in ENKTL patients [122]. Next-generation sequencing and proteomic analyses over the past decade have significantly improved our understanding of the molecular landscape that induces ENKTL tumorigenesis [23,28] and have revealed several novel therapeutic approaches. Given that EBV-driven DNA hypermethylation is ubiquitously observed in ENKTL, combinations of hypomethylating agents with other therapeutic agents, particularly immunotherapy, in which synergy has already been demonstrated, present promising opportunities for future clinical trials [17,104]. These innovations have also revealed new predictive biomarkers in newly-diagnosed and relapsed disease as well as refined prognostic and predictive scoring systems, including composite single-nucleotide polymorphism signatures [144] and identification of unique genetic clusters of ENKTL [28,145], which may enable a personalized medicine approach towards ENKTL therapy. Prospective trials which incorporate these biomarkers and novel therapeutics in rational combinations with existing standard-of-care treatment regimens will be vital to validate their clinical utility and new treatment paradigms for this aggressive malignancy.

PMC10000135

Xianbo Jia,Yang He,Zhe Kang,Shiyi Chen,Wenqiang Sun,Jie Wang,Songjia Lai

Comparison of Fecal Microbiota Communities between Primiparous and Multiparous Cows during Non-Pregnancy and Pregnancy

27-02-2023

cow,pregnancy,primiparous and multiparous,fecal microbiota,16S rRNA sequencing

Simple Summary An imbalance of the gut microbiota composition may lead to several reproductive disorders and physiological diseases during pregnancy. This study investigates the fecal microbiome composition between primiparous and multiparous cows during non-pregnancy and pregnancy to analyze the host-microbial balance at different stages. The results indicate that host-microbial interactions promote adaptation to pregnancy and will benefit the development of probiotics or fecal transplantation for treating dysbiosis and preventing disease development during pregnancy. Abstract Imbalances in the gut microbiota composition may lead to several reproductive disorders and diseases during pregnancy. This study investigates the fecal microbiome composition between primiparous and multiparous cows during non-pregnancy and pregnancy to analyze the host-microbial balance at different stages. The fecal samples obtained from six cows before their first pregnancy (BG), six cows during their first pregnancy (FT), six open cows with more than three lactations (DCNP), and six pregnant cows with more than three lactations (DCP) were subjected to 16S rRNA sequencing, and a differential analysis of the fecal microbiota composition was performed. The three most abundant phyla in fecal microbiota were Firmicutes (48.68%), Bacteroidetes (34.45%), and Euryarchaeota (15.42%). There are 11 genera with more than 1.0% abundance at the genus level. Both alpha diversity and beta diversity showed significant differences among the four groups (p < 0.05). Further, primiparous women were associated with a profound alteration of the fecal microbiota. The most representative taxa included Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, Christensenellaceae_R_7_group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, Methanobrevibacter, and [Eubacterium] coprostanoligenes group, which were associated with energy metabolism and inflammation. The findings indicate that host-microbial interactions promote adaptation to pregnancy and will benefit the development of probiotics or fecal transplantation for treating dysbiosis and preventing disease development during pregnancy.

Comparison of Fecal Microbiota Communities between Primiparous and Multiparous Cows during Non-Pregnancy and Pregnancy An imbalance of the gut microbiota composition may lead to several reproductive disorders and physiological diseases during pregnancy. This study investigates the fecal microbiome composition between primiparous and multiparous cows during non-pregnancy and pregnancy to analyze the host-microbial balance at different stages. The results indicate that host-microbial interactions promote adaptation to pregnancy and will benefit the development of probiotics or fecal transplantation for treating dysbiosis and preventing disease development during pregnancy. Imbalances in the gut microbiota composition may lead to several reproductive disorders and diseases during pregnancy. This study investigates the fecal microbiome composition between primiparous and multiparous cows during non-pregnancy and pregnancy to analyze the host-microbial balance at different stages. The fecal samples obtained from six cows before their first pregnancy (BG), six cows during their first pregnancy (FT), six open cows with more than three lactations (DCNP), and six pregnant cows with more than three lactations (DCP) were subjected to 16S rRNA sequencing, and a differential analysis of the fecal microbiota composition was performed. The three most abundant phyla in fecal microbiota were Firmicutes (48.68%), Bacteroidetes (34.45%), and Euryarchaeota (15.42%). There are 11 genera with more than 1.0% abundance at the genus level. Both alpha diversity and beta diversity showed significant differences among the four groups (p < 0.05). Further, primiparous women were associated with a profound alteration of the fecal microbiota. The most representative taxa included Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, Christensenellaceae_R_7_group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, Methanobrevibacter, and [Eubacterium] coprostanoligenes group, which were associated with energy metabolism and inflammation. The findings indicate that host-microbial interactions promote adaptation to pregnancy and will benefit the development of probiotics or fecal transplantation for treating dysbiosis and preventing disease development during pregnancy. Pregnancy is a wonderful and complex physiological process. In order to adapt to the growth and development of the fetus, drastic changes occur in maternal hormones, immunity, and metabolism before and after pregnancy. For mammals, progesterone (P4), estradioal (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), and Prolactin (PRL) are the main reproductive hormones to maintain and evaluate maternal pregnancy [1]. Growth hormone, thyroid hormone, and sex hormones could also change with maternal pregnancy [2]. The maternal immune system undergoes significant adaptations during pregnancy to avoid harmful immune responses against the fetus and to protect the mother and her future baby from pathogens [3]. For example, the number of T cells during pregnancy is lower than before pregnancy [4]. More nutrients are needed to be stored and consumed during pregnancy to meet the nutritional demands of the mother and fetus. Maternal metabolism changes to meet the nutritional requirements during pregnancy, the most obvious being the decrease in insulin sensitivity [5,6]. Additionally, compared to multiparous women, primiparous women have more exaggerated physiological responses, resulting in higher weight gain and body fat gain than that of multiparous women during pregnancy [7]. There are also many differences between primiparous and multiparous cows, including productivity, reproductive ability, energy balance, immune, metabolic, and hormonal responses [8,9]. Gut microbiota can produce a variety of nutrients, such as amino acids, fatty acids, and vitamins, which play an important role in regulating host metabolism, energy balance, and immune response [10,11,12,13]. With the changes of maternal hormones, immunity and metabolism during pregnancy, the composition and abundance of gut microbiota also shifted. The relative abundance of 21 genera of gut microbiota showed significant differences between non-pregnant and pregnant mice fed a standard diet. There were 4 abundant genera (present at greater than 1%) significantly increased and 5 rare taxa (present at lower than 0.5%) reduced during pregnancy compared to non-pregnant mice [14]. For dairy cows, the fecal microbial communities change dramatically in bacterial abundance at different taxonomic levels among the 12 distinctly defined production stages in a modern dairy farm, especially between virgin cows and parous cows [13]. Information on host-microbial interactions during pregnancy is emerging [15]. Recent studies showed that gut microbiota can impact the synthesis and metabolism of a variety of substances during pregnancy, regulating body weight, blood pressure, blood sugar, blood lipids, and other physiological indexes, and even leading to some pregnancy complications [16,17,18]. Parity has also been identified as one of the key determinants of the maternal microbiome during pregnancy. The difference in microbiome trajectories among different parities was significant in sows, with the greatest difference between zero parity and low parity animals. It was suggested that there are dramatic differences in the microbial trajectories of primiparous and multiparous animals [19]. Compared to multiparous sows, primiparous sows had a lower gut microbiota richness and evenness during the periparturient period [20]. Primiparous cows have different uterine and rumen microbiome compositions compared to multiparous cows [21,22]. However, it is still unclear if parity impacts the maternal cow’s gut microbiome during both non-pregnancy and pregnancy. In this study, the gut microbiome composition was investigated in fecal samples from primiparous and multiparous cows during non-pregnancy and pregnancy. It confirmed that there is an inherent shift in gut microbiota associated with pregnancy and differences in gut microbiota composition between primiparous and multiparous animals. The results will help develop strategies to improve the reproductive management of cows. The collection of biological samples and experimental procedures carried out in this study were approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, China (DKY20210306). A total of 24 healthy Holstein cows were selected from one dairy herd under the same conditions in southwestern China, with the same feeding processes, similar body conditions, and similar body weight. According to their reproductive stages, the cows were divided into four groups: the cows before their first pregnancy (13 months, n = 6, BG); at their first pregnancy (the 4th month of pregnancy, 18 months, n = 6, FT); open cows with more than three lactations (30 days after parturition, 57 months, n = 6, DCNP); and pregnancy cows with more than three lactations (the 4th month of pregnancy, 60 months, n = 6, DCP). Animals were fed the total mixed ration (TMR) made according to NRC (2012) with the same feed raw material. None of the cows had received antibiotics in the last 3 months. All 24 fecal samples were obtained once from cow rectum content on the same day, transferred to separate sterilized 2 mL tubes, and stored immediately in liquid nitrogen. All samples were then transported to the laboratory and stored at −80 °C for further analysis. Total genome DNA was extracted from fecal samples, the negative control (DNA free water), and the positive control (16S Universal E29), using a BIOMICS DNA Microprep Kit (Zymo Research, D4301, Irvine, CA, USA) according to the manufacturer’s instructions. DNA concentration and purity were tested on 0.8% agarose gels. DNA yield was detected with a Tecan Infinite 200 PRO fluorescent reader (Tecan Systems Inc., San Jose, CA, USA). The 16S rRNA amplification covering the variable region V4-V5 was carried out using the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 915R (5′-GTGCTCCCCCGCCAATTCCT-3′) by a Thermal Cycler PCR system (Gene Amp 9700, ABI, Foster City, CA, USA). PCRs were performed in triplicate in a 25 µL mixture. The PCR products were diluted six times, quantified with electrophoresis on 2% agarose gel, and then purified by the Zymoclean Gel Recovery Kit (Zymo Research, D4008, Irvine, CA, USA). About 100 ng of DNA were used for library preparation. The library was prepared using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA), followed by quality evaluation on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). Library was finally paired-end sequenced (2 × 300) on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA). The raw fastq files were merged using FLASH [23]. The raw tags were analyzed using the QIIME (v1.9.0) pipeline [24]. All tags were quality filtered. Sequences shorter than 200 nt with an average quality value less than 25, and those containing two or more ambiguous bases, were discarded. The clean tags were then mapped to the Gold database (http://drive5.com/uchime/uchime_download.html (accessed on 5 May 2021)) using UCHIME algorithm, followed by removal of the chimera sequences to identify the effective tags [25]. The operational taxonomic units (OTUs) table was created at 97% similarity using the UPARSE pipeline [26]. Representative sequences from each OTU were aligned to 16S reference sequences with PyNAST [27]. The phylogenetic trees were drawn using FastTree [28]. Annotation analysis was performed using the UCLUST taxonomy and the SILVA database [29,30]. The abundance of OTUs was normalized using a standard sequence number corresponding to the sample with the least sequence. The comparison of OTU numbers used a one-way analysis of variance (one-way ANOVA), followed by the Bonferroni multiple comparisons test. The alpha diversity was calculated to analyze the complexity of species diversity in the sample, including observed species, Chao1, Shannon, Simpson, coverage, and Faith’s PD. The beta diversity, weighted Unifrac and unweighted UniFrac, was calculated to evaluate the differences of samples in species complexity. Principal coordinate analysis (PCoA) was used to visualize differences in bacterial community composition among groups. The linear discriminant analysis coupled with effect size (LEfSe) was performed to identify the differentially abundant taxa between different groups. Pairwise comparisons were made using metagenomSeq. In order to evaluate the effect of reproductive status on the cow fecal microbiota, the V4–V5 hypervariable regions of the 16S rRNA gene were sequenced in the microbial communities of 24 samples. A total of 705,988 raw PE reads were generated from these 24 samples (average: 29,416 ± 4914, range: 21,956–36,765). After quality control, 632,192 effective tags were obtained from 24 samples (average: 26,341 ± 4408, range: 19,472–32,926), with an average of 407.67 ± 0.92 bps per tag after the merging of overlapping paired-reads, quality filtering, and removing of chimeric sequences. By the 97% sequence similarity, 6842 OTUs were computationally constructed with 1727.38 ± 405.39 (range: 999–2788) as the mean number of OTUs per sample, and the mean number of OTU in DCNP group was significantly lower than that of BG and FT group (p < 0.01) (Figure 1). These 6842 OTUs taxonomically assigned to microbial 2 Kingdom, 17 phyla, 25 classes, 38 orders, 67 families, 168 genera, and 117 species. According to OTUs’ number, the average abundance of each group at different category levels was evaluated (Figure 2). The fecal microbial communities were dominated by bacteria (84.58%), and archaea were only 15.42% abundant. The most abundant phyla across all 24 metagenomic libraries were Firmicutes (48.68%), followed by Bacteroidetes (34.45%), and Euryarchaeota (15.42%). Other less abundant phyla were Spirochaetes (0.85%), Tenericutes (0.42%), Proteobacteria (0.07%), Actinobacteria (0.06%), Fibrobacteres (0.02%), Cyanobacteria (0.02%), and Planctomycetes (0.01%) (Figure 3). At the genus level, there are 11 genera with more than 1.0% abundance, including Ruminococcaceae UCG-005 (21.91%), Methanobrevibacter (13.28%), Rikenellaceae RC9 gut group (10.13%), [Eubacterium] coprostanoligenes group (7.10%), Prevotellaceae UCG-004 (6.47%), Alistipes (5.52%), Ruminococcaceae UCG-013 (4.89%), Prevotellaceae UCG-003 (4.61%), Ruminococcaceae UCG-014 (1.78%), Methanocorpusculum (1.42%), Christensenellaceae R-7 group (1.12%) (Figure 4). The alpha diversity indexes, including observed species, Chao1, Shannon, Simpson, coverage, and Faith’s PD, for four groups were calculated to estimate species richness and diversity (Figure 5). Compared to the BG and FT groups, the observable species, Chao1, and Faith’s PD were significantly lower, and coverage was significantly higher in the DCNP group (p < 0.05, Kruskal–Wallis test), but without statistical significance in the DCP group (p > 0.05, Kruskal–Wallis test). Further, no statistically significant difference was shown among the four groups in Shannon and Simpson (p > 0.05, Kruskal–Wallis test). Based on the Jaccard and Bray–Curtis methods, principal coordinated analysis (PCoA) of beta diversity was further used to analyze compositional differences in fecal microbiota among four groups (Figure 6). The samples in the BG, FT, and DCP groups were clustered together according to their particular groups, while the samples in the DCNP group were spread out. The samples in the BG and FT groups tended to cluster together in accordance with PCoA results. Both Jaccard and Bray-Curtis distances showed significant differences among the four groups (ANOSIM, p < 0.01), except between groups DCP vs. DCNP (ANOSIM, p > 0.05). Linear discriminant analysis effect size (LEfSe) was used to discover the differential microbiota and estimate their effect size. Based on LEfSe, it restrictively analyzed the successfully annotated species and detected 60 taxa significantly different in abundance among four groups. There were 7 taxa significantly more abundant in the BG group, 17 in the FT group, 8 in the DCNP group, and 28 in the DCP group (Figure 7). The most representative taxa were Rikenellaceae and Rikenellaceae_RC9_gut_group in the DCP group, Prevotellaceae and Prevotellaceae_UCG_003 in the FT group, Christensenellaceae_R_7_group in the DCNP group, and Firmicutes, Clostridia, Clostridiales, and Ruminococcaceae in the BG group. The metagenomeSeq was further used to compare the abundance of OTUs between each group. The abundance of 4, 12, and 23 OTUs was significantly increased, while that of 1, 2, and 17 OTUs was significantly reduced in the FT, DCNP, and DCP groups compared with the BG group, respectively. In the three comparison groups, the abundance of six common genera (>1%), namely Prevotellaceae UCG-003, Ruminococcaceae UCG-013, [Eubacterium] coprostanoligenes group, Rikenellaceae RC9 gut group, Methanobrevibacter, and Ruminococcaceae UCG-005, was identified as a significant difference (Figure 8). There were 16 and 21 OTUs that were significantly increased, and 2 and 19 OTUs that were significantly reduced, in the DCNP and DCP groups compared with the FT group, respectively. A total of 8 common genera, such as the Christensenellaceae R-7 group, Ruminococcaceae UCG-014, Prevotellaceae UCG-003, Ruminococcaceae UCG-013, [Eubacterium] coprostanoligenes group, Rikenellaceae RC9 gut group, Methanobrevibacter, and Ruminococcaceae UCG-005, were observed to have significant differences (Figure 8). Furthermore, in the DCP group, the abundance of 4 OTUs decreased compared with the DCNP group. The relative abundance of 2 common genera, Methanobrevibacter and Prevotellaceae UCG-003, in the DCNP group was higher than that in the DCP group. The reproductive efficiency and health of cows have always been priorities. The gut microbiota composition plays an important role in the reproductive performance throughout a female’s lifetime. In humans, the gut microbiome has been considered to affect every stage and level of female reproduction, including follicle and oocyte maturation in the ovary, fertilization and embryo migration, implantation, the whole pregnancy, and parturition [31,32,33,34]. The gut microbial communities can influence reproductive success from mate choice to healthy pregnancy and successfully producing offspring in animals [35,36]. Recent studies reported that bovine vaginal and fecal microbiome associated with differential pregnancy outcomes [37,38]. The fecal microbiome predicted pregnancy with a higher accuracy than that of the vaginal microbiome [38]. In this study, the fecal microbiota were investigated in 4 different reproductive stages and revealed the dramatic changes in fecal microbiota diversity and composition among 4 groups using the sequencing of the 16S rRNA gene. In this study, Firmicutes, Bacteroidetes, and Euryarchaeota were the three most dominant phyla, and Ruminococcaceae UCG-005, Methanobrevibacter, and Rikenellaceae RC9 gut group were the three most dominant genera in the cow fecal samples. They were consistent with several earlier studies [39]. In previous studies, Bacteroidetes (51.6~59.74%) and Firmicutes (27.6~38.74%) together comprised up to 81.6~93.20% of the cow fecal bacterial abundance [13,40,41]. The phylum Euryarchaeota was predominant within the Archaea and accounted for around 0.25% of the cow fecal microbiota abundance [41,42]. Ruminococcaceae UCG-005, Methanobrevibacter, and Rikenellaceae RC9 gut groups predominate in the Firmicutes, Euryarchaeota, and Bacteroidetes phyla, respectively. Ruminococcaceae UCG-005 and Rikenellaceae RC9 gut group usually had a relative abundance >8% of fecal microbiota in dairy cows. The genus Methanobrevibacter comprised more than 80% of the phylum Euryarchaeota in cow fecal Archaea [13,43]. The age and pregnancy are two important factors contributing to the species richness and diversity of fecal microbiota. The alpha diversity index, observed species, Chao 1, coverage, and Faith’s PD were significantly different among the BG, FT, and DCNP groups in this study. However, the cluster among four groups was significant, separating BG and FT groups from DCNP and DCP groups by PCoA based on Jaccard and Bray–Curtis distances. These also showed that the greatest differences in microbiome trajectories occurred between nulliparous and primiparous animals [19]. Nulliparous animals had higher gut microbial diversity than that of primiparous animals, and pregnancy could increase gut microbial diversity [19,20]. The effect of age is more related to calving. The increase in alpha diversity during pregnancy could be due to an increase in nutrient requirements during lactation. The first birth is the most important physiological change in a cow’s life, and pregnancy increases metabolism. In order to further identify important taxa differed among groups, LEfse and metagenomeSeq analyses were conducted. LEfse analysis is helpful to discover the important differential taxa (biomarkers) and estimate their effect sizes. The LEfSe analysis revealed that the most differentially abundant taxa were in DCP, followed by FT, DCNP, and BG. The metagenomeSeq analyses showed that the comparisons with the most significant differences in microbial taxa are BG vs. DCP and FT vs. DCP, followed by FT vs. DCNP, BG vs. DCNP, BG vs. FT, and DCNP vs. DCP. These suggested that parturition experience is one of the most important factors to impact cattle gut microbiome trajectory. Previous study also reported that the most difference in microbiome trajectory occurred between nulliparous and low parity sows [19]. There was significant difference between multiparous and primiparous cows on vaginal and uterine microbiotas [44,45]. The most representative taxa were associated with energy metabolism and inflammation. Mice fed with high-fat diet increased the richness of gut microbial Rikenellaceae_RC9_gut_group. The high-fat diet also increased the risks of intestinal pathogen colonization and inflammation [46]. Supplementation of probiotics increased the relative abundance of Prevotellaceae_UCG_003, which improved the energy status of the beef steers [47]. Fibrolytic enzyme increased the relative abundance of Christensenellaceae_R_7_group, which improve the average daily gain and feed conversion ratio of lambs [48]. The ruminococcaceae family is the predominant acetogen in the cattle rumen, which is related to cellulose and hemicellulose degradation [49]. The carbohydrate resource and the fiber decomposition process in diet contribute to the different abundances of Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, and other Ruminococcaceae in cattle feces [49,50]. Methanobrevibacter is another common inhabitant of the cattle rumen, which can reduce CO2 with H2 to form methane [51,52].The serum cholesterol concentration tended to be lower after feeding Eubacterium coprostanoligenes to germ-free mice [53]. Thus, gut microbes are involved in changes in energy intake and immunity during cattle adaption to pregnancy. In conclusion, this study investigated the difference in fecal bacterial communities between primiparous and multiparous cows during non-pregnancy and pregnancy. The results revealed that pregnancy increased the relative abundance and diversity of fecal microbiota, while aging reduced those traits. In addition, primiparous were related to a profound alteration of the fecal microbiota. The most representative taxa included Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, Christensenellaceae_R_7_group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, Methanobrevibacter, and [Eubacterium] coprostanoligenes group, which were associated with energy metabolism and inflammation. In the future, further functional studies will be able to treat dysbiosis and prevent disease development during pregnancy by using probiotics or fecal transplantation.

PMC10000137

Fubin Wang,Xiaoyun Wu,Xiaoming Ma,Qi Bao,Qingbo Zheng,Min Chu,Xian Guo,Chunnian Liang,Ping Yan

The Novel Structural Variation in the GHR Gene Is Associated with Growth Traits in Yaks (Bos grunniens)

26-02-2023

Bos grunniens,growth hormone receptor (GHR) gene,growth traits,SV 246 bp,candidate molecular marker

Simple Summary Yak is the dominant animal species in China’s plateau regions. The development of the yak industry is conducive to protecting the ecosystem of the Qinghai–Tibet Plateau and promoting local economic development. However, the slow growth and development of yak have seriously affected the development of the yak industry. Structural variation (SV) has been widely applied in livestock breeding for growth traits. Therefore, it is of great significance to use SV to improve yak growth traits in yak breeding. Meanwhile, the GHR gene plays an important role in the formation and normal development of bones. This study associated the correlation between the yak GHR SV gene and growth traits and confirmed that GHR-SV can be used as a molecular marker for the early reproduction of yaks. This study provides a theoretical basis for the early growth and development of yaks. Abstract The growth hormone receptor (GHR) is a member of the cytokine/hematopoietic factor receptor superfamily, which plays an important role in the growth and development, immunity, and metabolism of animals. This study identified a 246 bp deletion variant in the intronic region of the GHR gene, and three genotypes, including type II, type ID, and type DD, were observed. Genotype analysis of structural variation (SV) was performed on 585 individuals from 14 yak breeds, and it was found that 246 bp deletion was present in each breed. The II genotype was dominant in all yak breeds except for SB yak. The association analysis of gene polymorphisms and growth traits in the ASD yak population showed that the 246 bp SV was significantly associated with body length at 6 months (p < 0.05). GHR messenger RNA (mRNA) was expressed in all the tested tissues, with significantly higher levels in the liver, muscle, and fat than in other organs. The results of transcription activity showed that the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). Additionally, the transcription-factor binding prediction results showed that the SV in the runt-related transcription factor 1 (Runx1) transcription-factor binding site may affect the transcriptional activity of the GHR gene, regulating yak growth and development. This study showed that the novel SV of the GHR gene could be used as a candidate molecular marker for the selection of the early growth trait in ASD yak.

The Novel Structural Variation in the GHR Gene Is Associated with Growth Traits in Yaks (Bos grunniens) Yak is the dominant animal species in China’s plateau regions. The development of the yak industry is conducive to protecting the ecosystem of the Qinghai–Tibet Plateau and promoting local economic development. However, the slow growth and development of yak have seriously affected the development of the yak industry. Structural variation (SV) has been widely applied in livestock breeding for growth traits. Therefore, it is of great significance to use SV to improve yak growth traits in yak breeding. Meanwhile, the GHR gene plays an important role in the formation and normal development of bones. This study associated the correlation between the yak GHR SV gene and growth traits and confirmed that GHR-SV can be used as a molecular marker for the early reproduction of yaks. This study provides a theoretical basis for the early growth and development of yaks. The growth hormone receptor (GHR) is a member of the cytokine/hematopoietic factor receptor superfamily, which plays an important role in the growth and development, immunity, and metabolism of animals. This study identified a 246 bp deletion variant in the intronic region of the GHR gene, and three genotypes, including type II, type ID, and type DD, were observed. Genotype analysis of structural variation (SV) was performed on 585 individuals from 14 yak breeds, and it was found that 246 bp deletion was present in each breed. The II genotype was dominant in all yak breeds except for SB yak. The association analysis of gene polymorphisms and growth traits in the ASD yak population showed that the 246 bp SV was significantly associated with body length at 6 months (p < 0.05). GHR messenger RNA (mRNA) was expressed in all the tested tissues, with significantly higher levels in the liver, muscle, and fat than in other organs. The results of transcription activity showed that the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). Additionally, the transcription-factor binding prediction results showed that the SV in the runt-related transcription factor 1 (Runx1) transcription-factor binding site may affect the transcriptional activity of the GHR gene, regulating yak growth and development. This study showed that the novel SV of the GHR gene could be used as a candidate molecular marker for the selection of the early growth trait in ASD yak. Yak (Bos grunniens) mainly lives in the Qinghai–Tibet Plateau at an altitude of 3000–5500 m [1]. In China, there are about 16 million yaks, accounting for about 95% of the world’s population [2]. It is an important and dominant animal species in plateau animal husbandry, which can provide meat, milk, fur, etc. [3]. Among them, yak meat is one of the main resources of the plateau animal husbandry economy [4]. Compared with cattle beef, yak meat is high in protein and energy, low in fat, and rich in amino acids, and the habitat of yak is least affected by humans. Therefore, yak meat is considered a natural green food, which meets people’s demand for green and healthy meat quality [5]. As a plateau variety, yak can protect the Qinghai–Tibet Plateau ecosystem and promote local economic development. Compared with cattle, the growth rate of yak is slow due to the lack of an efficient yak breeding program to improve growth traits, which affects the yield of yak meat. Therefore, it is of great significance to study the growth traits of yak. The growth hormone receptor (GHR) gene is an essential growth-related candidate gene that plays a critical role in the early stages of animal growth and development [6]. The growth hormone (GH)–insulin-like growth factor (IGF) growth axis is crucial for the growth and differentiation of skeletal muscle [7]. The GHR gene combines with GH to initiate the intracellular signal transduction mechanism, increase the expression of insulin-like growth factor 1 (IGF-1), promote cell proliferation, and affect the growth and development of animal skeletal muscle [8]. In mice, GHR knockouts exhibit impaired skeletal muscle development characterized by a reduced number and area of muscle fibers and concomitant functional defects. GHR gene variants are associated with growth and development in livestock. The GHR gene is considered to be a molecular genetic marker of growth performance in fertile pigs [9]. In sheep, the GHR gene variation is associated with growth traits [10]. According to recent studies, the presence of genetic variations in the GHR gene is associated with economic traits, such as cattle growth and production [11,12]. These studies suggest that genetic variation in the GHR gene plays an important role in regulating animal growth and development. Structural variation (SV) is an important and abundant source of genetic and phenotypic variation, including insertions/deletions (Indel), duplications, translocations, and copy-number variations (CNVs) [13]. Compared with a single nucleotide variant (SNV), SVs could have a greater impact on the function and expression of genes [14]. Up to now, many SVs are found to be associated with economic traits in domestic animals. For example, a deletion of 110 kb in the MER1 repeat containing the imprinted transcript 1 (MIMT1) gene is associated with abortion and stillbirth in cattle [15]. The lysine acetyltransferase 6A (KAT6A) gene is associated with the development of different body size traits in sheep [16]. In addition, a 225 bp deletion in the glutaminyl–peptide cyclotransferase-like (QPCTL) gene was associated with body weight and carcass traits in chickens [17]. Upadhyay et al. [18] detected an SV in the regulatory region of polypeptide N-acetylgalactosaminyltransferase-like 6 (GALNTL6) associated with feed efficiency and growth traits in cattle. However, there is no detailed study on SVs of the yak GHR gene. In this study, we found a 246 bp sequence deletion SV of the yak GHR gene. In order to identify the role of the GHR gene SV in yak populations in China, we analyzed the association between GHR 246 bp deletion variation and phenotypic traits to illustrate the genetic effect of GHR 246 bp deletion variation in yak breeds. This study provides useful information for the genetic protection and improvement of Chinese yak. This work has been approved by the Lanzhou Institute of Husbandry and Pharmaceutical Sciences; the grant number is No. LIHPS-CAAS-2017-115. In addition, we collected all blood samples and analyzed the data strictly following the guidelines for the Care and Use of Laboratory Animals. Blood samples were collected from a total of 585 individuals of 14 yak breeds. The numbers of samples from each breed were as follows: Datong yak (DT, n = 22); Xueduo yak (XD, n = 21); Huanhu yak (HH, n = 21); Gannan yak (GN, n = 21); Tianzhu White yak (TZB, n =21); Niangya yak (NY, n = 18); Leiwuqi yak (LWQ, n = 21); Sibu yak (SB, n = 18); Muli yak (ML, n = 23); Jiulong yak (JL, n = 21); Maiwa yak (MW, n = 21); Zhongdian yak (ZD, n = 21); Bazhou yak (BZ, n = 21); and Ashidan yak (ASD, n = 315). We tracked the phenotypic data of these Ashidan yaks (i.e., body weight (BW, kg), withers height (WH, cm), body length (BL, cm), and chest girth (CG, cm)) at four age groups of growth (i.e., 6 months old, 12 months old, 18 months old, and 30 months old). The phenotype was measured using the standard method of measurement [19]. Tissues, including the heart, muscle, liver, spleen, kidney, lung, and brain, were collected for expression profiling. Yak blood gDNA was extracted using the Tiangen whole-genome blood extraction kit (Beijing, China), and the DNA quality was detected using an ultra-microspectrophotometer (Thermo Fisher Scientific, USA). The gDNA was stored at −20 °C for further analysis. The primers for gene structure variation were designed by the National Center for Biotechnology Information (NCBI) online primer design software Primer-BLAST and synthesized by Qingke Zexi (Xi’an) Biotechnology company (Xi’an, Shaanxi, China) (Table 1). PCR was then performed. A total volume of 10 μL PCR mixture contained 1 μL of gDNA (50 ng/μL), 1 μL of each primer (10 μmol/L), 5 μL of Go Taq®Green Master Mix (Promega, Madison, WI, USA), and 2 μL of ddH2O. The PCR conditions were as follows: 95 °C for 3 min, 95 °C for 5 s, 58 °C for 30 s, 72 °C for 1 min for 30 cycles, and 72 °C for 5 min. The products were identified via 2% agarose gel electrophoresis. Six PCR products were selected and sequenced by Qingke Zexi (Xi’an) Biotechnology Company (Xi’an, Shaanxi, China). The total RNA of yak tissue was extracted using the Trizol method, and the integrity of RNA was identified with 1% agarose gel electrophoresis. cDNA was synthesized via reverse transcription using a translator first-strand cDNA synthesis kit (Roche, Shanghai, China) and stored at −20 °C for further analysis. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using Go Taq®qPCR and RT-qPCR Systems (Promega, Madison, WI, USA) to investigate the expression levels of the GHR messenger RNA (mRNA) in each tissue. Three replicates were selected for each sample. The primer information is provided in Supplementary Table S1. The RT-qPCR conditions were as follows: 95 °C for 3 min, 95 °C for 5 s, 64 °C for 20 s for a total of 40 cycles, and 72 °C for 30 s. The results were analyzed using the 2−ΔΔCT method [20]. The 293T cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). High-sugar DMEM (Hyclone, Logan, UT, USA), fetal bovine serum (Gibco, Waltham, MA, USA), and 1% streptomycin/penicillin (Invitrogen, Waltham, MA, USA) were used. Plasmids were transfected into cells using ViaFectTMTransfection Reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Based on the yak GHR 246 bp deletion locus, the luciferase reporter vector pGL4.10 was selected, and the pGL4.10-II (324 bp, containing the 246 bp deletion) and pGL4.10-DD (78 bp, lacking the 246 bp deletion) vectors were constructed to detect the transcriptional activity of GHR 246 bp deletion. In this study, the online software AnimalTFDB 3.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/) (accessed on 10 February 2022) and JASPAR (http://jaspar.genereg.net/) (accessed on 10 February 2022) were used for the prediction of TFBSs at the GHR 246 bp deletion locus. The runt-related transcription factor 1 (Runx1) binding sequence (TAAATGCAAA) was identified on the II genotype, and the pGL4.10-KO-Runx1 dual-luciferase reporter vector (324 bp) was constructed. The vector pGL4.75 (Promega, Madison, WI, USA) was used as an internal reference vector for determining the luciferase reporter system. Gene frequency and genotype frequency were calculated using the following equations: Genotype frequency (FAiAj) = AiAj number of individuals/total number of samples; The online website SHEsis was used to calculate Hardy–Weinberg equilibrium, heterozygosity, polymorphism information, and genetic variation content of GHR gene SV in the population. Meanwhile, we analyzed the association between the SV type of the GHR gene and the growth traits of Ashidan yak by using a one-way analysis of variance (ANOVA) and frequency-distribution histogram test. A mixed linear model used in the analysis is as follows: Yij = μ + Gij + eij, where Yij is the observed growth trait; μ is the population average of each trait; Gij is the fixed effect of SV genotype of the GHR gene; and eij is the random residual. The difference between the mean values was evaluated by using one-way ANOVA with a post-event least significant digit (LSD) multiple-comparison test. The software used for the statistical analyses was the IBM SPSS Statistics software (Version 23.0). Data are expressed as mean± standard error (SE). A 246 bp deletion variation was identified in intron 6 of the GHR gene (NW_005393449.1: 3052434-3052680). Based on the designed primers (F1, R1), GHR-SV246 was amplified with PCR using yak gDNA as the template. The GHR-SV246 polymorphism was analyzed via PCR amplification and agarose gel electrophoresis of the product (Figure 1). The product length matched the expectation, and three genotypes were identified, namely II (778 bp), ID (778 bp and 532 bp), and DD (532 bp). The sequencing analysis of the amplified products showed that the sequencing results were consistent with the reference genome sequence. The genotype frequencies and allele frequencies of GHR-SV246 in different yak breeds are shown in Table 2. The frequencies of different genotypes in Chinese yak populations were 0.723, 0.202, and 0.075, respectively. The II genotype was the dominant genotype in different populations except for SB yak; the allele frequencies were 0.824 and 0.176, respectively. Among them, the frequency of the I allele was the highest in the ML yak population (0.979) and the lowest in the SB yak population (0.500) (Table 2). The Ho ranged from 0.500 to 0.959, indicating high heterozygosity within different yak breeds, and the Ne ranged from 1.04 to 2.00. Classification based on the polymorphism information content (PIC value < 0.25, low polymorphism; 0.25 < PIC value < 0.5, medium polymorphism; PIC value > 0.5, high polymorphism) showed that the 246 bp deletion of the GHR gene exhibited relatively low polymorphism in ZD, ML, LWQ, NN, GN, and XD yak populations, while moderate polymorphism was observed in other yak populations (Table 3). In this study, the normal distribution of yak growth traits (Supplementary Figure S1–S4) and the average value of the growth parameters were statistically analyzed (Supplementary Table S1). We also analyzed the association of GHR gene polymorphisms with the growth traits in yaks using a linear model. As shown in Table 4, the polymorphisms in the GHR gene were significantly associated with body length in 6-month-old ASD yaks (p < 0.05), while there was no significant relationship with other growth traits (p > 0.05). To detect the transcriptional activity of different GHR-SV246 genotypes in cells, pGL4.10, pGL4.10-II, and pGL4.10-DD vectors were co-transfected into 293T cells with the internal reference plasmid pGL4.75. The fluorescence activity of the pGL4.10-II vector was significantly different from that of the blank control group pGL4.10 (p < 0.05) and was significantly lower than that of the pGL4.10-DD vector (p < 0.05) (Figure 2). In order to determine the effect of Runx1 transcription-factor binding with the 246 bp deletion on transcriptional activity in cells, the pGL4.10-KO-Runx1 luciferase reporter vector with mutant Runx1 binding site was constructed, and its fluorescence activity was identified. The luciferase activity of vector pGL4.10-KO-Runx1 was significantly lower than that of vector pGL4.10-II (Figure 3; p < 0.05), indicating that the transcription factor Runx1 could be combined with the II genotype sequence, thus increasing its transcriptional activity in cells. The expression levels of the GHR gene in different tissues were identified in yaks. There were significant differences in the expression of the GHR gene in various tissues of yaks, with the highest expression in fat, followed by the liver, muscle, and kidney tissues, and the lowest expression in the heart, spleen, and lung tissues (Figure 4). SV is a major determinant of the phenotypic diversity of animals. Studies on chickens [21], sheep [22], and cattle [10] have reported that SVs play a significant role in their genetic diversity. In poultry, the SVs of prolactin receptor (PRLR) [23] and Lpin1 [24] were significantly associated with chicken growth, carcass characteristics, and other economic traits. In cattle, the SVs of SIRT4 and NPM1 were significantly associated with bovine growth traits and meat quality [25,26]. These studies show that SV is an important class of molecular genetic markers, which is of great value in revealing the genetic mechanism of the economic traits of livestock and poultry. The GHR gene is a member of the cytokine receptor superfamily [27]. Some studies have pointed out that the GHR gene can regulate the growth of skeletal muscle by interacting with the growth hormone (GH) [22,28]. In this study, we analyzed the expression of the GHR gene in different tissues of yak and found that the GHR gene was expressed in different tissues, with high expression in adipose tissue and moderate expression in the muscle. This is consistent with previous studies on GHR expression profiles [29]. GHR mRNA expression levels in muscle and fat have a positive effect on post-weaning weight gain in cattle, while GHR mRNA expression in the liver has a negative effect on post-weaning weight gain in cattle [30]. A study reported that there is a natural antisense transcript (GHR-AS) on the GHR gene of chickens, and its overexpression can promote the expression of myogenic differentiation 1 (MyoD) and myosin heavy chain (MyHC) and the differentiation of myoblasts, thus enhancing the differentiation of myoblasts [31]. A previous study found associations between GHR gene polymorphisms and the growth traits in cattle at different ages [16], showing that GHR gene variation can affect the growth and production traits of cattle. However, few studies have reported an association between GHR gene variation and yak growth and production traits. In this study, we found a 246 bp structural variation site in intron 6 of the yak GHR gene. The genotyping results showed that the three genotypes of GHR genes existed in different yak breeds, and the analysis of genetic parameters suggested that the 246 bp deletion was in Hardy–Weinberg equilibrium in different yak breeds, which indicates that it is relatively stable in yak population. In ASD yak, this SV belongs to a moderate polymorphism, indicating that the degree of genetic variation is relatively large, and it has certain breeding potential. We found that individuals with the DD genotype showed higher body length at 6 months of age in ASD yak breeds. These data suggest that SVs in the GHR gene might influence the early development of yak. Introns are noncoding segments of genes that are removed via splicing during gene transcription and ultimately do not exist in mature RNA molecules [32]. Introns play an important role in the regulation of gene expression networks. Ostrovsky et al. [33] found that the intron 9 of the heparanase (HPSE) gene has a regulatory effect on its expression, and the existence of mutation sites in this intron has a significant impact on the regulatory effect of the intron. In this study, the transcriptional activity of the deletion sequence in the yak GHR gene was analyzed, and the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). This finding indicates that there may be repressive TFBSs in the II genotype sequence, which interferes with the expression of the GHR gene by binding to transcription factors and affects the growth and development of yaks. Genomic variation in introns can affect gene expression through cis-element targets or by binding to other functional genes [27]. Boriushkin et al. [34] found a Krüppel-like factor 4 (KLF4) transcription-factor binding site located in the intron of the delta-like canonical Notch ligand 4 (DLL4) gene, which is a key regulator of angiogenesis. They also found that KLF4 can inhibit the expression of DLL4 via this binding site. The appearance of gene variant sites may introduce novel transcription-factor binding sites or destroy existing binding sites [35,36]. In this study, in the II genotype sequence, we detected a Runx1 transcription-factor binding site, which was first discovered in acute myeloid leukemia [37]. Runx1 has an important regulatory role in the growth and development of animal muscles. In mouse muscle cells, the overexpression of Runx1 inhibits myogenic differentiation but promotes myoblast proliferation [38]. When skeletal muscle is injured, the expression of Runx1 is upregulated in skeletal muscle, thereby inhibiting the premature differentiation of primary myoblasts and promoting muscle regeneration [39]. Runx1 inhibits Spi1 gene expression by interacting with Spil introns [29]. In this study, we hypothesized that Runx1 has an effect on the transcriptional regulation of the GHR gene II genotype in yaks. This study found that changes in Runx1-TFBS reduced the fluorescence activity of the II genotype vector, indicating that the Runx1 could bind to the II genotype sequence and increase the transcriptional activity of the GHR gene. The luciferase activity of the pGL4.10-II vector was not significantly different from the pGL4.10 empty-vector fluorescence activity (p > 0.05). There may also be a binding site of an inhibitory transcription factor in the II genotype sequence. We detected and validated the SV of the GHR gene in yaks for the first time. The results indicated that SV of the GHR gene was significantly correlated with body length at 6 months of age in ASD yaks. Moreover, the transcriptional activity assay found significant differences among different genotypes, and transcription factor Runx1 promoted transcriptional activity in the II genotype. Our study provides primary evidence for the role of the GHR gene, which may be a molecular marker for early yak breeding in the future.

PMC10000145

Bo Deng,Jie Wu,Xuan Liu,Qian Ma,Xin Tao,Keke Qi,Xinping Diao,Ziwei Xu

Effects of Extruded Corn with Different Gelatinization Degrees on Feed Preference, Growth Performance, Nutrient Digestibility, and Fecal Microbiota of Weaning Piglets

03-03-2023

extrusion,corn gelatinization,choice feeding,palatability,growth rate,fecal microbiota,piglets

Simple Summary This study was conducted to evaluate the effects of extruded corn with different gelatinization degrees on the feed preference, growth performance, nutrient digestibility, and fecal microbiota of weaning piglets. The results showed that extruded corn can improve feed preference, increase growth performance and nutrient digestibility, and modify gut microbiota, and the ideal degree of gelatinization is approximately 41.82–62.60%. Abstract Preference and performance trials were conducted to investigate the effects of extruded corn with different degrees of gelatinization on the feed preference, growth performance, nutrient digestibility, and fecal microbiota of weaning piglets. In the preference trial, 144 piglets who were 35 days old were weighed and allotted to six treatments with four replications per treatment. Piglets in each treatment group were allowed to choose two of the following four corn-supplemented diets: conventional corn (NC) or extruded corn with low (LEC; 41.82% gelatinization), medium (MEC; 62.60% gelatinization), or high (HEC; 89.93% gelatinization) degrees of gelatinization for 18 days. The results showed that the piglets preferred diets supplemented with a low degree of gelatinization of extruded corn. In the performance trial, 144 piglets who were 35 days old were weighed and allotted into four treatments with six replications per treatment. Piglets in each treatment were fed one of the four diets for 28 days. The results showed that LEC and MEC decreased the feed:gain ratio at 14–28 days and 0–28 days, respectively, and increased the apparent total tract digestibility (ATTD) of crude protein compared with NC. Meanwhile, LEC increased the total protein and globulin content in the plasma on day 14, and MEC increased the ATTD of ether extract (EE) compared with NC. Extruded corn with low and medium degrees of gelatinization increased the abundance of Bacteroidetes at the phylum level and Lactobacillus, Alloprevotella, Prevotellaceae_UCG-03, and Prevotella_2 at the genus level. The results showed that extruded corn can improve feed preference, increase growth performance and nutrient digestibility, and modify gut microbiota, and the ideal degree of gelatinization is approximately 41.82–62.60%.

Effects of Extruded Corn with Different Gelatinization Degrees on Feed Preference, Growth Performance, Nutrient Digestibility, and Fecal Microbiota of Weaning Piglets This study was conducted to evaluate the effects of extruded corn with different gelatinization degrees on the feed preference, growth performance, nutrient digestibility, and fecal microbiota of weaning piglets. The results showed that extruded corn can improve feed preference, increase growth performance and nutrient digestibility, and modify gut microbiota, and the ideal degree of gelatinization is approximately 41.82–62.60%. Preference and performance trials were conducted to investigate the effects of extruded corn with different degrees of gelatinization on the feed preference, growth performance, nutrient digestibility, and fecal microbiota of weaning piglets. In the preference trial, 144 piglets who were 35 days old were weighed and allotted to six treatments with four replications per treatment. Piglets in each treatment group were allowed to choose two of the following four corn-supplemented diets: conventional corn (NC) or extruded corn with low (LEC; 41.82% gelatinization), medium (MEC; 62.60% gelatinization), or high (HEC; 89.93% gelatinization) degrees of gelatinization for 18 days. The results showed that the piglets preferred diets supplemented with a low degree of gelatinization of extruded corn. In the performance trial, 144 piglets who were 35 days old were weighed and allotted into four treatments with six replications per treatment. Piglets in each treatment were fed one of the four diets for 28 days. The results showed that LEC and MEC decreased the feed:gain ratio at 14–28 days and 0–28 days, respectively, and increased the apparent total tract digestibility (ATTD) of crude protein compared with NC. Meanwhile, LEC increased the total protein and globulin content in the plasma on day 14, and MEC increased the ATTD of ether extract (EE) compared with NC. Extruded corn with low and medium degrees of gelatinization increased the abundance of Bacteroidetes at the phylum level and Lactobacillus, Alloprevotella, Prevotellaceae_UCG-03, and Prevotella_2 at the genus level. The results showed that extruded corn can improve feed preference, increase growth performance and nutrient digestibility, and modify gut microbiota, and the ideal degree of gelatinization is approximately 41.82–62.60%. Weaning stress is normally associated with decreased feed intake and growth performance due to abrupt changes in the piglet’s environment and diet [1,2]. It takes up to three weeks for piglets to re-establish their pre-weaning levels of energy intake [3]. Thus, feed with high digestibility and good palatability is crucial to ensure a fast initiation of feeding after weaning and maintain a continuous supply of energy. The nutritional improvement of corn is of interest to producers because corn plays an important role in the piglet diet because it is rich in starch and a major source of energy. Extrusion is a thermal and mechanical process that results in several starch chemical changes, including changes to the crystalline structure and gelatinization degree that could increase enzyme susceptibility and flavor [4,5,6]. Therefore, corn extrusion may be an effective way to improve nutrition utilization and palatability in the piglet diet. Many experiments have focused on the effect of gelatinized vs. non-gelatinized corn or its supplementary ratio, yet few results have been published regarding the appropriate gelatinization degree. Different gelatinization degrees of extruded corn may result in different fermentable substrates in the hindgut which change the gut microbiota. Further, starch with different gelatinization degrees would change pellet properties, such as hardness and water absorption indexes, which may affect piglets’ chew work and thus influence palatability [7,8]. Our hypothesis is that the composition of gut microbiota and feed palatability may vary with changes in gelatinization degrees. We noticed that findings in previous studies were inconsistent. Kotara et al., found that the average daily gain (ADG), average daily feed intake (ADFI), and nutrient digestibility increased linearly when the gelatinization degree increased from 23.8% to 81.9% [9]. In contrast, Hongtrakul et al., showed a quadratic decrease in ADG and ADFI and a quadratic increase in the digestibility of dry matter (DM), nitrogen, and gross energy (GE) when the gelatinization degree increased from 14.5% to 89.3% [10]. Therefore, in the present study, we aimed to quantify the palatability of extruded corn with different degrees of gelatinization using a two-way choice–preference trial. The impact of extruded corn with different gelatinization degrees on the growth performance, nutrient digestibility, and fecal microbiota of piglets was also studied in a performance trial. All experimental materials of corn from northeast China were selected from a single patch. Corn was grounded using a hammer mill with a 2.0 mm screen and then extruded using a single-screw extruder (EXT-200S, Beijing Modern Yanggong Machinery S&T Development Co., Ltd., Beijing, China). To obtain low, medium, and high gelatinization degrees of the extruded corn, the samples were steam-cooked at 105 °C for 60 s, 80 s, and 100 s with 17%, 17.5%, and 17.5% moisture content and then extruded at 100 °C, 110 °C, and 120 °C for 5 s, respectively. After this, the corn samples were cooled using a counter-flow cooling procedure and ground though a 1 mm screen to generate mashed corn. A total of 144 piglets (35 days old; 72 barrows and 72 gilts; Duroc × Large White × Landrace) with 9.45 ± 0.76 kg body weight (BW) were used to perform an 18-day two-way choice test. The piglets were allotted to six dietary treatments with four replicates per treatment and 6 piglets per replicate (including three barrows and three gilts). The four diets used in the experiment included a basal diet containing 52.5% conventional corn (NC, 12.65% gelatinization) and three extruded corn diets containing 12.5% conventional corn and 40% extruded corn with low (LEC; 41.82% gelatinization), medium (MEC; 62.60% gelatinization), and high (HEC; 89.93% gelatinization) degrees of gelatinization. All four diets were tested in pairs to form six treatments: (i) NC vs. LEC, (ii) NC vs. MEC, (iii) NC vs. HEC, (iv) LEC vs. MEC, (v) LEC vs. HEC, and (vi) MEC vs. HEC. The pen used in the experiment had an area of 2.7 × 1.8 m and was equipped with two feeders on each side and an equidistant independent water supply at the opposite wall. The feeders in each pen were switched to the opposite side every three days to avoid any differences associated with the location. Water and feed were provided ad libitum throughout the experimental period. The diets were offered in pellet form, and the composition of the basal diet is presented in Table 1. Body weight and diet consumption were measured at the end of the experiment to calculate ADFI, ADG, and the feed:gain ratio (F:G). The relative preference for each individual test diet was the ratio of the amount of feed intake of that diet to the total sum of the amount of feed intake of the two diets offered simultaneously in the pair combination. A total of 144 piglets (34 days old; 72 barrows and 72 gilts; Duroc × Large White × Landrace) with 9.24 ± 0.55 kg BW were used in a 28-day performance trial. Piglets were weighed and allotted to four dietary treatments and fed with one of the four diets also used in Experiment 1. Each treatment consisted of six replicates, and each replication had 6 piglets (including three barrows and three gilts). The piglets were housed with separate feeders and drinkers. Water and feed were provided ad libitum throughout the experimental period. The diets were offered in pellet form, and the composition of the diets was the same as in Experiment 1, as presented in Table 1. Chromic oxide, which is an indigestible marker, was added to the diets at an inclusion rate of 4 g/kg feed during the last four days of the experimental period. Piglets were weighed on days 0, 14, and 28, and feed consumption was recorded at the same time on a pen basis to calculate ADFI, ADG, and F:G. Feces and feed were collected from each pen every day during the final four days. The whole feces obtained from each pen were pooled, and H2SO4 (10%) at 10% (v/w) was added to part of the feces to determine the crude protein (CP). All feces samples were then dried at 105 °C for 24 h and homogenized before sampling and analysis. The concentrations of nutrients in the feed and feces were analyzed according to the method of AOAC (2007) [11], including ether extract (EE; method 920.39; AOAC Int., 2007), CP (method 920.39; AOAC Int., 2007), DM (method 930.15; AOAC Int., 2007), ash (method 942.05; AOAC Int., 2007), and chromic oxide (method 930.15; AOAC Int., 2007). The organic matter (OM) was calculated as DM minus Ash. The apparent total tract digestibility (ATTD) of CP, EE, DM, and OM was calculated using the following equation: One barrow and one gilt from each pen were selected for the collection of blood samples from the anterior vena cava on day 14 and at the end of the experiment. The serum samples were centrifuged at 3000× g for 10 min at 4 °C and stored at –80 °C until analysis. The concentrations of total protein (TP), albumin (ALB), and total cholesterol (TC) were detected using biuret colorimetry, the bromocresol green method, and the cholesterol oxidase method, respectively. All of the above kits (TP, code no. A045-1-1; ALB, code no. A028-2-1; TC, code no. A111-1-1) were purchased from the Nanjing Jiancheng Bio-Engineering Institute (Nanjing, China). The concentrations of globulin (GLB) were calculated using TP minus ALB. Glucagon-like peptide-1 (GLP-1) and leptin were determined using porcine-specific ELISA kits (GLP-1, code no. H294-1, Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China; leptin, code no. H174, Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China). On day 28, fecal samples were collected via rectal massage from two pigs (one gilt and one ballow) per pen and pooled into one sample for high-throughput sequencing. The total DNA from each fecal sample was extracted using a MicroElute Genomic DNA kit (D3096-01, Omega Inc., Norwalk, CT, USA), according to the manufacturer’s instructions. The V3–V4 fragment of 16S rRNA was amplified using total DNA as a template, and the conserved primers used were 319F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGG GTWTCTAAT-3′). Raw data were processed using Greengenes, and sequential analysis, comparison, and annotation were performed using the Ribosomal Database Project (Version 11.3). Illumina MiSeq 2 × 300 bp paired-end was used for sequencing. Operational taxonomic units (OTUs) with a similarity threshold of 97% were determined using R version 3.1.0. Moreover, α- and β-diversity analyses based on the QIIME version 1.7.0-dev were performed to elucidate fecal microflora. Analyses of BW, ADG, ADFI, and F:G in experiment 1 were performed using one-way ANOVA with a line segment detector (LSD) using SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA). Feed consumption and relative preference in experiment 1 between two diets of a paired combination were analyzed using a t-test (SPSS 22.0). Feed consumption, relative preference associated with each individual diet in experiment 1 and growth performance, serum parameters, and nutrient digestibility in experiment 2 were analyzed using linear and quadratic regressions, and then the differences among the treatments were also analyzed using one-way ANOVA with LSD (SPSS 22.0). All statistics and data visualizations for the microbiota were analyzed using R statistical software and related R software or Qiime 1.7.0. Comparisons of alpha diversity and relative abundance of phyla and genera were performed using the Kruskal–Wallis and Wilcoxon rank sum tests. Principal coordinate analysis (PCoA) was performed using weighted UniFrac metrics. Linear discriminant analysis (LDA; >3.0) was performed using the linear discriminant analysis effect size (LEfSe). Data were presented as means and pooled SEM. For BW, ADG, ADFI, F:G, feed preference, ATTD of the nutrients, and fecal microflora, a pen was used as the experiment unit. For plasma parameters, the individual piglet was used as the experiment unit. Differences were considered significant at p < 0.05, and a tendency was revealed at 0.05 < p < 0.1. In the two-way choice test, piglets who were given a choice of the degree of gelatinization of the extruded corn showed limited effects on growth performance, including BW, ADG, ADFI, and F:G (Table 2). As shown in Table 3, the LEC piglets showed a higher relative preference and feed consumption when they were offered a combination of NC/LEC and LEC/MEC (p < 0.05). LEC showed a higher trend in relative preference and feed consumption when piglets were offered a combination of LEC/HEC (0.05 < p < 0.01). In the whole feed preference trial, diets supplemented with LEC were the most preferred corn (67.02%) with the highest feed consumption (408.95 g/d). Multiple comparisons showed that LEC markedly increased relative preference and feed consumption compared with that of NC, MEC, and HEC (p < 0.05). As the gelatinization degree increased, the relative feed preference and feed consumption quadratically increased (p < 0.05). As shown in Table 4, the gelatinization degree markedly affected F:G during the periods of 14–28 days and 0–28 days. Diets supplemented with extruded corn with low or medium gelatinization degrees markedly decreased F:G in the periods of 14–28 days and 0–28 days compared with conventional corn or extruded corn with a high degree of gelatinization (p < 0.05). As the gelatinization degree increased, F:G quadratically decreased in the period of 14–28 days (p < 0.05). No significant differences were observed in BW, ADG, or ADFI among the four treatments (p > 0.05). The nutrient digestibility results are summarized in Table 5. The piglets in the LEC and MEC groups showed a higher ATTD for CP than those in the NC and HEC groups (p < 0.05). The ATTD of EE also markedly increased in the MEC group compared with that of the NC and HEC groups (p < 0.05). As the gelatinization degree increased, the ATTD of CP and EE quadratically increased (p < 0.05). As shown in Table 6, the LEC group showed the highest level of TP and GLB content in the plasma and markedly increased compared with that of the NC group on day 14 (p < 0.05). On day 28, no significant difference was observed in the plasma contents of TP, ALB, GLB, and total cholesterol among the four groups (p < 0.05). The plasma levels of ORX, GLP-1, and LEP showed no differences on days 14 and 28 among all groups (p < 0.05). As the gelatinization degree increased, no linear or quadratic changes were observed in the serum indexes on days 14 and 28. The effects of the extruded corn on the microbial communities are shown in Figure 1. The MEC group tended to have an increased number of observed species (p = 0.090) and Chao-1 index (p = 0.106) (Figure 1A,B). Piglets in the four treatment groups shared 1795 OTUs of fecal microflora in the Venn diagram (Figure 1C). NC, LEC, MEC, and HEC contained 237, 45, 72, and 55 OTUs, respectively. The beta-diversity analysis by principal coordinate analysis showed that the microbiota in the NC group were separated from the LEC, MEC, and HEC groups (Figure 1D). The microbiota in the piglets who were fed the extruded corn were highly clustered, regardless of the degree of gelatinization (Figure 1D). The microbial community structure is presented in Figure 2. At the phylum level, the most abundant phyla were Bacteroidetes and Firmicutes, with an average relative abundance of over 95% of the total bacteria in the piglets. No significant difference was observed in the relative abundance of Firmicutes; however, the relative abundance of Bacteroidetes increased from 46.05% (NC) to 50.37% (LEC), 50.78% (MEC), and 51.92% (HEC) (p < 0.05) (Figure 2a). There was no significant difference in the relative abundance ratio of Firmicutes to Bacteroidetes between the groups (Figure 2b). At the genus level, Prevotella_9 (7.79%), Lactobacillus (7.70%), and Prevotellaceae_NK3B31_group (7.19%) were the top three genera (Figure 2c). Significant differences in the top 15 genera are shown in Figure 2d. The LEC group had an increased in the relative abundance of Lactobacillus, Prevotellaceae_NK3B31_group, Prevotellaceae_UCG-003, Allopreotella, Prevotella_1, and Prevotella_2 compared with the NC group (p < 0.05). The MEC group showed a marked increase in Lactobacillus, Allopreotella, Prevotellaceae_UCG-003, and Prevotella_2 abundance compared with that of the NC group (p < 0.05). The HEC group showed an increase in Prevotellaceae_NK3B31_group, Allopreotella, and Prevotella_1 abundance compared with that of the NC group (p < 0.05). LDA effect size was performed to detect specific bacterial taxa with significantly different abundances among the NC, LEC, MEC, and HEC groups (Figure 2d). Twelve distinct bacterial taxa were identified using a log LDA score threshold of 3.0. Prevotella 6 and Ignatzschineria were found to be enriched in the microbiota of the NC group. Lactobacillus, Prevotellaceae_UCG_003, Prevotellaceae_UCG_001, Campylobacter, and Succinivibrio spp. were enriched in the MEC group. Alloprevotella, Prevotella1, Prevotella_2, Coprococcus3, and Lachnospira were enriched in the LEC group. Prevotellaceae_NK3B31_group, Ruminococcaceae_UCG_008, Ruminococcus_2, Oscillibacter, Fournierella, and Ruminiclostridium_9 were enriched in the HEC group. Significant differences in the top 15 genera are shown in Figure 3. The LEC and MEC groups markedly increased their relative abundance of Lactobacillus, Alloprevotella, Prevotellaceae_UCG_003, and Prevotella_2 compared with the NC group (p < 0.05). Meanwhile, the LEC and HEC groups also showed a higher abundance of Prevotellaceae_NK3B31_group and Prevotella_1 than the NC group (p < 0.05). In the two-way choice preference tests, piglets preferred feed containing extruded corn, especially the feed with a 41.82% gelatinization degree. This result was similar to that of Solà-Oriol et al., who noted that extrusion increased pigs’ preferences for several cereals, including corn and naked oats, when the inclusion rate was 30% or 60% [12]. As expected, when the degree of gelatinization of the extruded corn reached 89.93%, the preference decreased to 41%, which was similar to that of conventional corn. This could be a result of the increased feed hardness. Hardness can indicate the maximum force that piglets must compress during chewing. The extrusion process gelatinizes starch and improves its adhesive property, which acts as a binder during pelleting and thus increases the pellet hardness [7,13]. As the degree of gelatinization increased, pellet hardness increased, which decreased feed preference [12]. Starch with a high degree of gelatinization is normally associated with a high water absorption index, which may stick to the mouth, and a greater amount of saliva may be needed to swallow the feed, which affects piglets’ chewing capability [14]. The addition of extruded starch to diets has beneficial effects on nutrient digestibility. Yong et al., reported that supplementary extruded corn increased the in vitro and in vivo apparent digestibility of CP, GE, DM, and starch, both in vitro and in piglets [15]. Liu et al., suggested that the extrusion process increases the ATTD of DM and GE in corn and broken rice in weaning piglets [16]. Several factors can be attributed to increased nutrient digestibility, such as increased enzyme susceptibility, reduced starch resistant content, and decreased starch granules [17,18,19]. In the present study, an increased ATTD of both CP and EE was observed in diets containing extruded corn. We also noted that diets with a medium degree of starch gelatinization (62.60%) resulted in the best digestibility, whereas a high degree of starch gelatinization contributed to low digestibility. These results aligned with Adb et al.’s study, which reported that CP, EE, and DM digestibility in pigeons increased when the starch gelatinization degree decreased from 73.6% to 53.1% [20]. Hongtragul et al., also noted significant quadratic variation between the degree of starch gelatinization and nutritional digestibility [10]. The decreased nutritional digestibility of highly gelatinized corn may be related to intestinal viscosity. Normally, extruded corn with a high degree of gelatinization has high water solubility which decreases the chyme viscosity [18]. The decreased viscosity reflects a short mean transit time, resulting in a reduction in nutrient digestion and subsequent absorption. Over-processing during extrusion may also promote a Maillard reaction between amino acids and glucose which enhances the encapsulation of starch granules via the protein and resistant starch content and thus reduces its digestibility [21,22]. The effects of extruded cereal on the growth performance of piglets varies. Lawlor reported no beneficial effects of extrusion on the growth performance of piglets with different initial body weights [23]. Similar results were reported in Mateos’s study, which used rice and maize diets [24]. Hongtragul’s study showed the positive effects of extruded corn as the piglets’ ADG and ADFI increased [10]. Kotara also found that ADG, ADFI, and G:F increased linearly when the degree of starch gelatinization increased from 23.8% to 81.9% in barley, wheat, and maize diets [9]. One possible explanation for these differences could be the variation in extruded corn quality caused by extrusion conditions, such as steam flow, temperature, and pressure. In the present study, although ADG and ADFI showed no differences among all groups, F:G markedly decreased when the degree of gelatinization of the extruded corn was 41.82% and 62.60%. The nutrient digestibility results were consistent with those of F:G and might be the main reason for the improved feed utilization in the LEC and MEC groups. Meanwhile, we also noted that feed preference had limited effects on ADFI when the piglets were fed a single feed. This difference may be because feed intake is influenced by multiple factors, such as nutritional composition, palatability, and nutritional availability [12]. In the present experiment conditions and diet compositions, nutritional requirements had a stronger effect on feed intake than palatability when piglets were fed the single feed. The TP is composed of ALB and GLB. ALB is synthesized by the liver and significantly correlated with nutritional status [25]. GLB is synthesized by immune organs and plays an important role in immune responses [26]. In the present study, TP and GLB showed significant differences on day 14, with the highest levels in the LEC group, followed by the MEC group. These results were consistent with the results of F:G and nutrient digestibility, which indicated an increased nutritional status and an increased immunity. GLP-1 and leptin are peripheral appetite regulatory peptides that play important roles in the regulation of feeding behavior. Zhuo et al., noted that extrusion ingredients, including corn and broken rice, elevated the plasma levels of GLP-1 and peptide YY [27]. However, the GLP-1 and leptin levels showed no differences among the four treatments in our present study. These differences may exist because the blood in the experiment was collected after fasting, and the hormone level had dropped to a low point, which made the difference not significant. The extrusion process elevates nutrient digestibility in the small intestine and alters fermentable substrates in the large intestine, thereby changing the gut microbial community structure. In weaning piglets, the extrusion process of corn or broken rice tends to increase the microbial diversity, as indicated by the increasing Chao-1 and ACE indices [27]. Our present study identified a similar result: extruded corn with a medium degree of gelatinization (62.60%) tended to increase the observed species and Chao-1 index. Generally, higher microbial diversity is associated with higher resilience to environmental challenges and better disease resistance [28]. A study conducted by Moen showed a converse result as cereal extrusion decreased the microbiota diversity in growing pigs [29]. These differences might be due to the growth period or extrusion condition and require further study. Bacteroidetes are Gram-negative bacteria that perform a main role in the maintenance of a healthy state and sophisticated homeostasis safeguarded by microbiota [30]. In the present study, extrusion increased the relative abundance of Bacteroidetes, regardless of the degree of gelatinization. Although Bacteroidetes are normally related to inflammation and obesity-driven dysbiosis [31,32], a study by Vadder et al., indicated that Bacteroidetes strongly correlate with increased levels of short chain fatty acid (SCFA) [33]. The increased abundance of Bacteroidetes can also provide anti-inflammatory effects with certain probiotics [34]. To further identify the effects of increased Bacteroidetes on piglets, we compared the microbial abundance at the genera level among the four groups. One important finding was that Prevotella, which is an important genus that belongs to Bacteroidetes, increased in the piglets who were fed extruded corn. A study by Mach et al., reported that the presence of Prevotella was associated with increased luminal secretory immunoglobulin A, indicating a positive influence on mucosal immunity [35]. Prevotella is also reported to be capable of metabolizing fibers into SCFAs, such as acetate, propionate, and butyrate [36]. Therefore, piglets with a high abundance of Prevotella may be better adapted to plant-derived feeds and may confer a performance advantage [37], which in turn may explain the improved feed conversion ratio of extruded corn in the present study. We also noted that extruded corn increased the fecal Lactobacillus abundance in piglets, and this effect was positively correlated with the degree of gelatinization. Lactobacillus is regarded as a beneficial bacterium that inhibits the growth of some pathogens, indicating healthier gut flora in extruded corn-treated piglets [38]. Wang also found a positive relationship between gelatinized starch and Lactobacillus in the sorghum-based diets of growing pigs [39]. The piglets in this study preferred diets containing extruded corn with a gelatinization degree of 41.82% when they were given a choice. Corn extrusion increased the feed conversion ratio, improved the ATTD of CP and EE, and increased the abundance of Bacteroidetes, Lactobacillus, and Prevotella in the piglets. Under the experimental conditions, the ideal gelatinization degree of extruded corn for the piglets was approximately 41.82–62.60%.

PMC10000146

Ying Yao,Wenjia Zhao,Guilin Xiang,Ruiqing Lv,Yanpeng Dong,Honglin Yan,Mingxi Li

Bamboo Plant Part Preference Affects the Nutrients Digestibility and Intestinal Microbiota of Geriatric Giant Pandas

25-02-2023

giant panda,bamboo part,aging,microbiome,nutrient digestibility

Simple Summary Bamboo part preference and a panda’s age have been shown to shift the gut microbiota composition of the giant panda, thus eliciting changes in their nutrient utilization capacity. The present study compared the differences in nutrient digestibility and fecal microbiota composition between adult and geriatric captive giant pandas when fed exclusively with a diet comprising of either bamboo shoots or leaves. Bamboo part preference exerted a significant effect on nutrient digestibility and fecal microbiota composition in both adult and aged giant pandas. Bamboo part dominated over age in shaping the nutrient digestibility and gut microbiota composition of giant pandas. Abstract Bamboo part preference plays a critical role in influencing the nutrient utilization and gastrointestinal microbiota composition of captive giant pandas. However, the effects of bamboo part consumption on the nutrient digestibility and gut microbiome of geriatric giant pandas remain unknown. A total of 11 adult and 11 aged captive giant pandas were provided with bamboo shoots or bamboo leaves in the respective single-bamboo-part consumption period, and the nutrient digestibility and fecal microbiota of both adult and aged giant pandas in each period were evaluated. Bamboo shoot ingestion increased the crude protein digestibility and decreased the crude fiber digestibility of both age groups. The fecal microbiome of the bamboo shoot-fed giant pandas exhibited greater alpha diversity indices and significantly different beta diversity index than the bamboo leaf-fed counterparts regardless of age. Bamboo shoot feeding significantly changed the relative abundance of predominant taxa at both phylum and genus levels in adult and geriatric giant pandas. Bamboo shoot-enriched genera were positively correlated with crude protein digestibility and negatively correlated with crude fiber digestibility. Taken together, these results suggest that bamboo part consumption dominates over age in affecting the nutrient digestibility and gut microbiota composition of giant pandas.

Bamboo Plant Part Preference Affects the Nutrients Digestibility and Intestinal Microbiota of Geriatric Giant Pandas Bamboo part preference and a panda’s age have been shown to shift the gut microbiota composition of the giant panda, thus eliciting changes in their nutrient utilization capacity. The present study compared the differences in nutrient digestibility and fecal microbiota composition between adult and geriatric captive giant pandas when fed exclusively with a diet comprising of either bamboo shoots or leaves. Bamboo part preference exerted a significant effect on nutrient digestibility and fecal microbiota composition in both adult and aged giant pandas. Bamboo part dominated over age in shaping the nutrient digestibility and gut microbiota composition of giant pandas. Bamboo part preference plays a critical role in influencing the nutrient utilization and gastrointestinal microbiota composition of captive giant pandas. However, the effects of bamboo part consumption on the nutrient digestibility and gut microbiome of geriatric giant pandas remain unknown. A total of 11 adult and 11 aged captive giant pandas were provided with bamboo shoots or bamboo leaves in the respective single-bamboo-part consumption period, and the nutrient digestibility and fecal microbiota of both adult and aged giant pandas in each period were evaluated. Bamboo shoot ingestion increased the crude protein digestibility and decreased the crude fiber digestibility of both age groups. The fecal microbiome of the bamboo shoot-fed giant pandas exhibited greater alpha diversity indices and significantly different beta diversity index than the bamboo leaf-fed counterparts regardless of age. Bamboo shoot feeding significantly changed the relative abundance of predominant taxa at both phylum and genus levels in adult and geriatric giant pandas. Bamboo shoot-enriched genera were positively correlated with crude protein digestibility and negatively correlated with crude fiber digestibility. Taken together, these results suggest that bamboo part consumption dominates over age in affecting the nutrient digestibility and gut microbiota composition of giant pandas. The giant panda (Ailuropoda melanoleuca) is a highly specialized herbivorous species of ursid that consumes bamboo as the primary and almost exclusive diet. Unlike most herbivores, the giant panda has no apparent internal gastrointestinal adaptions to its bamboo-dominated diet, and exhibits a short digestive tract with a rapid passage of digesta, which is similar to the gastrointestinal tract morphology of most carnivores [1]. The extremely high amount of bamboo consumption each day and low energy expenditure can partly explain how giant pandas persist solely on bamboo, a high fibrous plant with low nutritional value and digestibility [2]. However, the giant panda has been shown to lack homologs of the enzymes needed for the degradation of structural carbohydrates, the key component of bamboo [3]. It has thus been believed that the utilization and extraction of nutrients from the bamboo diet largely depends on the gut microbiome of the giant panda, as the giant panda gut microbiome has been found to exhibit a high abundance of putative genes involved in carbohydrate degradation, suggesting high utilization potential of structural polysaccharides [1,4]. Both wild and captive pandas exhibit seasonal changes in bamboo part preference, with shoots consumed in spring and summer, leaves in autumn and winter, and culms in the transition period, namely later winter and early spring [5,6]. Dietary changes are an important factor influencing the composition and function of the gut microbiome [7]. Evidences have been accumulated to show the giant panda’s gut microbiota are shaped by the seasonally-driven shifts in bamboo part preference, as the nutrient content in different parts of bamboo varies significantly, with higher cellulose, hemicellulose, and starch, as well as lower proteins, in the leaves and culms than in shoots [3,8,9]. Gut microbiota has been shown to significantly affect the nutrient utilization capacity and health status of the host [10]. In captive giant pandas, the apparent digestibility of bamboo parts differed significantly, resulting in different degrees of nutrient retention used by gut microbes in the hindgut [8]. Therefore, the changes in gut microbiome elicited by different bamboo part consumption would significantly affect the nutrient digestibility of the giant pandas. Aging is an inevitable biological process in an organism that leads to an increased risk of many diseases [11]. In terms of longevity, captive giant pandas generally have a lifespan of almost 30 years, and individuals older than 20 are considered to be “geriatric” because the reproduction process of the giant panda generally ends after this age [12]. Aging has been proven to significantly shape the structure of gut microbiota and affect the immune and metabolic functions of giant pandas [13]. Likewise, impaired digestive function and higher risk of gastrointestinal disorders have been recognized in aged giant pandas [12]. The seasonal variation in bamboo part consumption has been shown to significantly affect the nutrient digestibility of captive giant pandas [6]. However, little is known about the effects of bamboo part preference on aged giant pandas, especially the changes of gut microbiome and nutrients digestibility. To address this issue, the nutrients digestibility and gut microbiota composition were compared between adult and older captive giant pandas when fed exclusively with a diet comprising of either shoots or leaves. All protocols for the present study that involved animal care and treatment were approved by the Institutional Animal Care and Use Committee of Chengdu Research Base of Giant Panda Breeding (No. 2020010). A total of 11 adult (aged 9–17 years, average age was 13) and 11 geriatric (aged 20–37 years, average age was 25) captive giant pandas were the subjects of the present study. All subjects were singly housed at the Chengdu Research Base of Giant Panda Breeding (CRBGPB, Chengdu, Sichuan, China), and all were considered healthy and were not under any medical treatment during the study period. The ambient temperature was maintained at 15 °C–22 °C, and the air humidity was 65–75%. All giant pandas were fed according to the normal husbandry practices of the CRBGPB as described in Wang et al. [6]. Bamboo was provided to giant pandas three times each day (08:00, 14:00, and 20:00). In the present study, giant pandas were given free access to bamboo and water, and the specific bamboo part was offered according to the seasonal shifts. In CRBGPB, bamboo shoots of Phyllostachys nidularia Munro were consumed by pandas in autumn and bamboo leaves of Bashania fargesii were provided to pandas in winter. In addition to the supply of bamboo parts, dietary supplements were provided daily and of the same mass to all subjects. In this study, both adult and geriatric pandas were provided with bamboo shoots for 3 months and bamboo leaves for 3 months: bamboo shoot-fed adult (AS), bamboo leaf-fed adult (AL), bamboo shoot-fed old (OS), and bamboo leaf-fed old (OL) giant pandas. At the last day of each period during which pandas were offered the corresponding bamboo part, fecal samples were collected from each giant panda. For each panda, the spontaneous excreted fecal samples were collected within 10 min of defecation after the feeding in the morning. To avoid contamination, samples were collected only after the floor was cleaned and disinfected. Furthermore, the outer layer of feces that contacted the floor was discarded and only fecal parts that did not touch the floor were kept and stored at −80 °C pending further analysis. During the last three days of each single-bamboo-part consumption period, the apparent nutrient digestibility of the corresponding bamboo part was determined in both adult and older giant pandas. The amount of ingested food and excreted feces of each individual giant panda was weighed. The bamboo samples that pandas consumed and fecal samples were collected twice a day, weighed, and immediately stored at 4 °C. During the next day, corresponding proportions of fecal samples were kept and mixed according to the amount of daily excreted feces. Finally, about 1 kg of bamboo leaves and 1.5 kg of the corresponding fecal samples, as well as 5 kg of bamboo shoots and the corresponding fecal samples, were kept at −80 °C for long-term storage. The bamboo and fecal samples were dried, ground, and sieved through a 0.45 mm sieve, then mixed, sampled, and stored at −20 °C. The chemical components of the bamboo and fecal samples were determined according to the AOAC analysis method [14]. An oven drying method was adopted to measure the dry matter (DM) content, the Kjeldahl method was used to determine the crude protein (CP) content, the Soxhlet extraction method was applied to evaluate the ether extract (EE) content, the continuous extraction of samples by dilute acids and bases was used to measure crude fiber (CF), and lastly, the oxygen bomb calorimeter calorimetric method was used to analyze the gross energy (GE) concentration of bamboo and fecal samples. The calculation equation of apparent nutrient digestibility was as follows: The genomic DNA of each fecal sample was isolated with the QIAamp Fast DNA Stool Mini Kits (Qiagen, Beijing, China) following the manufacturer’s instructions. The integrity and concentration of obtained DNA samples were assessed visually by agarose gel electrophoresis or measured using a NanoDrop ND-1000 device. Sterilized water was used as a negative control sample, and was included in the DNA isolation process, which showed no detectable PCR product. The common primers 515F and 806R were used to amplify the V4 region of the bacterial 16S rRNA gene, and the resulting PCR products were pooled and purified by using the Agencourt AMPureXP beads (Beckman Coulter, Brea, CA, USA) along with the MinElute PCR Purification Kit (Qiagen, Beijing, China). After pooling and purification, these amplicons were then used to construct Illumina libraries with the Ovation Rapid DR Multiplex System 1-96 (NuGEN, San Carlos, CA, USA). All of the sample libraries were sequenced on the Illumina MiSeq platform with a PE250 sequencing strategy (Novogene, Beijing, China). The raw data were deposited in the NCBI BioProject database with the accession number PRJNA916390. The raw Illumina data were processed by Mothur software v1.3.6 (MI, USA) [15]. The high-quality paired-end sequences, which were obtained by removing the primer and barcode sequence, and also the low-quality reads, were assembled into tags with overlapping relationships. The library size of each sample was randomly subsampled into the minimum sequencing depth to minimize the biases caused by sequencing depth between samples. The USEARCH v7.0.1001 [16] was applied to cluster tags into OTUs based on 97% cut-off. The representative sequence of each OTU cluster was used for taxonomic classification against the Ribosomal Database Project database with RDP v2.6 [17]. The OTU abundance table and the OTU taxonomic assignment table laid out from the Mothur software were processed with R studio v3.4.1 [18] to calculate alpha diversity indexes of communities, as well as the beta diversity index and the Bray–Curtis distance [19]. The structural dissimilarity of the microbiota communities across the samples were visualized by non-metric multidimensional scaling (NMDS) analysis based on the Bray–Curtis distance matrix. For nutrient digestibility parameters, the statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Giant panda was considered the experimental unit for all analyses (n = 11 per treatment), and the results were expressed as means and SEM. The main effects of bamboo part and age, and the interaction between bamboo part and age were determined via two-way ANOVA. After transforming non-normal distributed data to approximately conform to normality by SAS software, the alpha indexes [20] including Observed species, Chao 1, Shannon and Simpson index as well as the relative abundance of top 10 phyla and top 30 genera were tested for significance with the one-way ANOVA, followed by Tukey’s test to evaluate the differences between treatments. Data were presented as mean ± SE. The intragroup statistic differences in beta diversity based on the Bray–Curtis distance were assessed using the one-way ANOSIM test with 10,000 permutations. Spearman’s correlation between the gut microbiota composition and nutrient digestibility parameters were calculated by the ggcor package within R software version 3.6.1 [18]. Only correlations with Spearman’s coefficient r > 0.5 and p < 0.05 were used to generate the network graph, which was visualized and manipulated by Gephi version 9.2 [21]. The differences were considered statistically significant when the p values were less than 0.05. A significant effect of age (F = 4.86, df = 1, p = 0.04) on the dietary gross energy utilization efficiency was observed showing that aged giant pandas had weaker energy extraction capacity from their diet compared to their younger counterparts (Table 1). There was a significant effect of bamboo part ((F = 203.23, df = 1, p < 0.001) for crude protein digestibility, indicating that bamboo shoot ingestion increased the crude protein digestibility of both adult and aged giant pandas (Table 1). There was a significant effect of bamboo part (F = 13.65, df = 1, p = 0.001) and age (F = 11.44, df = 1, p = 0.002) as well as a significant bamboo part × age interaction (p < 0.05) for ether extract digestibility (Table 1). This demonstrates that bamboo shoot feeding increased ether extract digestibility of aged rather than adult giant pandas when compared to bamboo leaf ingestion. Results indicated that bamboo shoot-fed giant pandas had lower crude fiber digestibility than bamboo leaf-fed counterparts (F = 16.06, df = 1, p < 0.001, Table 1). After the pre-processing of raw reads, high-quality tags were generated from all samples ranging from 57,136 to 91,531, which were subsampled to 57,136 to avoid the bias induced by the sequencing depth between samples. A total of 3,728 OTUs were obtained by clustering these tags at a 97% similarity cutoff. The fecal microbiome of the bamboo shoot-fed giant pandas exhibited greater observed species (F = 4.65, df = 3, p = 0.01), Chao1 (F = 56.08, df = 3, p < 0.001), Shannon (F = 62.11, df = 3, p < 0.001), and Simpson index (F = 5.01, df = 3, p = 0.005) values than the bamboo leaf-fed counterparts regardless of age (Figure 1). The inter-group Bray–Curtis distance was significantly higher than the intra-group when giant pandas were fed with different bamboo parts independent of age (F = 25.49, df = 5, p < 0.001), otherwise there was no difference in the inter-group and intra-group Bray–Curtis distances (Figure 2A). The NMDS-based map also showed that the fecal microbiome of giant pandas could be sorted into two clusters by bamboo part consumption rather than age (Figure 2B), indicating the dominant role of bamboo part consumption in shaping the fecal microbiome of both adult and old giant pandas. The predominant phyla in feces of AS, AL, OS, and OL pandas were Firmicutes and Proteobacteria (Figure 3A, Table S1). Bamboo shoot feeding was found to decrease the relative abundance of Firmicutes and increase the relative abundance of Proteobacteria in adult giant pandas rather than old giant pandas compared to bamboo leaf consumption (Figure 3B). Additionally, bamboo shoot feeding increased the relative abundance of Acidobacteriota, Actinobacteria, and Chloroflexi as well as decreased the relative abundance of Bacteroidetes in both adult and old giant pandas compared to bamboo leaf feeding (Figure 3C). At the genus level, Escherichia-Shigella and Clostridium_sensu_stricto_1 were the two most abundant bacteria in feces of all four groups (Figure 4A, Table S2). The relative abundance of Cellulosilyticum, Citrobacter, Enterococcus, Lactococcus, Pantoea, Ralstonia, Raoultella, Acinetobacter, Bradyrhizobium, Leuconostoc, Massilia, and Providenicia were higher in feces of bamboo shoot-feeding giant pandas than the bamboo leaf-feeding group regardless of age (Figure 4B,C). Bamboo shoot intake was found to decrease the relative abundance of Streptococcus, Lachnospiraceae_NK4A136_group, and Terrisporobacter in feces of both adult and old giant pandas compared to bamboo leaf consumption (Figure 4B,C). Bamboo shoot feeding increased the relative abundance of Helicobacter and decreased the relative abundance of Clostridium_sensu_stricto_1 in feces of adult giant pandas rather than the old group (Figure 4B,C). Compared to bamboo leaf consumption, the decreased abundance of Escherichia-Shigella and increased abundance of Turicibacter, Hafnia-Obesumbacterium, and Weissella were observed in bamboo shoot-fed old giant pandas rather than the adult group (Figure 4B,C). The genus Streptococcus and Lachnospiraceae_NK4A136_group were significantly positively correlated with crude fiber digestibility, whereas the genus Lactococcus, Turicibacter, Raoultella, Citrobacter, Enterococcus, Pantoea, Cellulosilyticum, Weissella, Providencia, and Hafnia-Obesumbacterium were significantly negatively correlated with crude fiber digestibility (p < 0.05, Figure 5). The genus Streptococcus, Terrisporobacter, and Lachnospiraceae_NK4A136_group were significantly negatively correlated with crude protein digestibility, whereas the genus Lactococcus, Turicibacter, Raoultella, Citrobacter, Enterococcus, Ralstonia, Pantoea, Cellulosilyticum, Weissella, Providencia, Helicobacter, Hafnia-Obesumbacterium, Massilia, Bradyrhizobium, Leuconostoc, and Acinetobacter were all significantly positively correlated with crude protein digestibility (p < 0.05, Figure 5). The genus Providencia was significantly positively correlated with ether extract digestibility (p < 0.05, Figure 5). Despite exhibiting a carnivore’s characteristic simple gastrointestinal tract, giant pandas acquire the majority of the required nutrients from bamboo. Because of the limited digestibility of plant cellulose by the giant panda genome, it was suggested that the gut microbiome may play a vital role in the digestion of this highly fibrous bamboo diet [22]. Seasonal dietary shifts in bamboo part selection have been observed in both wild and captive giant pandas, and have been shown to extensively shape the host microbiome [5]. The bamboo part preference during different seasons has been shown to significantly influence the nutrient digestibility of adult captive giant pandas, which is associated with changes in the gut microbiota composition [6]. Owing to the improvements in husbandry and veterinary care, the number of geriatric pandas in zoological institutions has increased in recent years. The aging process in giant pandas elicits a significant change in the gut microbiome, indicating that geriatric pandas exhibit a different gut microbiota composition than younger pandas [12]. While studies in humans and other animals have shown that there may exist an interaction between diet and aging in regulating host phenotype and shaping gut microbiota composition [23,24], such information in different bamboo part-fed geriatric and adult pandas remains unknown. Unlike studies in other animals showing similar nutrient digestibility between adult and senior individuals [25,26], lower energy digestibility was found in aged giant pandas compared to the adults in the present study, indicating the declined energy extraction capacity from food in aging giant pandas. Giant pandas feed almost exclusively on bamboo, of which the different plant parts exhibit significantly different nutrient compositions [4]. Wang et al. [6] showed that the bamboo part exerted a significant effect on nutrient digestibility in giant pandas. Bamboo shoots consumption has been shown to increase the crude protein digestibility and decrease the crude fiber digestibility of giant pandas [6]. Consistently, higher crude protein digestibility and lower crude fiber digestibility were observed in bamboo shoot-fed adult and geriatric giant pandas compared to those fed with bamboo leaves in the present study, which might be attributed to the inhibition of crude protein utilization induced by the higher level of fiber in bamboo leaves [27]. In rodent models, the aging process was found to decrease lipid absorption through reducing the pancreatic lipase activity [28]. In this study, bamboo shoot consumption increased the ether extract digestibility in aged giant pandas rather than in adults compared to bamboo leaf feeding. This finding might be related to the lower lipase activity in the small intestine of senior giant pandas and the higher ether extract content in bamboo leaves. Compared with adults, the ether extract in bamboo leaves was too high for aged giant pandas to fully digest, resulting in the lower digestibility of ether extract in senior pandas fed with bamboo leaves than those fed with bamboo shoots [6]. Accumulated evidences have demonstrated the possible role of the gut microbiota in the regulation of nutrient harvest in humans and monogastric animals [29,30]. More typically, as the giant panda lacks enzymes for the digestion of bamboo, it has thus been suggested that the giant panda appears to have no alternative but to rely on symbiotic gut microbes to extract nutrients from its highly fibrous bamboo diet [31]. A previous study contended that dietary shifts induced changes in nutrient digestibility in captive giant pandas and were associated with the alteration of the microbiota composition [6]. Both bamboo plant part and age have been shown to play a critical role in shaping the gut microbiota profile in captive giant pandas [7,8,12], however the interaction between bamboo plant part and age on intestinal microbiota composition, as well as the relationship between the interaction-induced gut microbiota shifts and nutrient digestibility of the captive giant pandas, remains unknown. Consistent with the previous study showing a more diverse gut microbiome in bamboo shoot-fed giant pandas than their counterparts [8], we found that bamboo shoot feeding increased the observed species, Chao1, Shannon, and Simpson indexes in both adult and old giant pandas. This indicates that there is a more abundant and diverse microbiome in bamboo shoot-fed giant pandas. Research showed that the elderly pandas exhibited lower bacterial species richness and diversity than the younger individuals [12,22]. However, in this study, the main effect of age on the alpha diversity indices of microbiome in giant pandas was not observed, which is inconsistent with findings in rodents in which the microbial composition was generally affected by age rather than diet [32]. This indicates the predominant role of dietary shifts rather than age in shaping the gut microbiota of giant pandas. The dissimilarity distance analysis in the present study also confirmed that the fecal microbiota of giant pandas could be sorted into two clusters by bamboo part independent of age. It has been demonstrated that phyla Firmicutes and Proteobacteria were the most predominant bacteria in the fecal microbiome of giant pandas [3,4]. In the present study, bamboo shoot feeding decreased the abundance of Firmicutes and increased the abundance of Proteobacteria in the adult group rather than the geriatric group compared to bamboo leaf feeding. This is contradictory with the previous finding that the relative abundance of Proteobacteria was the highest in the bamboo-leaf fed giant pandas [8]. However, in vivo studies in rodents revealed that bamboo shoot-derived components promoted the colonization of bacteria belonging to Proteobacteria and decreased the abundance of Firmicutes bacteria in the gut [33,34]. The contradictory results might stem from the different study subjects or use of different bamboo species. Previous studies in monogastric animals showed that the relative abundance of Acidobacteriota was positively correlated with the intake amount of dietary protein and the relative abundance of Bacteroidetes was negatively correlated with dietary protein level [35,36]. In the present study, the higher abundance of Acidobacteriota and lower abundance of Bacteroidetes were observed in bamboo shoot-fed giant pandas regardless of age, which might be attributed to the higher amount of protein in bamboo shoots than bamboo leaves [6]. Consistent with the previous findings [4], the genera Escherichia-Shigella and Clostridium_sensu_stricto_1 were predominantly present in the fecal microbiome of giant pandas in this study. Bamboo shoot consumption has been shown to decrease the abundance of Escherichia-Shigella and increase the abundance of Weissella in the feces of giant pandas [8]. Our study further revealed that the bamboo shoot feeding-induced changes in Escherichia-Shigella and Weissella abundances were only observed in aged giant pandas. In addition, the decreased abundance of Clostridium_sensu_stricto_1 was observed in bamboo shoot-fed adults rather than geriatric giant pandas compared to the bamboo leaf group. This finding was consistent with the previous study showing the higher abundance of Clostridium_sensu_stricto_1 in the bamboo leaf consumption stage versus bamboo shoot consumption stage [3]. The inconsistent findings demonstrate that the genus Clostridium_sensu_stricto was not significantly enriched in the bamboo leaf stage and showed low sensitivity to the host’s seasonal dietary changes [1]. These contradictory results regarding the effects of bamboo part consumption on predominant genera abundance in giant pandas further suggest that the distribution of bacteria at the genus level in giant pandas might be dependent on the interaction effect of dietary shifts and age of the host. Seasonal variations in bamboo part selection has been shown to shape the bacteria distribution at the genus level of giant pandas [1,3]. The abundances of genera Cellulosilyticum, Lactococcus, and Streptococcus were significantly affected by the consumption of different bamboo parts [8]. Consistently, in this study, bamboo shoot feeding significantly increased the abundance of Cellulosilyticum, Lactococcus and other genera as well as decreased the abundance of Streptococcus in feces of both adult and aged giant pandas compared with bamboo leaf ingestion. In monogastric animals, the shifts in gut microbiota composition were found to closely correlate with nutrient digestibility [37]. The genus Streptococcus was positively related to crude fiber digestibility in pigs [38]. In this study, the genera Streptococcus and Lachnospiraceae_NK4A136_group were positively correlated with crude fiber digestibility in giant pandas, indicating the critical role of these two genera in the utilization of crude fiber of bamboo. High protein diets and ingredient consumptions have been shown to increase the abundance of the genera Turicibacter and Lactococcus in rodents [39,40]. In the present study, the genera Turicibacter, Lactococcus, and other genera were positively correlated with the crude protein digestibility of giant pandas, which indicates that these bacteria may be important for the protein utilization of the bamboo parts. Taken together, the gut microbiota composition of giant pandas was mainly shaped by bamboo part consumption rather than age. In conclusion, bamboo shoot feeding increased the crude protein digestibility and decreased the crude fiber digestibility of giant pandas regardless of age. Bamboo part consumption dominated over age in shaping the gut microbiota composition of giant pandas. The shifts in taxa distribution at genus level might be responsible for the bamboo part-induced nutrient extraction alterations.

PMC10000151

Susan Lalondrelle,Jen Lee,Rosalind J. Cutts,Isaac Garcia Murillas,Nik Matthews,Nicholas Turner,Kevin Harrington,Katherine Vroobel,Emily Moretti,Shreerang A. Bhide

Predicting Response to Radical Chemoradiotherapy with Circulating HPV DNA (cHPV-DNA) in Locally Advanced Uterine Cervix Cancer

22-02-2023

cervical cancer,plasma HPV DNA,response prediction,circulating DNA,next generation sequencing

Simple Summary Cervix cancer is largely (95%) caused by human papilloma virus (HPV). Curative treatment for locally advanced cervical cancer (progressed beyond the cervix) is chemotherapy and radiotherapy (CRT), followed by internal radiation (brachytherapy). In approximately 30% of patients the cancer will return, usually within 2 years of completing initial treatment. Assessment after CRT is by clinical examination and MRI and PET/CT scans. None of these methods are particularly sensitive for detecting early relapse or discriminating between early relapse and treatment related scarring. Currently, there is no test that can reliably predict early relapse. We have developed a blood test which measures the presence of HPV-DNA fragments in the blood. In this initial feasibility study, we have demonstrated that cHPV-DNA can be accurately detected in the blood before CRT and if present at the end of treatment or detected during follow up, is indicative of tumour relapse. Abstract Background: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay—panHPV-detect—in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. Method: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. Results: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70–99%) and 100% (95% CI-30–100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. Conclusions: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort.

Predicting Response to Radical Chemoradiotherapy with Circulating HPV DNA (cHPV-DNA) in Locally Advanced Uterine Cervix Cancer Cervix cancer is largely (95%) caused by human papilloma virus (HPV). Curative treatment for locally advanced cervical cancer (progressed beyond the cervix) is chemotherapy and radiotherapy (CRT), followed by internal radiation (brachytherapy). In approximately 30% of patients the cancer will return, usually within 2 years of completing initial treatment. Assessment after CRT is by clinical examination and MRI and PET/CT scans. None of these methods are particularly sensitive for detecting early relapse or discriminating between early relapse and treatment related scarring. Currently, there is no test that can reliably predict early relapse. We have developed a blood test which measures the presence of HPV-DNA fragments in the blood. In this initial feasibility study, we have demonstrated that cHPV-DNA can be accurately detected in the blood before CRT and if present at the end of treatment or detected during follow up, is indicative of tumour relapse. Background: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay—panHPV-detect—in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. Method: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. Results: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70–99%) and 100% (95% CI-30–100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. Conclusions: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort. Locally advanced cervical cancer (LaCC) FIGO (2019) stage IB3-IVA is treated with external beam radiotherapy (EBRT) and concomitant cisplatin, followed by image guided adaptive brachytherapy (IGABT). In contemporary series this leads to excellent 5-year local control rates of 89%, and 5-year overall survival of 65% across all stages [1]. When relapse occurs, it is most often metastatic but can include cases of salvageable para-aortic lymph node relapse or oligometastatic disease. A smaller proportion of patients will also experience persistent or recurrent disease within the cervix or uterus, amenable to salvage surgery [2]. The identification of persistent disease and early relapse is challenging and largely relies on multiparametric functional imaging for which differentiating between disease and treatment related changes is difficult [3,4]. Often, recurrent or persistent disease may not be detected radiologically until wider metastasis has developed. On the other hand, sometimes, unnecessary salvage surgery is performed, which can lead to considerable morbidity. Therefore, there is a need to establish an alternative marker of active disease, for use as a monitoring tool to identify patients with either persistent or emerging disease, to complement and stratify existing clinical and radiological tools. The majority of LaCC are caused by the human papilloma virus (HPV). During carcinogenesis, the viral DNA that is present in the tumour cell (integrated into the tumour DNA or in episomal form) is released into the blood following tumour lysis and can be measured. Studies have been published demonstrating the utility of circulating tumour DNA as a method of investigating and monitoring tumour biology and clinical status [5,6]; circulating HPV-DNA (cHPV-DNA) can potentially be used as a detection marker for HPV-related LaCC. Published studies have used polymerase chain reaction (PCR) based techniques to detect cHPV-DNA in patients with LaCC at diagnosis; with variable sensitivity of 24–83% [7,8,9,10,11,12,13,14]. The potential of cHPV-DNA as a marker of disease response following radical chemo-radiotherapy in LaCC has not been extensively evaluated. We developed an ultra-sensitive HPV DNA next generation sequencing (NGS) assay, panHPV-detect, with the ability to comprehensively detect circulating DNA of high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52 and 58) and assess its relationship with disease status and response. We have previously validated this assay in patients with locally advanced anal cancer undergoing CRT. Here, we validate the assay in prospectively collected plasma DNA at serial time points in patients with LaCC treated with primary CRT. Informed consent was obtained from patients with LaCC planned to receive chemoradiation. The Institutional board (Ref. no. CCR 4157) and ethics committee (Ref. no. 14/NE/1055) approved the study. Treatment consisted of external beam radiotherapy to the pelvis (and para-aortic lymph nodes if common iliac lymph nodes involved) to a dose of 45 Gy in 25 fractions, concomitant cisplatin 40 mg/m2 weekly, and image guided adaptive brachytherapy to a combined target dose ≥85 Gy EQD2. Involved lymph nodes were treated to 55–57.5 Gy in 25 fractions using a concomitant boost technique. Pelvic examination, multiparametric MRI and FDG PET/CT were performed at baseline, end of external beam chemoradiotherapy (week 5) and at 3 months post completion of brachytherapy. In the case of complete local response, further imaging with MRI and CT thorax was deferred until 12 months from completion of treatment, although patients continued to be followed with pelvic examination every 3 months. Standard practice for patients with negative PET/CT but equivocal MRI was to perform repeat MRI 3 months later, i.e., at 6 months. To identify locally persistent or recurrent cervical disease any patients with persistent FDG avidity at any time and corresponding MRI changes underwent examination under anaesthetic and cervical biopsy. Patients with regional or distant metastatic relapse were identified on serial imaging at the timepoints described. Serial plasma samples (20 mL) were collected at baseline (before CRT), at week 6–7 (during brachytherapy) and 3 months following completion of treatment in Streck® tubes. Samples of pooled plasma from a set of five healthy pre-screened individuals with no cancer or history of cancer purchased from CTLS (Clinical Trial Laboratory Services), London and 19 patients with breast cancer enrolled in the prospective sample collection study (PlasmaDNA, CCR3297, REC Ref No: 10/H0805/50) were used as negative controls. None of the negative controls were known to have pre-cancerous lesions. Written informed consent was obtained from all control participants. 20 mL of blood was centrifuged at 1600× g for 10 min within 48 h of collection. The resulting plasma was then centrifuged again at 1600× g for 10 min, aliquoted and frozen at −80 °C. DNA was extracted from 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Manchester, UK) according to manufacturer’s instructions. DNA was eluted in 50 μL of AVE buffer and stored at −20 °C. Plasma DNA was quantified using a Bio-Rad QX200 ddPCR system, using ribonuclease P (RNase P) as a reference gene as previously described [15]. Formalin fixed paraffin embedded tumour blocks of the diagnostic biopsy samples were obtained. Eight 10 μm nuclear fast red-stained slides and two haematoxylin and eosin (H&E) stained slides were obtained from representative FFPE blocks. Tumour content, cellularity and suitable areas of tumour were marked for macro-dissection. DNA was extracted using the AllPrep DNA FFPE Kit (Qiagen, Manchester, UK) followed by cDNA synthesis using the Omniscript Reverse Transcription kit (Qiagen, Manchester, UK). PanHPV-detect was designed as follows. Representative sub-lineages [16] from each of the eight high risk HPV genotypes associated with 98% of cervical cancers (16, 18, 31, 33, 35, 45, 52 and 58) were aligned using CLUSTAL O (1.2.1) multiple sequence alignment programme (https://www.ebi.ac.uk/ accessed on 30 November 2020). Diagnostic single nucleotide polymorphisms (SNP) were identified for each sub-lineage [17,18,19] and used as a guide for primer design using the Ampliseq Designer (ThermoFisher Scientific, Manchester, UK). Eight primer sets targeting the human genes ACTINB, GAPDH, HPRT were also included in the panel to act as positive controls for library preparation and sequencing efficiency. A total of 2043 SNP targets were submitted; 235 targets were missed thus providing 88.5% coverage. The amplicon length of 140 bp was specified with the primary aim of detection of circulating free DNA fragments in plasma. The panel consisted of 548 primers divided into two pools (Table 1). Ion torrent libraries were prepared using an Ion Ampliseq library preparation kit 2.0 (ThermoFisher Scientific) according to manufacturer’s instructions using 5 ng of tissue DNA or 3 ng of plasma DNA per primer pool. Reads were aligned to an amalgamated reference containing scaffolds for each of the HPV genotypes as well as the reference human targeting genes using TMAP on the Ion Torrent machine. Bedtools v2.23.0 [20] was used to extract on-target reads from the aligned files with a minimum overlap of 90% with amplicons in the panel. Additionally reads with a mapping quality of <30 were removed using samtools v1.2 [21]. Reads were split into those covering human and HPV amplicons and coverage of each portion and each genotype was calculated individually. We performed an initial validation of panHPV-detect by testing its ability to correctly identify the HPV sub-type in cervical cancer tissue samples. We obtained tumour DNA extracted from cervical cancer tissue previously typed for the HPV sub-type using polymerase chain reaction (RT-PCR) for E6 and E7 mRNA using validated assays. Five samples for each of the eight high-risk HPV sub-types were obtained from the Scottish HPV archive. Samples from five HPV negative patients, with head and neck cancer recruited in the head and neck cancer cohort of this study and 19 patients with breast cancer enrolled in the prospective sample collection study (PlasmaDNA, CCR3297, REC Ref No: 10/H0805/50) were used as negative controls. HPV status was confirmed as negative with E7 RT-PCR performed on RNA extracted from the samples. None of the negative controls were known to have pre-cancerous lesions. Written informed consent was obtained from all participants. Following the initial validation, we investigated whether panHPV-detect was able to detect cHPV-DNA in pre-treatment plasma of patients recruited in the study. We then tested the concordance of HPV subtype identified by panHPV-detect in plasma with the HPV subtype identified in the tumour biopsies collected from the patients at diagnosis. The primary endpoint for this pilot study was the feasibility of detecting cHPV-DNA at baseline in patients undergoing CRT with radical intent. Our pilot study in HPV+ head and neck cancer demonstrated 100% sensitivity and 93% specificity in detecting cHPV-DNA in patients’ plasma at baseline (pre-treatment) using the original NGS assay (HPV-detect) [6]. To prove 85% sensitivity assuming that the true sensitivity is 99% and to have 80% power given two-sided type I error of 0.05, would require at least 19 HPV+ patients, all of them expected to be labelled as positive by NGS. Since ~85% of LaCC patients are HPV+, therefore, the study required 22 patients. The secondary endpoint was to assess the potential of cHPV-DNA to predict response to treatment. Sensitivity and Specificity were calculated using contingency tables. These were calculated with the tissue HPV status and sub-type obtained from the patient’s tumour biopsy as the gold standard test. All statistical analyses were performed using GraphPad Prism version 7 software. To classify HPV+ and HPV− samples using panHPV-detect in tissue, we set a threshold whereby a sample was classified positive if there were ten reads present from more than 30% of the different HPV amplicons for each sub-type. To assess the threshold for the number of amplicons needed for positive panHPV-detect readout in plasma—a ROC analysis was used. In the first step, HPV status and subtype was assigned in tissue using E7 mRNA to separate the two groups. The number of amplicons with greater than ten reads at baseline was input for each patient to find a suitable threshold for this parameter. Sorting the values in both HPV+ and negative groups and averaging adjacent values in the sorted list generated a list of thresholds. Based on the ROC analysis, a threshold that gave the greatest sensitivity and specificity for each HPV subtype was selected as the threshold for classification of plasma as HPV DNA positive. Based on the ROC analysis, a threshold of 6.5 amplicons with more than ten reads and 7.5 amplicons in more than ten reads was set for HPV16 and HPV18, respectively. These were the thresholds that gave the greatest sensitivity and specificity and were selected as thresholds for classification of plasma as HPV DNA positive. There were insufficient plasma samples for the other rarer HPV subtypes to provide a statistically robust threshold and therefore, an empirical and pragmatic (between 6.5 and 7.5 amplicons) threshold of seven amplicons with greater than ten reads were set for the other subtypes. Tumour DNA from 39/40 pre-typed tumour samples (five of each of the HPV sub-types 16, 18, 31, 33, 35, 45, 52 and 58) obtained for validation was of satisfactory quality for the initial tissue validation. PanHPV-detect demonstrated 100% sensitivity and specificity in correctly identifying the HPV sub-type in tumour DNA and negative controls (Table 2). Twenty-two patients were recruited into the study. The median age was 48 (range 33–88). Patient characteristics are described in Table 3. Tumour tissue and baseline blood samples were available for 20 patients. In two patients’ plasma samples were of poor quality and unsuitable for analysis. Tumour tissue in 18/20 patients was positive for HPV. cHPV-DNA was detected by panHPV-detect in 16/18 plasma samples. The tissue sample from one patient was positive for HPV59, which was not included in panHPV-detect. cHPV-DNA was not detected in plasma of HPV negative patients. Therefore, panHPV-detect demonstrated a sensitivity and specificity of 88% (95% CI-70–99%) and 100% (95% CI-30–100%), respectively. In 16 samples with detectable cHPV-DNA, the following sub-types were correctly identified as follows—HPV16—11 samples, HPV18—5 samples, HPV45—2 samples and HPV58—3 samples. Some samples had multiple HPV-subtypes. See Table 4. The median follow-up for all patients was 16 months (range 11–29). A total of 16 of the 22 patients recruited in the study had a complete response (CR) and six patients had equivocal disease according to MRI and PET-CT assessment at 3 months following completion of CRT. Of the 16 patients with detectable cHPV-DNA at baseline, cHPV-DNA was detected in plasma at 3 months in the two patients with residual disease and one patient with clinical CR. Of the two patients with detectable cHPV-DNA and radiological residual disease, one had disease in the para-aortic nodes and the other had residual disease in the cervix. The para-aortic disease was treated with radiotherapy. The other patient was referred for salvage surgery; however, this was not undertaken due to significant comorbidities. The patient with detectable cHPV-DNA and clinical CR at the 3-month time-point had clinical relapse at 14 months post-treatment. This was treated with salvage surgery. Four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point and eight patients with CR and undetectable cHPV-DNA at the 3-month time-point were disease free at the last follow-up appointment. One patient with CR and undetectable cHPV-DNA at the 3-month time-point relapsed at 14 months post-CRT. This was successfully treated with salvage surgery. Of the 16 patients with detectable cHPV-DNA at baseline, panHPV-detect accurately predicted presence/absence of residual disease (macroscopic/microscopic) in 15 out of the 16 patients. We have designed “panHPV-detect” a novel NGS based method for detection and tracking of cHPV-DNA from eight high-risk HPV genotypes. The assay was initially tested in banked HPV-typed cervical tissue samples. Following this, we tested this assay in a prospective observational biological sample collection study in patients undergoing radical CRT and we have demonstrated high sensitivity and specificity. Tracking cHPV-DNA in sequential samples through and after CRT accurately predicted response and residual disease suggesting the potential of panHPV-detect to enhance clinical decision-making. Compared to PCR-based assays, amplicon-based NGS of multiple regions of the viral genome is able to detect cHPV-DNA at baseline, with higher sensitivity and specificity. To our knowledge this is the first study to use NGS to detect cHPV-DNA in patients undergoing CRT for LaCC. Previously published studies used PCR for cHPV-DNA detection, with reported sensitivity of detecting cHPV-DNA at baseline of between 24–83% [7,8,9,10,11,12,13,14] (Table 5). The majority of these studies used primers specific to HPV16 and 18. Although, 16 and 18 are the oncogenic HPV subtypes in approximately 80% of the HPV associated LaCC, excluding the other high-risk sub-types reduces the sensitivity of the assay. Despite using an identical PCR primer set for the seven commonest high-risk sub-types, two studies demonstrate sensitivity of 12% and 65% [9,11]. Our NGS based assay which is able to detect multiple HPV genomic regions across the eight most common high-risk sub-types, demonstrates a superior sensitivity to the PCR based technique. We envisage several uses for cHPV-DNA in LaCC. Firstly, as an adjunct to standard-of-care MRI and/or PET-CT scans, cHPV-DNA could potentially confirm complete response at 3 months, precluding the requirement for repeated scans and biopsies. This would result in a significant health economic impact and avoid unnecessary patient morbidity and anxiety. This is supported in our study by the observation that 14 out of 16 patients with detectable baseline cHPV-DNA in our study had undetectable cHPV-DNA at 3 months post-CRT. Out of the 14, four had partial or equivocal radiological response; however, no evidence of disease relapse was observed on subsequent imaging and these patients remain relapse free at the last follow-up. Secondly, in other cases of equivocal imaging and where biopsy is either not possible or negative, cHPV-DNA could be used to confirm both local and metastatic disease. Three patients in our study had detectable cHPV-DNA at 3 months and subsequently had confirmed relapse. To our knowledge only three other studies correlated cHPV-DNA levels to disease response. In the study by Campitelli et al. [13] high cHPV-DNA levels in 2 patients (out of their 16 patients with post-CRT blood samples) correlated with disease relapse and in the study by Yang et al. [10] 5 patients (out of their 21 patients with post-CRT blood samples) had detectable cHPV-DNA all of whom had disease relapse. Elevated cHPV-DNA levels at 3-months following CRT in our study and the ones by Campitelli et al. [13] and Yang et al. [10] accurately predict residual disease and/or disease relapse (10/53 patients with detectable cHPV-DNA at 3 months, the three studies combined). More recently Leung et al. [22] evaluated an NGS technique with 100% sensitivity and 67% specificity for detection of recurrence. Patients considered to have residual or recurrent cervical disease may be evaluated for salvage surgery. Although repeat cervical biopsy is undertaken prior to surgery, in the post CRT setting this is not targeted and difficult to interpret. Some patients will therefore proceed to surgery without positive biopsy and ultimately will not have pathological evidence of disease on hysterectomy specimen. Surgical complications of wound infection and healing are increased after CRT. Another role for cHPV-DNA is in the monitoring or follow up phase. Patients with persistent cHPV-DNA at 3 months and negative imaging could be triaged to more intensive imaging follow up, hopefully to capture early emergence of relapse sites potentially amenable to targeted treatment. Furthermore, using more sensitive assays such as plasma cHPV-DNA as a marker for detection of true high-risk patients could aid the design of adjuvant therapy studies. Using panHPV-detect we were able to accurately identify the specific sub-type of the cHPV-DNA, which possibly indicates the causative oncogenic sub-type. Studies have suggested that the causative HPV sub-type can affect prognosis with the alpha-7 (HPV18, 39, 45) sub-types having inferior outcomes compared to alpha-9 (HPV16, 31, 33, 52, 58) sub-types following CRT [23] and worse prognosis following primary surgery in cervical cancers related to HPV 18 [24]. Therefore, identification of the correct sub-type driving the oncogenic process may provide prognostic information. This was a pilot study to examine the potential of a novel NGS assay to detect cHPV-DNA before radical therapy and identify the causative HPV sub-type. This study did not have adequate power to so. However, it confirms the findings from studies by Campitelli et al. [13] and Yang et al. [10] highlighting the potential of cHPV-DNA in predicting disease response following CRT. cHPV-DNA has significant potential as a biomarker of response following radical intent CRT and of recurrence during surveillance for patients with LaCC. Furthermore, panHPV-detect can identify cHPV-DNA with high sensitivity and specificity enabling the use of cHPV-DNA as a biomarker to become a reality, albeit further validation in larger multi-centre studies is required. Such validation studies of panHPV-detect in patients undergoing curative intent CRT in LaCC are currently in development.

PMC10000161

Vince G. Amoroso,Aishi Zhao,Isabel Vargas,Thomas J. Park

Naked Mole-Rats Demonstrate Profound Tolerance to Low Oxygen, High Carbon Dioxide, and Chemical Pain

24-02-2023

naked mole-rat,hypoxia,anoxia,pain,NaV1.7,HIF-1a,VEGF,Substance P,KCC2

Simple Summary Naked mole-rats live in crowded underground burrows where concentrations of oxygen can be low and concentrations of carbon dioxide can be high. Accordingly, this species is tolerant of low oxygen levels and high carbon dioxide levels, which would be deadly to most surface dwellers. The current article reviews what we know about these unusual tolerances and their underlying mechanisms. Understanding these mechanisms could lead to new strategies for treating human disorders related to low oxygen and high carbon dioxide, as experienced, for example, during a heart attack. Abstract Naked mole-rats (Heterocephalus glaber) are very unusual among subterranean mammals in that they live in large colonies and are extremely social, spending large amounts of time gathered together in underground nests more than a meter below the surface. Many respiring individuals resting in deep, poorly ventilated nests deplete the oxygen supply and increase the concentration of carbon dioxide. Consistent with living in that atmosphere, naked mole-rats tolerate levels of low oxygen and high carbon dioxide that are deadly to most surface-dwelling mammals. Naked mole-rats appear to have evolved a number of remarkable adaptations to be able to thrive in this harsh atmosphere. In order to successfully survive low oxygen atmospheres, they conserve energy utilization by reducing the physiological activity of all organs, manifest by reduced heart rate and brain activity. Amazingly, they resort to the anaerobic metabolism of fructose rather than glucose as a fuel to generate energy when challenged by anoxia. Similarly, high carbon dioxide atmospheres normally cause tissue acidosis, while naked mole-rats have a genetic mutation preventing both acid-induced pain and pulmonary edema. Together, these putative adaptations and the tolerances they provide make the naked mole-rat an important model for studying a host of biomedical challenges.

Naked Mole-Rats Demonstrate Profound Tolerance to Low Oxygen, High Carbon Dioxide, and Chemical Pain Naked mole-rats live in crowded underground burrows where concentrations of oxygen can be low and concentrations of carbon dioxide can be high. Accordingly, this species is tolerant of low oxygen levels and high carbon dioxide levels, which would be deadly to most surface dwellers. The current article reviews what we know about these unusual tolerances and their underlying mechanisms. Understanding these mechanisms could lead to new strategies for treating human disorders related to low oxygen and high carbon dioxide, as experienced, for example, during a heart attack. Naked mole-rats (Heterocephalus glaber) are very unusual among subterranean mammals in that they live in large colonies and are extremely social, spending large amounts of time gathered together in underground nests more than a meter below the surface. Many respiring individuals resting in deep, poorly ventilated nests deplete the oxygen supply and increase the concentration of carbon dioxide. Consistent with living in that atmosphere, naked mole-rats tolerate levels of low oxygen and high carbon dioxide that are deadly to most surface-dwelling mammals. Naked mole-rats appear to have evolved a number of remarkable adaptations to be able to thrive in this harsh atmosphere. In order to successfully survive low oxygen atmospheres, they conserve energy utilization by reducing the physiological activity of all organs, manifest by reduced heart rate and brain activity. Amazingly, they resort to the anaerobic metabolism of fructose rather than glucose as a fuel to generate energy when challenged by anoxia. Similarly, high carbon dioxide atmospheres normally cause tissue acidosis, while naked mole-rats have a genetic mutation preventing both acid-induced pain and pulmonary edema. Together, these putative adaptations and the tolerances they provide make the naked mole-rat an important model for studying a host of biomedical challenges. Naked mole-rats are small rodents endemic to the arid and semi-arid regions of the sub-Saharan northeastern horn of Africa. Here, they live in a complex maze of underground burrows reaching more than 3000 m in length [1] and reaching depths of more than 1.8 m [2]. They primarily forage and disperse through excavating tunnels, an energetically costly activity, exacerbated by food being sparse and patchily distributed [2]. Naked mole-rats meet all their energy and water requirements through the foods they consume and the metabolic water they generate [1]. In addition to having to contend with the daunting problems associated with foraging below ground, they may also encounter numerous physiological challenges imposed by underground atmospheric conditions. Both gas and heat exchange are dependent on the diffusion and porosity properties of the soil and may be impaired. As fossil evidence suggests they have occupied this subterranean niche since the early Miocene [3,4], it is not surprising that they have evolved a fascinating montage of biological traits well suited to the harsh conditions they encounter in their hot, humid, and poorly ventilated sealed maze of burrows (for a review, see [5]). These include having a lower body temperature (32 ℃) than most mammals and a reduced basal metabolic rate for their body size. Not surprisingly, when they are housed outside of the warm and humid confines of these equatorial burrows, and lacking an insulatory pelage, naked mole-rats are poor thermoregulators, unable to counter the high rates of heat loss with effective endothermic mechanisms and rather rely on communal huddling to keep warm [5,6]. Naked mole-rats live in large groups of up to 295 individuals [1]. Reproduction is confined to a single breeding female and 1–3 breeding males within the colony, and there is a high reproductive skew such that less than 1% of all individuals within the colony get the opportunity to breed over their lifetime, contributing to their description as one of only two eusocial mammals [7,8]. Living in large colonies, they have distinct, culturally transmitted vocal dialects [9], despite having a non-functional cochlear amplifier and associated poor auditory thresholds [10]. Many of their other adaptive traits to this environmental niche may also protect against numerous disease states, including cancer, neurodegeneration, and cardiovascular disease, contributing to prolonged healthy aging and extreme longevity [11]. Naked mole-rats are extremely long-lived for their body size, living approximately six times longer than predicted allometrically. Intriguingly, unlike every other mammalian species studied to date, in which after the age of sexual maturity there is an exponential increase in risk of dying, naked mole-rats do not. Rather, death is random, with similar likelihoods of dying at 2 and 20 years of age [11]. In other words, they do not show an increased risk of dying with advancing age, defying Gompertzian fundamental laws of mortality. This stochastic pattern of age-independent risk of dying is accompanied by a negligible senescence phenotype, maintaining physiological function well into their fourth decade [12], as well as retaining many youthful paedomorphic features, including high oxygen affinity fetal hemoglobin in adult life [13]. These and many other unusual qualities make the naked mole-rat an interesting model species with an abundance of recent genomic and epigenetic aging clock data available, facilitating the creation of explanatory models [14,15,16,17]. In this review, we will cover significant adaptations that the naked mole-rat has evolved, likely due to living in their unique subterranean niche [2]. Their communal lifestyle, while facilitating better foraging success, is not without problems. The animals rest and respire together in deep nests where diffusion through the soil is poor, creating an atmosphere that is both hypoxic and hypercapnic. Naked mole-rats are very tolerant of these environmental challenges [2,18,19], and here we report on some of the underlying mechanisms that have been identified to date [19,20,21,22,23]. Naked mole-rats demonstrate a profound tolerance to oxygen deprivation when compared to mice [19]. This includes resilience against exposure to low oxygen (hypoxia) atmospheres as well as no oxygen (anoxia). While all mice can survive a 30 s exposure to anoxia, none survive a 1 min exposure (Figure 1). In contrast, all naked mole-rats survived an 18 min exposure to anoxia, although no naked mole-rats survived a 30 min exposure. During anoxia, naked mole-rats drastically reduced physiological activities, presumably to conserve energy expenditure [19,24,25,26]. Within 30 s of exposure to anoxia, animals lose consciousness, and their respiration rate is drastically depressed (Figure 2A), suggestive of a lower metabolic rate [13,18]. The red line below the X-axis indicates the time course of anoxia exposure. After exposure to anoxia and a return to normoxic room air, the respiration rate increased toward baseline levels. During anoxic exposure, the heart rate of naked mole-rats decreased from about 200 beats per minute to about 50 beats per minute and then recovered to baseline levels of activity under normoxic recovery conditions (Figure 2B). This is consistent with data collected by Ilacqua et al. [24], showing that under acute hypoxia and anoxia, naked mole-rats reduce metabolic rates by decreasing locomotion and body temperature. For mice, the electrical activity in mouse hearts was not detectable in the electrocardiogram after 6 min of exposure to anoxia. Another physiological metric is brain activity as measured by electroencephalography (EEG) of the caudate putamen. It was found that brain activity in naked mole-rats was dramatically reduced during a 10 min exposure to anoxia [25] (Figure 2C). The EEG revealed that within 3–5 min, brain activity had declined and remained at these low levels for the duration of anoxic exposure. EEG recordings were acquired using a four-channel Pinnacle Technology EEG system, and electrodes were positioned stereotaxically. It is important to note that naked mole-rats in the wild are likely to encounter hypoxia, not full anoxia. Park et al. [19] showed that naked mole-rats tolerate 5% O2 for at least 5 h, whereas mice cannot survive for more than about 12 min. Additionally, Ilacqua et al. [24] tested naked mole-rats with 9%, 7%, 5%, and 3% O2 and showed an incremental decline in activity with increasing severity of hypoxia. Additional studies using electrophysiological assessments of hippocampal brain slices in an interface recording chamber reported intrinsic brain tolerance to anoxia in naked mole-rats [27,28], see Figure 3A. Evoked potential data for one slice from a mouse and one slice from a naked mole-rat are illustrated in Figure 3B, wherein the slices were stimulated alternately every 10 s. After reaching a stable baseline, the slices were exposed to anoxia until both slices lost function (shaded area). In these examples, the slice from a naked mole-rat took significantly longer to experience anoxic depolarization (AD) and loss of detectable functionality. After reoxygenation, naked mole-rat slices showed recovery of synaptic transmission, while mouse slices did not. Given that naked mole-rats and mice have disparate body temperatures (32 °C and 37 °C, respectively), studies were undertaken at waterbath temperatures approximating their divergent body temperatures (Figure 3C). While waterbath temperature greatly affects the time to loss of synaptic function and transmission, it should be noted that at both temperatures, the naked mole-rats showed a significantly longer time to loss of detectable functionality. Naked mole-rat brain cells show a much lower accumulation of intracellular calcium during hypoxia compared to cells from mice [29]. One detrimental effect of hypoxia is that it triggers an increase in intracellular calcium concentration, resulting in the activation of several toxic downstream signaling cascades [30,31]. Unlike the brain slices from mice that show a significant increase in calcium influx during hypoxic challenge, that observed in mole-rat brain slices is considerably attenuated (Figure 4A). One of the major avenues for neuronal calcium entry is the NMDA receptor (NMDAR). The NMDA receptor is a fundamental building block of neuronal processing, being a central mediator of Hebbian learning and integral to the induction of Long-term Potentiation (LTP) and Long-term Depression (LTD) of neurons. Once the NMDAR is opened, it is notable for its large calcium current that activates second messenger pathways within the neuron. Although calcium entry is necessary for many forms of synaptic plasticity and physiological behavior of the NMDAR, it is highly regulated in the cell, and errant entry during times of overactivation can quickly lead to excitotoxicity [32]. The canonical NMDAR is composed of several subunits, with each receptor being a tetramer that is composed of two requisite NR1 (NMDA receptor, subunit 1) subunits and two subunits of a combination of GluN2A, GluN2B, GluN2C, or GluN2D. Bickler et al. found that the GluN2D subunit reduces its mean open time during hypoxia, reducing hypoxia-induced calcium accumulation [33]. It was demonstrated that calcium accumulation and NMDAR currents were reduced under hypoxia in NMDARs composed of the GluN2D subunit. This alteration in the opening of these channels likely provides a mechanistic contribution to the greater tolerance of hypoxic conditions. NMDAR subunit composition is age- and region-dependent in most mammals, but globally the proportion of GluN2B and GluN2D decreases with maturity [34]. Adult mice retain only about 10% of the hypoxia-resistant GluN2D subunit compared to neonate mice (Figure 4B). On the other hand, adult naked mole-rats retain about 66% of these receptors with GluN2D subunits that close under hypoxia compared to neonate naked mole-rats [35]. It is believed that this neotenous feature of the naked mole-rat is an adaptation to the hypoxic environment described here, where evolution has favored the trade-off of kinetic characteristics idiosyncratic to the GluN2D subunit, such as lower conductance for a diminished entry of calcium, as more optimal at their point on the fitness landscape. Unique molecular adaptations to hypoxia have also been discovered in the naked mole-rat. These include findings by Xiao et al. that Hypoxia Inducible Factor 1 alpha (HIF-1a) and Vascular Endothelial Growth Factor A (VEGFA) are significantly upregulated in naked mole-rat tissues compared to ICR (Institute of Cancer Research) control mice (Figure 5A,B) [36]. Since the discovery in 1995 of HIF-1a by Semenza [37] and its functional role as a molecular sensor of oxygen tension, HIF-1a has played a central mechanistic role in hypoxia research. HIF is hydroxylated in an oxygen-dependent reaction on conserved prolines, allowing for its recognition by Von Hippel-Lindau (VHL), a substrate binder component of the E3 ubiquitin ligase, and thereby targeted for the rapid proteasomal destruction of HIF under normoxic conditions [38]. Under hypoxic conditions, this hydroxylation cannot occur, and HIF levels remain stable and translocate into the nucleus. This oxygen-dependent transcriptional activator regulates the expression of over 100 genes and thereby provides a central hub for deploying the appropriate hypoxic response in the various organ systems [39]. In this manner, HIF regulates biochemical responses to oxygen deprivation by modulating the expression of genes involved in glucose uptake, energy metabolism, as well as cell proliferation and cell death [40]. Xiao et al. have found that even under the normoxic conditions found in the laboratory, naked mole-rat expression of HIF-1a is significantly elevated in several tissues, with concomitant increases in VEGFA, which controls angiogenesis and oxygen delivery [36] (Figure 5B). These responses are undoubtedly of benefit to the naked mole-rat, which experiences bouts of hypoxia in their crowded nest environments and perhaps other burrow segments in the wild. Genetic analysis of the naked mole-rat genome by Kim et al. uncovered two intriguing mutations hardwired into the HIF-VHL system [16]. Within the HIF-1 sequence at location 407, the naked mole-rat has exchanged threonine for isoleucine (T407I). This point mutation was proposed to be significant as, within the 3D structure of HIF-1, it lies within the VHL-binding domain [16], altering substrate binding for ubiquitination. In addition, it was found that naked mole-rat VHL contains a mutation at residue 166 (V166I). This site is functionally important since other mutations found here are correlated with a pathological outcome, Von Hippel-Lindau disease, due to a weakened interaction in the HIF-VHL complex resulting in reduced HIF1a degradation, a longer HIF-1 half-life, and concomitantly higher constitutive levels [41,42]. These mutations may contribute significantly to the higher constitutive levels observed by Xiao et al. [36] and are consistent with an evolutionary adaptation to tolerate hypoxic conditions as described in this review. Naked mole-rats have another unusual systemic adaptation for dealing with hypoxia and anoxia that involves undergoing drastic metabolic rewiring, which enables the anaerobic glycolysis of fructose [19,43]. In this study, it was demonstrated that naked mole-rats express GLUT5 (solute carrier family 2 member 5), the fructose transporter, at significantly higher levels (>10-fold) in their brain and heart cells compared to mice (Figure 6). Naked mole-rats also showed a substantial and significant increase in fructose in the blood during anoxia, whereas mice did not. Levels of ketohexokinase (KHK), which phosphorylates fructose into fructose-1-phosphate (F1P), were significantly elevated in the naked mole-rat. Additionally, in brain slices exposed to hypoxia, a metabolic flux analysis demonstrated that fructose-derived carbons accumulated at a significantly higher level within many glycolytic intermediates in slices from naked mole-rats compared to slices from mice, indicating the metabolism and incorporation of fructose into naked mole-rat metabolism. Finally, in a demonstration of the physiological utility of this metabolic rewiring, brain slices and isolated hearts from naked mole-rats and mice were tested by switching glucose to fructose in the bath, and their functionality was measured. In both the brain and heart preparations, naked mole-rats retained significantly better performance with fructose compared to mice. A model was proposed in which glucose metabolism is blocked under near-anoxia by inhibition at the rate-limiting step of phosphofructokinase (PFK). This feedback inhibition is induced by protons, ATP binding, or downstream intermediates such as citrate [19]. The authors put forward that the naked mole-rat has evolved to efficiently interface with and make use of fructose under near-anoxic conditions to bypass the metabolic block at PFK that glucose suffers from and provide for a continued, albeit less efficient, energetic source under these trying periods. Another detrimental effect of hypoxia in most mammals is a rapid buildup of fluid in the lungs known as pulmonary edema. This is caused by altered sympathetic tone, vasoconstriction, and pulmonary hypertension, as well as altered alveolar endothelial and epithelial permeability to water and macromolecules, causing an increased permeability across the alveolar barrier and the capillaries to leak into the alveolar sacs and interstitial spaces that overwhelm the alveolar reabsorption capacity [44]. This is manifested by a change in the wet/dry weight of excised lung tissue. Naked mole-rats appear to be completely immune to this effect. Park et al. [19] exposed naked mole-rats and mice to normoxia (20% O2) or hypoxia (10%, 7.5%, or 5% O2) for 15 min. Mice showed severe pulmonary edema for hypoxic atmospheres of 7.5% and 5% O2, while naked mole-rats showed no edema at either of these low O2 concentrations. (Figure 7). Currently, the mechanisms facilitating the lack of hypoxia-induced edema in naked mole-rats are unknown. Room air contains about 0.03% CO2. Breathing higher concentrations of CO2 causes acidification of the tissues, with a variety of associated behavioral and physiological responses. For example, humans and laboratory mice find elevated CO2 concentrations to be painful to the upper respiratory tract [45,46]. On the other hand, naked mole-rats are less averse to elevated CO2 concentrations compared to mice. In an avoidance test, naked mole-rats and mice were placed in a rectangular arena where CO2 was infused at one end and room air at the other [19]. In that study, mice avoided all three of the CO2 concentrations tested: 2.5%, 5%, and 10% (Figure 8A), whereas naked mole-rats only avoided the highest concentration of 10% CO2 (Figure 8B), implying that the naked mole-rats did not find 2.5% or 5% CO2 to be aversive. Elevated CO2 concentrations also trigger an increase in respiration rate [46,47]. This can be observed in mice for 5% and 10% CO2 (Figure 8C). Naked mole-rats only showed an increase in respiration rate of 10% and greater concentrations of CO2 (Figure 8D). Johansen et al. [48] showed that naked mole-rat blood can buffer acid better than mouse blood. Consistent with this observation, Park et al. [19] showed that naked mole-rats do not show systemic acidification from breathing CO2 concentrations below 10%, whereas mice show acidification from breathing even 1% CO2 (Figure 8E). Zions et al. demonstrated that captive naked mole-rat habitats have a high degree of anisotropy in carbon dioxide levels that are tightly coupled to the location of the nest chamber [49]. The previously described eusocial nature of this species lends itself to individuals spending a higher fractional time respiring in the nest chamber than the surrounding area. This is reflected in a higher carbon dioxide load in the nest chambers and the surrounding areas. Figure 9A shows a drawing of one of the colony caging systems used in their study. The nest chamber, indicated by the hashtag symbol, was the area showing consistently the highest concentration of CO2 in the system and is the chamber in which the naked mole-rats prefer to crowd together, despite the high concentration of CO2 as shown in Figure 9B. Hypercapnia has been demonstrated to suppress neuronal activity [50]. Zions et al. report that the neuronal potassium-chloride cotransporter 2 (KCC2) has a loss-of-function point mutation at position 952 where a highly conserved arginine has been mutated to histidine in the naked mole-rat (R952H) [49]. This mutation results in less chloride being extruded from the neuron, altered chloride homeostasis, and ultimately a reduction in inhibitory currents through GABAergic ion channels (gamma-aminobutyric acid receptors). Zions et al., therefore, propose an elegant hypothesis in which hypercapnia’s ability to suppress neural activity acts to shift the excitation-inhibition balance in naked mole-rats’ neuronal circuits and allows them to conserve energy by reducing the expenditure necessary to drive KCC2 [49]. This hypothesis is supported by their finding of a similar mutation (R952C) in the eusocial relative of the naked mole-rat, the Damaraland mole-rat. In another study, LaVinka and Park [51] showed that naked mole-rats do not avoid fumes from 10% ammonia (household cleaning ammonia) or 20% acetic acid (Figure 10). In contrast, laboratory rats, mice, and Damaraland mole-rats significantly avoided these irritants. Breathing high concentrations of CO2 also induces rapid and life-threatening pulmonary edema. CO2 induces changes in the alveolar permeability and reabsorption properties of the lung, and pulmonary edema happens when CO2 acidifies the tissues of the lungs, activating acid-sensitive sensory nerves in the tissues that trigger neurogenic inflammation and edema. Park et al. measured pulmonary edema from CO2 in naked mole-rats and mice [19]. Naked mole-rats showed no edema even at the highest concentration of CO2 tested (50%) (Figure 11). In contrast, mice showed significant edema at 15% and robust edema at 20–50% CO2 concentrations. A mutation in the voltage-gated sodium channel NaV1.7 likely contributes to the lack of CO2-induced pulmonary edema in naked mole-rats [23]. Unlike the positive charge of a highly conserved loop motif (the KKV motif in humans) in domain IV observed in most species, this motif in naked mole-rats has a net negative charge (Figure 12). This mutation of residues 1718 and 1720 imparts an altered charge landscape in this region through the placement of two mutated glutamate (E) residues flanking a conserved lysine (the EKE motif). As a result, the sensory nerves that normally respond to acid become significantly less sensitive, blocking the presence of acid-induced neural signaling [23]. The authors suggest that there is a greater level of proton inhibition in naked mole-rat NaV1.7 that alters the excitation dynamics of these neurons under acidic conditions. This in turn serves to balance depolarization from TRPV1 (transient receptor potential cation channel subfamily V member 1) and ASIC (acid-sensing ion channel) receptors and inhibit the propagation of this signal via NaV1.7. Ultimately, this mutation likely reduces the level of signaling, for under acidic conditions, ASIC receptors will be continually activated by protons. This mutation is likely an evolutionary adaptation to living in an underground hypercapnic niche. Consistent with a gene mutation in the nerves that sense acid, naked mole-rats are completely insensitive to acid pain in their skin [22]. Figure 13A shows the behavioral response of mice and naked mole-rats that received an injection of acidic saline in the skin of one hind paw. Mice responded with robust licking of the injection site, whereas naked mole-rats had virtually no response. Consistent with acid insensitivity being associated with a gene mutation, naked mole-rat pain-sensing neurons also showed no response to an acidic bath solution (Figure 13B). Naked mole-rats were also behaviorally insensitive to the injection of capsaicin, the spicy substance found in “hot” chili peppers (Figure 13C). However, unexpectedly, pain-sensing neurons from naked mole-rats demonstrated robust responses to capsaicin in the bath solution (Figure 13D). Because the mutation in NaV1.7 is specific for sensing acid pain, there must be a different mechanism associated with insensitivity to capsaicin. Park et al. [53] showed that naked mole-rats lack the neuropeptide transmitters Substance P and Calcitonin Gene-Related Peptide from their pain-sensing peripheral nerves. In a subsequent study, Park et al. [22] showed that introducing exogenous Substance P alone was capable of rescuing pain behaviors from capsaicin (Figure 14). The researchers used two techniques to introduce Substance P: the application of a transgenic herpes virus expressing the preprotachykinin (PPT1/TAC1) gene for Substance P (Figure 14A, top) and the intrathecal injection of Substance P directly into the spinal cord (Figure 14A, bottom). Figure 14B shows pain behavior for the virus-treated foot and the untreated foot (labeled ‘No Virus’ in Figure 14). In this test, the naked mole-rats were lightly anesthetized, and the feet were heated with a calibrated heat source. The first three data points show foot withdrawal latencies from the heat source of about 12 s for both feet. The remaining data points show withdrawal latencies after topical application of capsaicin to both feet. The untreated foot shows withdrawal latencies that are similar to the latencies prior to capsaicin application of about 12 s. However, the virus-treated foot shows robust sensitization to capsaicin in the form of significantly faster withdrawal latencies of about 6 s. Figure 14C shows the results of introducing Substance P via intrathecal injection. The two bars on the left show the results of testing with capsaicin. In this test, one hind paw was injected with a capsaicin solution prior to Substance P application (labeled Pre in Figure 14). As noted previously, there was virtually no response. However, following the introduction of Substance P, the naked mole-rats spent a significant amount of time licking the injection site, supporting the theory that exogenous Substance P could rescue capsaicin pain. The two bars on the right show the results for acid pain. In this case, the application of Substance P had no effect on acid pain insensitivity, supporting the hypothesis that acid insensitivity was mediated by another mechanism, namely the mutation in NaV1.7. It is noteworthy that naked mole-rats respond to acute mechanical pain (pinch) and acute thermal pain (heat) in the same way as mice (Figure 15). Both naked mole-rats and mice react to a pinch with a tail clip in about 6 s (Figure 15, left), and both naked mole-rats and mice react to heat pain in about 12 s (Figure 15, right). The naked mole-rat was the first mammal identified to lack acid and capsaicin pain. However, a subsequent study found that some other African subterranean rodents also showed pain insensitivities [20]. Figure 16 shows the results of behavioral tests on eight African mole-rat species, the East African root rat, and the laboratory mouse. Animals were tested with diluted solutions of capsaicin (the chili pepper drawing in Figure 16), acid (the lemon drawing), and allyl isothiocyanate (AITC), the active ingredient in mustard oil and wasabi root (the wasabi root drawing). Pain insensitivity is indicated by an “X” over the drawings. In addition to the naked mole-rat, there was one other species, the Natal mole-rat, that was insensitive to capsaicin, and two other species that were insensitive to acid: the Cape mole-rat and the East African root rat. An interesting finding was that the Highveld mole-rat, while sensitive to acid burn, was insensitive to AITC, which activates TRPA1 (transient receptor potential cation channel, subfamily A, member 1) receptors [54]. The Highveld mole-rat shares its burrows with the Natal droptail ant, whose normally painful sting is known to activate TRPA1 receptors, likely suggesting a co-evolutionary history. Eigenbrod, et al. [20] showed that, in addition to being insensitive to AITC, the Highveld mole-rat was insensitive to the sting of the Natal droptail ant. Other species tested in the same study showed pain behaviors from the sting. Additional experiments showed that the Highveld mole-rat overexpresses the leak channel NALCN (sodium leak channel, nonselective), which could act to dampen excitation in pain nerves after TRPA1 activation, thus rendering Highveld mole-rats insensitive to the ant’s sting [20]. In addition to pain insensitivities, the somatosensory system of the naked mole-rat has another interesting feature. Although naked mole-rats lack fur, their bodies have 10 rows of sensitive vibrissae that form a sensory array [55]. One row of vibrissae can be seen in the photograph in Figure 17. Crish et al. [55] found that deflecting a single vibrissa triggered an accurate and repeatable orientation of the snout to the position of the deflected vibrissa. Interestingly, simultaneously deflecting two vibrissae on the same side of the body triggered an orientation response to a position that is the average of the two stimulated vibrissae. For example, stimulating a vibrissa at the shoulder and hip triggered an orientation of the snout to a position halfway between the shoulder and the hip (Figure 17A). This result indicates that the naked mole-rat nervous system is capable of making a computation about touch location. However, this is not the case for simultaneously deflecting two vibrissae on opposite sides of the body, which triggers an orientation to either one or the other deflected vibrissa in a winner-take-all strategy (Figure 17B). Naked mole-rats have evolved under extraordinary environmental challenges, resulting in the evolution of a suite of exceptional adaptations to deal with those challenges. The current review article highlights some of those adaptations related to the naked mole-rat’s tolerance to hypoxia, hypercapnia, and pain, including some of the underlying mechanisms. Although, there is still much to be learned about naked mole-rat adaptations to the atmospheric conditions under which they have evolved, elucidating the protective mechanisms they employ may highlight new directions for the treatment of human pathological conditions such as inflammation and concomitant pain, as well as ischemia/reperfusion injury as occurs in cardiac infarctions and stroke.

PMC10000162

Feng Cheng,Hui Wang,Lei Zhou,Ganqiu Lan,Hanchun Yang,Lixian Wang,Ligang Wang,Jing Liang

Systematic Identification and Comparison of the Expressed Profiles of Exosomal MiRNAs in Pigs Infected with NADC30-like PRRSV Strain

28-02-2023

PRRSV,serum exosome,miRNAs

Simple Summary Exosomes play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry. Here, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs, isolate serum exosomes, and identify 33 significantly differentially expressed (DE) exosomal miRNAs between infection and control groups, and 18 DE miRNAs associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes. Abstract Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3′ UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3′ UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes.

Systematic Identification and Comparison of the Expressed Profiles of Exosomal MiRNAs in Pigs Infected with NADC30-like PRRSV Strain Exosomes play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry. Here, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs, isolate serum exosomes, and identify 33 significantly differentially expressed (DE) exosomal miRNAs between infection and control groups, and 18 DE miRNAs associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes. Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3′ UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3′ UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes. Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-strand RNA virus with an envelope structure belonging to the order Nidovirales, family Arteriviridae, genus Betaarterivirus [1,2]. It is spherical or ellipsoidal with a diameter of 50–65 nm under a freezing electron microscope [3,4]. The PRRSV genome is about 15 kb in length with a 5′ cap and a 3′ polyA-tail and contains at least 10 open reading frames (ORFs) flanked by untranslated regions (UTRs) at both the 5′ and 3′ termini [5,6], and is wrapped by nucleocapsid protein, with lipid double-layer coating to form virus particles. Exosomes belong to vesicles with monolayer membrane structures and have the same topological structure as cells [7]. The shape is “cup-shaped” or “disc-shaped” under an electron microscope [8,9]. Exosomes can exist in the circulatory system for a long time, and substances in exosomes can be absorbed by adjacent cells or distant receptor cells and then regulate the receptor cells to participate in the exchange of genetic materials between cells [10,11]. They are mainly composed of membrane surface substances and carried contents, including cell surface receptors, membrane proteins, soluble proteins, lipids, RNA (mRNA, miRNA, lncRNA, and viral RNA, etc.), genomic DNA, mitochondrial DNA [12,13,14]. MicroRNAs (miRNAs) are a class of 18–25 nucleotides (nt) evolutionarily conserved endogenous non-coding single-stranded small RNAs, which inhibit the translation process by inducing the degradation of target mRNA or by binding with 3′ UTR of target mRNA, leading to post-transcriptional gene silencing, then regulating the gene expression at the post-transcriptional level [15,16,17]. It is estimated that miRNAs regulate more than 60% of mammalian genes post-transcriptionally [18,19]. MiRNAs play an important role in intercellular communication and can also be used as a potential functional molecule for disease and virus infection, transmission, and defense [20]. A growing number of studies have shown that miRNAs can be present in body fluids, such as saliva, urine, breast milk, and blood, and act through the body’s fluid circulatory system [21,22]. Exosomal miRNAs are considered to be endogenous regulators of gene expression and metabolism and can indicate various pathological conditions [23,24]. Over the past two decades, it has been shown that miRNAs have crucial roles in the regulation of immune cell development, innate immune responses, and acquired immune responses. Some other miRNAs are reported to impair PRRSV infection through the following ways, directly target the PRRSV genome or PRRSV receptor, or play a role by regulating the host’s innate immune response. The miR-26 family can significantly damage virus replication, and miR-26a can inhibit the replication of type 1 and type 2 PRRSV strains in porcine alveolar macrophages (PAMs) by regulating the type I interferon (IFN) pathway, which is more efficient than miR-26b [25,26]. miR-30c and miR-125b are identified to modulate host innate immune response by targeting the type I IFN pathway and NF-κB pathway, respectively [27,28,29]. MiR-23, miR-378, and miR-505 are antiviral host factors targeting PRRSV and have conservative target sites in type 2 PRRSV strains [30]. At the same time, host miR-506 has been identified to inhibit PRRSV replication by directly targeting PRRSV receptor CD151 in MARC-145 cells [31]. miR-181 also can indirectly inhibit PRRSV replication by down-regulating PRRSV receptor CD163 in blood monocytes and PAMs [32]. In addition, miRNAs can promote PRRSV replication by interfering with basic cell physiology. MiR-24-3p and miR-22 directly target 3′UTR of HO-1 during PRRSV infection to escape the inhibition of heme oxygenase-1 (HO-1), a heat shock protein (also known as HSP32) on PRRSV [33,34]. Pigs are known to be more susceptible to PRRSV and less able to defend themselves against the entry of this pathogen into the organism [35]. In the present study, the innate immunity and acquired immunity of pigs infected with this virus were studied at the molecular level using a strain prevalent in the field. A serum exosome isolation kit, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB) were used to isolate and identify serum exosomes before and after infection with PRRSV, followed by small RNA sequencing analysis, identification, and analysis of differential expression results using bioinformatics methods to obtain a number of PRRSV-associated serum exosome miRNAs, followed by identification of data results using quantitative real-time PCR (qRT-PCR). Six PRRSV antigen and antibody double-negative healthy 42-day-old large white pigs were placed in the pig clean feeding system for isolation, healthcare, and environmental adaptation. All pigs were free to eat and drink without restrictions. When they were familiar with the conditions in the isolator, the pigs were nasally inoculated with 2 mL 105 TCID50/mL PRRSV NADC30-like CHsx1401, which was mentioned by predecessors [36,37]. The blood of the pigs before (control group, n = 6) and 7 days after (treatment group, n = 6) virus inoculation was collected from the anterior vena cava for serum isolation. The cellular debris in the serum was removed by centrifugation at 3000 g for 15 min. All animal experiments in our study were approved by the Animal Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS) (Beijing, China), IAS2022-130. Exosome isolation and purification were carried out using the exoEasy Maxi kit (QIAGEN, Hilden, Germany, cat. no. 76064) according to the manufacturer’s protocol. Extracted exosome suspensions were spotted onto the formvar carbo-coated copper mesh, and the exosomes were rinsed with PBS and subjected to standard uranyl acetate staining for 3 min at room temperature. After drying for several minutes at room temperature, the grid was visualized and photographed at 100 kV by transmission electron microscope (HT-7700, Hitachi-High Tech, Tokyo, Japan). Extracted exosomes were diluted with 1 × PBS by changing the volume from 10 to 30 μL. After the sample was tested, the concentration and size of serum exosomes were analyzed by an N30E flow nano-analyzer following the manufacturer’s instructions (NanoFCM, Xiamen, China). The extracted exosome samples were added to RIPA lysate mixed with protease inhibitor (Invitrogen, Waltham, MA, USA) and phenylmethylsulfonyl fluoride (PMSF) to extract the exosome protein, which was lysed on ice for 30 min. Then, according to the instructions of the Bradford kit, we quantified the concentration of serum exosome protein. Exosome proteins underwent thermal denaturation. The same amount of protein was separated on 12% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). It was soaked in TBST containing 5% skimmed milk powder and sealed for 1 h at room temperature. We soaked the membrane in the diluted primary antibody (anti-CD9 antibody, Abcam, Boston, MA, USA, #ab92726; anti-CD81 antibody, Abcam, Boston, MA, USA, #ab109201) overnight at 4 °C, and recovered the primary antibody. We soaked the membrane in the diluted secondary antibody, incubated it at room temperature for 1 h, and recovered the secondary antibody. We laid the washed film of PBST on the fresh-keeping film, added equal volume mixed ECL a/b chromogenic solution, and placed it in the chemiluminescence imager. Total RNA from the exosomes was extracted with Trizol according to the manufacturer’s instructions. We then detected the RNA concentration and optical density (OD) value and detected the degradation and purity of RNA with 1% agarose gel electrophoresis. Meanwhile, Agilent Bioanalyzer 2100 was used to detect the integrity of RNA. We used the total RNA of exosomes after quality inspection. According to the manufacturer’s instructions, we used NEB NEXT multiplex small RNA library prep set for Illumina® (Illumina, San Diego, CA, USA). The kit prepared a small RNA cDNA library and sequenced it to produce 50 nt single-end reads by the Illumina Novaseq 6000 platform. All the procedures for small RNA library preparation were accomplished by Novogene (Beijing, China). The data after quality control were aligned to the porcine reference genome (Sus scrofa 11.1) using bowtie. Known miRNAs were identified by the miRbase (v22.0) database [38] (https://www.mirbase.org, accessed on 14 January 2022), miRdeep2 (v0.0.5) [39], and miRevo (v1.1) [40] and were used to predict new miRNAs. At the same time, the differential expression analysis for miRNAs was performed by DESeq (v1.24.0) [41], requiring |fold change| > 1.6 and p < 0.05. Alignment was performed using MEGA (V11) [42] followed by single base scoring using PHAST (v1.6.9) [43] and evaluation of the most conserved regions of 10 virus genes, including WUH3 (GenBank accession no. HM853973), VR2332 (GenBank accession no. U87392), JXA1 (GenBank accession no. EF112445), CH-1a (GenBank accession no. AY032626), NADC30 (GenBank accession no. HN654459), HUN4 (GenBank accession no. EF635006), HLJZD22-1812 (GenBank accession no. MN648450), SC/DJY (GenBank accession no. MT075480), and Lelystad (GenBank accession no. M96262.2). RNAhybrid (V2.0) [44] was used to predict the binding of the identified miRNA sequence to the 3′ UTR of the CHsx1401 virus genome. MiRanda (v3.3a) and RNAhybrid were used to target gene prediction. The clusterProfiler [45] R package was used for GO (Gene Ontology) functional enrichment analysis of target genes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. Total RNA was isolated from serum exosomes using Trizol (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. The isolated RNA was verified by RT-qPCR on samples (n = 6 per group). cDNA was synthesized according to the instructions of miRNA 1st strand cDNA synthesis (by stem-loop) kit (Vazyme, Nanjing, China), and the fluorescence quantification was performed using ABI 7500 according to the instructions of miRNA universal SYBR qPCR master mix (Vazyme, Nanjing, China). The thermal cycle parameters used were as follows: the first stage: 95 °C for 30 s; Stage 2: 95 °C for 5 s, 60 °C for 34 s, and 40 cycles; Stage 3: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Primer sequences of miRNAs, the U6 gene, were used as a reference [46] and listed in Supplementary Table S1. All qRT-PCR verifications were performed using three biological replicates and with three replicates for each sample. The relative abundance of transcripts was calculated by the 2−ΔΔCt method, and SPSS (v22.0) and GraphPad Prism (v8.0) were used for data analysis and mapping, respectively. p < 0.05 means the difference is statistically significant. The results of PRRSV antigen and antibody tests before (day 0) and after the (day 7) challenge are shown in Table 1. The serological detection of the PRRSV antigen and antibody before the challenge was negative, and the antigen was positive after the challenge, indicating that the pigs were successfully infected with CHsx1401. The vesicles isolated from serum were discovered by TEM. Most vesicles can clearly see the concave saucer- or disc-shaped exosomes in the middle. The membrane edge of exosomes is clearly visible, and the morphology is relatively complete (Figure 1A,B). The nanoparticle tracking analysis showed that 95.73% of the exosomes had a diameter of 30–150 nm, mainly around 72.25 nm, with an average diameter of 76.22 nm, which was consistent with the size characteristics of exosomes (Figure 1C). This size range was similar to that detected by TEM and further confirmed the identity of these vesicles as exosomes. Western blot analysis showed that the vesicles isolated from the serum samples were positive for CD9 and CD81 proteins (Figure 1D). The above characteristics conform to the exosome identification standards formulated by the international society for extracellular vesicles (ISEV) in MISEV2018 [47]. For each sample, the clean data reached 0.5 Gb, and the Q30 base percentage was above 96.20%. The clean reads of each sample were aligned with the pig reference genome. Among the 12 samples, the control group obtained 10,920,887, 10,248,696, 10,109,117, 10,655,494, 9,217,285, and 9,782,523 reads, respectively. The treatment group obtained 11,889,518, 10,593,504, 10,593,504, 12,846,080, 10,105,325, 11,729,451, and 9,789,542 reads, respectively. On average, 77.96% of the total clean reads comprised 19–22 nucleotides (nt) in length (Figure 2A). The reads after quality control accounted for more than 92.59% of the total reads. The processed clean reads were aligned to the porcine reference genome, and the mapped rate of 12 libraries on the genome was more than 92.30%, and the mapped rate was 94.98% (Figure 2B). It indicated that the constructed serum exosomal miRNA library was of high quality and suitable for further analysis. Details are listed in Supplementary Table S2. After quantitative analysis of the identified miRNA expression, miRNAs were screened by the thresholds described previously in Section 2.6. A total of 305 miRNAs were obtained before and after inoculation of the CHsx1401 strain (control, n = 6; treatment, n = 6). A total of 33 differentially expressed (DE) miRNAs were identified between the two groups, 13 DE miRNAs were upregulated, and 20 DE miRNAs were downregulated in the treatment group (Figure 3 and Supplementary Table S3). A total of 7283 target genes were predicted by 33 DE miRNAs, and the functions of target genes were mainly concentrated in the positive regulation of MAPK cascade, lipid metabolism process, regulation of intracellular signal transduction, ERK1 and ERK2 cascade, etc. (Figure 4A). In terms of molecular functions, the differentially expressed miRNAs target genes mainly focus on GTP-enzyme regulatory activity, kinase activity, nucleoside triphosphatase regulatory activity, and other functions related to signal transduction and energy metabolism (Figure 4B). In addition, among the cell components, the target genes mainly participate in the biological functions of supramolecular polymers, Golgi, autophagosomes, cell surface, early endosomes, etc. (Figure 4C). The functions of these components are closely related to the formation of exosomes, which also explains the accuracy of the sequencing. KEGG pathway enrichment analysis showed that the target genes were significantly enriched in endocytosis, the MAPK signaling pathway, the Rap1 signaling pathway, the sphingolipid signaling pathway, and the PI3K Akt signaling pathway (p < 0.05) (Figure 5A). At the same time, the enriched pathways were classified and analyzed. The results showed that the KEGG pathway of the target gene was mainly enriched in environmental information processing, human diseases, and biological systems (Figure 5B). According to the phastCons score of a single base after alignment by PHAST, a total of eight most conserved segments (black bands above the peak map) were obtained among the viral genomes (Figure 6). A total of 31 DE miRNAs were found to bind to the conserved segment by predicting the miRNAs bound to the conserved segment. Among them, in the conserved region (14,644–15,020 nt) closest to the 3′ UTR (14,870–15,020) of CHsx1401 genome, 16 DE miRNAs are predicted to bind to it, including 5 miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529) that can bind to the 3′ UTR of CHsx1401. Among these miRNAs, only ssc-miR-223 was upregulated after infection, and other miRNAs were downregulated after infection. See Supplementary Table S4 for details. A variety of differentially expressed miRNAs related to the function of exosomes and PRRSV were found by functional enrichment analysis of target genes. Among them, 11 DE miRNAs such as ssc-miR-4331-3p, ssc-miR-744, and ssc-miR-320 are involved in exosome uptake, and their target genes are mainly concentrated in the Ras gene family, annexin family, and ADP ribosylation gene family. Eighteen DE miRNAs, including ssc-miR-10b, ssc-miR-124a, and ssc-miR-128, participate in immune-related pathways, and their target genes are mainly concentrated in the MAPK gene family, PIK3 gene family, and protein phosphatase gene family. While 11 DE miRNAs are involved in virus invasion, the related target genes are mainly concentrated in the MAPK gene family and protein phosphatase gene family. Furthermore, multiple differentially expressed miRNAs, such as novel_102. Six DE miRNAs, including ssc-miR-320, ssc-miR-423-5p, ssc-miR-4331-3p, ssc-miR-7137-3p, and ssc-miR-744, are co-expressed in exosome function, PRRSV virus invasion, and immune-related pathways, as shown in Figure 7. Details are shown in Supplementary Table S5. Five DE miRNAs were randomly selected for verification. According to the qRT-PCR results, the expression of ssc-miR-19a and ssc-miR-32 increased in the treatment group, while ssc-miR-124a, ssc-miR-375, and ssc-miR-34c showed higher expression in the control group, consistent with the sequencing data (Figure 8). PRRSV is still a stubborn pathogen in the global pig industry, causing huge economic losses in the world. At present, vaccination is mainly used to prevent and control PRRSV, among which the modified live (MLV) virus vaccine is the most widely used [48]. Although this vaccine was effective in reducing PRRS outbreaks and incidence, it also greatly increased genetic variation and diversity of the virus and led to viral recombination between wild and live vaccine viruses in the field [49,50]. In recent years, the spread and prevalence of the recombinant virus NADC30-like PRRSV strain have caused multiple outbreaks of porcine reproductive and respiratory syndrome in China. The similarity between CHsx1401 and NADC30 used in this study remained at 92.2–99.1%. Since then, it has become an epidemic strain in China. Exosomes, as mediators of cell communication, are widely found in various body fluids and have unique advantages in disease diagnosis and treatment [51,52]. According to previous reports, exosomes play an important communication role in antigen presentation [53], immune response [53,54], virus replication [54], cancer [55], neurodegenerative diseases [56], angiogenesis [57], tumor cell migration [58] and invasion [59], and have high research value. In this study, high-throughput sequencing technology was used to construct the miRNA expression profile of serum exosomes, and 33 DE miRNAs were identified. As we all know, the host-encoded miRNA can bind with the viral genome and then regulate the replication, synthesis, and release of the virus to limit infection and affect the pathological process [15]. Studies of miRNAs targeting the viral genome have also been repeatedly reported in animals. gga-miR-454 and gga-miR-130b in chicken infectious bursal disease can target the viral genome to inhibit viral replication, while gga-miR-21 directly targets the viral protein VP1 to inhibit viral protein translation [60,61]. In PRRSV studies, ssc-miR-181 specifically binds to a highly conserved region downstream of the viral genome ORF4 and strongly inhibits PRRSV replication [62]. In this study, the expression difference of ssc-miR-181 between the two groups did not reach a significant level. In our study, the genomes of nine different PRRSV viruses were compared with those of the CHsx1401 strain, and the eight most conserved segments were identified. It was predicted that 31 DE miRNAs could bind to the 8 most conserved segments of CHsx1401, and 16 DE miRNAs could bind to the conserved sequences close to the 3′ UTR of CHsx1401. Among them, 5 DE miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529) can simultaneously bind to the CHsx1401 3′ UTR. In addition, the upregulated expression of ssc-miR-223 was predicted to bind to the 3′UTR target of the PRRSV genome. The results showed that the conserved sequences of the virus genome might play a key role in its pathogenicity, and the miRNAs that can bind to the conserved sequences between the genomes of different PRRSV strains may have important significance in controlling the pathogenicity of the virus. Some differentially expressed miRNAs have been proven to be related to PRRSV by previous studies and even directly involved in the regulation of PRRSV, including ssc-miR-10b [63], ssc-miR-378 [30], ssc-miR-124a [64], let-7f-5p [65], ssc-miR-744 [66], and ssc-miR-19a [67]. PRRSV can evade host defense by interfering with innate immune response. This process is regulated by many signaling pathways, including the MAPK signaling pathway, PI3K Akt signaling pathway, autophagy, chemokine, and TNF signaling pathway. At present, the MAPK signaling pathway includes three main pathways: ERK1/2, JNK, and p38 pathway. Activation of the MAPK cascade can promote host cell apoptosis, assist the virus in escaping the host immune defense response and promote PRRSV replication [68]. Moreover, the activation of c-Jun N-terminal kinases (JNKs) and p38 can also promote the release of the inflammatory factor IL-10 [68,69,70] and enhance the inflammatory effect. In addition to inducing apoptosis, PRRSV can also induce autophagy, which can promote PRRSV replication. The activation of PI3K/Akt is necessary for virus entry and promotion of virus replication, and PRRSV-activated Akt inhibits host cell apoptosis by negatively regulating the JNK pathway [71]. TNFα It can play an important role in the induction and regulation of inflammatory response together with other inflammatory factors, but TNF α Expression is affected by the negative regulation of PRRSV replication [72]. In the present study, miRNAs (ssc-miR-10b, ssc-miR-122-5p, ssc-miR-124a, ssc-miR-128, ssc-miR-129a-5p, etc.) enriched in these pathways are involved in PRRSV-induced apoptosis, autophagy, and inflammation and are closely associated with viral immune response, immune evasion, and replication. The cell plasma membrane is rich in a variety of lipid rafts, and sphingolipid- and cholesterol-rich in sphingolipids (sphingomyelin and glycosphingolipids) are key molecules of lipid rafts. The recognition of lipids by some proteins of the virus may be a necessary condition for the entry of the virus [73]. Envelope viruses insert viral envelope glycoproteins into lipid rafts at the stage of virus entry, interact with receptors located in lipid rafts, or change from their natural state to activated form to initiate or promote viral internalization/fusion, such as HSV, SARS coronavirus, and piglet epidemic diarrhea virus [73,74]. Previous studies found that the removal of cholesterol from the surface of MARC-145 cells significantly reduced PRRSV infection, demonstrating that inhibition of PRRSV infection was specifically mediated by the removal of cellular cholesterol. Depletion of cell membrane cholesterol significantly inhibited virus entry, particularly virus attachment, and release [75]. Obviously, sphingolipid metabolism can regulate membrane structure and adhesion, which is of great significance in PRRSV virus invasion. Endocytosis was the most significant enrichment in this study. Endocytosis is an important mechanism of exosome uptake by target cells. Previous studies have shown that exosome uptake is an energy-demanding and cytoskeleton-dependent process, which highlights the potential role of endocytosis in this process [76]. It has been proved that there are several pathways that can mediate this process, including phagocytosis, macropinocytosis, clathrin, etc. [77,78], which led to different classifications and roles of endocytosed substances. The enrichment of differentially expressed exosomal miRNAs in this pathway indicates that exosomes play an important role in PRRSV infection, and the regulation of content transport and uptake in exosomes may lead to pathophysiological changes in target cells and organs. Through the identification and bioinformatics analysis of serum exosomal miRNAs from PRRSV-infected pigs, a variety of PRRSV-related pathways and differentially expressed miRNAs were obtained in this study, such as ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc., which play potential functional roles in PRRSV-induced immune response, invasion, and exosome uptake. In addition, because a single miRNA can target multiple genes and a single gene is also regulated by multiple miRNAs, there are a number of miRNAs that perform multiple functions in the above pathways. Some miRNAs have been verified to regulate PRRSV infection by acting on key receptors or directly targeting the virus genome, such as ssc-miR-10b, ssc-miR-378, miR-124a, let-7f-5p, ssc-miR-744, ssc-miR-19a, etc. Meanwhile, the present study also predicted a variety of miRNAs that can bind to the most conserved fragment of the 3′ UTR of the CHX1401 virus genome, including ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529, which may be important for regulating viral pathogenicity.

PMC10000164

Giovanni De Benedetto,Fabiano Capparucci,Sabrina Natale,Serena Savoca,Kristian Riolo,Claudio Gervasi,Marco Albano,Alessia Giannetto,Gabriella Gaglio,Carmelo Iaria

Morphological and Molecular Identification of Mullet Helminth Parasite Fauna from Ganzirri Lagoon (Sicily, Southern Italy)

25-02-2023

Acanthocephalan,trematode,Chelon labrosus,Chelon auratus,Oedalechilus labeo

Simple Summary We investigated the role of mullet as a biological tag to evaluate environmental status, mainly in a specific and circumscribed area, between March and June 2021, by conducting a survey of 150 mullet caught from Ganzirri Lagoon (Messina, Sicily, Italy), in order to describe the helminth fauna. All retrieved parasite specimens were investigated by morphological and molecular analysis. Morphological evaluation allowed us to describe the Acanthocephalan Neoechinorhynchus agilis; however, identification of the digenean trematode Haploporus benedeni was only possible using molecular analysis. None of the parasite species isolated in the present study presents a risk to human health. Abstract Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host–parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus (n = 99), Chelon auratus (n = 37), and Oedalechilus labeo (n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at −80 °C for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites (Neoechinorhynchus agilis) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes (C. labrosus, 49.5 %; C. auratus, 27%, and O. labeo, 50%), molecularly identified as Haploporus benedeni. This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon.

Morphological and Molecular Identification of Mullet Helminth Parasite Fauna from Ganzirri Lagoon (Sicily, Southern Italy) We investigated the role of mullet as a biological tag to evaluate environmental status, mainly in a specific and circumscribed area, between March and June 2021, by conducting a survey of 150 mullet caught from Ganzirri Lagoon (Messina, Sicily, Italy), in order to describe the helminth fauna. All retrieved parasite specimens were investigated by morphological and molecular analysis. Morphological evaluation allowed us to describe the Acanthocephalan Neoechinorhynchus agilis; however, identification of the digenean trematode Haploporus benedeni was only possible using molecular analysis. None of the parasite species isolated in the present study presents a risk to human health. Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host–parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus (n = 99), Chelon auratus (n = 37), and Oedalechilus labeo (n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at −80 °C for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites (Neoechinorhynchus agilis) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes (C. labrosus, 49.5 %; C. auratus, 27%, and O. labeo, 50%), molecularly identified as Haploporus benedeni. This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon. Fish are increasingly becoming the subjects of research studies, above all for their importance as a valuable source of protein. However, severe parasitic infections can cause functional disturbances, such as growth reduction and sensitivity to many secondary bacterial infections, as well as giving fish an unsightly appearance, thereby reducing their economic value. Fish populations play a key role in aquatic biological communities; moreover, they regulate the dynamics of the food chain, maintaining the balance of nutrients [1,2,3]. Thanks to the study of diet and migratory movements, fish fauna provide important data on anthropogenic action on all aquatic ecosystems [4,5], including reliable information on the possibility of accumulating pathogen organisms, such as bacteria, viruses, and parasites, as well as pollutants that pose a risk to human health [6,7]. Mugilidae, also known as mullets, are a large teleost family, divided into seventeen genera that include a high number of species [8,9,10,11,12,13]. They belong to the Actinopterygii and are characterized by a significant presence in shallow costal brackish waters, such as lagoons, rivers, estuaries, and lakes, between temperate, sub-tropical, and tropical areas, in both hemispheres [8]. They are also considered a secondary consumer, occupying an intermediate position in the food chain due to their eating habits [14]. In the Mediterranean Sea, four mullet genera (Chelon, Liza, Mugil, Oedalechilus) and six species (Chelon labrosus, C. auratus, L. ramada, L. saliens, Mugil cephalus, O. labeo) have been described [15]. Ganzirri Lagoon (Messina, Sicily, South Italy) is connected by several canals to the northern part of the Strait of Messina, forming two small ecosystems characterized by brackish water and high biodiversity levels in comparison with the other areas in the Strait, making it suitable for the exploitation of biological resources and for shellfish farming, an activity that has been practiced for several centuries. The lake is approximately 338,000 m2, 1.7 km long and 250 m wide, with a maximum depth of approximately 7 m and an average temperature that varies between 11° in January and 30° in August [16]. Mullet parasites associated with large-scale freshwater and seawater polyculture have been described worldwide since 1970 [17,18]. Mullets are economically important teleosts and are often farmed or caught for human consumption. However, like many species of fish, mullet can also be affected by parasites. Among the protozoan parasites that affect the Mugilidae, Cryptocaryon irritans [19,20], Trichodina sp. [21,22]. Trichodinella sp. [23], Tetrahymena pyriformis, and T. pediculus [24] have been reported globally. Dinoflagellata, Amyloodinium ocellatum, agent of the “velvet disease”, represents one of the most dangerous parasites in both wild and cultured mullets, causing high mortality rates [25,26]. Zoonotic Heterophidae metacercariae attributable to Ascocotyle sp., Heterophyes heterophyes and Stictodora sp., have been isolated from the muscle tissue of C. labrosus caught off the island of Sardinia and then morphologically [27] and molecularly identified [28]. The same parasite species have been isolated in M. cephalus from the Atlantic and Pacific Oceans [29] and in L. ramada from the Black Sea [30]. Monogenean flukes, such as Microcotyle mugilis, Ligophorus sp., and Ergenstrema sp. were found in M. cephalus sampled from the Marmara Sea (Turkey) [31] and L. ramada gills from Cabras Lagoon (Sardinia, Italy) [27,32]. Adult digenean trematodes, such as Saccocoelium cephali, S. obesum, S. tensum, and Haploporus benedeni were molecularly identified from C. auratus and M. cephalus gastrointestinal tracts caught along the Spanish Mediterranean coasts [33] and L. ramada (p = 36%) and C. labrosus (p = 67%) [27], where the Haploporidae was the most retrieved parasite family in all investigated species. Neoechinorhynchus agilis (Acanthocephala: Neoechinorhynchidae), which attaches to the intestinal submucosa layer, has mainly been observed in M. cephalus from the island of Sardinia (p = 91%) [27], and in other areas worldwide [34], as well as in other species such as C. labrosus and C. auratus [35]. Nematode infections in mullets are represented mainly by Anisakidae [36,37]. Contracaecum sp. larvae and Capillaria sp. have been described in Mugilids (C. labrosus and C. auratus) encysted in the celomic organ serosa and inside the gastrointestinal lumen [27], in the Mediterranean Sea, and particularly from the island of Sardinia. The lack of information on the Mugilid gastrointestinal parasitic fauna in the study area is strictly related to the complexity of the environmental characteristics. Due to many variables, such as water movement and the prey present, it is difficult to improve current data on the control of parasites in wild fish. Thus, the aim of the current study is to characterize the gastrointestinal helminth parasite fauna of mullets from Ganzirri Lagoon (southern Italy). From March to June 2021, during two different sampling trips in the same sampling site, a total of 150 mullet specimens were caught by a throwing net, also known as sparrow hawk, in the Nature Reserve of Ganzirri Lagoon (38°15′41″ N, 15°37′35″ E) (Messina, Sicily, Italy). After the sampling, all specimens were stored at +4 °C and transferred to the Experimental Fish Pathology Centre of Sicily (CISS), at the Department of Veterinary Sciences, University of Messina, Italy. All specimens were morphologically identified according to the key suggested by [8]. Fish were sexed, measured (total length, TL), and weighed (body weight, BW) (PBA220, Mettler Toledo, Columbus, OH, USA, accuracy of 1 g). All biometric data were recorded and subsequently reported as main values (mean length, ML; mean weight, MW). The specimens included in this manuscript were not part of any experiment and were commissioned by fish farmers for diagnostic purposes, specifically for fish disease control. For this reason, no ethical committee approval was needed, but all animal handling was, in any case, performed under European and Italian guidelines on animal welfare. All the sampling activities were authorized by the Regional Agency for the Protection of the Environment (ARPA Sicilia authorization n.1138/A of 15.03.2021). The evaluation of organs was carried out according to [38], modified according to requirements. The gastrointestinal (GI) tract was opened using scissors and the gastric and intestinal mucosa were gently scraped with the aid of a microscope slide; pyloric caeca were considered as part of the intestine; the organs were analyzed separately. GI contents and mucous were transferred into 1l graduated conical beakers and filled with saline solution for the subsequent sedimentation phase; the supernatant was replaced hourly to clarify the sediment. After 2 or 3 water changes, depending on supernatant clarity, the sediment was transferred to a Petri dish and monitored with the aid of a stereomicroscope (SteREO Discovery.V12 Zeiss, Jena, Germany). The collected parasite specimens were preserved in 70% ethanol until morphological identification. Other specimens were stored at −80 °C for subsequent molecular analyses. All retrieved specimens were diaphanized by immersion in glycerin for 24h, mounted and then identified with morphological keys [39,40,41]. Due to the unclear trematode identification by the morphological keys used, molecular analysis was performed only for these parasites. The morphological evaluation was performed under a light microscope (Axioskop 2 plus, Zeiss, Jena, Germany) and all pictures were taken using a digital camera (Axiocam Mrc, Zeiss, Jena, Germany) supported by a digital system (Axiovision, Zeiss, Jena, Germany). Only the trematode specimens were used for molecular identification. After morphological evaluation and confirmation of the morphological characteristics, the samples were collected in three different pools and placed in 1.5 mL (Eppendorf, Hamburg, Germany); three series of 20 min washes were performed with a phosphate buffer saline (PBS) buffer solution. DNA was extracted using a Wizard SV Genomic DNA Purification System (Promega, Madison, WI, USA). Concentration and purity were verified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The amplification of different rDNA regions was performed using a GoTaq® Colorless Master Mix (Promega, Madison, WI, United States) and different primers for trematode identification: 28S, ITS-2 and 18S: forward primer 5′ GTCCGATAGCGAACAAGTACCGT 3′ and reverse primer 5′ AGCATAGTTCACCATCTTTCGGGTCTCAA 3′ for 28S; forward primer 5′ GTCGTAACAAGGTAGCTGTA 3′ and reverse primer 5′ TATGCTTAAGTTCAGCGGGT 3′ for the partial ITS-2 [41], while for the oligonucleotide primers, C-For ATGGCTCATTAAATCAGCTAT and A-Rev TGCTTTGAGCACTCAAATTTG were used for 18S amplification [42]. The thermal cycling conditions were the following: initial denaturation for 30 s at 94 °C, 35 cycles of denaturation for 30 s at 94 °C, annealing at 58 °C for 30 s, elongation for 60 s at 72 °C with final extension of 10 min at 72 °C for 28S rDNA; initial denaturation for 30 s at 94 °C, 35 cycles of denaturation for 30 s at 94 °C, annealing at 56 °C for 90 s, elongation for 90 s at 72 °C with final extension of 10 min at 72 °C for ITS 2; initial denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, elongation at 72 °C for 60 s repeated 30 cycles, followed by a final 60 s elongation at 72 °C for 18 s [42,43]. Positivity was assessed on 1% (w/v) agarose gel. Once the presence of bands had been ascertained, a gel extraction was carried out using the Promega kit Wizard® SV Gel and PCR Clean-Up System. The concentration and purity were verified using the Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples were sent to Genechron Biotech for sequencing in both forward and reverse directions using the same primers used for amplification. Epidemiological infection indices of prevalence (p, %), mean abundance (MA), and mean intensity (MI) were calculated according to [44]. All data are presented as the mean ± standard deviation (SD). A chi-squared test was performed to assess parasite occurrence among investigated Mugilid species. The non-parametric Kruskal–Wallis’s test, followed by Dunn’s multiple comparisons test, were performed to highlight any significant difference in parasite abundance between districts and investigated Mugilid species. Furthermore, a Spearman correlation analysis was performed to detect any possible correlation between parasite abundance and Mugilid morphological features. The level of significance was set at p values < 0.05. A statistical analysis was performed using the software Prism V.9.0.0 (Graphpad Software Ldt., La Jolla, CA, USA). A total of 150 mullets were examined. Identification of Mugilidae sampled species revealed a heterogeneous group composed mainly of C. labrosus (99/150), followed by C. auratus (37/150) and O. labeo (14/150). All biometric ML and MW data on the three retrieved species are reported in Table 1. Of the 150 sampled fish GI tracts, 67 (44.7%) presented at least one parasite species. Sixty-six (44%) specimens were affected by adult digenean trematodes, both in stomach and gut. Of these, 2 C. labrosus specimens (1.3%) were affected by a total number of 16 Acanthocephala in the gut, identified according to [41] as Neochinorhynchus agilis (Neoechinorhynchidae, Rudolphi, 1819) (Figure 1). Trematode loads per species are reported in Table 2. The results of the chi-squared test did not show any significant difference in parasite occurrence between the three Mugilidae species (χ2 = 5.7; df = 2; p = 0.056). A different result was observed for the abundance data, which varied significantly between C. auratus and C. labrosus (p <0.05), as shown in Table 3 and Figure 2. No significant differences were found between districts (p > 0.05). Finally, Spearman’s correlation was performed to detect any possible correlation between parasite abundance and morphometric fish features, i.e., total length (TL) and body weight (BW). The results did not show any significant correlation between parasite abundance detected in CA and CL with these parameters. However, OL parasite abundance was negatively correlated both to the TL (r = −0.68; p = 0.009) and BW (r = −0.64; p = 0.015) of the specimens. The nucleotide sequences of the amplified products were identical among biological replicates. The sequences obtained for 18S (727 bp), ITS-2 (401 bp), and 28S (871 bp) were analyzed by BLAST similarity search against the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 14 July 2022) database to calculate the statistical significance of the matches found. In particular, BLAST analysis of the sequences obtained from ITS-2 showed a similarity of 99.5% (E-value 0.0; query cover 100%) with the relative sequence from Haploporus benedeni (accession number FJ211247.1) while there was a similarity of 100% for 28S (Accession number FJ211237.1) and 99.86% for 18S (accession number FJ211228); Haploporus benedeni (Stossich, 1887) was found with an E-value of 0.0 and a Query cover of 100% for both sequences. Amplification of the 28S rDNA gene included the D2 domain, the most robust domain, since it could provide a higher resolution of the species. The representative DNA sequence for 18S, ITS-2, and 28S was submitted to GenBank (accession numbers OQ283825, OQ283827, OQ283826, respectively). The present study represents the first survey on mullet helminth parasite fauna from a Special Protection Area (SPA) in the central Mediterranean Sea. Digenean parasites in mullets from different areas of the Mediterranean Sea have been reported [27,35]. Several digenean parasites were collected in the intestinal mucosa [45,46], as reported in L. saliens and L. ramada [45]. In the present study, the most abundant collected species was C. labrosus; nevertheless, the most parasitized species was O. labeo, although it is the least representative collected species and the high prevalence is probably related to a specific ecological characteristic of O. labeo to host the trematode herein described, localized mainly in the intestine, rather than the stomach. Another relevant aspect of the investigated area, characterized by regular movement of water, is the difference in the salinity rate in comparison with the nearby sea. Salinity is strictly related to mollusk and crustacean evolution, regulating both growth and intermediate host vulnerability to the predators [16]. The likelihood that the sampling spot is distant from the infection point, even by a couple of kilometers, in an unknown area, characterized by different environmental conditions, should be highlighted [47]. The parasitological examination and subsequent molecular confirmation allowed us to identify the trematode Haploporus benedeni (Haploporidae, Stossich 1887) using the ITS2, 18S, and 28S ribosomal RNA genes. Despite the high number of reports of H. benedeni in most Mugilid species from the Mediterranean basin [48,49,50,51], few studies provide a molecular identification of this species [33]; therefore, our study improves the current knowledge about the molecular description of this digenean trematodes. The H. benedeni life cycle is characterized by the free-swimming miracidium and an intermediate host belonging to the genus Hydrobia, as reported by [52]. In particular, Hydrobia sp. is a small aquatic gastropod mollusk belonging to the family Hydrobiidae. The presence of Hydrobia sp. inside the mullet stomach could be associated to the H. benedeni prevalence in each of the three studied species, which also indicates a high distribution of snails belonging to the Hydrobia sp. in the investigated area. The H. benedeni prevalence reported by [27] in C. labrosus (67%) was higher than the present study, where the most infected species was O. labeo. Neoechinorhynchus agilis is a widespread parasite of grey mullets worldwide [27,41]. In [47], N. agilis specimens attached to the intestinal mucosal and submucosal layers of L. saliens and L. ramada (41.6%) at a higher prevalence than in the present study (1.3%). This Acanthocephalan has also usually been considered as a M. cephalus parasite [46]. C. labrosus was found in the eastern Atlantic Ocean and Mediterranean Sea as one of the most infected species (up to 54%) by N. agilis [27,41], a prevalence higher than our study (1.3%), which is probably related to the difference in fish size. The identified parasites, H. benedeni and N. agilis, previously reported from the Mistras Lagoon (Sardinia—Italy—western Mediterranean Sea) [14], were reported for the first time in the central Mediterranean Sea, showing an equal distribution in two different Mediterranean areas, as well as in Libya and Egypt [53]. This ecological evaluation is also supported by the current information on the diversity and distribution of parasites, such as H. benedeni and N. agilis in mullets. In conclusion, this study improves the current knowledge on mullet helminth parasitic fauna from the Ganzirri Lagoon in the central Mediterranean Sea. Mullet, while not characterized by a high commercial value, are increasingly being appreciated both for their greater production in aquaculture and for their use in traditional food preparation in the Mediterranean basin. Hydrobia spp., a common mollusk found in Ganzirri Lake, and considered a host for a wide range of trematode species that can infect several marine species such as mullet, can be used as a biological tag for further studies in the same area. Moreover, even if mullets are considered a host of parasites which are potentially dangerous for human health, the parasitic species identified in the examined GIT are not considered zoonotic agents.

PMC10000167

Jennifer R. Weinert-Nelson,Amy S. Biddle,Harini Sampath,Carey A. Williams

Fecal Microbiota, Forage Nutrients, and Metabolic Responses of Horses Grazing Warm- and Cool-Season Grass Pastures

22-02-2023

equine microbiome,glycemic response,non-structural carbohydrates,rotational grazing

Simple Summary Incorporating warm-season grasses into traditional cool-season grass equine rotational grazing systems can increase pasture availability during hot, dry months and bridge the “summer slump” forage gap. The objective of this study was to evaluate the impacts of this pasture management practice on the equine microbiome and to explore relationships between the fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Results of this study indicate that distinct changes in microbial community structure and composition occur as horses adapt to different forages and that shifts in the microbial community were most influenced by forage non-structural carbohydrates and crude protein, rather than fiber. Interrelationships were found between these nutrients, glycemic responses, and Akkermansia and Clostridium butyricum. These bacteria were also found to be enriched in horses adapted to warm-season grasses. While the results of this study suggest that integrating warm-season grasses may not offer substantial metabolic benefits in healthy adult horses, this study did reveal new insights and targets for future research necessary to better understand the function of Akkermansia and Clostridium butyricum in the hindgut microbiome of grazing horses and possible roles in modulation of equine metabolic health. Abstract Integrating warm-season grasses into cool-season equine grazing systems can increase pasture availability during summer months. The objective of this study was to evaluate effects of this management strategy on the fecal microbiome and relationships between fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Fecal samples were collected from 8 mares after grazing cool-season pasture in spring, warm-season pasture in summer, and cool-season pasture in fall as well as after adaptation to standardized hay diets prior to spring grazing and at the end of the grazing season. Random forest classification was able to predict forage type based on microbial composition (accuracy: 0.90 ± 0.09); regression predicted forage crude protein (CP) and non-structural carbohydrate (NSC) concentrations (p < 0.0001). Akkermansia and Clostridium butyricum were enriched in horses grazing warm-season pasture and were positively correlated with CP and negatively with NSC; Clostridum butyricum was negatively correlated with peak plasma glucose concentrations following oral sugar tests (p ≤ 0.05). These results indicate that distinct shifts in the equine fecal microbiota occur in response different forages. Based on relationships identified between the microbiota, forage nutrients, and metabolic responses, further research should focus on the roles of Akkermansia spp. and Clostridium butyricum within the equine hindgut.

Fecal Microbiota, Forage Nutrients, and Metabolic Responses of Horses Grazing Warm- and Cool-Season Grass Pastures Incorporating warm-season grasses into traditional cool-season grass equine rotational grazing systems can increase pasture availability during hot, dry months and bridge the “summer slump” forage gap. The objective of this study was to evaluate the impacts of this pasture management practice on the equine microbiome and to explore relationships between the fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Results of this study indicate that distinct changes in microbial community structure and composition occur as horses adapt to different forages and that shifts in the microbial community were most influenced by forage non-structural carbohydrates and crude protein, rather than fiber. Interrelationships were found between these nutrients, glycemic responses, and Akkermansia and Clostridium butyricum. These bacteria were also found to be enriched in horses adapted to warm-season grasses. While the results of this study suggest that integrating warm-season grasses may not offer substantial metabolic benefits in healthy adult horses, this study did reveal new insights and targets for future research necessary to better understand the function of Akkermansia and Clostridium butyricum in the hindgut microbiome of grazing horses and possible roles in modulation of equine metabolic health. Integrating warm-season grasses into cool-season equine grazing systems can increase pasture availability during summer months. The objective of this study was to evaluate effects of this management strategy on the fecal microbiome and relationships between fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Fecal samples were collected from 8 mares after grazing cool-season pasture in spring, warm-season pasture in summer, and cool-season pasture in fall as well as after adaptation to standardized hay diets prior to spring grazing and at the end of the grazing season. Random forest classification was able to predict forage type based on microbial composition (accuracy: 0.90 ± 0.09); regression predicted forage crude protein (CP) and non-structural carbohydrate (NSC) concentrations (p < 0.0001). Akkermansia and Clostridium butyricum were enriched in horses grazing warm-season pasture and were positively correlated with CP and negatively with NSC; Clostridum butyricum was negatively correlated with peak plasma glucose concentrations following oral sugar tests (p ≤ 0.05). These results indicate that distinct shifts in the equine fecal microbiota occur in response different forages. Based on relationships identified between the microbiota, forage nutrients, and metabolic responses, further research should focus on the roles of Akkermansia spp. and Clostridium butyricum within the equine hindgut. Integrating warm-season grasses into traditional cool-season grass rotational grazing systems can provide productive pasture for grazing horses during the hot, dry months of the “summer slump” period [1,2]. Despite reported benefits for pasture yield [2,3] the impacts of this practice on equine metabolic health and the hindgut microbiome have not been previously investigated. Warm- and cool-season grasses have different mechanisms for storage of soluble carbohydrates [4], with non-structural carbohydrate (NSC = sugars + starch + fructans) concentrations in cool-season grasses typically greater than that of warm-season grasses [1,5]. These differences in NSC content are of interest in equine management as current feeding recommendations for horses with existing metabolic dysfunction include limiting dietary NSC concentrations [6,7,8]. Thus, lower-NSC warm-season grasses have been suggested as an alternative forage source [1,9]. Feeding supplemental concentrate higher in NSC has been shown to lower insulin sensitivity in horses [10,11,12], but over the relatively smaller range of NSC concentrations observed in forages, potential benefits of limiting NSC intake are less clear [13,14,15]. However, glycemic and insulinemic responses of horses grazing low-NSC warm-season grasses have not been extensively evaluated. In addition to the potential metabolic effects, transitioning horses between forage types may also have implications for equine gastrointestinal health. Diet has been identified as a dominant factor shaping the community structure of the gut microbiota both in humans and across animal species including horses [16,17]. However, prior studies on the influence of diet on the equine hindgut microbiome have focused primarily on concentrate vs. concentrate or concentrate vs. forage diets [18,19,20,21]. Recent studies have demonstrated that type of hay (alfalfa vs. grass) and transitions between hay and pasture grass can impact equine cecal or fecal microbial community composition [22,23]. Overall, few studies have evaluated the hindgut microbiome of grazing horses and only one previous study has been conducted in horses grazing cool- vs. warm-season pasture grasses [24]. A number of studies have begun to explore associations between the equine hindgut microbiota and metabolic health [25,26,27,28], but there is a lack of information on the interplay between forage nutrients, the hindgut microbiome, and metabolism of grazing horses. Biddle et al. [28] explored relationships between abundance profiles of fecal microbial taxa, feed types (pasture, hay, or hay supplemented with concentrate), and blood analytes including circulating glucose and insulin, finding negative correlations between insulin and over 50 taxa. Studies conducted in mouse models have conclusively demonstrated that changes in diet influence host metabolism in a microbiome-dependent manner [29,30]. Thus, there is a need for future research to better understand potential roles of the gut microbiota in modulating metabolism of grazing horses and the influence of specific forage nutrients on these interactions. While shifts in hindgut microbial communities have been documented in pastured horses over time, little is known regarding relationships between these changes in bacterial composition and forage nutrient profiles or metabolic responses of grazing horses. Therefore, the aims of this study were to characterize shifts in glucose metabolism and the fecal microbiota of horses adapted to different forage types and to explore relationships between forage nutrients, microbial composition, fecal metabolites, and metabolic responses of grazing horses. Research was conducted in 2018 at the Ryders Lane Environmental Best Management Practices Demonstration Horse Farm (Rutgers, The State University of New Jersey; New Brunswick, New Jersey, NJ, USA). Weather data for the study period and historical averages are presented in Table S1 [31]. Two separate 1.5 ha integrated warm- and cool-season rotational grazing systems were utilized in this study. Grazing system design and management were as published in previous companion studies [2,24]. In brief, warm-season grass pasture sections contained either Wrangler bermudagrass [BER; Cynodon dactylon (L.) Pers.; Johnston Seed Company, Enid, OK, USA] or Quick-N-Big crabgrass [CRB; Digitaria sanguinalis (L.) Scop.; Dalrymple Farms, Thomas, OK, USA]. Mixed cool-season grass sections contained Inavale orchardgrass [Dactylis glomerata (L.)], Tower tall fescue (endophyte-free) [Lolium arundinaceum (Schreb.) Darbysh.], and Argyle Kentucky bluegrass [Poa pratensis (L.)] (DLF Pickseed, Halsey, OR, USA). Grazing management was guided by established best management practices [32]. Use of animals in this study was approved by the Rutgers University Institutional Animal Care and Use Committee protocol #PROTO201800013. Eight adult Standardbred mares (age: 18 ± 0.71 yr; body weight (BW): 537 ± 17 kg; body condition score (BCS): 5–7)) were used in this study, with horses grouped by BW and BCS (Henneke Body Condition Score scale [33]. Horses were then randomly assigned to each system (n = 4 horses system−1). Prior to the study, oral sugar tests (OST) were administered to screen horses for impaired insulin sensitivity [34]. Insulinemic responses of all horses were found to be normal (peak insulin ≤45 mIU/L) [7]. Animal care and management have been previously detailed in companion studies [1,24]. Horse condition measures over the course of the study can be found in Table S2. Manual grab fecal samples were collected (0800 h) rectally from horses after 21 d adaptation to the initial hay diet in the spring (HAY-SP), cool-season grass pasture in the spring (CSG-SP), warm-season grass (WSG)—either BER or CRB during the “summer slump” period, and again following a return to cool-season grass in the fall (CSG-FA) and a final cool-season grass hay at the end of the grazing season (HAY-FA). See Figure S1 for a diagram of experimental design and sampling protocol. Due to delayed establishment and grazing of BER, only 17 days of grazing were possible prior to sample collection. Upon collection, samples were immediately placed on ice, transported to the laboratory, and then stored at −80 °C. To determine impact of forage type within integrated systems on glycemic and insulinemic responses of grazing horses, OST [34] were conducted following adaptation to CSG-SP, WSG, and HAY-FA. On the evening prior to each collection period, horses were confined to dry lots at 2000 h with no access to either hay or pasture. Following the overnight fast, horses were moved into the barn facility at 0800 h. Two consecutive baseline blood samples were collected (15 min apart) by venipuncture at 0800 and an oral dose of Karo Syrup (0.25 mL/kg BW; modified dosing as utilized by Jacob et al. [12]) was immediately administered after the second baseline sample. Subsequent blood samples were collected at 30, 60, 90, 120, 180, and 240 min following Karo Syrup administration. Whole blood was collected into sodium heparin Vacutainer tubes, which were inverted several times before being placed on ice. Due to logistics of distance and travel time (~10 min from the barn to the laboratory), samples were transported to the laboratory after the 120, 180, and 240 min samples. In the laboratory, whole blood was centrifuged at 3700 rpm for 7 min. Following centrifugation, plasma was harvested from the Vacutainer tubes and aliquoted into microcentrifuge tubes, which were then stored at −80 °C. Representative hand-clipped forage samples were also collected (0800–1000 h) on three days per period for analysis of nutrient composition. Pasture samples were collected according to previously published procedures [1,24,32] and dried (60 °C; 36 h minimum) in a Thelco oven (Precision Scientific, Chicago, IL, USA). Samples were ground (1 mm) and submitted to a commercial laboratory (Equi-Analytical Laboratories, Ithaca, NY, USA) for analysis by near-infrared spectroscopy. The mean nutrient composition of hay diets and pasture forages are shown in Table 1. Plasma glucose was analyzed by colorimetric assay (Glucose C-2, Wako Chemicals, Richmond, VA, USA), with the commercial kit adapted for microplate assay following manufacturer instructions. Plasma insulin was evaluated using an enzyme-linked immunoassay (Mercodia Equine Insulin ELISA, Mercodia, Winston-Salem, NC, USA) previously validated in horses [35]. Inter-assay and intra-assay coefficients of variation for glucose were 4.0% and 2.9%, respectively. Inter-assay and intra-assay coefficients of variation for insulin were 7.8% and 3.4%, respectively. Fecal pH was measured in duplicate with a handheld Accumet pH meter (Fisher Scientific; Waltham, MA, USA) using a previously published protocol for preparation of fecal slurries [24,36]. Short-chain and branched-chain fatty acid (SCFA and BCFA, respectively) concentrations were determined by GC-MS analysis of fecal samples. The SCFA analyzed included acetate, propionate, butyrate, and valerate. The BCFA analyzed included isobutyrate, isovalerate and isocaproate. Sample preparation was performed according to a previously published protocol [37]. In brief, frozen fecal samples were weighed and deposited in bead tubes over dry ice. Feces were then resuspended in 1 mL of 0.5% phosphoric acid per 0.1 g of sample and tubes were beaten for 5 min at 22.5 rpm in a cold case. Samples were again frozen at −80 °C until analyzed by Gas Chromatography and Mass Spectrometry system GC-MS (Agilent Technologies, Santa Clara, CA, USA). Prior to GC-MS analyses, thawed fecal suspensions were re-homogenized and centrifuged (10 min at 17,949× g), with the aqueous phase extracted using diethyl ether in a 1:1 volume to volume ratio. Before analysis, 2-methyl hexanoic acid (Thermo Fisher Scientific, Dallas, TX, USA) was added to the organic phase extract as an internal standard. Samples were analyzed in duplicate, with independent extractions for each replicate. The specifications of the GC-MS system and analyses as well as data acquisition and procedures for SCFA/BCFA quantitation have been previously described by Honarbakhsh et al. [37] and Garcia-Villalba et al. [38]. Quick-DNA Fecal/Soil Microbe Kits (Zymo Research; Irvine, CA, USA) were used for DNA extraction (in triplicate). The highest yielding replicate (quantified with a Qubit 2.0 Flourometer [Invitrogen; Carlsbad, CA, USA]) was submitted to a commercial laboratory (RTL Genomics; Lubbock, TX, USA). Amplification of the V4-V5 region of the 16S rRNA gene was conducted using region specific primers (515F/926R) [39]; sequencing was conducted by Illumina MiSeq. Sequence and statistical analyses were performed in QIIME 2 (Quantitative Insights into Microbial Ecology, v. 2020.8) [40] and R (v. 4.0.2) [41]. Network mapping was conducted in Cytoscape (v. 3.8.0) [42]. Animal was considered the experimental unit. Quality and chimera filtering of forward reads was conducted using DADA2 (read length = 185) [40,43,44]. Mafft and FastTree (q2-phylogeny plugin) were used to create trees for diversity analyses [45,46,47]. The lower quartile of Amplicon Sequence Variants (ASV) based on absolute abundance were removed (minimum frequency = 16; minimum samples = 4). The feature table was rarefied to a minimum sampling depth of 10,600 prior to α- and β-diversity analyses. The α-diversity metrics were analyzed by Kruskal–Wallis tests [48,49,50,51,52]. The β-diversity metrics analyzed included Weighted and Unweighted UniFrac by permutational ANOVA (PERMANOVA) [53,54,55,56,57,58,59]. Benjamini and Hochberg FDR adjustments for multiple pairwise comparisons were applied for all diversity analyses. Permutational multivariate analysis of dispersion (PERMDISP) was used to test homogeneity of dispersion [60]. To further explore differential abundances, ASV were then grouped into bacterial co-abundance groups (BCG) based on abundance profiles using Sparce Cooccurrence Network Investigation for Compositional Data (SCNIC) [61,62]. A random forest classifier with nested cross validation was applied to determine if forage type could be predicted based on BCG composition [63,64]. Features (BCG) were removed from the model based on importance scores generated by the random forest classifier in an iterative process (serial reduction with increments of 5) until the point at which model accuracy began to decline [65]. Relative abundances of remaining BCG and any uncorrelated ASV retained in the model following feature reduction processes were then analyzed by linear discriminant analysis effect size (LEfSe) to identify BCG specific to each forage type, with significance set at an LDA score >2.0 [66]. Taxonomy was then assigned using the latest SILVA database (SSU 138) [63,67,68,69,70]. Glucose and insulin response variables as well as SCFA/BCFA concentrations and fecal pH were analyzed by mixed model ANOVA in R, with grazing system, forage type and their interactions set as fixed factors and horse as the random factor in the initial model. The area under the curve (AUC) was calculated based on the trapezoid rule as the positive incremental AUC utilizing a published macro [71] in SAS (v.9.4 SAS Institute, Cary, NC, USA). Proxies for insulin sensitivity (fasting glucose-to-insulin ratio (FGIR); reciprocal of the square root of insulin (RISQI)) and insulin secretory response (modified insulin-to-glucose ratio (MIRG)) were calculated using baseline (fasting) values as previously described [72,73]. The relationship between forage nutrients and SCFA/BCFA fermentation metabolites (as well as pH) and fecal microbial community composition was then explored using random forest regression with nested cross validation to determine if nutrient concentrations and/or metabolite concentrations could be predicted based on bacterial abundance profiles. Spearman correlations between BCG and forage nutrients and fecal metabolites were analyzed in R. Relationships between forage nutrients/fermentation metabolites and BCG were then visualized in Cytoscape. Spearman correlations between metabolic variables and the BCG, as well as between forage nutrients and metabolic responses were also evaluated in R. For all variables analyzed by mixed model, model residuals were analyzed for normality using the Shapiro–Wilk test. Log, square root, or inverse data transformations were applied where appropriate for non-normal data. Means were separated using Tukey’s method. When analyzing pairwise comparisons of transformed data, means and standard errors were back-transformed to the original variable scale following application of Tukey’s method with the delta method. For all analyses which generated p-values, results were considered significant at p ≤ 0.05, with trends considered at p ≤ 0.10. Data for variables analyzed by mixed model are presented as means ± SEM. Overall analysis of microbiome, fecal metabolite, and glucose/insulin data did not reveal differences by grazing system. Therefore, grazing system was removed from models and results for combined data are presented with n = 8. A summary of 16S rRNA gene sequencing reads before and after quality and chimera filtering as well as after filtering of low abundance features in the 40 samples analyzed for this study is shown in Table 2. There were 1264 distinct ASV in the final dataset (taxonomy can be found in Table S3). All α-diversity metrics evaluated, including the Shannon Diversity Index, Faith’s Phylogenetic Diversity, Pielou’s Evenness, and Observed ASVs, differed by forage (Kruskal–Wallis tests with Benjamini and Hochberg FDR adjustments; p < 0.03; Figure 1a–d). Shannon Diversity was greater when horses were adapted to WSG than CSG-SP (p < 0.05), and there was a trend for greater diversity when adapted to HAY-SP compared to CSG-SP (p < 0.08) and WSG vs. CSG-FA (p = 0.09). However, the only differences in evenness (Pielou’s Evenness) were trends for greater evenness in WSG and CSG-SP than in CSG-FA (p < 0.06). Conversely, richness (Observed ASVs) was greater in horses adapted to WSG, CSG-FA, and HAY-FA than CSG-SP (p < 0.02), but did not differ between horses adapted to HAY-SP and CSG-SP. Similarly, phylogenetic differences (Faith’s Phylogenetic Diversity) were also found between CSG-SP and subsequent forages (WSG, CSG-FA, HAY-FA; p < 0.02). Principal coordinate analysis of β-diversity metrics including Weighted and Unweighted UniFrac did not reveal distinct clustering by forage (Figure 2a,b). However, statistical analysis by PERMANOVA (with Benjamini and Hochberg FDR adjustments) found significant differences in these measures (p ≤ 0.02). Subsequent PERMDISP analysis confirmed that these differences were not due to differences of variance or dispersion within groups. Unweighted UniFrac differed for all pairwise comparisons of forages (p < 0.02), with the exception of WSG vs. CSG-FA and WSG vs. HAY-FA for which there were trends for differences (p < 0.10). In contrast, there were no significant differences in any pairwise comparisons of forages for Weighted UniFrac. The percent of variation explained by PC1 was almost 3 times greater for Weighted than for Unweighted UniFrac, indicating an influence of abundance profiles in addition to phylogenetic differences. Application of SCNIC identified 333 BCG, with 224 individual ASV remaining ungrouped. Iterative reduction of features (BCG) based on random forest model importance scores revealed that model accuracy increased through reduction to the top 65 features (0.90 ± 0.09). Further feature reduction to the top 25 features (based on importance scores) did not impact random forest model accuracy (0.90 ± 0.09 with the top 25 features retained). All retained features were BCG; no ungrouped ASV remained in the reduced feature set. These 25 BCG were retained for further analysis, with 11.54% of the total microbial community abundance represented by the reduced 25 BCG feature set. The strength of the random forest model accuracy score indicated that forage type could be predicted based on bacterial composition, and that, conversely, bacterial community composition was influenced by forage. Subsequent Linear discriminant analysis Effect Size (LEfSe) analysis conducted on features retained from the random forest classification modelling identified 6 BCG as markers of HAY-SP, 3 BCG enriched in CSG-SP, 5 BCG for WSG, 5 BCG for CSG-FA, and 5 BCG for HAY-FA (LDA > 4.0; p < 0.03). Forage-specific BCG markers as well as taxonomic classifications of subsequent linear discriminant analysis Effect Size analysis conducted on features retained from the random forest classification modelling identified 6 BCG as markers of HAY-SP, 3 BCG enriched in CSG-SP, 5 BCG for WSG, 5 BCG for CSG-FA, and 5 BCG for HAY-FA (LDA > 4.0; p < 0.03). Forage-specific BCG markers as well as taxonomic classifications of individual ASV within each BCG are presented in Table 3, Table 4 and Table 5. Numerous taxa were represented in BCG markers for multiple forages. At the family level, the BCG markers for HAY-SP contained twice as many ASV mapped to the Lachnospiraceae family as any other forage (CSG-SP: 0; WSG: 2; CSG-FA: 3; HAY-FA: 2 ASV). The family Oscillospiraceae also had ASV members of BCG markers for all forages but CSG-SP (HAY-SP: 3; WSG: 3; CSG-FA: 2; HAY-FA: 2 ASV). At the genus level, ASV assigned to Christensenellaceae R-7 group were present in BCG markers of both hay diets (HAY-SP: 2; HAY-FA:1 ASV) and WSG (2 ASV); ASV assigned to the NK4A214 group of Oscillospiraceae were among members of BCG identified as markers of HAY-SP (2 ASV), WSG (1 ASV), and CSG-FA (1 ASV). The BCG markers of both hay diets and CSG-FA each included an ASV mapped to Rikenellaceae RC9 gut group, and BCG markers of HAY-SP and WSG each contained ASV assigned to Fibrobacter and Papillibacter. The BCG markers of HAY-SP and CSG-FA contained ASV within Catenisphaera and Lachnospiraceae UCG-009. Amplicon sequence variants mapped to the genus Clostridium sensu stricto 1 were found in BCG markers of CSG-SP and WSG. The BCG markers of WSG and CSG-FA included ASV assigned to Bacteriodales RF16 group and the Family XIII AD3011 group of Anaerovoracaceae. The BCG markers of CSG-FA and HAY-FA each contained an ASV assigned to Coprostanoligenes group, Lachnospiraceae XPB1014 group and Marvinbryantia, and ASV assigned to Treponema were assigned to BCG markers of WSG and HAY-FA. Amplicon sequence variants assigned to Ruminococcus, Pseudobutyvibrio, Anaerovibrio, probable genus 10 of Lachnospiraceae, and the p-251-o5 genus and family of the order Bacteroidales were only present in BCG markers identified for HAY-SP; no other ASV assigned to these taxa were found in BCG markers of other forages. An ASV within Bacteroidales BS11 gut group was the only taxa specific to BCG markers of CSG-SP. Amplicon sequence variants mapped to the genera Akkermansia, Mogibacterium, and the Hallii group of Lachnospiraceae as well as UCG-005 metagenome of Oscillospiraceae and Clostridium butyricum were only found in BCG markers of WSG. The BCG markers of CSG-FA contained ASV assigned to Alloprevotella, Erysipelatoclostridium, Bacteroidales UCG-001, the WCHB1-41 genus, family and class within the class Kiritimatiellae, and Denitrobacterium detoxificans; these taxa were not identified in BCG markers of other forages. Taxa specific to only BCG markers of HAY-FA included ASV from the Incertae Sedis genus of Ethanoligenenaceae as well as Streptococcus. Fecal pH differed by forage type (mixed model ANOVA with Tukey’s post hoc adjustment; p < 0.0001). Fecal pH was greater in horses adapted to WSG (7.56 ± 0.18) and HAY-FA (7.57 ± 0.18) than in HAY-SP (6.73 ± 0.18), CSG-SP (6.58 ± 0.18), or CSG-FA (6.53 ± 0.18; p ≤ 0.02; Figure 3). Fecal concentrations of short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) also differed by forage (mixed model ANOVA with Tukey’s post hoc adjustment; p < 0.001; Table 6). Unlike fecal pH, differences in SCFA and BCFA were primarily between pasture forages and the hay diets, with horses adapted to WSG often intermediate (numerically) between cool-season pasture and hay diets. Total BCFA were greater in CSG-SP, WSG, and CSG-FA than for either HAY-SP or HAY-FA (p < 0.05). Total SCFA were greater in CSG-SP and CSG-FA than HAY-SP or HAY-FA; WSG did not differ from CSG-SP and CSG-FA or HAY-SP, but was greater in comparison to HAY-FA (p < 0.002). Similarly, acetate was greater in CSG-FA than HAY-SP or HAY-FA, while WSG only differed from HAY-FA (p < 0.02). Acetate concentrations for CSG-FA also differed with HAY-FA (p = 0.0003), but there was only a trend for a difference between CSG-SP and HAY-SP (p = 0.09). Fecal butyrate was lower for HAY-FA than when horses were adapted to any of the pasture forages (p < 0.03), but there was no difference between HAY-SP and any other forage. Fecal propionate was lowest in horses adapted to HAY-FA (p < 0.002), but propionate concentrations were also lower for HAY-SP than either CSG-SP or CSG-FA (p < 0.03). Propionate did not differ between horses adapted to WSG and all other forages. Fecal valerate was lower for horses adapted to HAY-FA than CSG-SP or CSG-FA (p < 0.03) but did not differ from WSG or HAY-SP. Horses adapted to HAY-SP also had fecal valerate concentrations lower than CSG-SP or CSG-FA (p ≤ 0.0002). Isobutyrate was greater for all pasture forages in comparison to both HAY-SP and HAY-FA (p < 0.02). Isovalerate was greater in CSG-SP and CSG-FA vs. HAY-SP and HAY-FA (p ≤ 0.002), while concentrations in horses adapted to WSG were once again intermediate. Isocaproate was detected in all fecal samples, but concentrations were negligible and below the limit of quantification (<1.0 ug g feces−1). Random forest regressors were applied to determine if fecal pH and metabolite concentrations could be predicted based on microbial composition. Random forest regression did not support a strong influence of bacterial composition of the full microbial community on these fecal variables. Relatively weak predictive accuracy was found for isovalerate, valerate, hexanoate, and heptanoate (model R2 and p-values are shown in Table 7). Major SCFA including acetate, butyrate, and propionate could not be predicted based on BCG abundance profiles. However, when random forest regressors were applied to only the top 25 BCG identified as most predictive of forage type, model accuracies improved for all fecal variables with the exception of valerate (Table 7). While predictive accuracy was still relatively weak, all metabolites (and pH) could be predicted with statistically significant accuracy (p ≤ 0.001). Individual correlations were found between 19 of these BCG and at least one fecal metabolite or pH (Spearman correlation; rs ≥ |0.30|; p ≤ 0.05; Figure 4), with most BCG correlated with multiple fecal variables. There were positive correlations between BCG_300 and total SCFA and total BCFA as well as with acetate, butyrate, propionate, valerate, isobutyrate, and isovalerate, while this BCG was negatively correlated with hexanoate, heptanoate, and pH. The ASV members of this BCG were assigned to the genera Lachnospiraceae XPB1014 and Streptococcus. There were also positive correlations between both BCG_9 and BCG_259 and total SCFA and BCFA in addition to acetate, butyrate, propionate, valerate, isobutyrate, and isovalerate; these BCG were negatively correlated with heptoanate. These BCG included multiple ASV assigned to Lachnospiraceae as well as ASV within the genera Christensenellaceae R-7 group, Rikenellaceae RC9 gut group, Papillibacter, and the NK4214 group of Oscillospiraceae. Positive correlations were also found between BCG_173 and BCG_201 and total SCFA and BCFA, acetate, propionate, valerate, isobutyrate, and isovalerate, with negative correlations between these BCG and both hexoanate and heptoanate. These BCG included ASV assigned to Treponema, Rikenellaceae RC9 gut group, the NKA214 group of Oscillospiraceae, and the p-251-o5 and F082 genera and families of Bacteroidales. Total SCFA and BCFA as well as acetate, butyrate, propionate, isobutyrate, and isovalerate were positively correlated with BCG_113, while hexanoate was negatively correlated. This BCG contained ASV assigned to taxa including Synergistaceae, Bacteroidales RF16 group, Papillibacter, Christensenellaceae R-7 Group, Mogibacterium, and Clostridium butyricum. Seven BCG were positively correlated with total BCFA and some combination of propionate, valerate, isobutyrate, and isovalerate, while negative correlations were found between these BCG and hexanoate and/or heptanoate. These BCG included ASV members assigned to the family level for Anaerovoracaceae, Oscillospiraceae, and Lachnospiraceae. Other ASV within these BCG were assigned to Lactobacillus equigenerosi and the genera Akkermansia, Fibrobacter, Treponema, Sphaerocheata, Phasolarctobacterium, Christensenellaceae R-7 group, Lachnoclostridium, Cellulosilyticum, Marvinbryantia, Coprostanoligenes group, Lachnospiraceae XPB1014, Lachnospiraceae UCG-009, Erysipelatoclostridium, Clostridium sensu stricto 1, and Bacteroidales BS11 gut group as well as Family XIII AD3011 group of Anaerovoracaceae, the UCG-004 genus within Erysipelatoclostridiaceae, the UCG-002 and UCG-005 genuses of Oscillospiraceae, and the UCG-010 genus and family of Oscillospirales. Fecal pH was positively correlated with five BCG (in addition to BCG_300) (rs ≥ |0.30|; p ≤ 0.05), which contained ASV from taxa including Anaerovoracaceae, the Family XIII AD3011 group of Anaerovoracaceae, Akkermansia, Christensenellaceae R-7 group, Fibrobacter, Treponema, Catenisphaera, Alloprevotella, Bacteroidales RF16 group, Marvinbryantia, Coprostanoligenes group, Bacteroidales UCG-001, the NK4A214 group of Oscillospiraceae, and the WCHB1-41 genus, family, and order within Kiritimatiellae. Fecal pH was negatively correlated with BCG_94. This BCG included ASV assigned to Prevotella, Sarcina, and Clostridium sensu stricto 1. Application of random forest regressors demonstrated that forage nutrient concentrations could be predicted based on bacterial community composition (BCG composition of the full microbial community), including water-soluble carbohydrate (WSC) and NSC at a predictive accuracy > R2 = 0.50 and crude protein (CP) with a predictive accuracy of R2 = 0.41 (p < 0.0001; Table 7). Weaker, but still statistically significant, predictive accuracy was found for digestible energy (DE), acid detergent fiber (ADF), neutral detergent fiber (NDF), ethanol-soluble (ESC), and starch (p < 0.04). Similar to results for fecal metabolites, when random forest regressors were applied only to the top 25 BCG identified as most predictive of forage type, model accuracies improved (Table 7). Forage CP, NSC, and WSC remained as the nutrients most accurately predicted by the bacterial composition of this subset of BCG, all with R2 > 0.60 (p < 0.0001). Model predictive capacity with the reduced feature set was also above R2 = 0.40 for starch; weaker, but statistically significant, predictive accuracy was found all other nutrients (p < 0.0001). Subsequent correlation analysis found 23 BCG (of the 25 in the reduced feature set) were correlated with at least one forage nutrient (Spearman correlation; rs ≥ |0.30|; p ≤ 0.05; Figure 5). Nine BCG were correlated with ADF, NDF, CP, and DE. In all cases, opposite correlations were seen between ADF/NDF and CP/DE (i.e., if ADF and NDF were positively correlated with a BCG, CP and DE were negatively correlated). There were distinct ASV from the taxa Christensenellaceae R-7 group and NK4A214 group of Oscillospiraceae that were members of separate BCG which were positively and negatively responding to these nutrients (i.e., these taxonomic classifications were found across all positive and negative responder groups). The BCG negatively correlated with ADF/NDF, and thus positively correlated with CP/DE included ASV members assigned to Clostridium butyricum and genera Papillibacter, Lachnoclostridium, Cellulosilyticum, Fibrobacter, Treponema, Catenisphaera, Bacteroidales RF16 group, Synergistaceae, Mogibacterium, Rikenellaceae RC9 gut group, the Hallii group of Lachnospiraceae, the p-251-o5 genus and family of Oscillospiraceae, and the F082 genus and family of Bacteroidales, as well as multiple ASV assigned only to the family level for Lachnospiraceae. The BCG positively correlated with ADF/NDF and negatively correlated with CP/DE included ASV within Sphaerochaeta, Phascolarctobacterium, Bacteroidales UCG-001, Rikenellaceae RC9 gut group, Coprostanoligenes group, the UCG-002 genus within Oscillospiraceae, and the UCG-010 genus and family within Oscillospirales. Four additional BCG were correlated with some combination of ADF/NDF and CP/DE. The BCG negatively correlated with ADF/NDF, and thus positively correlated with CP/ DE contained ASV members assigned to additional taxa including Ruminococcus, Anaerovibrio, Pseudobutyvibrio, Lachnospiraceae UCG-009, probable genus 10 of Lachnospiraceae, and the WCHB1-41 genus, family and order of Kiritimatiellae. The BCG positively correlated with ADF/NDF, and negatively correlated with CP/DE included ASV within Bacteroidales BS11 gut group and Clostridium sensu stricto 1. Forage ADF and NDF were also negatively correlated with BCG_300 (Streptococcus and Lachnospiraceae XPB1014), but this BCG was not correlated with either CP or DE. Forage CP was also positively correlated with BCG_35, for which there was no relationship with ADF, NDF, or DE, but for which there was also a negative correlation with NSC and WSC. The ASV members of BCG_35 were assigned to taxa including Akkermansia, Fibrobacter, Treponema, Christensenellaceae R-7 group, and Family XIII AD3011 within Anaerovoracaceae. Forage NSC and WSC were correlated with 13 and 12 BCG, respectively (rs ≥ |0.30|; p ≤ 0.05). In addition to the above-mentioned taxa from ASV within BCG_35, ASV within BCG negatively correlated with both NSC and WSC were assigned to Lactobacillus equigenerosi and genera including Lachnoclostridium, Cellulosilyticum, Catenisphaera, Marvinbryantia, Erysipelatoclostridium, Mogibacterium, Lachnospiraceae XPB1014 group, Lachnospiraceae UCG-009, Coprostanoligenes group, UCG-005 metagenome within Oscillospiraceae, the Hallii group within Lachnospiraceae, the NK4A214 group of Oscillospiraceae, the Incertae Sedis genus of Ethanoligenenaceae, the WCHB1-41 genus, family, and order within Kiritimatiellae, as well as Denitrobacterium detoxificans. Additional ASV were assigned only to the family level for Synergistaceae, Lachnospiraceae, Oscillospiraceae, and Anaerovoracaceae. Forage NSC and WSC were positively correlated with BCG_94 (Clostridium sensu stricto 1, Prevotella, and Sarcina). Forage ESC was correlated with 9 of the same BCG as NSC and WSC, but also had a negative correlation with BCG_173, which contained multiple ASV assigned to Lachnospiraceae, probable genus 10 within Lachnospiraceae, Pseudobutyvibrio as well as to the p-251 genus and family of Bacteroidales. Starch was positively correlated with BCG_113 and BCG_300 and negatively correlated with two BCG, BCG_43 and BCG_48, containing ASV from Sphaerochaeta, Phascolarctobacterium, Christensenellaceae R-7 group, Coprostanoligenes group, Bacteroidales UCG-001, Rikenellaceae RC9 gut group, the NK4A214 group of Oscillospiraceae, the UCG-002 genus within Oscillospiraceae, and the UCG-010 genus and family within Oscillospirales; these BCG were also among those positively correlated with ADF/NDF and negatively correlated with CP/DE. Relationships between fecal variables and forage nutrients were also evaluated through correlation analysis (Figure 6). Acetate, butyrate, propionate, valerate, isobutyrate, isovalerate and total SCFA and BCFA were positively correlated with DE (Spearman correlation; rs ≥ 0.62) and CP (rs ≥ 0.41), but were negatively correlated with NDF (rs ≤ −0.48) and ADF (rs ≤ −0.57; p ≤ 0.008). There was also a weaker positive correlation between these fecal metabolites and starch (rs ≥ 0.39; p ≤ 0.01). Total BCFA, isobutyrate, and isovalerate were negatively correlated with NSC, WSC, and ESC (rs ≤ −0.37; p ≤ 0.02), with a weaker negative correlation between valerate and NSC and WSC (rs ≤ −0.34; p ≤ 0.03). Hexanoate and heptanoate were negatively correlated with DE, CP, and starch (rs ≤ −0.39; p ≤ 0.01), but were positively correlated with NDF, ADF, NSC, WSC, and ESC (rs ≥ 0.37; p ≤ 0.02). Conversely, fecal pH was positively correlated with ADF (rs = 0.47; p = 0.002), but negatively correlated with DE, NSC, WSC, ESC, and starch (rs ≤ −0.32; p ≤ 0.04). Plasma glucose responses to OST administration differed by forage (mixed model ANOVA with Tukey’s post hoc adjustment; p ≤ 0.01). The AUC for glucose was lowest when horses were adapted to WSG (42.4 ± 6.8 mg/dL*h) vs. CSG-SP (70.0 ± 6.8 mg/dL*h) and HAY-FA (76.3 ± 6.8 mg/dL*h; p ≤ 0.03; Figure 7a). Peak plasma glucose was also lower for WSG (110 ± 3 mg/dL) in comparison to HAY-FA (123 ± 3 mg/dL; p = 0.01), and there was a trend for lower peak plasma glucose for WSG than for CSG-SP (120 ± 3 mg/dL; p = 0.06; Figure 7b). Forage did not, however, impact OST insulin responses. Neither AUC (CSG-SP: 36.1; WSG: 29.1; HAY-FA: 32.7 ± 6.4 mIU/L*h) or peak plasma insulin (CSG-SP: 28.8; WSG: 25.5; HAY-FA: 26.6 ± 3.3 mIU/L) varied by forage (Figure S2). Fasting plasma insulin (CSG-SP: 3.69; WSG: 5.04; HAY-FA: 5.20 ± 0.64 mIU/L) and glucose (CSG-SP: 81.4; WSG: 78.7; HAY-FA: 81.3 ± 1.4 mg/dL) also did not differ by forage (Figure S3). There were trends for differences by forage in proxies for insulin sensitivity including the fasting glucose-to-insulin ratio (FGIR) and reciprocal of the square root of insulin (RISQI; mixed model ANOVA with Tukey’s post hoc adjustment; p ≤ 0.10; Figure S4). Differences in FGIR and RISQI were limited to WSG (FGIR: 25.2 ± 16.4; RISQI: 0.58 ± 0.04) vs. HAY (FGIR: 16.4 ± 2.1; RISQI: 0.46 ± 0.04; p = 0.08). However, the modified insulin-to-glucose ratio (MIRG), a proxy for the insulin secretory response, did not vary by forage (Figure S4). Glucose and insulin dynamics were only assessed after three of the forages (CSG-SP, WSG, and HAY-FA), and this smaller dataset precluded the use of random forest regression modelling for these variables. As the primary metabolic differences in grazing horse metabolism were AUC and peak plasma glucose in response to OST administration, correlation analysis was conducted to explore relationships between these metabolic variables and BCG. Only one BCG (of the 25 BCG in the reduced feature set) was correlated with AUC (rs = −0.48; p = 0.04), with members of BCG_124 including ASV within Papillibacter, Christensenellaceae R-7 group, and Synergistaceae. Four additional BCG were correlated with peak plasma glucose. Positive correlations were found between peak glucose and BCG_51 and BCG_305 (rs ≥ 0.43; p ≤ 0.04). These BCG included ASV mapped to genera including Lachnospiraceae XPB1014, Lachnospiraceae UCG-009, Erysipelatoclostridium, Catenisphaera, and Fibrobacter as well to the family level for Anaerovoracaceae. Conversely, BCG_113 (Clostridium butyricum, Papillibacter, Bacteroidales RF16 group) and BCG_259 were negatively correlated with peak glucose (rs = −0.44; p = 0.03). Members of BCG_259 included ASV assigned at the family level to Lachnospiraceae in addition to the Hallii group within Lachnospiraceae. While relationships between AUC and peak plasma glucose and forage nutrients were identified through correlation analysis (Spearman correlation; rs ≥ |0.41|; p ≤ 0.04; Figure 7c), these metabolic variables were not correlated with any fecal variables including SCFA, BCFA, and pH. Forage NSC, WSC, and ESC were positively correlated with AUC (rs ≥ 0.53; p ≤ 0.007) and peak glucose (rs ≥ 0.41; p ≤ 0.04). There was also a negative correlation between CP and glucose responses to OST administration (rs ≤ −0.50; p ≤ 0.01). Forage DE, NDF, ADF, and starch, however, were not correlated with AUC or peak glucose. Warm-season grasses can be utilized to bridge the “summer slump” forage gap in cool-season grass grazing systems [1,2], and differences in NSC between these forage types could have implications for equine metabolic health [1,5,9]. However, there is limited information on the impacts of grazing warm-season grasses on the equine hindgut microbiome as well as potential associations between forage nutrients, the hindgut microbiome, and equine metabolic responses. Therefore, this study aimed of to characterize the fecal microbiota of horses adapted to different forage types and to explore relationships between forage nutrients, microbial composition, fecal metabolites, and glycemic responses of grazing horses. Results of this study clearly demonstrated that shifts in fecal microbiome structure and species composition occur as horses are adapted to different forages within an integrated warm- and cool-season grass rotational grazing system. This was supported by statistical differences in both α- and β-diversity. Furthermore, random forest classification modelling was able to predict forage type based on microbial community composition, indicating the influence of forage type on the fecal microbiome. Finally, forage-specific BCG were identified through LEfSe analysis, but the 25 BCG identified as most predictive of forage type through random forest modeling and subsequently analyzed by LEfSe represented only ~10% of the total fecal microbiota across all forages. This indicates that distinct and identifiable shifts in microbial composition do occur as horses adapt to different forage types. However, the majority of the microbiome (~90% in the current study) is resistant and/or resilient to potential perturbations induced by transitioning among forage types with different physical and chemical properties, including between cool-season and warm-season grass pastures. The BCG identified as markers specific to HAY-SP included multiple ASV assigned to taxa to which fibrolytic and butyrate-producing functions have been previously ascribed [74,75,76]. The BCG specific to this forage contained a heavy representation of ASV within the Lachnospiraceae family as well as Fibrobacter, Ruminococcus, and Pseudobutyvibrio. Prior studies have also reported increased prevalence of Lachnospiraceae in horses fed hay vs. pasture [19,77]. Conversely, ASV assigned to Anaerovibrio were also identified in BCG markers of HAY-SP. Increases in this genus in response to abrupt inclusion of dietary starch have been documented [78] as well as in temporal proximity to oral administration of oligofructose in experimental laminitis induction models [79,80]. The co-occurrence of bacteria in these taxonomic groups could potentially be reflective of the nutrient composition of HAY-SP, which was the highest in both fiber and NSC of all forages. Co-occurrence of these bacteria may also reflect cross-feeding relationships between bacteria and/or metabolic plasticity of bacterial populations. These factors may also account for unexpected associations revealed by analysis in the current study, such as ASV assigned to Streptococcus within BCG markers of HAY-FA, despite the relatively low NSC content of this forage. However, Zhu et al. [77] also reported a lower abundance of species within Streptococcaceae in horses maintained on pasture vs. horses fed hay or silage [77]. Overall, the fecal microbiota of horses adapted to cool-season pasture was characterized by bacteria capable of utilizing rapidly fermentable fibers, which could explain the greater fecal SCFA concentrations for CSG-SP and CSG-FA. Capacity for hemicellulose fermentation and SCFA production have been previously documented in the Bacteroidales BS11 gut group [81], which was identified only in BCG markers of CSG-SP. Equine studies have found increased relative abundance of Bacteroidales BS11 gut group in horses fed barley vs. a hay diet [82] and in horses presenting with colic [83,84]. For CSG-FA, BCG markers included genera such as Alloprevotella and Erysipelatoclostridium, which are also associated with degradation of fermentable fibers [85,86,87]. The BCG markers of CSG-FA also included ASV assigned WCHB1-41 clade within Kiritimatiellae. Prior studies have found enrichment of the phylum Kiritimatiellaeota in the fecal microbiota of horses with equine metabolic syndrome and insulin dysregulation [26,27]. In contrast to results of the present study, Fitzgerald et al. [27] reported increased abundance of this phylum in response to a change from pasture to a hay diet in both healthy and insulin dysregulated ponies, and Ericsson et al. [88] similarly found increased abundance of the class Kiritimatiellae when horses with equine metabolic syndrome were transitioned from pasture to a hay diet. These conflicting results reinforce that nutritional composition of specific forages, rather than form of forage alone, need to be considered when evaluating interstudy effects of diet on the equine microbiome. A number of interesting relationships were found between BCG identified as markers specific to WSG and fecal variables, forage nutrients, and horse glucose metabolism. Warm-season grasses are characteristically lower in soluble carbohydrates than cool-season grasses [1,5], and of all forages evaluated in the current study, NSC and WSC were lowest in WSG. Four of the five BCG markers of WSG were negatively correlated with forage NSC and WSC, including BCG_35, which contained ASV assigned Akkermansia as well as Fibrobacter, Treponema, Christensenellaceae R-7 group, and the Family XIII AD3011 group of Anaerovoracaceae. Akkermansia has been linked to metabolic health via regulation of inflammatory responses and has also been explored for probiotic use in animal species [89,90,91,92]. Lindenberg et al. [93] recently demonstrated immune (and inflammatory) modulation by specific hindgut microbiota in horses, including Akkermansia spp. Furthermore, supplementation with mannanoligosaccharides and fructooligosaccharides led to increased abundance of Akkermansia muciniphilia in a subsequent study [94], and improvements in insulin sensitivity have been previously documented in horses supplemented with low doses of fructooligosaccharide [95,96]. Markers of WSG also included BCG_113, which contained an ASV assigned to Clostridium butyricum. Clostridium butyricum is a prolific butyrate producer that has also been investigated for probiotic use due to its capacity to promote anti-inflammatory responses and improve gut barrier function and metabolic health [97,98,99]. BCG_35 was positively correlated with CP as well as isobutyrate and total BCFA, which reflects the proteolytic capacity of this BCG. BCG_113 was also positively correlated with total SCFA and all three major SCFA (acetate, butyrate, and propionate) in addition to isobutyrate, isovalerate, and total BCFA. This BCG was also negatively correlated with peak plasma glucose. These findings suggest that this bacterial group including Clostridium butyricum could play a role in modulation of equine metabolic health. While limited connections between the gut microbiota and metabolic responses were observed in the current study, three of the five BCG correlated with AUC and peak plasma glucose were WSG-specific BCG markers. Further research is necessary to determine if other WSG species or varieties would produce similar effects as those observed in the current study. While Akkermansia and Clostridium butyricum have been investigated in mice and other species, comparatively little research has been conducted to understand the function of these bacteria in the equine hindgut. The relationships found between ASV in these taxa and forage nutrients, fecal metabolites, and equine glycemic responses in addition to associations with low-NSC warm-season grasses in the current study support further research to determine the role of these bacteria, factors including dietary interventions that can promote prevalence in the hindgut, and potential probiotic applications. Results of this study also confirmed the strong influence of dietary nutrients on the equine microbiome and provided insights into the complex relationships between forage nutrients and the gut microbiota. The concentrations of several nutrients could be predicted based on microbial community composition. Furthermore, regression model accuracy improved when the reduced feature set identified through prior forage classification modeling was utilized for prediction of nutrient concentrations. This indicates that differences in forage nutrients were driving the shifts in equine fecal microbial communities as horses adapted to various forages within the integrated grazing system. Results of this study revealed the influence of soluble carbohydrates including NSC and WSC on fecal microbial community composition. In contrast, fiber was not identified as a key nutrient shaping differences in microbial communities. Forage NSC and WSC concentrations could be predicted based on microbial community composition with an accuracy of R2 = 0.61 and R2 = 0.67, respectively, while predictive accuracy for ADF and NDF was < 0.40 (R2). Random forest modeling also revealed that in addition to soluble carbohydrates, the gut microbiota were also influenced by CP (R2 = 0.62). The impact of CP on the gut microbial community was interesting, but unsurprising, as two-thirds to three-fourths of total tract nitrogen digestion and absorption occurs in the equine hindgut [100], and many microbial species are capable of proteolytic fermentation [101]. Prior studies in horses have found distinct effects of high-fiber vs. lower-fiber diets on the hindgut microbial community, with benefits of increased fiber including greater microbial diversity [102], reduction of lactic-acid producing bacteria [101,103], and less-acidic hindgut pH [104,105]. However, these previous studies have been primarily conducted in horses fed concentrate vs. forage-based diets in which there was broad variation in fiber concentrations between treatments. In the current study, horses were maintained on a forage-only diet throughout, and there were relatively high concentrations of NDF and ADF found across all forages. It is possible that only minimal shifts occur in bacterial populations most responsive to fiber above a certain concentration threshold, and that these populations remained relatively stable throughout the study (and thus fiber concentration was not able to be predicted with strong accuracy by random forest modeling). However, it should be noted that the concentrations of NSC, WSC, and CP in forages is low in comparison to that of fiber. Additionally, in contrast to fiber, these nutrients are all subject to digestion in the equine foregut, and only a fraction of the total soluble carbohydrates and CP ingested would be available for bacterial fermentation in the hindgut. Thus, only a small amount of these nutrients were capable of exerting influence on gut microbial communities. Digestibility was not evaluated in the current study, however, and a more in-depth analysis of total digestibility and digestibility of specific nutrients would be necessary to fully understand the interactions between forage nutrients and the gut microbiota. Fecal pH is commonly utilized as a marker of microbial activity in the equine hindgut [20,103]. Differences in fecal pH in the current study indicated that functional changes occur in the equine gut microbiota, in addition to shifts in microbiome structure and composition, as horses adapt to different forages. Fecal pH was highest in horses adapted to WSG and HAY-FA, which were lower in NSC than CSG-SP, CSG-FA. Accordingly, fecal pH was negatively correlated with forage NSC. Lower fecal pH is broadly associated with hindgut dysfunction in horses [106,107], and thus the higher fecal pH in horses adapted to WSG in comparison to cool-season pasture could suggest some benefit to grazing horses on warm-season grass pastures. However, mean pH was above 6.5 for CSG-SP, CSG-FA, and HAY-SP, which is within ranges previously documented in healthy forage-fed horses [33,108]. Therefore, while statistically significant, the difference in fecal pH between horses adapted to WSG vs. cool-season pasture may not be of physiological relevance. Differences in fermentation metabolites across forages did not mirror results for fecal pH, as differences between forages for SCFA and BCFA were primarily between pasture forages and the hay diets. Numerically, SCFA and BCFA concentrations in horses adapted to WSG were intermediate between the cool-season pasture (greatest) and hay (lowest), but these differences were not statistically significant. Furthermore, while statistically significant, the predictive accuracy of random forest regressors for fecal metabolites and pH were lower in comparison to those found for forage nutrients. Numerous studies have noted that while the gastrointestinal tract harbors a phylogenetically diverse community, many unrelated microorganisms are capable of performing similar functions [109,110,111,112,113]. The relatively poor predictive accuracy of regressors for fecal metabolites in the current study is likely due to this functional redundancy within the equine microbial community. Functional redundancy has been previously suggested as a contributing factor in stability of the gut microbial communities [111,113] including within the equine microbiome during transitions between warm- and cool-season grasses [24]. While there were distinct shifts in the fecal microbiota across forages in the integrated system, forage type exerted minimal effects on glucose metabolism. A large body of research, primarily conducted in mouse models, has established that diet (and dietary intervention) modulates metabolic health in a microbiome-dependent manner [29,30,114]. However, shifts in gut microbial structure and function may precede changes in metabolic outcomes [115]. The adaptation period utilized in the present study was of a similar duration as used in prior studies evaluating the microbiome of forage-fed horses [22,23,116], and is considered sufficient for stabilization of microbial communities [95]. However, longer treatment periods are often required to assess metabolic adaptations to diet [13,103,117,118]. The lower AUC and peak plasma glucose in horses adapted to WSG in the current study does suggest that glucose may be cleared more rapidly in horses adapted to this forage, but a longer adaptation period would be necessary to confirm these results. Regardless, glucose and insulin responses to the OST for all forages evaluated in the current study were within normal and previously reported ranges [12,34], indicating that substantial metabolic changes are unlikely within the context of integrated rotational grazing management even with the observed changes in the fecal microbiota, fecal metabolites, and pH. Additionally, the lack of correlation between any fermentation metabolites and glucose responses, as well as the relatively small number of BCG correlated with AUC and peak plasma glucose, suggest that any difference in glycemic responses of horses in the current study may not have been heavily dependent on shifts in the hindgut microbiome. Seasonal and environmental factors should be considered when interpreting results of this study. Prior studies have reported seasonal changes in microbial diversity and species composition [119,120,121] but also lacked a true seasonal control, and thus, the effect of seasonality on the equine hindgut microbiome requires further investigation. Seasonal variance in insulin sensitivity has been documented in grazing horses [15,122], but when fed controlled diets without nutrient fluctuations seen in pasture forages, minimal seasonal differences in circulating glucose and insulin and insulin sensitivity have been found in healthy horses [15,123,124]. Environment and management factors beyond season can also impact pasture forage nutrient composition. Characterizing the microbiome of a larger number of grazing horses over multiple years and in multiple locations/regions would allow a more robust evaluation of the impacts of cool- versus warm-season grasses on the equine hindgut microbiome. Finally, it should be noted that the analytical approach in this study differs from more conventional taxon-based analysis. Rather, this study implemented a guild-based approach, grouping individual microbial ASV by co-abundance to subsequent analysis of abundance profiles [123,124,125,126,127]. This strategy has been previously utilized as an alternative to grouping bacteria by taxonomy [24,29,30,92,121]. Substantial genetic variation is possible within taxa, even at the species level, and therefore bacteria with similar taxonomic assignments may not represent a functionally homologous group [29,92,127]. Results of the current study also illustrate this concept, as ASV with the same assigned taxonomy were found in BCG characteristic of different forages as well as in separate BCG that positively and negatively responded to forage nutrients and with both positive and negative relationships with fecal metabolites and grazing horse metabolism. In conclusion, distinct shifts in equine fecal microbial community structure and composition occur as horses adapt to different forages within an integrated warm- and cool-season grass rotational pasture system, but a substantial impact of this management practice on glucose metabolism in healthy adult grazing horses is unlikely. Forage NSC, WSC, and CP were the most influential nutrients driving these shifts in microbial composition. Results of this study underscore the potential for relatively small amounts of NSC to influence hindgut microbial composition and also that protein utilization may be an important ecological niche within the microbiome of forage-fed horses. Fecal BCFA and SCFA concentrations were higher in horses adapted to all pasture forages versus hay, but in comparison to forage nutrients, bacterial community composition did not have as strong an impact on fermentation metabolites, likely reflecting functional redundancy of the microbial community. The guild-based analytical approach utilized in this study also identified key relationships between specific bacterial groups associated with adaptation to warm-season grass pasture and forage nutrients, fecal metabolites, and equine glycemic responses to administration of oral sugar tests. Relationships identified in this study revealed new insights and targets for future research necessary to better understand the function of Akkermansia spp. and Clostridium butyricum in the hindgut microbiome of grazing horses, as these bacteria may play a role in modulation of equine metabolic health.

PMC10000172

Md. Ariful Islam,Md. Bayazid Hossen,Md. Abu Horaira,Md. Alim Hossen,Md. Kaderi Kibria,Md. Selim Reza,Khanis Farhana Tuly,Md. Omar Faruqe,Firoz Kabir,Rashidul Alam Mahumud,Md. Nurul Haque Mollah

Exploring Core Genes by Comparative Transcriptomics Analysis for Early Diagnosis, Prognosis, and Therapies of Colorectal Cancer

21-02-2023

colorectal cancer,gene expression profiles,core genes,early diagnosis,prognosis,therapies,integrated statistics and bioinformatics approaches

Simple Summary Colorectal cancer (CRC) is a complex disease that has a high mortality rate. This study explored CRC-related core genes (CGs) from multiple microarray gene-expression profiles in the NCBI-GEO database by combining some statistics and bioinformatics techniques. It also disclosed their molecular functions, biological processes, cellular components, signaling pathways, and transcriptional and post-transcriptional regulatory factors by using different online bioinformatics tools and databases. The prognostic power of CGs was investigated from the independent TCGA database by using survival probability curves and box plots of CGs-expressions in different stages (control, Stage 1, Stage 2, Stage 3, and Stage 4) of CRC. Finally, a few CGs-guided drug molecules were suggested for the treatment of CRC by molecular docking and dynamic simulation studies. Therefore, the findings of this study would be useful resources for early diagnosis, prognosis, and therapies of CRC. Abstract Colorectal cancer (CRC) is one of the most common cancers with a high mortality rate. Early diagnosis and therapies for CRC may reduce the mortality rate. However, so far, no researchers have yet investigated core genes (CGs) rigorously for early diagnosis, prognosis, and therapies of CRC. Therefore, an attempt was made in this study to explore CRC-related CGs for early diagnosis, prognosis, and therapies. At first, we identified 252 common differentially expressed genes (cDEGs) between CRC and control samples based on three gene-expression datasets. Then, we identified ten cDEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the CGs, highlighting their mechanisms in CRC progression. The enrichment analysis of CGs with GO terms and KEGG pathways revealed some crucial biological processes, molecular functions, and signaling pathways that are associated with CRC progression. The survival probability curves and box-plot analyses with the expressions of CGs in different stages of CRC indicated their strong prognostic performance from the earlier stage of the disease. Then, we detected CGs-guided seven candidate drugs (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) by molecular docking. Finally, the binding stability of four top-ranked complexes (TPX2 vs. Manzamine A, CDC20 vs. Cardidigin, MELK vs. Staurosporine, and CDK1 vs. Riccardin D) was investigated by using 100 ns molecular dynamics simulation studies, and their stable performance was observed. Therefore, the output of this study may play a vital role in developing a proper treatment plan at the earlier stages of CRC.

Exploring Core Genes by Comparative Transcriptomics Analysis for Early Diagnosis, Prognosis, and Therapies of Colorectal Cancer Colorectal cancer (CRC) is a complex disease that has a high mortality rate. This study explored CRC-related core genes (CGs) from multiple microarray gene-expression profiles in the NCBI-GEO database by combining some statistics and bioinformatics techniques. It also disclosed their molecular functions, biological processes, cellular components, signaling pathways, and transcriptional and post-transcriptional regulatory factors by using different online bioinformatics tools and databases. The prognostic power of CGs was investigated from the independent TCGA database by using survival probability curves and box plots of CGs-expressions in different stages (control, Stage 1, Stage 2, Stage 3, and Stage 4) of CRC. Finally, a few CGs-guided drug molecules were suggested for the treatment of CRC by molecular docking and dynamic simulation studies. Therefore, the findings of this study would be useful resources for early diagnosis, prognosis, and therapies of CRC. Colorectal cancer (CRC) is one of the most common cancers with a high mortality rate. Early diagnosis and therapies for CRC may reduce the mortality rate. However, so far, no researchers have yet investigated core genes (CGs) rigorously for early diagnosis, prognosis, and therapies of CRC. Therefore, an attempt was made in this study to explore CRC-related CGs for early diagnosis, prognosis, and therapies. At first, we identified 252 common differentially expressed genes (cDEGs) between CRC and control samples based on three gene-expression datasets. Then, we identified ten cDEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the CGs, highlighting their mechanisms in CRC progression. The enrichment analysis of CGs with GO terms and KEGG pathways revealed some crucial biological processes, molecular functions, and signaling pathways that are associated with CRC progression. The survival probability curves and box-plot analyses with the expressions of CGs in different stages of CRC indicated their strong prognostic performance from the earlier stage of the disease. Then, we detected CGs-guided seven candidate drugs (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) by molecular docking. Finally, the binding stability of four top-ranked complexes (TPX2 vs. Manzamine A, CDC20 vs. Cardidigin, MELK vs. Staurosporine, and CDK1 vs. Riccardin D) was investigated by using 100 ns molecular dynamics simulation studies, and their stable performance was observed. Therefore, the output of this study may play a vital role in developing a proper treatment plan at the earlier stages of CRC. Cancer is a complex disease caused by multiple alterations at the genetic and epigenetic levels that increasingly lead to abnormal cell division and cellular transformation [1,2]. Colorectal cancer (CRC) is the third most common solid malignancy and the second deadliest tumor worldwide [3]. The CRC incidence is expected to rise by 60%, with 2.2 million new cases and 1.1 million deaths globally by 2030 [4]. The number of new incidences and mortalities is increasing due to insufficient evidence about diagnostic biomarkers and the molecular mechanism of CRC [4]. Early detection of CRC is associated with lower morbidity and mortality and a higher survival rate compared with late detection. For example, the five-year survival rate increases from 11% (late detection) to 90% at early detection of CRC [5]. However, the survival rate significantly decreases, and the cost of treatment increases, whereas CRC is identified in later stages compared to earlier stages [3,6,7]. Therefore, early diagnosis, prognosis, and therapies leads to reduce CRC-related mortality [7]. Several factors, including excessive alcohol consumption, obesity, unhealthy dietary habits, and an abnormal lifestyle, are all considered non-causal risk factors for CRC development. However, these non-causal risk factors cannot be used for CRC detection at an earlier stage. Generally, differentially expressed genes (DEGs) between cancer and control samples are considered as the cancer-causing/stimulating genes. A gene may show a differential expression pattern between cancer and control samples for several reasons, including mutation, DNA methylation, and other epigenetic stimulations. The genes that are associated with the development of cancer, are known as oncogenes (upregulated DEGs) and tumour-suppressor genes (down-regulated DEGs) [1,2,8]. Thus, cancer incidence, development, recurrence, and non-recurrence are associated with pathogenetic processes of DEGs [9]. Several studies reported some dysregulating genes in the CRC cases compared to non- CRC cases that are associated with CRC proliferation, differentiation, apoptosis, metastasis, recurrence, and lower survival [10,11,12,13]. Many earlier transcriptomics studies explored the pathogenetic processes of CRC through DEGs [14,15,16,17,18,19,20,21]. However, none of them discussed rigorously about early diagnosis, prognosis, and therapies for CRC. Patil et al. (2021) [22] identified forty CRC-causing/stimulating core DEGs and recommended their application for the early diagnosis of CRC. Though the number 40 is much smaller than the whole genome size, it may also not be suitable for further investigation by the wet-lab researchers, since wet-lab experiments with 40 DEGs might be costly, time-consuming, and laborious. So, a smaller set of core DEGs might be required for further experimental investigation. On the other hand, this study did not provide any recommendations about suggested core DEGs guide any drug molecules for therapies for CRC. Therefore, the present study attempted to discover CRC-causing/stimulating core-DEGs, highlighting their pathogenetic processes for early prognosis, diagnosis, and therapies of CRC. The pipeline of this study is given in Figure 1. The necessary datasets that were analyzed in this study are described below. We downloaded three microarray datasets of CRC using the accession IDs GSE106582, GSE110223, and GSE74602 from the NCBI Gene Expression Omnibus (GEO) database. The GPL10558 platform was used for the GSE106582 dataset, which contains 194 samples, including 77 cancer and 117 adjacent tissue samples. The GPL96 platform was used for the GSE110223 dataset, which contains 26 samples, including 13 cancer and 13 adjacent tissue samples. The GPL6104 platform was used for the GSE74602 dataset, which contains 60 samples, including 30 cancer and 30 adjacent tissue samples. For identifying candidate repurposed drugs, we collected target receptor-guided drug molecules from the DSigDB [23] database (Table S1). We used the GEO2R web tool [24] based on the LIMMA (linear models for microarray data) approach to identify DEGs between cancer and adjacent tissue samples for each of the three datasets. The LIMMA method uses modified t-statistics to calculate p-values. We used the Benjamini–Hochberg (BH) approach [25] to adjust the p-values. The log2 fold-change (Log2FC) and adjusted p-values were used to separate the up- and down-regulated DEGs by the following cut-offs: We considered common DEGs (cDEGs) from three datasets to identify core CGs. All DEGs were visualized using a Venn diagram using FunRich 3.1.3 [26]. The protein-protein interaction (PPI) network was utilized to detect core genes (CGs) from cDEGs. We considered the STRING database [27] with a median confidence score (MCS) of 0.4 to produce a PPI network of cDEGs and Cytoscape software for better visualization of the network [28]. The CGs were selected from the PPI network using the CytoHubba plugin in Cytoscape [28,29]. The present study used maximal clique centrality (MCC) topology analysis methods to identify the CGs. To investigate the association of CGs with the different stages of CRC based on independent databases, we performed box-plot analysis based on their expression levels in different CRC progression stages (Normal status, Stage 1, Stage 2, Stage 3, and Stage 4) through the UALCAN web tool with the TCGA-COAD and TCGA-READ databases [30,31]. To investigate the prognosis power of CGs by multivariate Kaplan–Meier survival probability curves, we considered the SurvExpress web tool based on the TCGA-COAD and TCGA-READ databases (https://portal.gdc.cancer.gov/exploration, accessed date: 2 January 2022) [30,32]. The log-rank test was used in SurvExpress, and the risk group hazard ratio with a 95% confidence interval was included in the Kaplan-Meier survival plot [32]. The p-value < 0.05 was used as the cut-off. CGs-set enrichment analysis (cGSEA) determines the classes of genes or proteins that are over-represented (enriched) in a predefined large set of genes or proteins that are associated with the terms of interest, including gene ontology (GO), pathways, diseases, chemicals, drugs, biomolecules (miRNA, TFs), and so on. To detect the significantly enriched terms of interest, let Ai be the predefined gene-set in the ith term of interest, and Mi denotes the number of CGs in Ai (i = 1, 2,…, r); T is considered as an enriched gene number that created a combined set A such that and where is considered as the complement-set of Ai. Again, suppose t represents the number of CGs and mi denotes the number of CGs subset of Ai. Table 1 summarizes these results. The Enrichr web tool [33] was considered to investigate the association of CGs with terms of interest. This web tool uses the Fisher exact test to examine the significance of the association between CGs and ith term of interest. We considered the Enrichr web tool [33] to verify the association of CGs with different diseases, including CRC, using the DisGeNET database, which was constructed based on 21,671 genes and 30,170 diseases [34]. It measures the association of a disease with a group of CGs that are overlapped (common) with the reference gene set of that disease (see Table 1). To investigate the pan-cancer role of CGs, we performed the pan-cancer analysis of each CG by the TIMER 2.0 web tool [35] with the TCGA database [36]. In both cases, the p-value < 0.05 was selected as the cut-off for statistical significance. To disclose the pathogenetic processes of CGs, we performed CGs-set enrichment analysis with GO terms and pathways by using the Enrichr web tool [33]. Biological process (BPs), molecular function (MFs), and cellular component (CCs) were investigated to explore potential GO terms and pathways based on the KEGG database as displayed in Table 1. A p-value < 0.001 was used as the cut-off for the statistical significance. A gene regulatory network (GRN) provides information about molecular regulators that connect to regulate the gene expression level of mRNA. Transcription factors (TFs) and microRNAs (miRNAs) are considered the major regulators of gene expression. TFs proteins are regarded as the significant contributors to GRN because they bind to a particular region of DNA (enricher/promoter) and influence gene expression at the transcriptional level. A miRNA is a non-coding RNA considered a central post-transcriptional regulator of gene expression. The human genome contains up to 1600 TFs and 1900 miRNAs. A TFs vs. CGs network is considered an undirected graph, where nodes represent TFs or CGs and edges depict interactions between TFs and CGs, respectively. A TF-node is considered the major regulatory factor for CGs if it contains the largest number of interactions with CG nodes. We considered regulatory analysis of CGs (transcription factors (TFs) vs. CGs and micro-RNAs (miRNAs) vs. CGs) through Network Analyst [37] platform-based JASPAR [38] and TarBase [39] databases, respectively, to detect the core transcriptional and post-transcriptional regulators of CGs. For better illustration, we used Cytoscape software [28]. The core regulators were chosen by utilizing degree [40] and betweenness [41] scores. We conducted molecular docking studies of receptors and drug molecules to explore FDA-approved repurposable drugs for CRC. CGs-mediated proteins and related TFs proteins were considered drug target receptors (p = 14). The online database DSigDB was used to extract CGs-guided drug agents. The 3-dimensional (3D) structures of AURKA, TOP2A, CDK1, PTTG1, CDC20, MAD2L1, CKS2, MELK, TPX2, YY1, and SRF targets were retrieved from the Protein Data Bank (PDB) [42] with IDs 6VPM, 5NNE, 5LQF, 7NJ1, 4GGC, 2V64, 5LQF, 5M5A, 6VPM, 4C5I, and 1HBX, respectively. The remaining targets, FOXC1, CDKN3, and NFIC, were retrieved from the SWISS-MODEL [43] with the Uniport IDs Q12948, P08651, and Q16667, respectively. Using the PubChem database [44], we retrieve the 3D structures of all (q = 158) drug molecules. The visualization of the receptor proteins and co-crystal ligands were performed via the Discovery Studio Visualizer 2019 [27]. The receptor proteins were processed using AutoDock tools [45] and the Swiss PDB viewer by adding the structural charges and reducing the energy of receptors, respectively [45,46]. The docked complexes were analyzed through Discovery Studio Visualizer 2019. Let Bij be the binding affinity (BA) score of ith receptors (i = 1, 2,…, p) and jth drugs (j = 1, 2,..., q). The receptors and drug molecules were sorted by the decreasing order of their average BA score for selecting the top-ordered few potential candidate repurposable drugs. We performed MD simulations of the top-orderedprotein–ligand complexes (TPX2–Manzamine A, CDC20–Cardidigin, MELK–Staurosporine, and CDK1–Riccardin D) through the YASARA software (Version: 22.8.22) [47] based on the AMBER14 force field [48]. Prior to simulation, the hydrogen-bonding network of each complex in a simulated cell was optimized using a TIP3P water model [45]. The periodic limit conditions were kept constant at 0.997 gL-1 of solvent concentration. The primary energy was minimized in each simulation by considering the steepest gradient technique with 5000 cycles. The complexes “TPX2–Manzamine A”, “CDC20–Cardidigin”, “MELK–Staurosporine”, and “CDK1–Riccardin D” consist of a total of 56,287, 35,859, 81,347, and 45,153 atoms, respectively. At the Berendsen thermostat [49] and constant pressure, a 100 ns MD simulation was examined. Please see our previous publications for details about the MD simulation strategy [50,51,52,53]. For subsequent analysis, we took snapshots of the trajectories every 250 ps, ran them via the built-in script of YASARA [54] macro, and calculated the binding free energy of the MM-Poisson–Boltzmann surface area (MM-PBSA) by analyzing all the snapshots [55]. To calculate binding-free energy, we used the following formula: More positive binding energy represents stronger binding. To identify DEGs from each of three microarray gene-expression datasets, we used the statistical LIMMA approach through the GEO2R web tool, with the cut-off at adjusted p-value < 0.05 and |log2(fold change)| > 1. In the GSE106582 dataset, we identified 594 DEGs, including 213 upregulated and 381 downregulated genes. In the GSE110223 dataset, we identified 625 DEGs that contain 260 upregulated and 365 downregulated genes. In the GSE74602 dataset, we identified 1674 DEGs, including 673 upregulated and 1001 downregulated genes. The Venn diagram in Figure 2 visualizes the common DEGs among the three datasets. The Venn diagram exhibits 252 cDEGs among the three datasets. We construct the PPI network of cDEGs and visualize the PPI network to identify the potential genes most significantly associated with the development of CRC. The PPI network contains 216 nodes and 616 edges, with a confidence score of 0.40. Then, the MCC topology analysis method of CytoHubba was performed to calculate the top-ranked CGs within the network. We found the ten top-ranked (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) genes (see Figure 3). These top ten CGs were identified as major controllers of CRC and considered for subsequent analysis. The box-plot analysis based on an independent database represents a high difference between normal expression and every CRC progression stage (Stage 1, Stage 2, Stage 3, and Stage 4) expression of all CGs (see Figure 4A and Figure S1). So, our proposed CGs have strong prognostic power to identify CRC at an earlier development stage. A survival analysis was performed to examine the prognosis power of CGs. The multivariate Kaplan–Meier survival plot of CGs expressions using the TCGA-COAD and TCGA-READ databases represents a significant difference (p-value < 0.001) between lower-risk and higher-risk groups (see Figure 4B). The enrichment analysis of the CGs-set with different diseases based on the DisGeNET database showed that the CGs-set is significantly associated with various diseases (p-value < 0.05). Figure 3A and Table S2 show the top-ranked 20 diseases, all of which are different types of cancer, including CRC. We observed that 3 CGs (MELK, CKS2, CDC20) and 2 CGs (MELK, CDC20) do not overlap with the reference gene-sets of colon carcinoma and colorectal carcinoma, respectively (Figure 5A and Figure S2, and Table S2). These results suggested a pan-cancer role for CGs (Figure S3). To investigate the pan-cancer role of CGs, we also performed pan-cancer analysis based on the TCGA database. We selected the top-ranked 20 cancers as displayed in Figure 5B. Figure 5A,B commonly showed that eight cancers (colon adenocarcinoma, bladder urothelial carcinoma, esophageal carcinoma, glioblastoma multiforme, liver hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, and stomach adenocarcinoma) are significantly associated with CGs. Enrichment analysis of the CGs was performed using the Enrichr web tool. Table 2 shows the annotated GO terms in three categories (BPs, CCs, and MFs). In the case of biological processes (BPs), CGs were mainly involved in mitotic cell-cycle phase transition (GO:0044772), anaphase-promoting complex-dependent catabolic process (GO:0031145), regulation of G2/M transition of mitotic cell cycle (GO:0010389), mitotic spindle organization (GO:0007052), regulation of mitotic cell cycle (GO:0007346). In molecular function (MFs), CGs were mainly involved in histone kinase activity (GO:0035173), RNA polymerase II CTD heptapeptide repeat kinase activity (GO:0008353), protein kinase binding (GO:0019901), CXCR chemokine receptor binding (GO:0045236), cyclin-dependent protein kinase activity (GO:0097472), etc. In cellular components (CCs), CGs were mainly involved in the spindle (GO:0005819), cyclin-dependent protein kinase holoenzyme complex (GO:0000307), serine/threonine-protein kinase complex (GO:1902554), intracellular non-membrane-bounded organelle (GO:0043232), mitotic spindle (GO:0072686), etc. The KEGG pathway enrichment analysis results for CGs were also shown in Table 2. The KEGG pathways of CGs were enriched in the cell cycle, bladder cancer, oocyte meiosis, human T-cell leukemia virus one infection, progesterone-mediated oocyte maturation, etc. TFs proteins and miRNAs play a fundamental role in the modification of gene expression at the transcriptional and post-transcriptional levels, respectively. To explore the major transcriptional regulatory factors of CGs, we constructed a TFs vs. CGs interaction network where round nodes with red color represent the CGs and square nodes with green/purple color represent the TFs (see Figure 6A). TFs proteins vs. CGs regulatory analysis revealed four highest-ranking significant candidate TFs modifiers (NFIC, FOXC1, YY1, and GATA2) that may regulate the expression of CGs at the transcriptional level (see Figure 6A). Similarly, we constructed an undirected interaction network of miRNAs vs. CGs to reveal the post-transcriptional regulator of CGs, where red color nodes represent the CGs and green/blue color nodes illustrate the miRNAs (see Figure 6B). The miRNAs vs. CGs regulatory network analysis revealed six highly interacted non-coding RNAs (miRNAs) such as hsa-mir-147a, hsa-mir-129-2-3p, hsa-mir-124-3p, hsa-mir-34a-5p, hsa-mir-23b-3p, and hsa-mir-16-5p that act as gene expression regulators at the post-transcriptional level (see Figure 6B). So, those identified TFs and miRNAs may influence the gene expression of CGs at the transcriptional and post-transcriptional levels, respectively. We considered 10 CG-guided proteins and 4 TFs proteins as target-receptor proteins for molecular docking. A structural interaction was carried out between target-receptor proteins and 158 drug agents by molecular docking studies, which computed the receptor-drug binding affinities (BA) for each interaction (see Figure 7). Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D were shown to have strong BA against all of the target receptors, and their average BA lies between −9.20 and −8.2 (kcal mol−1). Among those drugs, Manzamine A was shown to have the highest BA against almost every target protein, with an average BA of −9.2 kcal mol−1. Therefore, we proposed those seven drugs (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) as candidate drug agents and displayed them in red color in Figure 7. We also revealed the structural interaction profiles of four top-ordered receptor proteins and drug complexes in Table 3. TPX2–Manzamine A, CDC20–Cardidigin, MELK–Staurosporine, and CDK1–Riccardin D complexes have shown the highest BA in molecular docking analysis (Table 3). So, we considered those complexes for examining their binding stability through MD simulations. We observed that each protein-ligand complex (TPX2–Manzamine A, CDC20–Cardidigin, MELK–Staurosporine, and CDK1–Riccardin D) showed significant stability in a 100 ns MM-PSSA simulation (see Figure 8A). RMSD values corresponding to each complex were calculated (see Figure 8A). RMSD values showed lower flexibility around 1.5 Å to 3.0 Å for all four complexes. The average RMSD values for TPX2-Manzamine A, CDC20–Cardidigin, MELK–Staurosporine, and CDK1–Riccardin D complexes were 2.592 Å, 1.724 Å, 2.235 Å, and 2.516 Å, respectively. The CDC20–Cardigin complex showed a more substantial structural rigidity than the other three complexes, gained equilibrium at four ns, and displayed good stability after that. MELK–Staurosporine showed a gradual increase in RMSD before 22 ns, and after this time point, the RMSD score of the complex illustrated almost stable movement between 2.2 Å and 2.50 Å for 68 ns. After that, there were irregular fluctuations in the RMSD. On the contrary, CDK1-Riccardin D complexes exhibited instability, and the RMSD displayed an upward trend from 2.0 Å to 3.2 Å over time. Similarly, TPX2–Manzamine A showed irregular oscillation in RMSD between 1.7 Å and 3.3 Å. In addition, the MM-PBSA binding energy for four complexes was also computed. Figure 8B illustrates the binding energies of the complexes. On average, TPX2–Manzamine A, CDC20–Cardidigin, MELK–Staurosporine, and CDK1–Riccardin D complexes produced MM-PBSA binding energies of 84.39 KJ mol−1, −95.07 KJ mol−1, −235.86 KJ mol−1, and 154.39 KJ mol−1, respectively. To explore core genomic biomarkers and their mechanisms in the CRC progression, firstly, we identified 252 cDEGs between CRC and control samples, out of around 40,000 genes in three gene expression datasets of the NCBI-GEO database with accession numbers GSE106582, GSE110223, and GSE74602. Though the number of DEGs is much smaller than the total number of genes, it may still be a large number for further investigation by wet-lab experiments since it would be laborious, time-consuming, and costly. Therefore, a smaller set of DEGs that are representative of all DEGs is required to reduce time, cost, and labor during further experiments by the wet-lab researchers. Though total DEGs are more informative than any smaller set of DEGs, a smaller representative set of DEGs would be more beneficial from the viewpoints of time, cost, and labor. Therefore, in this study, we proposed 10 top-ranked DEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the core genes (CGs) for early diagnosis, prognosis, and therapies of CRC. The survival probability curves and box-plot analyses with the expressions of CGs in different stages (control, Stage 1, Stage 2, Stage 3, and Stage 4) of CRC with the TCGA database indicated their strong prognostic performance from the earlier stages of the disease. It should be mentioned here that Patil et al. (2021) [22] identified CRC-causing 40 CGs for early diagnosis, which might be a large number for further investigation by the wet-lab researchers since it would be laborious, time-consuming, and costly, as mentioned earlier. In our proposed 10 CGs, 9 genes (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, MELK, and TPX2) were overlapped/common with their 40 CGs. In addition, we also investigated the association of our proposed CGs with different diseases. We found they have a strong pan-cancer role, including CRC. The literature review also supported the association of our proposed CGs with CRC. For example, AURKA is considered an oncogene that significantly impacts the proliferation and progression of colorectal carcinoma from colorectal adenoma [56]. Generally, AURKA is overexpressed and amplified in CRCs [57,58,59,60,61,62]. TOP2A is highly expressed during tumor development and responds to drug therapy for CRC [63]. CDK1 is also overexpressed and sensitive to apoptosis in CRC cells [64]. CDKN3 is highly expressed in CRC tissues and remarkably related to patients’ diagnoses [65]. CKS2 overexpression is correlated with aggressive tumor development in CRC, meaning that CKS2 might function as a decent CRC biomarker [66]. CKS2 is a promising biomarker contributing to CRC tumor development [66]. CDC20, PTTG1, and MAD2L1 might be CRC stage-related genes [67]. MELK might play a role as an effective therapeutic target for CRC [68]. TPX2 is highly upregulated in CRC tissues [69]. We identified the top-ranked five GO terms and KEGG pathways of CGs to reveal their molecular mechanisms in CRC progression. The identified GO term ‘cell cycle’ is one of the most important biological processes in the human body [70]. It has four sequential phases. Arguably, the most important phases are the S phase (DNA replication occurs) and the M phase (cell divides into two daughter cells) [71]. The fundamental task of the cell cycle is to ensure that DNA is faithfully replicated once during S phase and that identical chromosomal copies are distributed equally to two daughter cells during M phase. Usually, in adult tissue, there is a delicate balance between cell death and proliferation (cell division), producing a steady state. Disruption of this equilibrium by loss of cell cycle control may eventually lead to tumor development [72], including colorectal cancer [73], colon cancer [74], liver cancer [75], glioblastoma [76], breast cancer [77,78], lung cancer [79,80], gastric cancer [81], etc. So, the cell cycle is considered a vital cancer progression process. TOP2A is related to tumor development and poor survival outcomes by regulating cell proliferation and the CRC cell cycle [82,83]. CDK1 controls the cell cycle and aids in the development of colorectal tumors via an iron-regulated signalling axis [64]. In most CRCs, chromosomal variability resulting in an abnormal chromosome number, aneuploidy, was systematically related to a mitotic checkpoint’s loss of function [84,85]. The GO term ‘mitotic spindle orientation’ can influence tissue organization and control the placement of daughter cells within a tissue. Spindle misorientation greatly affects cancer development and progression [86], including CRC [87]. The top-ranked five MFs (cyclin-dependent protein kinase activity, CXCR chemokine receptor binding, protein kinase binding, RNA polymerase II CTD heptapeptide repeat kinase activity, and histone kinase activity) play a vital role in CRC development and proliferation [12,88,89,90]. Similarly, the enriched five CCs, including the spindle, mitotic spindle, cyclin-dependent protein kinase holoenzyme complex, intracellular non-membrane-bounded organelle, and serine/threonine-protein kinase complex, are strongly related to the progression of CRC [91,92,93,94,95]. Chromosomal instability happens in 80%–85% of CRCs and is considered the most common subtype of CRC [94]. Subsequent research showed that chromosomal instability is caused by mutations in the genes that govern the mitotic spindle checkpoint [93]. The consecutive activation of a group of serine-threonine kinases controls eukaryotic cell-cycle checkpoints [95]. We identified the top 5 enriched common KEGG pathways (cell cycle, bladder cancer [96], oocyte meiosis [22], human T-cell leukemia virus 1 infection, progesterone-mediated oocyte maturation) that are also reported by some other studies [22,96,97]. We also identified key regulators of CGs, such as four TFs (NFIC, FOXC1, YY1, and GATA2) and six miRNAs that played a significant role in CRC development. FOXC1, a member of the forkhead box family, has been connected to the growth and progression of numerous diseases [98,99], particularly CRC [100,101]. YY1 is a multipurpose TF protein that can stimulate or suppress gene expression [102] and plays a significant role in CRC tumor growth [103]. In CRC, the high-level expressions of GATA2 were linked with a poor prognosis and recurrence in solid tumors [104]. Nuclear factor 1 C-type (NFIC) regulates PFKB3 in response to CRC [105]. To find effective repurposable drugs against CRC, we performed molecular docking and computed binding scores among 158 CGs-associated drug agents and CGs-guided receptors. Then we proposed seven top-ordered drugs (Manzamine A, Cardidigin, Staurosporine, Benzo[a]pyren, Sitosterol, Nocardiopsis sp., and Riccardin D) based on binding affinities as the candidate repurposable drug. Manzamine A [106], Cardidigin [107], Staurosporine [108], Benzo[a]pyrene [109], Sitosterol [110], Nocardiopsis sp. [111], and Riccardin D [112] also suggested by some other studies for the treatment of CRC. Finally, the binding stability of top-docked four complexes (TPX2 vs. Manzamine A, CDC20 vs. Cardidigin, MELK vs. Staurosporine, and CDK1 vs. Riccardin D) was investigated by molecular dynamics (MD)-based MM-PBSA simulation and found their performance to be stable [113,114]. Thus, our findings may play a vital role in early diagnosis, prognosis, and therapies for CRC. The present study identified the 10 top-ranked DEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the core genes (CGs), which showed Strong prognostic performance in the earlier stages of CRC. The CGs-disease and pan-cancer analysis commonly showed that CGs have a robust pan-cancer role, including CRC, bladder urothelial carcinoma, esophageal carcinoma, glioblastoma multiforme, liver hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, and stomach adenocarcinoma. The CGs regulatory network analysis detected some essential TFs proteins (NFIC, FOXC1, YY1, and GATA2) and miRNAs (hsa-mir-147a, hsa-mir-129-2-3p, hsa-mir-124-3p, hsa-mir-34a-5p, hsa-mir-23b-3p, and hsa-mir-16-5p) as the transcriptional and post-transcriptional regulators of CGs. The enrichment analysis also revealed some important CRC-causing GO terms and signaling pathways. For example, cell cycle and mitotic spindle pathways have a significant association with CRC progression. Finally, we recommended our proposed CGs-guided 7 candidate drug molecules (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) for the treatment against the CRC by molecular docking analysis. Thus, the findings of this study may be more useful compared to the previous computation study results for early diagnosis, prognosis, and therapies forf CRC.

PMC10000176

Angeli Ambayya,Rozaimi Razali,Sarina Sulong,Ezzanie Suffya Zulkefli,Yee Yee Yap,Jameela Sathar,Rosline Hassan

Genomic Alterations, Gene Expression Profiles and Functional Enrichment of Normal-Karyotype Acute Myeloid Leukaemia Based on Targeted Next-Generation Sequencing

22-02-2023

acute myeloid leukaemia,next generation sequencing,gene expression,somatic variants,functional enrichment

Simple Summary The characterisation of normal-karyotype acute myeloid leukaemia (AML-NK) requires further refinement based on genetic variations. In this study, we ascertained genomic biomarkers via DNA and RNA sequencing in eight AML-NK patients during diagnosis and after achieving complete remission. We discovered putative variants affecting gene regulation and functional enrichments dysregulating transcription and DNA-binding transcription activator activity RNA polymerase II-specific in our cohort. Abstract Characterising genomic variants is paramount in understanding the pathogenesis and heterogeneity of normal-karyotype acute myeloid leukaemia (AML-NK). In this study, clinically significant genomic biomarkers were ascertained using targeted DNA sequencing and RNA sequencing on eight AML-NK patients’ samples collected at disease presentation and after complete remission. In silico and Sanger sequencing validations were performed to validate variants of interest, and they were followed by the performance of functional and pathway enrichment analyses for overrepresentation analysis of genes with somatic variants. Somatic variants involving 26 genes were identified and classified as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variants of unknown significance, 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Nine novel somatic variants were discovered, of which three were likely pathogenic, in the CEBPA gene with significant association with its upregulation. Transcription misregulation in cancer tops the affected pathways involving upstream genes (CEBPA and RUNX1) that were deregulated in most patients during disease presentation and were closely related to the most enriched molecular function gene ontology category, DNA-binding transcription activator activity RNA polymerase II-specific (GO:0001228). In summary, this study elucidated putative variants and their gene expression profiles along with functional and pathway enrichment in AML-NK patients.

Genomic Alterations, Gene Expression Profiles and Functional Enrichment of Normal-Karyotype Acute Myeloid Leukaemia Based on Targeted Next-Generation Sequencing The characterisation of normal-karyotype acute myeloid leukaemia (AML-NK) requires further refinement based on genetic variations. In this study, we ascertained genomic biomarkers via DNA and RNA sequencing in eight AML-NK patients during diagnosis and after achieving complete remission. We discovered putative variants affecting gene regulation and functional enrichments dysregulating transcription and DNA-binding transcription activator activity RNA polymerase II-specific in our cohort. Characterising genomic variants is paramount in understanding the pathogenesis and heterogeneity of normal-karyotype acute myeloid leukaemia (AML-NK). In this study, clinically significant genomic biomarkers were ascertained using targeted DNA sequencing and RNA sequencing on eight AML-NK patients’ samples collected at disease presentation and after complete remission. In silico and Sanger sequencing validations were performed to validate variants of interest, and they were followed by the performance of functional and pathway enrichment analyses for overrepresentation analysis of genes with somatic variants. Somatic variants involving 26 genes were identified and classified as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variants of unknown significance, 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Nine novel somatic variants were discovered, of which three were likely pathogenic, in the CEBPA gene with significant association with its upregulation. Transcription misregulation in cancer tops the affected pathways involving upstream genes (CEBPA and RUNX1) that were deregulated in most patients during disease presentation and were closely related to the most enriched molecular function gene ontology category, DNA-binding transcription activator activity RNA polymerase II-specific (GO:0001228). In summary, this study elucidated putative variants and their gene expression profiles along with functional and pathway enrichment in AML-NK patients. Acute myeloid leukaemia (AML) is a clonal haematopoietic stem cell disorder characterised by genetic heterogeneity with diverse clinical outcomes in patients. For decades, cytogenetics has been crucial in diagnosing AML, as it provides a snapshot of genome-wide structural and copy number changes. Established recurrent structural variations such as t(8;21), t(15;17), inv(16) and del(5/7) are pivotal in the diagnosis and prognosis of AML, as these abnormalities partake in leukaemogenesis [1]. However, almost half of all adult AML cases are diagnosed with a normal karyotype (NK) without structural aberrations identified during cytogenetic analysis. Varying frequencies of normal karyotypes were reported by different countries, and those frequencies ranged between 25% to 70% [2,3]. Although the European Leukaemia Network (ELN) in 2017 and 2022 classified AML-NK as an intermediate prognosis, the clinical outcomes of the patients in this group are diverse and arduous to define [4,5,6,7]. In recent years, the advent of novel and more sensitive platforms such as next-generation sequencing enabled the interrogation of the AML-NK genome to detect cryptic and subtle genomic aberrations that are otherwise undetectable with cytogenetics analysis. These genomic aberrations were indicated to be useful, and several findings were applied to the National Comprehensive Cancer Network (NCCN) guidelines [8]. Some of the genomic aberrations that are included in the diagnosis and prognosis of AML-NK are FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations that confer poor prognoses [6,9,10] if detected without nucleophosmin (NPM1) [6,11,12] concomitant mutations. NPM1 mutations without FLT3-ITD mutations are associated with good prognoses, and CCAAT enhancer-binding protein alpha (CEBPA) mutations also indicate a good prognosis if biallelic mutation [6,11,12,13,14] is present. This is also true of other myeloid gene-related mutations. Although some genomic aberrations have been established as diagnostic and prognostic markers, some genetic aberrations, such as Runt-related transcription factor 1 (RUNX1) [12] and Tet methylcytosine dioxygenase 2 (TET2) [15,16], are still controversial, as their role in clinical studies is yet to be fully understood. On the other hand, gene expression profiling improves the understanding of the AMK-NK subgroup and further refines risk stratification and clinical outcomes. Several studies elucidated the distinct gene expression profiles between the AML-NK patients and refinement based on genomic aberrations such as the mutational status of prognostic genes (FLT3-ITD, CEBPA and NPM1) [17,18,19,20]. The study of differentially expressed genes (DEG) profiles provides insights into the perturbed pathways associated with genomic markers in AML-NK. The discovery of activated and inactivated pathways enables prediction of a patient’s response to specific targeted therapies. For example, the perturbed RAS signalling pathway inspired the development of RAS-targeted therapy in AML patients [21]. Although considerable progress has been made in discovering genomic biomarkers in AML-NK, elucidating the genetic heterogeneity of this disorder, the backbone of induction chemotherapy with cytarabine (Ara-C) and anthracyclines has not changed in the last three decades. Monitoring patients’ responses to chemotherapy was limited in AML-NK patients due to the paucity of suitable markers for minimal residual disease (MRD) assessment in the past. Nevertheless, now, NGS technologies enable deciphering the clonal heterogeneity of the AML-NK genome to identify specific genomic biomarkers suitable for MRD monitoring [22,23]. The persistence of the genomic biomarkers of MRD at the time of complete remission (CR) serves as an independent prognosis for survival, as reported by Jongen-Laverencic et al. They analysed 482 AML patients using a targeted NGS panel at two time points: at presentation and CR after induction chemotherapy. In that study, the authors reported that about 50% of the mutations detected at presentation persisted after CR and that most of these mutations conferred increased relapse risk [24,25]. Other studies suggested that NGS-based MRD monitoring has better sensitivity and specificity than conventional MRD monitoring techniques such as morphological review and flow cytometry immunophenotyping [26,27]. To our knowledge, no comprehensive study on AML-NK integrating DNA and RNA sequencing with functional enrichment has been published. Therefore, we explored the genomic aberrations in the AML-NK genome using a targeted NGS panel and RNA sequencing for gene expression profiling to disclose DEGs. Next, we elucidated functional and pathway enrichment by performing overrepresentation analysis (ORA) in AML-NK patients affected by genomic aberrations and DEGs. Subsequently, we identified genomic biomarkers that could be useful in MRD monitoring for AML-NK patients. A cohort of eight matched de novo AML-NK patient samples at two time points, presentation (DX1-8) and after remission attainment (RX1-8) following completion of consolidation chemotherapy, were included in this study. All patients were diagnosed based on the latest WHO [12] criteria, and their responses to treatment were classified based on the International Working Group Criteria [28,29]. Patients were included if their cytogenetic findings were normal at presentation using the standard G-banding karyotype analysis of 20 metaphase chromosomal spreads obtained from bone marrow aspirates. Multiplex RT-qPCR was performed using a Leukaemia Q-Fusion Screening Kit (QuanDx, San Jose, CA, USA) for simultaneous detection of 30 fusion genes to rule out chromosomal aberrations. All samples were screened for prognostic markers using qualitative PCR and high-resolution melting analysis to detect FLT3-ITD and NPM1 mutations. The remission statuses of patients at CR1 were assessed based on post-induction chemotherapy that included a combination of cytarabine and anthracycline (daunorubicin) and consolidation chemotherapy that included high-dose cytarabine (HIDAC), a combination of mitoxantrone and cytarabine (MIDAC) or a combination of fludarabine, high-dose cytarabine and granulocyte colony-stimulating factor (FLAG). Patients’ remission at CR2 was assessed after salvage therapy using a combination of fludarabine, cytarabine, granulocyte-colony stimulating factor and idarubicin (FLAG-IDA). MRD monitoring was based on a morphological leukaemia-free state (bone marrow blast count of less than 5%) and either negativity of genetic markers (NPM1 and/or FLT3-ITD) or multiparametric flow cytometry for cases without genetic markers (absence of a leukaemia-associated immunophenotype) [6]. These samples were retrospectively selected from the Clinical Haematology Referral Laboratory, Hospital Ampang. Ethical approvals were obtained from the Medical Research Ethics Committee of the Ministry of Health Malaysia (NMRR 17-1929-36614) and Universiti Sains Malaysia (USM/JEPeM/21010107). Patients’ gDNA was extracted from blood collected in K2-EDTA using a Qiamp DNA Blood Minit Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Genomic DNA concentration and purity were assessed by using a NanoDrop ND-1000 UV-VIS Spectrophotometer. Library preparation was performed using Archer HGC and VariantPlex Myeloid (Boulder, CO, USA) (Table SW1, Supplementary File S1). The following Illumina’s adapter sequences were used for library preparation: read 1 (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) and read 2 (AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT). Libraries were then quantified and normalised using qPCR (Kapa Biosystems, Wilmington, MA, USA). Sequencing was performed by Theragen Etex Inc. (Seoul, Korea) using Novaseq 6000, 150 bp paired-end analysis with a minimum 7M read per sample (presentation sample) and 30M (CR1/CR2 sample). Data analyses that included read quality cleaning, error correction, genome alignment and variant detection and annotation were performed on the ArcherDx platform (version 6.2) (Table S1, Supplementary File S2). The settings were optimised to include coding and non-coding sequences with default read filtering criteria, including a cut-off of allele frequency (AF) ≥ 0.027 and gnomAD AF ≤ 0.05. Raw sequencing data in Fastq format were used as the input file, and reads were mapped to the Hg 19 build of the human genome. The variant calling was conducted using three algorithms: Vision, Freebayes and LoFreq tools. A vision variant caller was used to call the somatic variants confined within the targeted mutation genes (Table SW1). The Freebayes variant caller utilised somatic variants, whereas Lofreq was used to detect somatic variants with AF frequencies. Variant annotation was done using the ArcherDx platform and verified using the Variant Effect Predictor tool (Ensembl) [30]. The variant allele frequency (VAF) was used to deduce the origin of a variant: germline or somatic. A variant was considered to be of germline origin if the VAF was between 50% to 100%, and it was then excluded from the analysis to ensure only somatic variants were included in this study [31]. All annotated variants were reviewed manually, and a series of filtering and prioritising was applied based on the following criteria: (1) variant consequences as defined by Sequence Ontology (SO) [32] based on Ensembl’s variation calculation filtering to include high-impact and selected moderate-impact variants (i.e., missense and protein-altering variants) [33], (2) exclusion of variants with minor allele frequencies (MAFs) of ≥ 1% in healthy populations’ databases (1000 Genomes Project, ExAC, gnomAD, and ESP6500) and (3) exclusion of likely sequencing errors (variants with VAF < 5%) [34]. Next, filtered variants were manually inspected using Integrated Genome Viewer (IGV) software (Broad Institute, Cambridge, MA, USA) and scrutinised against Varsome [35], dbSNP [36], Clinvar [37] and COSMIC [38] to assess previous reports on pathogenicity. Novel putative variants that were not reported were then evaluated based on computational variant effect prediction tools for coding regions (SIFT [39], Provean [40], Mutation Assessor [41], FATHMM [42], LRT [43]) and non-coding regions (CADD [44]). All variants were classified based on the American College of Medical Genetics (ACMG)’s recommendations and those of the Genomics and the Association for Molecular Pathology (AMP) and the American Society of Clinical Oncology [45,46]. Variants were categorised into five main groups: (1) pathogenic, (2) likely pathogenic, (3) uncertain significance, (4) likely benign and (5) benign. The ArcherDx platform provided AMP guideline-specified somatic categories as Tier I to IV, with the first tier indicating strong clinical significance and the fourth tier deemed benign/likely benign [45]. Sanger sequencing was performed on a recurrent ASXL1 c.1934dup G646WfsTer12 variant detected in 75% of the samples to verify if the variants with VAF < 5% were genuinely sequencing errors in this study (Supplementary S1: ASXL1 NM_015338.5:c.1934dup (VAF < 5%) validation, Supplementary File S1). The IDT Primer Quest (Integrated DNA Technologies, Inc. was used to design the primers for the variant of interest (ASXL1c.1934dup) and checked for specificity using the NCBI Primer-BLAST. Optimisation of the annealing temperature DNA input was performed, followed by mastermix preparation and PCR cycles. The PCR thermocycling protocols were as follows: 98 °C for 10 s for initial denaturation, 60 °C for 5 s and 72 °C for 15 s (30 cycles), followed by a final extension at 72 °C for 1 min. The PCR products were visualised using Agilent’s D1000 Screentape with Agilent’s 2200 TapeStation system. PCR product purification was performed using Geneall Exspin PCR SV (103-102) according to the manufacturer’s instructions, and the samples were assessed for purity using the Nanodrop spectrophotometer. Samples with acceptable concentration and purity ratios were sent for Sanger sequencing. The Sanger sequencing data were reviewed using the Chromas software (version 2.6.6) (C. McCarthy; accessed on 20 January 2022), http://www.technelysium.com.au/chromas2.html). Total RNA from the whole blood was isolated using QIAamp® RNA Blood Mini and processed according to the manufacturer’s instructions. The concentration and purity of the extracted RNA was assessed using the Nanodrop ND-1000 UV-VIS Spectrophotometer. RNA integrity numbers (RIN) were determined using an Agilent RNA 6000 Nano Kit with the Agilent 2000 Bioanalyser. Only samples with RIN > 7 were selected for RNA sequencing. Messenger RNA (mRNA) library preparation was done using Agilent’s SureSelect Strand-Specific RNA library preparation for Illumina Multiplex Sequencing. The steps for library preparation were as follows: (1) poly(A) RNA purification from total RNA; (2) first-strand cDNA synthesis and purification using AMPure XP beads; (3) second-strand cDNA synthesis, end repair and purification using AMPure XP beads; (4) dA-Tail of the cDNA 3′ ends; (5) ligation of the adapters, followed by their purification using AMPure XP beads; (6) amplification and indexing of the adapter-ligated cDNA library and purification with AMPure XP beads; (7) quality assessment using DNA 1000 chip (Bioanalyser 2100) and ScreenTape (using Tapestation system). The library concentration was visualised using an electropherogram, which displayed a single peak with a size between 200–600 bp. All library preparations were sent to Theragen Etex Inc. (Seoul, Korea) for transcriptome sequencing, which was done using Illumina’s NovaSeq 6000 (150 bp paired-end reads; 200 million reads per sample). Results were received in a raw format and processed in-house. The following Illumina adapter sequences were used in the library preparation: read 1 (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) and read 2 (AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT). The quality of the sequencing raw data was inspected using FastQC (version 0.11.8). Low-quality reads (reads with Phred scores less than Q20), adapter and poly G sequences were removed using Fastp (Supplementary File S2, Table S2(a): RNA sequencing reads and alignments, Supplementary File S2, Table S2(b): RNA Sequencing FASTP summary). The cleaned reads were referenced against Ensembl’s Human Reference Genome (version 38, Ensembl, Cambridge, United Kingdom) using HISAT2 (version 2.1.0). Aligned reads were quantified using featureCounts (version 1.6). Differential gene expression analyses were performed using DESeq2. Raw read and internal normalisation were performed across all samples, followed by customised sample-level and gene-level quality controls. Principal component analysis (PCA) was conducted to exclude potential outliers that could affect downstream analyses. Next, DEGs were filtered based on the following criteria: baseMean > 100, p-adjusted value (padj) < 0.01 and log2 fold change (lfc) > 1 and <−1. DEG profiles for genes with variants discovered in the ArcherDx targeted DNA sequencing panel were selected, and expression profiles were inspected during disease presentation and after attaining CR1/CR2. A heatmap to represent the DEG of the genes with variants was generated with hierarchical clustering based on a Euclidean distance metric. WebGestalt was utilised to perform gene ORA on genes present with somatic variants that were deregulated in this cohort [47]. ORA analysis was conducted to determine if the a priori gene set with variants in this study was present more than would be expected by chance [48]. A total of 26 genes with somatic variants were included in the ORA analysis for pathway and gene ontology analysis using WebGestalt. Customised filtering parameters were applied for ORA analysis as follows: (1) a minimum of three genes for a category, (2) Benjamini–Hochberg multiple test adjustment was chosen and (3) categories were first ranked based on their FDR, and then the most significant categories were selected for significance-based filtering. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) database was utilised to identify affected pathways based on a p-value < 0.05 and a false discovery rate of ≤0.05. Based on the filtering criteria, seven out of ten pathways were inferred as significant, as summarised in Table S3, Supplementary File S2 [49]. Next, gene ontology for biological processes (BP), cellular components (CC) and molecular functions (MF) was conducted using WebGestalt for the 26 genes present with somatic variants. Customised filtering parameters were applied for ORA analysis as follows: (1) a minimum of three genes for a category, (2) Benjamini–Hochberg multiple test adjustment was chosen and (3) categories were first ranked based on their FDR, and then the most significant categories were selected for significance-based filtering based on p-value <0.05 and a false discovery rate of ≤0.05. A total of 10 BP categories were significantly enriched, as summarised in Table S4, Supplementary File S2. As for the MF, 1/10 and 3/10 for the CC categories met the filtering criteria (Tables S5 and S6, Supplementary File S2). Descriptive statistics were generated using IBM SPSS Statistics 22 software (SPSS, Chicago, IL, USA). The gene mutations and gene expression profiles were assessed using the Fisher exact test, and a p-value of less than 0.05 was considered significant to scrutinise if there was concordance between the mutational and gene expression profiles of these genes. A total of eight de novo AML-NK patients whose DNA and RNA were collected at presentation and after CR1/CR2 were retrospectively included in this study. The median age of diagnosis was 40.5 years (range: 21–55 years). Six out of eight patients were female. All patients were negative for 30 fusion genes tested using a Leukaemia Q-Fusion Screening Kit (QuanDx, San Jose, CA, USA). The genotype for the FLT3-ITD and NPM1 mutations revealed that four patients’ genotypes were FLT3-ITD+/NPM1+, two were NPM1+FLT3wt, and two were NPM1wt FLT3wt. Aberrant antigen expressions were seen in four cases (three CD2positive and one CD56positive). The remission status of patients at CR1 was assessed based on post-induction chemotherapy that included a combination of cytarabine and anthracycline (daunorubicin) (DA 3+7) and consolidation chemotherapy that included HIDAC, MIDAC or FLAG. The patient’s remission at CR2 was assessed after salvage therapy using FLAG-IDA. MRD monitoring was based on either negativity for genetic markers (NPM1 and/or FLT3-ITD) or multiparametric flow cytometry for cases without genetic markers [6]. Based on the ELN 2022 risk classification by genetics, 50% (4/8) of the patients had intermediate prognoses, and the others had good prognoses. Patients (DX1-7) were given the standard induction chemotherapy of daunorubicin and cytarabine (DA 3+7) followed by HIDAC/MIDAC/FLAG as the consolidation for patients who attained CR1. Patient DX8 was given salvage chemotherapy with FLAG-IDA, and they attained CR2. Four patients underwent stem cell transplantation, and all of the patients had an overall survival of more than five years in this study. The patients’ demographic, clinical and laboratory details are listed in Table 1. An initial total of 208 variants (122 in the presentation and 86 in the CR1/CR2) were detected across the eight AML-NK samples. Rigorous filtering criteria were applied to exclude variants with MAF ≥ 1% and potential sequencing artefacts. Sanger sequencing was performed on the ASXL1 c.1934dup G646WfsTer12 variant recurrently seen in 12/16 samples to confirm if variants with VAF < 5% and/or that were recurrent in most samples were true findings. Sanger sequencing confirmed that this finding was a sequencing artefact. Hence for subsequent analyses, only cases with VAF > 5% were included to avoid the false positive inclusion of variants. After filtering, 131 recurrent (23/42) and non-recurrent (23/42) somatic variants involving 26 genes were identified, and they are summarised in Table S7, Supplementary File S2. Of these 42 variants, 21 missense, seven frameshifts, one splice donor variant, two in-frame insertions, and 11 untranslated regions (UTR)/intronic variants were affirmed. The oncogenic effects and role of the somatic variants were assessed in various databases (dbSNP, Clinvar and COSMIC) and published in the literature. Novel putative somatic variants were assessed using computational variant effect prediction tools. Based on ACMG and AMP classification, the pathogenicity of the somatic variants was determined as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variant of known significance (VUS), 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Overall, the types of somatic variants that were detected were SNVs (26/42,61.9%), insertions (11/42, 26.2%) and deletions (5/42, 11.9%). The pathogenic variants were mainly SNVs (12/22, 54.5%) and insertions (8/22, 36.4%), and the rest were deletions (2/22, 9.1%). A total of nine novel somatic variants were discovered in this study (three likely pathogenic, one VUS and four benign/likely benign (Table S7, Supplementary File S2). Three likely pathogenic novel somatic variants were detected in the CEBPA gene (DX5 and DX8) involving regions that are within previously reported pathogenic variants in the COSMIC database [38]. We detected FLT3-ITDs in four patients in this cohort (DX1, DX4, DX6 and DX7), as summarised in Table 1. The length of the ITDs ranged between 42–144 bp, with a median ITD length of 49.5 bp. The longest ITD was selected for patients with more than one ITD. The samples in this cohort appeared to harbour between seven to thirteen somatic variants per sample; the minimum number of pathogenic somatic variants per sample was two, and the maximum was six. The mutational spectra of all somatic variants and their corresponding functional groups at disease presentation and after CR1/CR2 are depicted in Figure 1. Most recurrent somatic variants were detected belonging to cell signalling (FLT3), transcription (RUNX1 and CEBPA), methylation (DNMT3A) and nucleoplasmin (NPM1) functional groups. A total of 28 pathogenic/likely pathogenic variants (15 non-recurrent and 7 recurrent) were detected during disease presentation in all patients. Five somatic pathogenic/likely pathogenic variants were persistent in the CR1/CR2 samples (DNTM3A p.Arg659His, IDH2 p.Arg140Gln, LUC7L2 p.Glu253ArgfsTer34, NOTCH1 p.Ala1740Val and RUNX1 p.Leu56Ser genes). About 82% (23/28) of pathogenic/likely pathogenic variants seen during the presentation were lost after CR1/CR2. The VAF of pathogenic/likely pathogenic variants detected during the presentation and after CR1/CR2 in this study are depicted in Figure 2. A total of twelve clonal haematopoiesis driver mutations were seen in eight genes, according to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours. The myeloid and histiocytic/dendritic neoplasms that were detected in this study that contribute to the increased risk of developing AML, as reported in COSMIC and published in the literature, are listed in Table S8, Supplementary File S2 [12,38]. Of the twelve CH driver mutations, the persistently detected mutations in CR1/CR2 samples were DNMT3A p.Arg659His in patient DX1, IDH2 p.Arg140Gln in patient DX3 and NOTCH1 p.Ala1740Val in patient DX5. The mutated genes were then classified into mutational classes (Class I, II and III) based on their putative effects that led to leukaemic transformation based on established leukaemogenesis models. Class I mutations confer proliferation and survival, such as activation of signal transduction pathways, whereas Class II gene aberrations affect differentiation and apoptosis, and Class III gene alterations promote epigenetic modifications. A total of 13/42 somatic pathogenic variants were classified into Class I (n = 1, gene: FLT3), Class II (n = 7, gene: CEBPA, NPM1, WT1) and Class III (n = 3, gene: DNMT3A, IDH2, TET2) as depicted in Figure 3. All of the patients had at least one mutation in one of these classes; five patients had mutations in Class II and Class III genes, one patient had mutations in all three gene classes and two patients had mutations in the Class II gene. Only two mutations in Class III, those in DNMT3A c.1976G > A and IDH2 c.419G > A that encode epigenetic modifiers, were retained, with VAFs of 41.68% and 44.83%, respectively, at disease presentation and 9.55% and 30.4%, respectively, after CR1. Somatic variants in recurrently mutated genes in AML (FLT3, NPM1, CEBPA and IDH2) were detected in all patients implicated as classic AML drivers and/or CH driver and hotspot mutations. Two VUS were recurrently detected in three patients (RUNX1 NM_001754.4:c.1270T > C) and two patients (ZRSR2 NM_005089.3:c.283G > A) that need further investigation to elucidate their roles in the leukaemogenesis of AML. Differential gene expressions for the 26 variants were determined from the RNA sequencing data to compare the expression profiles of patients during disease presentation and after attaining CR1/CR2 based on the normalisation and fold change information obtained from FeatureCounts (version 1.6) and DESeq2. Findings from the DEG profiles of patients during the presentation and after CR1/CR2 are summarised in Table 1 and depicted in a heatmap (Figure 4). The relationship between the presence of mutations and gene expression was investigated. We observed no significant relationship between mutations and gene expression in all genes except in the biallelic mutations in the CEBPA genes detected in patients DX5 and DX8. Although upregulation of the CEBPA gene was observed in other patients during the presentation, in cases with biallelic CEBPA gene mutations (DX5 and DX8), the fold changes were almost three times higher than in other cases with overexpression of the CEBPA gene. RUNX1 gene mutations were detected in patients DX1-2 and DX7-8, and overexpression was detected in patients DX1, DX3-4 and DX6-7, indicating no concordance between the mutation and gene expression profiles of this gene. Upregulation of the FLT3, WT1 and NPM1 genes was seen in all presentation samples, though a heterogeneous level of expression was observed across disease presentation and CR1/CR2. In terms of overall DEG expression (presentation samples versus CR1/CR2 samples), log2-fold changes of 5.07, 7.06 and 1.37 (Padj 0.01 for both) were observed in FLT3, WT1 and NPM1 genes, respectively. On the other hand, downregulation of genes was observed among the presentation samples compared to the CR1/CR2 samples in CBL, NOTCH1, PPM1D, RAD21, STAG2 and TET2. The normalised data for the presentation and CR1/CR2 samples, fold change and padj values generated from the DESeq2 are summarised in Tables S9 and S10, Supplementary File S2. Mutations in the CEBPA gene (known as transcription factor CCAAT/enhancer binding protein α) were detected in the frequently reported two prototypical classes of mutations: N-terminal mutations and C-terminal basic leucine zipper (bZIP) regions. Patients DX5 and DX8 had double (bi-allelic) mutations in both of these regions, as depicted in Figure 5. Further evaluation of the DX5 and DX8 patients’ CEBPA-specific gene expression profiles revealed significant upregulations in both patients (Figure 4). Fisher’s exact test statistically confirmed a significant association between the biallelic CEBPA mutation and its expression (p = 0.036). To elucidate the relevance of all of the genes present with somatic variants, over-representation analysis (ORA) using the WebGestalt tool for GO and KEGG pathway enrichment. Enriched KEGG pathways are summarised in Table S3, Supplementary File S2. Pathway analysis of the KEGG pathway is depicted in Figure 6 (transcriptional misregulation and pathways in cancer). Other significantly enriched pathways are appended in Supplementary Materials documents (Figure SW1 for the acute myeloid leukaemia pathway, Figure SW2 for central carbon metabolism in cancer, Figure SW3 for pathways in cancer and Figure SW4 for the RAS signalling pathway). Gene ontology depicting the genes’ biological processes, cellular components and molecular functions are available in Figure SW5, Supplementary File S1. All significantly enriched GO terms in BP, MF and CC are summarised in Tables S4–S6, Supplementary File S2. All of the enriched GO terms in BP, MF and CC are also illustrated using volcano plots in Figures SW6–SW8, Supplementary File S1. The genetic heterogeneity of AML could explain the varying responses of patients to the treatment regime, necessitating the need for tailored therapy based on the patient’s genomic findings. However, the challenges lie in identifying the oncogenic effect of specific genetic aberrations and their role in the progression of leukaemogenesis. With the advent of NGS technology and bioinformatics tools, more sensitive assessments of genomic aberrations and MRD genomic markers are available. Recent evaluations of genomic MRD markers and their associations with patient outcomes and survival were explored and reported in multiple studies [27]. This study compared the mutations detected in eight matched presentation and CR1/CR2 samples using a high-throughput 75-gene panel by Archer HGC VariantPlex Myeloid. We performed paired-end deep sequencing on the samples collected at disease presentation (a minimum of 7 million reads) and increased the sequencing depth to 10X (a minimum of 30 million reads) to ensure mutations with low VAFs and minor leukaemic cell clones were not missed in the CR1/CR2 samples. As suggested by Yan et al. (2021), Sanger sequencing was performed on the selected variant (ASXL1 c.1934dup G646WfsTer12) with VAF < 5% [34]. Sanger sequencing disconfirmed this variant as a sequencing artefact; hence, for all of the subsequent analyses, only variants with VAF > 5% were included in this study. We disagree with Wang et al. (2020), as they have included all variants with VAF > 1%, suggesting that there are no standardised cut-offs [16]. We affirm that the laboratory’s internal validation can determine cut-offs for VAFs by performing Sanger sequencing for validating low VAFs in samples as performed in this study. We also added another facet by performing high throughput transcriptomic deep sequencing (200 million reads, PE, 150 bp) for the same cohort of patients during disease presentation and CR1/CR2. We then compared the gene mutations and DEG profiles of the genes to discover the impact of pathogenic mutations on gene expression. We found that cases with biallelic CEBPA mutations were significantly associated with its upregulation in two patients (DX5 and DX8). Nevertheless, no significant association was observed between mutation and expression profiles in other genes investigated in this cohort. Next, gene enrichment was assessed via ORA using the WebGestalt tool to investigate the pathways that are affected among KEGG pathways. As an outcome, five pathways were significantly enriched, affecting several hub genes: FLT3, AML1 (also known as RUNX1) and HRAS. This cohort consisted of AML-NK patients below 60 years of age with good prognoses (n = 4) and intermediate prognoses (n = 4) according to the ELN 2022 classification. All the patients were treated with the same treatment protocol during induction therapy (DA 3+7), including personalised HIDAC/MIDAC/FLAG for consolidation/salvage therapy, and they attained CR (n(CR1) = 7; n(CR2) = 1). Four patients had undergone SCT: n(allo-SCT = 3); n(auto-SCT = 1). All patients had an excellent OS of above five years with no indication of relapse (Table 1). The number of pathogenic somatic variants detected in the patients ranged between two and six per patient with no association with age, unlike previous reports by other studies that disclosed the number of mutations was lower in younger patients (18 to 39 years old) than in those above 40 years of age [16,50]. This study’s findings concur with another local study that performed whole-exome sequencing (WES) and reported fewer somatic variants in their AML-NK cohort (n = 6) [10,23,28]. Although they performed WES sequencing with greater genome coverage, the number of somatic variants was relatively low, especially in the myeloid-related genes included in our targeted NGS panel. The number of somatic mutations detected in our cohort was lower than in other studies with similar targeted myeloid panel designs [16,51]. In this study, the overall ratio of indels to SNVs was 1.58 at presentation and 2.4 at CR1/CR2, whereas the ratio of pathogenic somatic indels to SNVs was 1.15 at presentation and 0.25 at CR1/CR2, in contrast to previous publications [52,53]. The higher overall ratios of indels to SNVs in our study are due to recurrent benign indels seen in FLT3, RAD21 and EZH2 genes, as illustrated in Table S1. Our findings did not concur with another WES study conducted on AML-NK in another local study that exhibited a lower ratio of indels to SNVs, with SNVs being almost twice as common as indels [53]. The findings could be due to differences in experimental design, such as WES versus the 75-myeloid gene panel utilised in this study. The variants detected in this study are depicted in Figure 1 based on their types and functional groups. This study saw mutations in the signalling and kinase pathway-related genes that caused abnormal activation and proliferation of cellular signalling pathways, also known as Class 1 mutations, seen in 6/8 cases in this study, which agrees with other studies [28,54]. Similar to other studies, we also identified mutations in the epigenetic modifiers that include regulation of DNA methylation and chromatin modification in 6/8 cases in this cohort, which are recognised as drivers in leukaemogenesis. Mutations in epigenetic modifiers result in clonal outgrowth but require subsequent mutations to initiate leukaemic transformation [28,54]. Mutations in the myeloid transcription factors were seen in 3/8 cases, which concords with other studies that reported their presence in approximately 20% to 25% of AML cases [54]. CEBPA is an essential transcription factor for haematopoietic lineage-specific myeloid differentiation. Studies have reported that CEBPA gene mutations were found in 10–15% of de novo AML cases, especially in AML-NK patients [55,56]. Typically, CEBPA mutations cluster in two focal hotspots: N-terminal frameshifts insertions/deletions and/or C-terminal in-frame insertions/deletions. Mutations in the N-terminal give rise to the truncated p30 protein, which presides negatively to other full-length p42 proteins, whereas mutations in the C-terminal will obstruct the binding of CEBPA to DNA or dimerisation. In this cohort, we found biallelic CEBPA mutations in two cases (patients DX5 and DX8) at the hotspot regions of the N-terminal (within the first 357 bp of coding sequence) and the C-terminal bZIP region (between c.834–1074) which likely yielded the truncated p30 protein and led to the disruption of the binding of CEBPA to DNA or to dimerization [57]. Upregulated CEBPA genes were observed distinctly in cases with biallelic CEBPA mutations, as discovered in other studies [58,59]. AML with a CEBPA mutation is still retained as a unique entity in the 2022 WHO classification, and patients with biallelic CEBPA mutations often have good prognoses [7,60]. CEBPA gene upregulation and the outcomes of AML patients were inconclusive in several studies [14,61]. Although ELN 2022 suggested in-frame mutations affecting the bZIP region of CEBPA, irrespective of whether monoallelic or biallelic occurrence is the favourable prognosis, we could not ascertain whether biallelic mutations accompanied with the upregulation of the CEBPA gene is a favourable prognostic factor because the DX5 patient attained CR1 following induction and had OS > 5 years without SCT, whereas the DX8 patient only attained CR2 and had undergone allo-SCT in this study. The length of internal tandem duplication in the FLT3-ITD cases in this cohort was above the cut-offs (>30) used in several studies for prognosis based on the ITD’s length [62,63,64,65]. There are contradictory findings about the size of FLT3-ITD insertion length and the patient’s clinical outcome. Several studies have shown poor OS [65,66,67] with increasing ITD length, whereas increased ITD insertion conferred better OS in some studies [62,68]. However, some studies did not agree with either of these findings and indicated that the ITD length has no significance in the prognosis or clinical application of AML [69,70,71]. In this study, all patients had a good OS of above five years with concomitant NPM1 mutation; hence, their prognoses were categorised as intermediate based on the ELN 2022 classification. In addition, we did not observe any relationship between the presence of FLT3-ITD and the ITD’s length with the expression of FLT3 genes, as FLT3 was overexpressed in all cases in this cohort. Moreover, the sample size was insufficient to infer the relationship between ITD insertion length and patient prognosis in this study. Of the five pathogenic/likely pathogenic somatic variants that were persistent in the CR1/CR2 samples, three variants (DNMT3A p.Arg659His, RUNX1 p.Leu56Ser in Patient DX1 and NOTCH1 p.Ala1740Val in Patient DX5) were reported as having a germline predisposition in AML [37,72,73,74]. Two other recurrent pathogenic somatic variants that persisted after CR1/CR2, variants in IDH2 p.Arg140Gln (seen in Patients DX3 and DX6) and LUC7L2 p.Glu253ArgfsTer34 (seen in Patients DX2, DX3 and DX8), were reported as somatic in the Clinvar database and in other publications [37,72,75,76]. We deduced that the pathogenic somatic variant detected in NPM1 p.Trp288CysfsTer12, which was recurrent in six cases (Patients DX1, DX2, DX3, DX4, DX6, DX7), is suitable for MRD monitoring for treatment response in this cohort where the VAF at presentation ranged between 25–29% and was not detectable at CR1/CR2. We also observed interesting findings in the DEG profiles of FLT3, WT1 and NPM1 that exhibited upregulation and those of CBL, NOTCH1, PPM1D, RAD21, STAG2 and TET2 that were downregulated at presentation versus the CR1/CR2 patients. Studies revealed the utility of FLT3, WT1 and NPM1 expression for MRD monitoring in AML, which supports the findings in this study [77,78,79]. Functional enrichment analysis using ORA (WebGestalt) revealed that five pathways were significantly enriched (p-value < 0.01, FDR < 0.05), as illustrated in Table S3, Supplementary File S2. In addition, several hub genes were identified in several pathways, such as FLT3, RUNX1 and HRAS (Figures SW2–SW5, Supplementary File S1). Transcription misregulation in cancer tops the list of affected pathways, as some of the upstream genes in the transcriptional regulation, such as CEBPA and RUNX1, were deregulated in most cases, as shown in Figure 6. The overexpression of FLT3 impacted JAK-STAT signalling and cytokine–cytokine receptor interaction, as this gene is also located upstream of the pathway (Figures SW2 and SW4, Supplementary File S1). Overexpression of RUNX1, which is a hub-gene crucial in haematopoietic differentiation, was observed in 62.5% (5/8) of presentation samples (Figure 6). Overexpression of CEBPA genes was observed in 88% (7/8 of the presentation samples) of the patients with concurrent biallelic mutations (Patients DX5 and DX8), supporting the notion that haematopoietic resistance is evident in the pathogenesis of AML-NK (Figure 6). The variant in the HRAS gene detected in this study (Sample DX7) was benign, occurred in the 5′ UTR region, and did not affect gene expression in this study. However, two patients (DX1 and DX2) exhibited upregulation of this HRAS gene, as depicted in Figure 4. Upregulation of the HRAS gene could impact the deregulation of several pathways: PI3K-Akt signalling pathway, Jak-STAT signalling pathway, MAPK signalling pathway and calcium signalling pathway, as HRAS is a hub-gene in the RAS signalling pathway (Figure SW5). The most significantly enriched pathway in this study was transcriptional misregulation in cancer (hsa05202) (p < 0.01, FDR = 0.001), which was closely related to the most enriched GO MF category, DNA-binding transcription activator activity related to RNA polymerase II-specific (GO:0001228) (p < 0.01, FDR = 0.4), with four overlapping genes that includes hub genes such as FLT3 and CEBPA, as listed in Tables S4 and S6, Supplementary File S2. The ancestral GO:0140110 (Transcription regulation activity) for GO:001228 (Figure SW9a,b, Supplementary File S1) ensures transcriptional regulators are modulating the gene expression at the right cells at the right time, which were disrupted in this study as the upstream genes (AML1/RUNX1 and CEBPA) at the hsa05202 (Figure 6) were dysregulated, as depicted in the DEGs of genes during disease presentation and after CR1/CR2 in Figure 4. This explains the transcriptional dysregulation because the majority of the signalling pathways target transcription machinery, which provides insights into the mechanism of AML-NK leukaemogenesis [80]. Primarily, this study classified mutations discovered in AML-NK patients based on various classification guidelines (ACMG/AMP), databases (dbSNP, Clinvar and COSMIC) and variant effect prediction tools. Based on the ACMG/AMP recommendation, the variants were classified as benign, likely benign, VUS, likely pathogenic and pathogenic. The variants were then assessed based on mutational classes (Class I, II and III) and their functional classes to explicate their putative effects that enabled leukaemic transformation based on established leukaemogenesis models. Then, the variants detected during disease presentation and after CR1/CR2 were assessed to identify potential MRD biomarkers for treatment monitoring. Next, gene expression profiles for 26 genes with somatic variants were determined to assess the effect of these variants on their DEG profiles. Next, DEG profiles for 26 genes with somatic variants were assessed during disease presentation and after CR1/CR2. Finally, ORA analysis was conducted using the WebGestalt tool for GO and KEGG pathway enrichment analyses of the 26 genes with their variants in this study. To recapitulate, this study demonstrated the mutational profiles depicting AML’s genomic landscape heterogeneity. Each patient had a unique list of somatic pathogenic variants that belonged to Class I/II/III mutations. In some cases, CH-related variants were identified that conferred an increased risk of developing AML. The deregulation of genes with mutations impacted the pathways in cancer and various signalling pathways, as these genes are either hub genes or were upstream of pathways, as described earlier. Based on ELN 2022 classifications, the two patients (DX5 and DX8) with biallelic CEBPA gene mutations affecting the bZIP regions were assigned to a favourable prognosis group (Table 1). We also verified the NPM1 p.Trp288CysfsTer12 mutation as a recurrent biomarker that conferred a favourable prognosis in our cohort. In addition, we also exemplified the usefulness of the DEG profiles of FLT3, WT1 and NPM1 for MRD assessment in AML-NK patients. This study elucidated the genomic mutations and gene expression profiles of myeloid genes using a 75-gene targeted DNA panel (Archer HGC VariantPlex Myeloid) during disease presentation and after CR1/CR2. Diverse mutations and expression profiles were observed in each patient during disease presentation, indicating that AML-NK is indeed a heterogeneous disorder requiring further risk-based stratification. This study discovered three novel pathogenic somatic variants at the reported prototypical regions, N-terminal and bZIP regions of the CEBPA gene that were significantly associated with its upregulation. Although the cohort size is relatively small, we identified potential biomarkers that indicate favourable prognoses and are suitable for MRD monitoring, including NPM1 p.Trp288CysfsTer12, which was recurrent in 75% (6/8) of the patients and is in line with the ELN 2022 recommendation. We also suggested DEG profiles for FLT3, WT1, and NPM1 for MRD as potential biomarkers for MRD assessment in AML-NK patients. This multifaceted study comprehensively integrated DNA and RNA sequencing findings with functional enrichment in AML-NK that has not been described elsewhere. Our next step is to expand this study into a larger cohort of AML-NK patients to formulate a hierarchical prognosis model with unique mutations and gene expression profiles.

PMC10000182

Hesham Ismail,Doaa Ibrahim,Shorouk El Sayed,Ali Wahdan,Reham M. El-Tarabili,Waleed Rizk El-Ghareeb,Bassam Abdullah Alhawas,Badr Abdul-Hakim Y. Alahmad,Sherief M. Abdel-Raheem,Marwa I. Abd El-Hamid

Prospective Application of Nanoencapsulated Bacillus amyloliquefaciens on Broiler Chickens’ Performance and Gut Health with Efficacy against Campylobacter jejuni Colonization

21-02-2023

probiotic-loaded nanoparticle,performance,C. jejuni,colonization,barrier function,poultry

Simple Summary The emergence of antibiotics resistance is a warning sign to limit antibiotics usage as growth promoters in the poultry industry. Probiotics served as superior candidates for replacing antibiotics in the poultry sector. However, the beneficial functions of probiotics did not reach their targeted outcomes owing to the harsh environment in the poultry gut. In this context, evolution of biotechnological aids offers new avenues for increasing bioavailability and beneficial efficacy of in-feed additives including probiotics. Therefore, encapsulation of Bacillus amyloliquefaciens (B. amyloliquefaciens) into nanocarriers boosted its growth-promoting purposes and consequently modulated the functions of digestive enzymes and kept the microbiota homeostasis towards the beneficial ones. The strengthening capability of B. amyloliquefaciens-loaded nanoparticles for broilers’ gut barrier limited Campylobacter jejuni (C. jejuni) colonization and shedding. This superior outcome would in turn interrupt the transmission cycle of C. jejuni through the food chain and consequently protect against its adverse consequences in humans. Abstract Probiotics as novel antibiotics’ substitutes are verified to provide barriers for hindering the colonization of enteric bacterial pathogens with nutritional benefits. For enhancement of the probiotics’ effectiveness, their integration within nanomaterials is a paramount tool to support the progress of new compounds with functional features. Therefore, we addressed the impact of effective delivery of probiotics (Bacillus amyloliquefaciens) loaded nanoparticles (BNPs) on performance and Campylobacter jejuni (C. jejuni) shedding and colonization in poultry. Two hundred Ross broiler chickens were divided into four groups fed various BNP levels: BNPs I, BNPs II, BNPs III, and BNPs-free diets for 35 days. Nanoparticles delivery of probiotics within broiler diets improved growth performance as reflected by higher body weight gain and superior feed conversion ratio, especially in BNPs II- and BNPs III-fed groups. In parallel, the mRNA expression levels of digestive enzymes encoding genes (AMY2a, PNLIP, CELA1, and CCK) achieved their peaks in BNPs III-fed group (1.69, 1.49, 1.33, and 1.29-fold change, respectively) versus the control one. Notably, with increasing the levels of BNPs, the abundance of beneficial microbiota, such as Bifidobacterium and Lactobacillus species, was favored over harmful ones, including Clostridium species and Enterobacteriaceae. Birds fed higher levels of BNPs displayed significant improvement in the expression of barrier functions-linked genes including DEFB1, FABP-2, and MUC-2 alongside substantial reduction in cecal colonization and fecal shedding of C. jejuni. From the aforementioned positive effects of BNPs, we concluded their potential roles as growth promoters and effective preventive aids for C. jejuni infection in poultry.

Prospective Application of Nanoencapsulated Bacillus amyloliquefaciens on Broiler Chickens’ Performance and Gut Health with Efficacy against Campylobacter jejuni Colonization The emergence of antibiotics resistance is a warning sign to limit antibiotics usage as growth promoters in the poultry industry. Probiotics served as superior candidates for replacing antibiotics in the poultry sector. However, the beneficial functions of probiotics did not reach their targeted outcomes owing to the harsh environment in the poultry gut. In this context, evolution of biotechnological aids offers new avenues for increasing bioavailability and beneficial efficacy of in-feed additives including probiotics. Therefore, encapsulation of Bacillus amyloliquefaciens (B. amyloliquefaciens) into nanocarriers boosted its growth-promoting purposes and consequently modulated the functions of digestive enzymes and kept the microbiota homeostasis towards the beneficial ones. The strengthening capability of B. amyloliquefaciens-loaded nanoparticles for broilers’ gut barrier limited Campylobacter jejuni (C. jejuni) colonization and shedding. This superior outcome would in turn interrupt the transmission cycle of C. jejuni through the food chain and consequently protect against its adverse consequences in humans. Probiotics as novel antibiotics’ substitutes are verified to provide barriers for hindering the colonization of enteric bacterial pathogens with nutritional benefits. For enhancement of the probiotics’ effectiveness, their integration within nanomaterials is a paramount tool to support the progress of new compounds with functional features. Therefore, we addressed the impact of effective delivery of probiotics (Bacillus amyloliquefaciens) loaded nanoparticles (BNPs) on performance and Campylobacter jejuni (C. jejuni) shedding and colonization in poultry. Two hundred Ross broiler chickens were divided into four groups fed various BNP levels: BNPs I, BNPs II, BNPs III, and BNPs-free diets for 35 days. Nanoparticles delivery of probiotics within broiler diets improved growth performance as reflected by higher body weight gain and superior feed conversion ratio, especially in BNPs II- and BNPs III-fed groups. In parallel, the mRNA expression levels of digestive enzymes encoding genes (AMY2a, PNLIP, CELA1, and CCK) achieved their peaks in BNPs III-fed group (1.69, 1.49, 1.33, and 1.29-fold change, respectively) versus the control one. Notably, with increasing the levels of BNPs, the abundance of beneficial microbiota, such as Bifidobacterium and Lactobacillus species, was favored over harmful ones, including Clostridium species and Enterobacteriaceae. Birds fed higher levels of BNPs displayed significant improvement in the expression of barrier functions-linked genes including DEFB1, FABP-2, and MUC-2 alongside substantial reduction in cecal colonization and fecal shedding of C. jejuni. From the aforementioned positive effects of BNPs, we concluded their potential roles as growth promoters and effective preventive aids for C. jejuni infection in poultry. Feed additives as growth promoters have a robust impact on the production cost of broilers for covering alterations in profits due to instability in feed costs [1,2]. The extensive use of antibiotics as feed additives for enhancing poultry growth performance and treatment of bacterial infections leads to the emergence of multidrug resistant (MDR) bacterial strains [3,4,5,6,7,8]. Up until now, research is in progress exploring feed additives that can substitute antibiotics as prophylactics and growth promoters in poultry production. Probiotics became prospective antibiotic alternative additives in broilers owing to their impact on growth performance and the well-being of broilers compared to antibiotics [9,10,11]. The main favorable effects of probiotics correlate largely with improved feed bioavailability and digestibility, boosting the immune system, saving health, and providing superior carcass composition [12,13]. They have immunomodulatory roles in enhancing immunity against microbes and preventing exaggerated inflammatory responses, and they serve as biological barriers to protect the epithelial cells from being breached by pathogenic bacteria and to maintain epithelial integrity [14]. As well, probiotic bacteria are able to impede the growth of pathogenic microflora in the gastrointestinal tract of birds owing to their roles in nutrient depletion, blockage of pathogens’ target receptors on epithelial cells, and the creation of natural antibacterial products known as bacteriocins [15,16,17,18]. In addition, the early institution of probiotics in the gut can provide a barrier against foodborne pathogen colonization [19]. Bacillus-based probiotics have unique properties comprising growth promotion, immunomodulation, competitive exclusion [20,21], and production of a variety of extracellular substances and antimicrobial peptides against a wide range of pathogens [22,23]. Bacillus amyloliquefaciens (B. amyloliquefaciens) is a strong Bacillus species that produces numerous extracellular enzymes such as cellulase, metalloproteases, proteases, and α-amylases that augment digestibility and nutrient absorption in addition to overall gut immune functions [24,25]. Moreover, B. amyloliquefaciens produces bacteriocins [26] with a consequence of gut resistance to infection, and it reduces noxious gases emission in chickens [27]. Additionally, B. amyloliquefaciens CECT 5940 possesses a wide range of antimicrobial activity that can ultimately improve broilers’ health [28]. Disruption in the elegant interaction between gut microbiota, intestinal epithelium barrier, and host immunity plays a crucial role in the development of acute bacterial enteritis including campylobacteriosis [29,30], which is caused by Campylobacter jejuni (C. jejuni) and is considered one of the most serious foodborne pathogens causing zoonotic bacterial gastroenteritis in humans. Poultry are a major reservoir for C. jejuni as they provide it with an optimal temperature (42 °C) in their gastrointestinal tract, which is required for its colonization and proliferation [19,31]. Campylobacter jejuni inhabits avian cecum with approximately 106–108 colony forming unit (CFU)/g without clinical illness [32]. Pondering this situation, exploring ongoing promising strategies to reduce C. jejuni colonization in poultry and therefore in humans is needed. Administration of beneficial live probiotics can rescue the intestinal microbiota ecosystem balance, inhibit exaggerated immune responses against innocuous antigens, and hinder pathogenic bacterial colonization by competitive exclusion [33]. Recently, B. amyloliquefaciens CECT 5940 could probably improve growth rate, nutrient intake, gut function, and negative impacts of necrotic enteritis challenge [34]. However, its usage as an alternative to antibiotics is still limited because a harsh gut environment of gastric acids and bile salts may kill it and decrease its bioavailability with a consequent demand for high doses and long-term use of probiotics to exert their curative functions [35,36,37,38]. Therefore, to overcome these limitations and use the advantages of available advancement in technology, recent studies have investigated different formulation methods for effective oral delivery of probiotics to protect them from harsh gut environments and sustain their therapeutic effects [35,39]. Nanoparticle formulations provide a new gateway for achieving a safe delivery system for the ingredients used in feed by increasing their potency and concentration at their target sites [36,40,41]. Moreover, using natural biodegradable biopolymers for encapsulation of these ingredients is widely applied [42]. Based on this technology, co-encapsulation of probiotics strains for their engineering to improve their therapeutic efficiency has been reported [43]. However, the effect of using B. amyloliquefaciens-loaded nanoparticles (BNPs) as alternatives to antibiotics for growth promotion and competing against campylobacteriosis needs to be defined. Therefore, we investigated for the first time in this study the effect of feeding poultry with BNPs on growth performance, barrier function, and immunity of broiler chickens in addition to its efficiency on cecal colonization of C. jejuni and its shedding in poultry excreta. All experimental procedures, bird rearing, and management were conducted upon approval from animal resources at the Faculty of Veterinary Medicine, Zagazig University, Egypt according to the rules of the Institutional Animal Care and Use Committee (ZU-IACUC/2/F/337/2022 approval number). The strain of B. amyloliquefaciens probiotic CECT 5940 obtained from Evonik Nutrition and Care GmbH was propagated in Luria–Bertani (Oxoid, Hampshire, UK) broth and incubated at 37 °C. Then, B. amyloliquefaciens was stored in the bacterial cryopreservation fluid at −80 °C for further experiments. The BNPs were prepared through incorporation of the bacteria into chitosan (0.4%, w/v) (Sigma-Aldrich, St. Louis, MO, USA) nanoparticles as previously described [44]. The prepared BNPs were stored by freezing at −20 °C in a cryoprotectant agent and were then dried and condensed for 18 h at −40 °C using LyoBeta 25™ freeze-dryer (Telstar, Terrassa, Spain). Finally, the dried cells were kept at 4 °C to be protected from light. The nano size and shape of the formulated BNPs were confirmed using transmission electron microscopy and Fourier-transform infrared spectroscopy characterization (Figure 1). Two hundred Ross 308 male broiler chickens (42.21 g average initial weight) at 1 day old were provided from a commercial hatchery (Dakahlia Poultry Company, Dakahlia, Egypt). The birds were housed in divided floor pens for separation between groups at the Animal Care Unit at the Faculty of Veterinary Medicine, Zagazig University. The birds were divided randomly into 4 groups; each group had 5 replicates (10 birds each). The first group defined as control was fed the conventional diet and the three other groups were offered the conventional diet supplemented with B. amyloliquefaciens-loaded nanoparticles (BNPs) with three different doses [BNPs I (2.5 × 105 CFU/g of feed), BNPs II (5 × 105 CFU/g of feed) and BNPs III (7.5 × 105 CFU/g of feed)]. Both control and tested groups were fed the conventional or BNPs-containing diets for 35 days during the experimental period. All chicks in control and tested groups were co-housed at the same rearing conditions: temperature (33 ± 1 °C), which was gradually decreased every week until it reached 24 ± 2 °C at the end of the experimental period and humidity (around 60%) was constant during the whole experimental period. The diet was formulated according to nutrition specification of a Ross broiler handbook [45] as presented in Table 1. Chemical analyses of all feed ingredients were performed using the standard method as endorsed by the Association of Official Analytical Chemists, AOAC77 [46]. All animals were provided with ad libitum access to water and food during the whole experimental period. Individual birds were weighed and feed residues were determined to calculate feed intake for evaluation of different growth parameters of broiler chickens within the growth phases (1–35 days) including body weight gain for each phase [final body weight (g/bird) − initial body weight (g/bird)] and feed conversion ratio (FCR) [feed intake (g/bird)/weight gain (g/bird)] [18,47]. Moreover, mortality rate was recorded during the period from 1 to 35 days. At the end of the experimental period, the birds (5 per replicate) were weighed and euthanized by cervical dislocation. The intestinal contents and cecal and fecal samples were then aseptically placed in sterile Eppendorf tubes and kept at −80 °C for further quantification of intestinal microbial populations and evaluation of C. jejuni colonization and shedding via real-time (quantitative) polymerase chain reaction (qPCR) strategies. Moreover, pancreatic and jejunal samples (around 1 cm each) were excised, flushed three times with phosphate buffered saline, and subjected later to genes expression analysis utilizing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays. The expression of genes associated with digestive enzymes [alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP), cholecystokinin (CCK), and chymotrypsin-like elastase family member 1 (CELA1)], barrier functions and antimicrobial defense [beta-defensin 1 (DEFB1), fatty acid-binding protein-2 (FABP-2) and mucin-2 (MUC-2)], and proinflammatory cytokines [interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α)] was done. Total RNA was extracted via RNeasy Mini kits (Qiagen, Cat. No. 74104) following the kits’ instructions. RNA purity and concentration were determined using NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was then synthesized using RevertAidTM H Minus kits (Fermentas Life Science, Pittsburgh, PA, USA) according to manufacturer’s instructions. The RT-qPCR assays were done using SsoAdvanced™ Universal SYBR Green® Supermix (Bio-Rad, 1725274) according to the manufacturer’s instructions and analyzed using Stratagene™ MX3005P qPCR thermocycler (Agilent Technologies, Santa Clara, CA, USA). The sequences of primers of target genes are illustrated in Table 2. Target genes expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Relative fold changes in the target genes’ expression were determined using 2−ΔΔCT method [48]. A pandrug-resistant (PDR) and multi-virulent field C. jejuni strain was used in this experimental trial. It was previously recovered from cloacal swabs of recently slaughtered broiler chickens based on a previous research by one of the co-authors [51]. The strain was inoculated into Bolton broth (Oxoid, UK) for 48 h at 42 °C in microaerophilic conditions (10% CO2, 85% N2 and 5% O2) using an anaerobic jar (Sigma-Aldrich) with CampyGen sachets (Oxoid). The inoculated broth was then streaked onto modified charcoal cefoperazone deoxycholate agar (Oxoid, UK) plates that were incubated under the previous microaerophilic conditions. The strain was then evinced to be resistant to 24 antimicrobials (amoxicillin, ampicillin, sulbactam-ampicillin, amoxicillin-clavulanic acid, cephalothin, cefoxitin, cefoperazone, cefepime, imipenem, aztreonam, nalidixic acid, ciprofloxacin, trimethoprim-sulfamethoxazole, doxycycline, erythromycin, azithromycin, clarithromycin, tobramycin, gentamicin, amikacin, linezolid, chloramphenicol, colistin, and clindamycin) of 10 different antimicrobial categories being identified as PDR. The stain was also proved to harbor three substantial virulence factors, which have vital roles in its pathogenesis (wlaN, virB11, and flaA) as previously detailed) [52]. The challenge inoculum was adjusted to provide a viable concentration of 108 CFU/mL [4]. At 30 days of age, the birds in all groups including control were orally infected through the crop gavage with 108 CFU/mL of C. jejuni (1 mL per bird). The experimental infection was then affirmed following the appearance of clinical signs via re-isolation and identification of the infecting C. jejuni strain in addition to re-investigating its antimicrobial resistance pattern and virulence genes profiling. Quantitative PCR assays were carried out to quantify the microbial populations including Bifidobacterium, Enterobacteriaceae, Lactobacillus, and Clostridium species in the avian intestinal contents at the end of the experimental period and to determine C. jejuni colonization in chickens’ cecal samples and its shedding in their fecal samples 3 and 7 days post-infection (33 and 35 d of age). Genome DNA was extracted with QIAamp DNA fast DNA stool kit (Qiagen, Hilden, Germany) adopting the guidance of the manufacturer. Purity and concentration of extracted DNA were determined using NanoDrop (Thermo Fisher Scientific). The sequences of the primers used for quantification of intestinal microbial populations and C. jejuni are illustrated in Table 2. The number of copies of DNA was determined, in triplicate, using Stratagene MX3005P® RT-PCR instrument considering the created standard calibration curves prepared from serially diluted pure bacterial cultures and then the bacterial quantities were expressed as log10 of the CFU/g of the sample. The results were evaluated via one-way ANOVA test using SPSS Inc. program version 20 (IBM Corp., Armonk, New York, NY, USA). Differences among the results were expressed as the standard error of the mean (SEM) and variations between means were assessed at a probability level of 5% using Tukey’s test. All graphs were made using GraphPad Prism program Version 8 (San Diego, CA, USA). The effect of adding BNPs in different doses to broilers’ diets within the whole experimental period (35 days) is shown in Table 1. Our data revealed that broiler chickens fed BNPs with higher doses (BNPs II and BNPs III) for 35 days exhibited significant (p ˂ 0.05) increases in their body weight gain (2499 and 2569 g/bird, respectively) compared to those fed BNPs I (2430 g/bird) and control diets free of BNPs (2297 g/bird). While there was a reduction in food intake in the groups of birds fed BNPs compared to those in the control group, their efficiency for food utilization was improved (p ˂ 0.05) specifically with higher BNPs doses as indicated by lower FCR in these groups compared to that in the control one. Notably, inclusion of BNPs with higher doses significantly (p ˂ 0.05) decreased mortality rates compared with the challenged group (Table 3). The analysis of genes expression associated with digestive enzymes including AMY2a, PNLIP, CELA1, and CCK is illustrated in Figure 2. The results indicated that the expression levels of AMY2a and CCK genes attended their peaks (p ˂ 0.05) following supplementation of BNPs III. A concentration-dependent manner upregulation of PNLIP gene (p ˂ 0.05) was detected following BNPs’ dilatory inclusion. Significant (p ˂ 0.05) upregulation of relative expression of CELA1 gene was noticed in broilers fed diets supplemented with higher levels of BNPs compared to control ones fed diets free of BNPs. The abundance of intestinal microbial populations following BNPs supplementation is illustrated in Figure 3. As expected, there were increases in the abundance of Lactobacillus and Bifidobacterium and reductions in Clostridium and Enterobacteriaceae as indicated by qPCR in groups of birds fed BNPs compared to control ones. Although the differences among the three groups of birds fed different doses of BNPs did not reach statistical significance in reduction of Enterobacteriaceae and Clostridium species abundance unlike the control group, PNPIII showed the highest tendency in their reduction. Conversely, a high tendency in increasing Bifidobacterium populations was noticed in BNPs-fed groups compared to the control one. Likewise, BNPs III-fed birds had significant (p ˂ 0.05) highest counts for both Lactobacillus and Bifidobacterium species. Analysis of the relative mRNA expression of genes related to intestinal barrier and antimicrobial defense including DEFB1, FABP-2 and MUC-2, and proinflammatory cytokines comprising IL-β and TNF-α is depicted in Figure 4. We observed an overwhelming increase in the expression of MUC-2 gene after supplementing BNPs in broiler chickens’ diets in a dose-reliant way. In addition, a proportional (p ˂ 0.05) increase in the expression of FABP-2 gene was demonstrated in broiler chickens with increasing the dose of dietary BNPs when compared with the control group. The expression of DEFB1 gene achieved its highest peak in BNPs III-fed birds. Notably, prominent downregulation (p < 0.05) was noticed regarding IL-1β and TNF-α genes, especially after feeding higher BNPs levels when compared to the control group. Investigating C. jejuni shedding and colonization in fecal and cecal samples 3 and 7 days post-infection using qPCR assay is shown in Figure 5. After 3 and 7 days of C. jejuni infection, there was a tendency of fecal and cecal C. jejuni counts toward reduction within groups of birds fed BNPs with different doses (BNPs I, BNPs II, and BNPs III) compared to those in the control group fed diets free of BNPs. Notably, a lower trend of C. jejuni counts was detected in fecal samples than cecal ones 3 and 7 days post-infection. At both intervals, inclusion of BNPs III in birds’ diets remarkably (p ˂ 0.05) reduced C. jejuni populations considering both sample types unlike the control group. Seven days post-infection, C. jejuni counts were significantly (p ˂ 0.05) reduced in fecal and cecal samples of birds fed BNPs III compared to those in the control group (2.93 and 4.22 CFU/g vs. 6.79 and 8.16 CFU/g), respectively. Higher productivity in the poultry industry has been complemented by the balance between nutrition, intestinal health, and animal welfare. Additionally, there are many impacts threatening this productivity such as the emergence of a large assortment of pathogens and bacterial resistance [53]. The concept of using in-feed live probiotics strains in the poultry industry not only exerts a beneficial effect on overall growth performance parameters [54], but also signifies their use in reducing and eliminating the colonization of pathogenic bacteria, such as C. jejuni [19]. Nevertheless, gut colonization and effectiveness of supplemented probiotics depend on various aspects involving the specificity of the strains relative to the host, digestive enzymes, bile acids, availability, nutritional status of the host, intestinal pH, stress, and form of substrate (prebiotics) [55]. Thus, offering probiotic living cells with a physical barrier for escaping from unfavorable environmental conditions is a crucial consequence, which currently has obtained substantial interest [56]. In this study, encapsulation of B. amyloliquefaciens in chitosan nanoparticles had beneficial effects on broiler chicken growth performance and reduced the colonization and shedding of C. jejuni. C. jejuni represents the most prevalent species accountable for 84% of cases diagnosed recently in Europe [57]. Poultry is the main carrier of the bacteria and is responsible for the most outbreaks of campylobacteriosis through consumption of contaminated poultry meat [43]. The bacteria can colonize and persist in the chicken gut during their lifetime without causing diseases [31]. The emergence of MDR, extensively drug-resistant, and PDR strains [58,59], especially C. jejuni, which is resistant to the main drugs used in the treatment of severe post-acute sequelae of campylobacteriosis (fluoroquinolone and macrolides) evoked a warning sign about hazardous uses of antibiotics for enhancing poultry growth performance and treatment of bacterial infections [60]. The use of probiotics such as Lactobacillus, Bacillus, and Bifidobacterium has been shown to be a promising alternative solution for reduction of pathogen burden in the avian gut and consequently breaks the transmission cycle to humans through the food chain. However, the effects of inclusion of BNPs in poultry diets to decrease the [60] colonization and shedding of C. jejuni need to be defined. In this study, we have shown that supplementation of B. amyloliquefaciens coated within chitosan nanoparticles, especially at high doses in broilers’ diets for 35 days, promoted their growth performance and increased their body weight gain. Moreover, feed utilization efficiency of broilers was improved as indicated by significant (p < 0.05) reduction in FCR and increase in BWG, especially in BNPs III-fed group. These results are consistent with previous data regarding the improvement in growth performance of broilers fed B. licheniformis spores supplement either alone or combined with mannan oligosaccharide feed additives [61]. Correspondingly, probiotics can modify the intestinal ecosystem by supplying digestion enzymes, reducing pH, and increasing the activity of enzymes in the gastrointestinal tract [62,63]. Similarly, [64] reported a significant improvement in FCR and growth performance of broilers fed dried brewer grain (10%) subjected to probiotics fermentation. Different formulations used for probiotics delivery would affect their absorption; therefore, in this study, chitosan nanoencapsulated probiotics improved their resistance against gastric and bile acids, which consequently increased their bioavailability, absorption, and sustained effectiveness during the overall period of experiments (35 days). The growth performance of birds was strongly correlated with the digestibility and utilization efficiency of feed [16]. Moreover, it has been known that dietary composition of broilers’ rations could modulate the expression of digestive enzymes and nutrient transport-related genes [57,65]. Therefore, our study revealed that a group of birds fed BNPs III for 35 days exhibited significant increase in relative mRNA expression of pancreatic digestive enzyme-linked genes, such as AMY2A, PNLIP, CELA1, and CCK. Aligned with our data, [64] stated that feeding microbially fermented dried brewer grains not only improved growth performance and carcass dressing, but also stimulated expression of pancreatic digestive enzymes encoding genes as AMY2A, PNLIP, CELA1, and CCK. Microbiota and their metabolites prompt gut enteroendocrine cells to secrete gut hormones, which consequently influence metabolism [66]. Our results indicated the ability of BNPs to stimulate gut hormones, such as CCK, which plays important roles in gastric motility, appetite, bile acids, and pancreatic enzymes release [66,67]. Although less is known about how probiotics regulate CCK secretion, a recent research showed that feeding broilers for 42 days on microbially fermented olive pomace via two stages of solid fermentation using Bacillus subtilis followed by Lactobacillus casei upregulated the genes encoding pancreatic enzymes: CCK, AMY2A, PNLIP, and CELA1 [18]. Furthermore, dietary inclusion of microbially fermented soybean meal enhanced pancreatic enzymes’ activities in broilers [68]. Moreover, dietary supplementation of B. amyloliquefaciens had significant efficacy in enhancing chymotrypsin, amylase, and lipase activities compared to groups fed antibiotics alone [69]. Similarly, it has been reported that B. amyloliquefaciens (2 × 105 CFU/g diet) pretreatment either alone or combined with mannan oligosaccharides improved metabolic activity of intestinal cell energy-related genes through upregulation of mRNA genes expression of enzymes implicated in protein digestion. The mechanism by which probiotics regulate digestive enzymes secretion needs to be deciphered. However, this may be attributed to the role of probiotics in restoring intestinal normal architecture and achievement of larger healthy surface area for nutrient assimilation [70]. Moreover, the role of probiotics in increasing the relative mRNA expression of pancreatic digestive enzymes could be related to their vital role in the activity of enteroendocrine cells to express the pancreatic enzymes encoding genes. Additionally, increasing the expression of genes encoding pancreatic lipase associated enzymes (PNLIP and CCK) could be secondary to the production of probiotics’ metabolites including short chain fatty acids [71]. Our data indicated increases in the abundance of Bifidobacterium and Lactobacillus species, which was accompanied by reduction in Enterobacteriaceae and Clostridium species populations. In parallel, probiotics not only exert their beneficial effects on host resistance through improving its immunity, epithelial function, and competitive exclusion of pathogens burden within the avian gut, but also through their effects on rescuing the normal microbial populations of the intestine via favoring the growth of beneficial commensals and inhibiting that of the opportunistic ones [72]. Moreover, Bao et al. [73]. reported an increase in the abundance of Lactobacillus species combined with reduction in Enterobacteriaceae and Clostridiales counts after inclusion of B. amyloliquefaciens in poultry diet compared to poultry fed a basal diet [73]. The increase of Lactobacillus and Bifidobacterium within the cecal contents of broilers may be attributed to the ability of BNPs to produce extracellular enzymes as phytase, amylase, xylanase, and β-glucanase, which could improve food digestibility and utilization [74]. In addition, accumulation of short chain fatty acids produced within microbial metabolic processes confers a suitable pH that would support the growth of beneficial bacteria and prevent that of harmful ones [73,74]. Both Lactobacillus and Bifidobacterium provide substrates for augmenting the growth of butyrate-producing bacteria, which stimulates epithelial regeneration and improves host energy metabolism [61]. Improvement of epithelial barrier functions as indicated by upregulation of MUC2 and IgA genes expression was observed following administration of Bacillus coagulans to chickens [75]. Consistent with this finding, we found that improvement of growth performance of birds was correlated with upregulation of epithelial barrier functions-related genes including MUC2, DEFB1, and FABP2, specifically, in birds supplemented with BNPs III in their diet for 35 days. Avian defensin represents the innate antimicrobial peptide with a wide range of spectrum against bacteria, fungi, and protozoa [76,77]. Moreover, its ability to kill bacteria is through alteration in cell membrane permeability and immunomodulatory functions involving chemoattraction for leukocytes to the injury site and elimination of bacterial lipopolysaccharides and lipoteichoic acids triggering proinflammatory response [76]. The upregulation of avian defensin following BNPs supplementation suggests its role as an antimicrobial peptide against MDR bacteria as proved previously [78]. Mucus is the first physical barrier protecting epithelial cells from microbial translocation and inflammatory response [79]. Likewise, intestinal barrier is strengthened by a glycosylated mucin-rich layer produced by goblet cells [80]. Supporting this view, probiotics have been proven to strengthen the integrity of the intestinal barrier by elevating the number of goblet cells that support the mucus layer [81]. A previous study described that many Bacillus species were evidenced to upregulate the expression of intestinal mucin [82] as demonstrated in our study. FABP-2 gene expression has been indicated as a biomarker for intestinal barrier in broilers’ gut and its higher expression signifies its superior role in intestinal function [83]. Similarly, our data showed upregulation of FABP-2 relative mRNA expression, which suggested that BNPs enhanced epithelial integrity and functions. In the present study, downregulation of intestinal gene expression levels of proinflammatory cytokines (TNF-α and IL-1β) were observed in broilers fed higher levels of BNPs. This observation indicated their suppressive role on proinflammatory cytokines, which in turn counteracted the inflammation [80,84,85]. Several studies evaluated the in vitro and in vivo anti-campylobacter activities of different probiotics strains [15,19]. However, the effect of dietary inclusion of probiotics-loaded nanocarriers on the improvement of birds’ resistance against C. jejuni and their interaction with host epithelial cells need to be explicitly defined. Our data further demonstrated that the aforementioned beneficial effects of BNPs, especially with higher doses, led to significant reduction in cecal colonization and fecal shedding of C. jejuni. These data suggested the ability of BNPs to competitively exclude C. jejuni from broilers’ intestine and prevent its colonization. From what has been declared above, our data explained several possible mechanisms by which BNPs could mediate the exclusion and inhibition of C. jejuni colonization and translocation within the avian gut. These mechanisms are mediated firstly through the improvement of nutrient digestibility and assimilation and secondly via their effects on improving intestinal morphology and functions through upregulation of FABP2 and MUC2 genes, which increased mucus secretion and prevented bacterial adhesion. Lastly, they exert antimicrobial impacts either through secretion of antimicrobial peptides, such as β-defensin, that breaks down the bacterial cell wall and consequently activates the innate immune response to control inflammatory process and prevent bacterial translocation. Our data suggested that encapsulation of B. amyloliquefaciens as a probiotic bacterium by chitosan nanoparticles enhanced its bioavailability and maintained its beneficial effects in the broiler chicken’s gut. The augmented growth promoter properties of BNPs were detected in our study and supported by higher expression of digestive enzymes-related genes. Friendly gut bacterial species including Bifidobacterium and Lactobacillus species outnumbered the unfriendly ones following inclusion of dietary BNPs. Supplementation of BNPs could also enhanced gut barrier functions and consequently decrease C. jejuni colonization with a superior outcome of minimizing its fecal shedding.

PMC10000190

Manuel Blonç,Jennifer Lima,Joan Carles Balasch,Lluis Tort,Carlos Gravato,Mariana Teles

Elucidating the Effects of the Lipids Regulators Fibrates and Statins on the Health Status of Finfish Species: A Review

22-02-2023

lipid-lowering agents,fibrates,statins,fish,gemfibrozil,atorvastatin

Simple Summary Pharmaceuticals used to treat abnormal cholesterol levels in the blood are known as lipid regulators (fibrates and statins), and their use is in constant growth. Treatment plants are usually unable to efficiently remove these compounds from wastewater, where their degradation rate is considerably slow, making them an emerging concern for aquatic systems. The present work reviews previously published research concerning the effects of these pharmaceuticals on several finfish species worldwide. Results suggest that both short- and long-term exposure to lipid regulators may have negative effects on fish health, affecting their metabolism and immune system and causing reproductive and developmental disorders. However, the information on these compounds in the available literature is still limited, and additional research is needed to fully understand the threat that their presence in aquatic systems may pose to the production of finfish by the aquaculture industry. Abstract The most documented fibrates are gemfibrozil, clofibrate and bezafibrate, while for statins, the majority of the published literature focuses on atorvastatin and simvastatin. The present work reviews previously published research concerning the effects of these hypocholesterolaemic pharmaceuticals on fish, with a particular focus on commercially important species, commonly produced by the European aquaculture industry, specifically in recirculated aquaculture systems (RAS). Overall, results suggest that both acute and chronic exposures to lipid-lowering compounds may have adverse effects on fish, disrupting their capacity to excrete exogenous substances, as well as both lipid metabolism and homeostasis, causing severe ontogenetic and endocrinological abnormalities, leading to hampered reproductive success (e.g., gametogenesis, fecundity), and skeletal or muscular malformations, having serious repercussions on fish health and welfare. Nonetheless, the available literature focusing on the effects of statins or fibrates on commonly farmed fish is still limited, and further research is required to understand the implications of this matter on aquaculture production, global food security and, ultimately, human health.

Elucidating the Effects of the Lipids Regulators Fibrates and Statins on the Health Status of Finfish Species: A Review Pharmaceuticals used to treat abnormal cholesterol levels in the blood are known as lipid regulators (fibrates and statins), and their use is in constant growth. Treatment plants are usually unable to efficiently remove these compounds from wastewater, where their degradation rate is considerably slow, making them an emerging concern for aquatic systems. The present work reviews previously published research concerning the effects of these pharmaceuticals on several finfish species worldwide. Results suggest that both short- and long-term exposure to lipid regulators may have negative effects on fish health, affecting their metabolism and immune system and causing reproductive and developmental disorders. However, the information on these compounds in the available literature is still limited, and additional research is needed to fully understand the threat that their presence in aquatic systems may pose to the production of finfish by the aquaculture industry. The most documented fibrates are gemfibrozil, clofibrate and bezafibrate, while for statins, the majority of the published literature focuses on atorvastatin and simvastatin. The present work reviews previously published research concerning the effects of these hypocholesterolaemic pharmaceuticals on fish, with a particular focus on commercially important species, commonly produced by the European aquaculture industry, specifically in recirculated aquaculture systems (RAS). Overall, results suggest that both acute and chronic exposures to lipid-lowering compounds may have adverse effects on fish, disrupting their capacity to excrete exogenous substances, as well as both lipid metabolism and homeostasis, causing severe ontogenetic and endocrinological abnormalities, leading to hampered reproductive success (e.g., gametogenesis, fecundity), and skeletal or muscular malformations, having serious repercussions on fish health and welfare. Nonetheless, the available literature focusing on the effects of statins or fibrates on commonly farmed fish is still limited, and further research is required to understand the implications of this matter on aquaculture production, global food security and, ultimately, human health. The daily use of man-made products such as pharmaceuticals, cleaning agents and plastics is in constant and accelerating growth [1], and many of these contaminants have been detected in a variety of environments [2,3]. This uncontrolled anthropogenic activity has triggered several potential risks for aquatic environments [4,5,6] since several contaminants have been quantified in considerable amounts both in surface and ground waters, as well as within organisms [7,8]. Conventional wastewater treatment processes are still unable to efficiently remove all contaminants from influents, and a substantial share of these pollutants, known as contaminants of emerging concern (CECs), remain in the effluent due to their high persistence and low degradation rates [1,9,10,11,12,13]. The input of many compounds classified as personal care products (PPCP), flame-retardants, endocrine-disrupting chemicals, polyfluoroalkyl substances and pharmaceuticals from wastewater treatment plants are not monitored in water, potentially subjecting different organisms to chronic exposures to various chemicals [14,15,16]. Improvements in analytical techniques allow for the detection of an increasing number of these compounds in a variety of bodies of water (e.g., stagnant and running water; surface and ground waters; salt, fresh and brackish water; and wastewater treatment plant influents, effluents and sludge) [17,18,19,20], as well as in food items [16,21]. Pharmaceuticals play a major role in life quality and welfare, but the fast increase in their use has led to significant pressure on natural ecosystems [20,22,23,24]. The over- and misuse of pharmaceuticals, combined with their persistence in the environment, and their proven ability to bioaccumulate on both organisms and sediments [25], make these compounds, and their respective bioactive metabolites, potential chemical stressors to aquatic and cultured fish, and, consequently, a possible threat to humans. Common aquaculture practices face a number of challenges (e.g., disease outbreaks, feed components shortages, droughts, unexpected flow of contaminants and xenobiotics) and present multiple threats to natural environments, such as eutrophication, habitat destruction and organisms escaping from aquaculture facilities into the wild [26,27,28]. To overcome the effects of those stressors on farmed fish, over the past few decades, recirculated aquaculture systems (RAS) have caught the attention of seafood farmers all over the world and have been increasingly developed at an international scale [27]. RAS are based on the concept of reusing or recirculating, over 90% of the production water, depending on the specific design, therefore minimising water exchange with the surrounding environments compared to traditional aquaculture practices [29,30]. The recirculated water is repeatedly treated with biological, mechanical, and often UV filters in order to maintain appropriate water quality [30,31]. It is now widely accepted that the implementation of RAS provides a wide range of benefits and that this technology may be increasingly adopted to raise all fish species. Firstly, the considerable reduction in water use allows for RAS to be installed in a variety of environments, including areas where water is scarce [31]. Second, being a virtually closed system, RAS enable the farmers to hold full control of water parameters that could naturally fluctuate in a body of water, such as oxygen levels, the concentration of nitrogenous compounds, temperature, and, eventually, the presence of pathogens [30,32,33]. In addition, RAS are usually installed indoors, therefore being sheltered from external factors such as climatic events and the potential predation of cultured organisms from wild animals [27]. Last, but not least, RAS significantly reduce the impact of aquaculture farms on neighbouring natural ecosystems. Indeed, by isolating the facility from the surrounding environments, the probabilities of eutrophication, escapees with potentially deleterious effects on wild populations, pathogen transfer or contamination outflow is minimised [27,30]. The low rates of water exchange, which directly translate to a considerable reduction in water usage, prevent surrounding rivers or groundwater to be heavily polluted or depleted as a consequence of this activity [28,33], and minimise the risk of pathogens entering the systems, translating to a decreased use of antibiotics [31,34]. These factors, in conjunction with the versatility of RAS regarding the species and life stages suitable for culture in such systems [27,31], as well as the different possible stocking and production densities [30], make RAS an appropriate approach to deal with the growing food demand while minimising the environmental impact of the food production industry [27,30,35]. Nonetheless, the array of technicalities incurred by RAS entails farmers developing further skills and expertise and must therefore undertake specific training in order to properly manage the entire system [30]. Furthermore, the different components of RAS, including power supply and sensory systems, are particularly expensive, and consequently, it has been observed that these constraints may restrict the development of these aquaculture practices to areas with greater financial power, such as European countries, North America or some Asian countries [27,36,37,38]. Therefore, further technological advances are required to make RAS truly cost-effective, and to enable its expansion and implementation all over the world [27]. In Europe, RAS farms are principally used to culture aquatic invertebrates (e.g., molluscs and crustaceans), and although the vast majority of farmed fish are salmonids (i.e., Salmo trutta, Oncorhynchus mykiss), other fish such as Anguilla anguilla (European eel) are also widely produced [37]. As pharmaceuticals are now recognised as ubiquitous in aquatic systems [39], and with treatment plants proving unable to remove these from influents [40], it can be assumed that these contaminants are also present in RAS and will affect cultured fish, and consequently aquaculture production. Here we will focus on the effects of lipid regulators, a group of pharmaceuticals engineered to treat dyslipidaemias in humans, acting as lipid-lowering agents, and as primary prevention methods for cardiovascular diseases [39,41]. Lipid regulators are usually manufactured from a variety of compounds such as atorvastatin, bezafibrate, clofibrate, ezetimibe, fenofibrate, fluvastatin, gemfibrozil, lovastatin, mevastatin, pravastatin, rosuvastatin or simvastatin. The first drug of its kind introduced to the market was clofibrate, followed by bezafibrate, fenofibrate and gemfibrozil [42]. Due to the rising need for anti-cholesterol treatments, with earlier-stage treatments and higher dosages [43], lipid regulators are widely prescribed all around the world, and consequently, comprise some of the most reported pharmaceuticals in drinking and wastewater [42,44,45]. The prescription and consumption of lipid regulators multiplied between 2000 and 2017 in countries members of the OECD, with the United Kingdom, Denmark and Belgium displaying the highest consumption rates [42]. Lipid regulators can be sub-classified into statins and fibrates [39], and, even if both are ubiquitous in water bodies, fibrates are often found at higher concentrations [42]. The present work aims to review the currently available literature on the presence of lipid-regulating agents (i.e., fibrates and statins) in the aquatic environment, and their effect on fish, including those commonly farmed in European RAS. Fibrates decrease levels of fatty acids, triglycerides and low-density lipoproteins, mainly by stimulating the peroxisomal, and partly the mitochondrial, β-oxidation pathways, lowering blood cholesterol levels [39]. On the other hand, fibrates act as agonists of the peroxisome proliferator-activated receptors alpha (PPAR-α), expressed in the liver, heart and skeletal muscle [46]. In the literature, the most commonly investigated fibrates are gemfibrozil, bezafibrate and clofibric acid, with the latter being the most commonly detected in drinking water and food [42]. The most common lipid regulators in water bodies, namely, atorvastatin, bezafibrate, clofibrate, gemfibrozil and simvastatin, have been described to have similar effects in aquatic organisms, particularly vertebrates, as they do in humans, including the induction of changes in lipid levels [42]. Hence, marine and freshwater organisms are vulnerable to the potentially deleterious effects that the presence of such contaminants might incur, making this an issue of major ecological concern. It is known that exposure to lipid regulators generally present in water (e.g., wastewater treatment effluents and drinking water) can have a range of consequences on fish, affecting physiology, metabolism, reproduction, behaviour and immunity [44,47,48,49]. For instance, previous studies have shown that exposure to gemfibrozil may lead to decreased levels of cholesterol in plasma [48], and trigger the organism’s antioxidant response, as well as tamper with a fish’s capacity to efficiently swim in counter-current [47]. Moreover, gemfibrozil, like many other compounds, can bioconcentrate in different tissues in fish [50], potentially having deleterious effects on human health through the ingestion of contaminated food. Gemfibrozil, is resistant to photodegradation, has a long half-life in water [51] and is not completely removed by wastewater treatment plants (WWTP) [40]. In Spain, for instance, removal rates of this pollutant from WWTP ranged between 10–75% [52]. Its persistence in water allows us to find concentrations up to 4760 ng/L in effluents [53], and up to 758 ng/L in coastal waters [54]. Moreover, values ranging between 6.69 and 10.34 ng/L were recorded in Portuguese surface waters [55] and up to 0.8 and 1.7 ng/L in Italian tap and surface waters, respectively [56]. In comparison, in Spain, values of up to 70.27 and 77.8 ng/L were detected in different sites of the Llobregat river [57,58]. Bezafibrate was detected in wastewater treatment facilities effluents in Spain at concentrations ranging between 2 and 132 ng/L, and in estuarine environments at concentrations ranging between 2 and 67 ng/L [59]. In comparison, Portuguese surface waters were contaminated with values of bezafibrate ranging from 11.86 to 15.52 ng/L [55]. Moreover, bezafibrate, gemfibrozil and clofibric acid, an active metabolite of clofibrate, have been detected in groundwater in Barcelona, being found in concentrations of maximum 25.8, 751 and 7.57 ng/L, respectively [18]. Nonetheless, the most predominantly detected fibrate in the aquatic environment is clofibric acid [60,61]. Statins lower cholesterol levels through the suppression of its biosynthesis as a result of competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), which is responsible for cholesterol biosynthesis in the liver [39]. Statins currently available on the market include atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin [62,63]. However, data on the toxicity of statins on different organisms are very limited and are generally restricted to atorvastatin and simvastatin, the two most common statins [63,64]. Few studies have focused on the environmental concentrations of statins despite the evident increase in their consumption. Statins appear to have more stable removal efficiencies compared to fibrates, but their presence has already been reported in both untreated and treated sewage water samples at values ranging from 4 to 117 ng/L and 1 to 59 ng/L, respectively [65]. Atorvastatin, for example, was found in low amounts, although its metabolites, namely p-hydroxy atorvastatin, and o-hydroxy atorvastatin, were found in greater amounts in wastewater. A similar trend occurs with simvastatin, where this statin was not directly detected in surface water and sediments unlike its specific metabolite (i.e., simvastatin hydroxy carboxylic acid) and was found up to 108 ng/L in Norwegian cities [66]. The inefficiency of wastewater treatment plants when it comes to the removal of these pharmaceuticals is translated by an inevitable exposure of both wild and cultured aquatic animals to these contaminants of emerging concern, even in closed systems, such as recirculated aquaculture systems (RAS), where they might accumulate in water. In both wild and laboratory-reared fishes, the consequences of exposure to fibrates and statins have been studied to varying extents, depending on compound and species, indicating the potentially deleterious effects of these contaminants on the overall health and welfare of fish (Figure 1). Here, we aim to compile existing knowledge on the effects of lipid-regulating agents, their environmental distribution, as well as their effects on fish, since these pharmaceuticals are likely to influence their lipid metabolism and nutritional quality, with a special interest in organisms that are highly relevant to aquaculture, and, therefore, directly linked to global food security and human health. We will also focus specifically on the most documented drugs, namely gemfibrozil, clofibric acid (a metabolite of clofibrate) and bezafibrate, representing the fibrates, and atorvastatin and simvastatin representing the statins. Articles published from 2011 onwards were selected in July–October 2022, using “lipid regulators”, “lipid-lowering agent”, gemfibrozil”, “clofibrate”, “clofibric acid”, “bezafibrate”, “fibrates”, atorvastatin”, “simvastatin” and “statins” as keywords. Moreover, the combinations of the aforementioned keywords, in conjunction with “effects in fish”, “aquaculture” and both the common and scientific names of the main species of interest for the European aquaculture industry (i.e., Salmo salar, Oncorhynchus mykiss, Anguilla anguilla and Clarias gariepinus) were also used. Due to the scarcity of recent studies focusing on the environmental presence and distribution of these pharmaceuticals in European countries, as well as their effects on the previously specified species of interest to the present work, articles related to these matters were selected, even if the publication was dated prior to 2011. An increasing number of studies focus on the potentially deleterious effects that the presence of lipid-regulating agents may have on non-target organisms. These suggest that lipid regulators can act similarly in mammals (e.g., humans) and other vertebrates, such as fish, when exposed to these compounds through contaminated waters. This factor, combined with the high use of these drugs, their incorrect discard and their low removal rates, imply a major environmental problem. A considerable quantity of the published literature has focused on the effects of gemfibrozil in aquatic organisms as it is now an issue of high concern (Table 1). In humans, gemfibrozil is capable of increasing serum aminotransferase, and, in isolated cases, of causing liver damage. The reduction in lipids is associated with the peroxisome proliferator-activated receptor (PPARα), involved in the gene expression of enzymes for the oxidation of fatty acids [45]. In fish, as in other animals, an increase in levels of lipoprotein lipase can occur, subsequently increasing triacyl glyceride-rich lipoproteins [51]. Danio rerio (zebrafish) exposed to gemfibrozil through diet for 30 days displayed decreased plasma levels of triglycerides and overall cholesterol levels [44]. These results corroborate the idea that gemfibrozil can operate in similar ways in fish as in humans, notably generating changes in lipid homeostasis and altering the availability of energy resources. Nonetheless, a study with fathead minnow (Pimephales promelas) showed an inconsistent effect of chronic exposure to gemfibrozil on lipid metabolism, steroidogenesis and reproduction, and no effect when employing environmentally relevant concentrations [48]. In gilthead seabream (Sparus aurata), gemfibrozil was found to affect swimming behaviour [47,90], with the ability to swim in counter-current for extended periods of time decreased by up to 65%. These behavioural effects were further associated with a significant increase in both catalase (CAT) and glutathione reductase (GR) activities in the gills of fish exposed to concentrations of the pharmaceuticals [47]. Furthermore, exposure to gemfibrozil appeared to have genotoxic effects on this species, causing nuclear anomalies [74]. In addition, exposure to gemfibrozil did not change gill total antioxidant capacity (TAC) but increased total oxidative status (TOS) and altered mitochondrial RNA of certain genes such as glutathione peroxidase 1 (GPx) and interleukin 1β in both gills and head kidney of the same species [49]. The transcription of key genes involved in lipid homeostasis was also affected by exposure to gemfibrozil, being characterized as a stress-inducing agent. Furthermore, exposure of S. aurata to 15,000 ng/L of gemfibrozil induced the activation of the inflammatory response in the liver [72]. Altogether, these studies suggest that, in S. aurata, this compound alters biochemical and gene functions involved in the immune and oxidative stress responses, having serious implications for the health and welfare of this species. The bioaccumulation of gemfibrozil in both muscle and adipose tissue of juvenile rainbow trout (Oncorhynchus mykiss) following an 8-day waterborne exposure to 3000 ng/L of gemfibrozil has been previously demonstrated using space-resolved solid-phase microextraction (SR-SPME) [50]. Prindiville et al. [45] investigated the effects of exposure to gemfibrozil on the lipoprotein metabolism of O. mykiss. The authors injected female individuals with 100 mg/kg of gemfibrozil every 3 days over a 15-day period and reported a significant decrease in the concentration of plasma lipids, as well as in lipoprotein concentrations. Such results might translate into a hindered ability to reproduce, to efficiently undergo homeoviscous adaption to changes in water temperature, as well as tampered locomotor capacities, impeding long-distance, constant swimming behaviours [45]. Nonetheless, this is mostly speculative work and required additional research to be carried out to confirm these hypotheses. Furthermore, it was reported that exposure to pharmaceutical doses of gemfibrozil significantly reduced the concentration of n-3 fatty acids, which is likely to have direct repercussions on the nutritional quality of the fish [45,91]. Gagné et al. [92] further corroborated the presence of gemfibrozil in municipal wastewater treatment plants effluent in Canada, once again emphasizing the importance of studying the effects of emergent contaminants on the health of both wild and cultured fishes. However, the authors classified this specific drug as of little-to-no concern to fish, as they observed minimal toxicity of this pharmaceutical in rainbow trout hepatocytes. This is in accordance with previously published findings, also indicating that gemfibrozil had no significant effect on the hepatocytes of O. mykiss [93]. On the other hand, Donohue et al. [93] described a significant increase in bioindicators for oxidative stress on hepatocytes, caused by clofibric acid, the metabolite of clofibrate. Similarly, Triebskorn et al. [94] reported a moderate reaction of the liver of rainbow trout to exposure to clofibric acid, including dilation of blood vessels, and no significant reaction in the kidney of the exposed fish. However, results indicated a more severe response from the gills of O. mykiss to this pollutant, as the authors described epithelial lifting, increased cell production (hyperplasia) and growth (hypertrophy) of mucus cells. Nonetheless, these reactions are non-specific, as they tend to occur when the gills enter into contact with waterborne contaminants and are often regarded as a measure to preserve ion balance [95]. In addition, through an experiment focusing on the exposure of primary hepatocytes of rainbow trout to clofibric acid alone and combined with female sex hormone, it was demonstrated that this pharmaceutical is not a significant PPAR activator in hepatocytes in this species. In addition, neither lipid metabolism nor estrogenic activity resulted altered from this exposure [96]. The available information on the impact that exposure to lipid-lowering compounds has on the European eel (Anguilla anguilla) is extremely limited. According to Lyssimachou et al. [69], gemfibrozil is not a significant PPARα activator in this species. Nonetheless, injections of gemfibrozil significantly decreased the activity of certain pathways directly linked to cytochrome P450, which are responsible for the metabolism of xenobiotics, as well as the oxidation of fatty acids. This could indicate a tampered ability of the fish to efficiently extract energy from their food source, and to develop and function efficiently [97], having potentially serious implications for aquaculture production. In zebrafish, chronic exposure to low concentrations of gemfibrozil can have transgenerational effects [77]. In adults, reproductive ability is reportedly altered, accompanied by histological changes in ovaries and kidneys. Furthermore, adult zebrafish chronically exposed to 500 and 10,000 ng/L of this pharmaceutical displayed a decrease in reproductive output, with atretic oocytes and altered kidney histology [67,68]. On the other hand, unexposed offspring of parents exposed to this contaminant display sex-dependent alterations in courtship activity and swimming behaviour, and abnormal sperm morphology, significantly affecting their fecundity and breeding success [70], although this seems limited to the first generation of offspring only [77]. Similarly, in a long-term exposure (i.e., 155 days) experiment with Japanese Medaka (Oryzias latipes), a significant decrease in fecundity was observed, tied with significant decreases in both testosterone levels in female fish and hatchability of F1 fish. Moreover, the expression of genes related to oestrogen receptors and vitellogenin was increased in both gonads and livers [78]. This suggests that, in some species, gemfibrozil can act as an endocrine disruptor, affecting reproduction and ontogeny. In male individuals of the same species, short-term exposure to gemfibrozil lowered levels of 17β-estradiol, 11-ketotestosterone and plasma cholesterol [78]. The authors argued that the endocrine disruption observed as a consequence of the challenge might be directly linked to the hypocholesterolaemic nature of the medicine [78], altering the reproductive success of individuals, and having varying effects depending on the exposure duration. The inconsistency in the published results is likely linked to the dependence of the gemfibrozil’s mechanisms of toxicity to exposure duration, administration method, species and life stage, as well as organ analysed. However, it appears as if exposure to gemfibrozil may have strong effects on reproduction (e.g., fecundity, gametogenesis), excretion of exogenous substances (i.e., detoxification process), lipid metabolism and the overall homeostasis. In addition, the presence of this pollutant in aquatic systems is a potential factor affecting larval development, leading to ontogenetic and behavioural anomalies in both adults and larval stages, and, to some extent, in the subsequent generations. Clofibric acid, the main metabolite of clofibrate, is a PPAR-α agonist that increases beta-oxidation and decreases triglyceride secretion in humans, but it is known to affect aquatic organisms [60,86]. Even though some of the previous literature investigated the consequences of exposure to clofibrate itself, a major part of the studies hereby considered focused on the effects of clofibric acid (Table 1). In grass carp (Ctenopharyngodon idellal), it was observed that clofibrate decreased triacylglycerol, plasma cholesterol and overall lipid concentrations. Similarly, it increased hepatic enzymes involved in the synthesis of fatty acids, as well as lipoprotein lipase expression. Furthermore, a decrease in lipogenic enzymes, previously increased through feeding with a high carbohydrate diet, was observed [81]. This suggests that clofibrate, like gemfibrozil, can act as a hypolipidemic agent and alter the lipid metabolism of some fishes. In zebrafish, exposure to clofibrate through ingestion leads to a modulation of genes such as fatty acid-binding proteins (fabp) and acyl-CoA oxidase 1 (acox1), a marker of PPAR-α activation in many tissues. Nonetheless, this process proved to be tissue dependent. Clofibrate is potentially unable to cross the blood–brain barrier of zebrafish, hence the lack of significant alterations in the transcription of acox1 in the brain [80]. In zebrafish larvae, the exposed group displayed shorter body lengths compared to the control group, in addition to changes in morphological characteristics and lethargic behaviour. When fed with this compound, an upregulation of peroxisomes in liver and heart mitochondria in was observed [81], suggesting that waterborne exposure to clofibrate affects both the metabolism of adults and larvae, and may cause strong alterations in the development of embryos, leading to severe deformities and behavioural changes. On the other hand, it has been reported that clofibric acid can cause behavioural and metabolic alterations in adults and larvae of D. rerio both after acute and chronic exposures but does not act as an endocrine disruptor. This drug can influence the enzymatic activities involved in counteracting oxidative stress, like superoxidase dismutase (SOD), CAT and GPx, although the level of alterations depends on the pollutant’s concentration. In addition, it has been found that this compound may cause alterations in biotransformation enzymes, as well as in lipid peroxidation levels [88]. Zebrafish embryos have a high potential for metabolizing xenobiotics, involving a variety of enzymatic activities, and generating derivate products such as clofibric acid [87]. It has been previously stated that a lifelong exposure of zebrafish to clofibric acid significantly affected the reproduction, spermatogenesis, growth and gene expression of fabp, in a transgenerational manner [86]. It was determined that the effects are dependent on the individual’s sex, and on the organ considered, and can express inter-generational variations, influencing fecundity, mortality and the occurrence of skeletal deformities. With the purpose of investigating key enzymatic activities with involvement in reproduction, an in vitro study in common carp (Cyprinus carpio) showed that clofibrate had an inhibitory effect upon CYP17, related to a synthesis of active androgens on gonads [79]. In an experiment challenging C. carpio the results showed alterations in haematological parameters (decreased red blood cells count and increased white blood cell counts), biochemical endpoints (increased levels of glucose and proteins), ion regulation (decreased plasmatic sodium, increased Na+/K+ ATPase in gills) and on enzymatic responses (i.e., Lactate dehydrogenase, LDH; glutamic-oxaloacetic transaminase, GOT; and glutamate pyruvate transaminase, GPT) [82]. In a different study from the same research group, Cirrhinus mrigala was exposed to the same concentrations resulting in alterations in enzymatic parameters, such as GOT and GPT after short-term exposure, and changes in the ion homeostasis of gills (Na+/K+ ATPase activity) after both short- and long-term exposures to this clofibric acid [83]. On the same species, clofibric acid influenced biochemical parameters, causing an increase in plasmatic glucose and Na+, and K+, as well as a decrease in plasma protein and Cl− following short-term exposures. On the other hand, long-term exposure to this contaminant lead to increased levels of plasmatic Na+ and Cl− and oscillations in the levels of plasma glucose, protein and K+ [84]. Through a similar experiment, the authors also reported changes in thyroid hormones in the plasma of C. mrigala [85]. The published literature focusing on the effects of these lipid-regulating agents on S. salar is extremely scarce. Rørvik et al. [98], briefly described a significant decrease in lipid content in Atlantic salmon following ingestion of a feed complemented with clofibrate when compared to a control group. Overall, these studies suggest, to a certain extent, inter-species variation regarding the effects of clofibric acid. However, the consequences of either chronic or acute exposure to this pharmaceutical often include strong alterations in lipid metabolism and homeostasis, ontogenetic disturbances, and effects on behaviour, haematological parameters and the detoxification process. Furthermore, and similarly to gemfibrozil, clofibric acid has been described as being, to a certain extent, an endocrine disruptor, and as having effects on fish displaying inter-generational and sex-dependent differences. Finally, it appears evident that rising environmental concentrations of this pollutant may have serious implications for the health and welfare of wild fish populations. Despite being one of the first lipid regulators to be introduced in the pharmaceutical market, there are not many studies investigating the effects of bezafibrate on aquatic organisms when compared to other fibrates, such as gemfibrozil and clofibrate, or its metabolite, clofibric acid. To the best of the authors’ knowledge, the only article available that investigates the effects of this compound reported that exposure to bezafibrate through diet can act like an endocrine disruptor in male zebrafish. This drug can lower plasma cholesterol, decrease 11-ketotestosterone (11-KT), and induce changes at the histological and molecular level in the testis. In addition, the expression of a variety of genes was affected, being either up- or down-regulated depending on exposure time (Table 1). Therefore, bezafibrate affects the gonadal steroidogenesis and spermatogenesis of this organism, consequently having strong implications on the reproduction of the species and affecting it at the population level [89]. It is clear from the literature reviewed that the effects of fibrates in fishes depend strongly upon the still unresolved influence of species-specific physiologies and lifecycles, developmental stage, dosage differences across studies and the amount of time exposed to environmentally relevant concentrations of fibrates. To date, gemfibrozil seems to be the focus of most studies, but overall, the main deleterious effects of fibrates on fish physiology seem to be restricted primarily to alterations in cell and tissue lipid metabolism. This, in turn, may affect the timing and functionality of the reproductive axis and vitellogenesis and may result in abnormalities during the larval development, affecting normal swimming, and perhaps foraging, performance. From the data reviewed, it is not clear if these effects impair the immune responses in adults, but the few reports that indicate alterations in the expression of genes related to inflammation processes suggest that fibrates do not damage long-term immune reactivity. The antioxidant response associated with exposure to fibrates seems, in this sense, mostly related to aerobic metabolic outbursts as a byproduct of lipid oxidative alterations rather than to energy-consuming inflammatory responses. However, this may not be the case in cultured fishes, even in more advanced RAS systems, fed with lipid-enriched formulations, prone to elicit oxidative stress and inflammatory responses [99]. It seems safe to assume that a short-life antioxidant response may dysregulate the metabolic budget during development but not the lifelong (adaptive) performance of adults. However, it is particularly worrying that some of the effects on lipid metabolism can be transgenerational as mentioned in zebrafish studies [77]. Although chronic exposures are hard to reproduce in non-model, free-living species, in fishes, the long-term impact of stressors (including pharmaceuticals, endocrine disruptors and persistent changes in physical variables such as temperature), at the population level seems to rely, partly, on the transgenerational persistence of stress-related disruptive effects in the thyroidal-, estrogenic/androgenic- and growth-related axis [100,101,102,103] all of them highly sensitive to alterations in the metabolism of lipids. Moreover, sex plasticity in fishes depends on the concerted influences of lipid-, temperature-, seasonality- and stress-responsive gene pathways regulating gonadal maturation and fate [104,105,106]. Pharmaceutical-related alterations in the availability and amount of lipid substrates may result in an impairment of fertility output and biases sex ratios. Of special concern are the changes of Na+/K+ ATPase transporters and activity resulting from exposure to fibrates in Cyprinus carpio and Cirrhinus mrigala [82,83]. In salmonids, these changes have still not been described, but if present, they may interfere with the osmoregulatory adjustments (smoltification) required for the migratory phase of diadromous fishes, in which branchial mitochondrial-rich ionocytes and the management of hepatic lipid stores determine the onset of smoltification [107], a complex process still understudied. In Atlantic salmon, prior to sea migration, lipid metabolism becomes less plastic to changes in lipid diets as demonstrated by the variations in the expression of genes linked to lipid uptake in the gut and hepatic lipogenesis and lipid transport [108]. These shifts in the metabolism of lipids are life-stage dependent, and constrict the growth rate that, in turn, acts as a trigger to undergo, or not, smoltification. In this sense, it would be worth studying the effects that exposure to fibrates during phases of major osmoregulatory and morphological changes may have on the obligate or facultative development of smoltification in migrant salmonids, a process that requires behavioural changes and modifications of swimming performance that may be influenced by the presence of gemfibrozil in the brain circuitry (see Figure 1). Statins inhibit an enzyme called 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) that converts 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) into the cholesterol precursor mevalonate. Thus, statins decrease cholesterol synthesis as they compete with HMG CoA for the active site of the enzyme, altering its conformation and inhibiting its function [109]. Although waterborne statins are expected to be bioavailable to fish due to their high input rate and persistence, there are indications that they will not bioaccumulate in organisms [25,50]. In addition to uptake via passive diffusion through membranes, statins may be taken up and excreted by membrane transporters. The activity and expression of membrane transporters in the gills of aquatic organisms may therefore influence their bioavailability by affecting their influx and/or efflux rates [110]. Therefore, the effects of this contaminant on an aquatic organism may vary greatly between species, or life stages within the same species (Table 2), but might be less severe than those observed for fibrates. The prescription rate of statins continues to increase, mainly with the high sale of atorvastatin, often under the brand name “Lipitor” [44,124], representing a pioneering treatment for hypercholesterolemia [125] but increasing the concentration of this pollutant in aquatic systems. Exposure to atorvastatin in rainbow trout was reported to alter genes involved in membrane transport, oxidative stress response, apoptosis and metabolism in gills at low concentrations. However, no effect was observed on genes involved in cholesterol biosynthesis or peroxisomal proliferation [111]. In contrast, an experiment focusing on primary rainbow trout hepatocytes showed that this environmental pollutant may alter lipid synthesis and reduce cholesterol biosynthesis [113]. In zebrafish, exposure to this emergent contaminant was directly linked to alterations in cholesterol metabolism, lipid regulation, and steroid production. Specifically, an observed reduction in cholesterol was associated with decreased levels of cortisol and sex steroids. Moreover, a reduction in triglyceride was associated with changes in mRNA levels involved with lipid regulation [44]. Additionally, exposure to this compound resulted in signs of skeletal muscle breakdown, indicating rhabdomyolysis, and strongly tampering with the welfare of male fish. Interestingly, this effect on skeletal muscle seemed to be gender-specific, as different response between male and female was observed [44]. Besides the effects observed on adult individuals, a strong, dose-dependent, myotoxic effect of this drug was reported in zebrafish larvae [112]. In addition, exposure to atorvastatin significantly reduced the response to tactile stimuli, and caused a decrease in enzyme activity, like citrate synthase (CS) and cytochrome oxidase (COX). This suggests involvement in metabolism in larvae, thus, affecting the early development of the fish, and, ultimately, success in adults. [112]. The effects of atorvastatin seem somewhat similar to those described for fibrates, with important implications for an individual’s development, and lipid, as well as overall metabolism, also acting as an endocrine disruptor. As with the other lipid-regulating agents mentioned before, exposure to simvastatin has been reported to have a variety of consequences in fish. For instance, exposure of fewer than 7 days was able to cause significant alterations in the antioxidant system of mosquitofish (Gambusia affinis) and related enzymatic activity, such as nuclear factor erythroid 2–related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) [122]. Furthermore, the presence of this contaminant in aquatic environments is reportedly responsible for strong histological changes in hepatic cells of the same species [119,122]. Similarly, histological changes were reported to occur in Mugilogobius abei following short-term exposure to this statin. Furthermore, increased expression of genes related to xenobiotic resistance and detoxification (pregnane X receptor, PXR), including cytochromes P450 (i.e., CYP1A, CYP3A), has been shown to be somewhat induced by short-term exposure to this pollutant, concomitantly with alterations in enzymatic activity [120]. In zebrafish, it has been reported that chronic exposure to simvastatin at environmentally relevant concentrations, may have, in certain parameters, both dose-dependent, and sex-dependent consequences [118,121]. Indeed, following a 90-day waterborne exposure to this contaminant, the authors observed significant differences in the expression of genes responsible for energy metabolism (e.g., fatty acids synthesis and metabolism, glucose metabolism) in the brain [121]. Further analyses performed during the same experiment indicated significant alterations in the mevalonate pathway (e.g., directly involved in the synthesis of cholesterol), as well as a sex-dependent variation in body weight and body length at the highest exposure concentrations. However, a significant increase in reproductive success (fertilisation %) was observed at 200 ng/L, although this was followed by an increase in developmental anomalies. In addition, chronic exposure to simvastatin resulted in significant changes in cholesterol and triglyceride levels, although this response was not dose dependent. Moreover, the authors described a significant decrease in the heartbeat rate of embryos following parental exposure to 8 ng/L of this statin [118]. This agrees with what was previously described by Campos et al. [115], who also reported significantly slower heartbeat in zebrafish larvae as a consequence of exposure to simvastatin. Additionally, the authors reported decreased movement and significant structural alterations (e.g., changes in septum shape and size of somites, essential for locomotion in fish). The authors further speculate that exposure to this lipid-regulator may directly affect DNA replication, apoptosis, and muscle function [115]. Zebrafish embryos exposed to high concentrations of simvastatin experienced important mortality, and sever morphological anomalies, compared to low concentrations, which induced barely any mortality, and no morphological alterations [114]. In addition, it has been observed that exposure to this compound caused strong developmental issues, leading to abnormalities in the septum, somites and myofibrils and most likely tampering with the individual’s ability to swim, further corroborating previous findings [114]. On the other hand, Cunha et al. [117] described no significant effects of simvastatin exposure on cholesterol levels of D. rerio larvae, although the experimental methods differed with regard to the developmental stages challenged, and the concentrations tested, potentially indicating dose-dependent differences in the effects of simvastatin. Furthermore, the authors reported that exposure to this lipid-lowering agent may cause either up- or down-regulation of nuclear receptors (i.e., PPAR; PXR; constitutive androstane receptor, CAR; retinoid X receptors, RXR), as well as in aryl hydrocarbon receptor (AhR) in zebrafish, depending on the dose of simvastatin, and the exposure duration [117]. In that regard, the authors had previously reported that waterborne exposure to simvastatin could cause severe developmental abnormalities in zebrafish embryos, particularly tail deformities [116]. Moreover, the authors detected significant upregulation of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferases (GST) and CAT as well as different ATP-binding cassette (ABC) transporters and cytochrome P450, indicating the induction of the cell detoxification process, and the related antioxidant procedure. Furthermore, the authors demonstrated the potential for simvastatin to interact with other chemical environmental pollutants, possibly having synergistic effects [116]. In contrast, Rebelo et al. [123] observed a significant decrease in SOD, GST and GPx, as well as thiobarbituric acid reactive substances (TBARS), in zebrafish embryos and juveniles following both acute and chronic exposures to simvastatin, indicating a reduction in antioxidant defences. In addition, significant behavioural changes were detected throughout these experiments, but no alterations in gonad development or sex determination were noted. The caveats mentioned above for fibrates are equally relevant for the effects of statins in the physiology and behaviour of fishes, with an amendment: statins are even less studied and in fewer species. Due to the pleiotropic nature of the effects of statins [126], some unrelated to their ability to reduce cholesterol levels, it is hard to say if the intensity and scope of statins’ effects in fish are comparable to those described for fibrates beyond changes in lipid metabolism, abnormal development in larvae and oxidative stress. Either way, even conflicting results seem to indicate no major effects on long-term inflammatory outcomes. However, unexpected systemic alterations related to interferences with metabolic and mitochondrial oxidative pathways should not be ruled out. In mammals, statins have been linked to dose-dependent alterations in angiogenesis [127], and in this sense it would be worth further analysing their putative myotoxic effects during cardiovascular development in fishes [118], widening the focus beyond the classical zebrafish ecotoxicology resources [128] to cover non-model species. The manufacturing and use of lipid-regulating agents are constantly growing, and many of these compounds (e.g., statins and fibrates) are ubiquitous in aquatic systems. However, limited information is available on the effects of lipid-regulating agents on the most common species of fish in European RAS, with most research having focused on O. mykiss. Nonetheless, the published literature offers a worrying insight into the implications of having aquaculture fishes exposed to such hypocholesterolaemic agents. Indeed, it has been reported that fish might be continuously exposed to these contaminants, and that, although not all these compounds are particularly likely to bioaccumulate, prolonged contact may potentially lead to modified swimming, feeding and social behaviour in these organisms. Furthermore, it has been suggested that lipid-lowering agents may tamper with fish’s ability to reproduce and develop properly, causing deformities if exposed during early life stages, and therefore, greatly affecting the welfare of farmed animals. In addition, preliminary findings indicate that the nutritional quality of cultured fish may be significantly decreased by these contaminants of emerging concern, which may have serious implications for food security, as fish represents a considerable proportion of protein sources worldwide. The low-level activation of inflammatory responses in fishes exposed to fibrates is intriguing and deserves more attention, considering the mutual influence between lipid and immune tissues [129]. Some regulators of key metabolic pathways, such as the mTOR anabolic signalling pathway, participate not only in the hepatic balance between lipolysis and lipogenesis, but also regulate the onset of inflammatory responses [130]. These pathways, together with the lipid tissue-dependent regulation of local immune responses in fishes [131] enforce the crosstalk between visceral adipose masses and immune cell trafficking and antigen presentation in fishes, similar to what has been observed in mammals. More chronic studies are needed to ascertain the effects of lipid-regulating agents at the population level in free-living fishes, including those that, such as salmonids, have experienced several rounds of genome duplication, gained a duplicated set of genes related to lipid regulation and management, and, thus, may be more resilient to pharmaceutical-derived metabolic alterations [132]. Seasonal trends in lipid allocation for vitellogenesis and gonadal maturation, coupled with transgenerational alterations due to chronic exposure to environmental stressors have been described in three-spined sticklebacks (Gasterosteus aculeatus) in an attempt to dissect the complexity of reproductive allocation of resources during the breeding season [133]. These, and other species with context-dependent lifecycles deserve more studies concerning the synergistic or antagonistic effects of lipid regulators, acting alone or coupled with other stressors, on normal and maladaptive physiology of fishes. In this sense, the activation of the hypothalamic-pituitary-interrenal (HPI) stress axis in fishes responds not only to environmental cues but also to the presence of pharmaceuticals, xenobiotics and other contaminants that may bias the effect of HPI byproducts on the sex plasticity pathways [134], compromising the maintenance of healthy populations. All these factors suggest that, if the environmental concentrations of statins and fibrates continue to rise, the effects on both wild and cultured organisms will intensify. Therefore, it appears that both the health and welfare of fishes in aquaculture systems will be at risk. Nonetheless, the effects of lipid-regulating agents seem to be somewhat dependent on a variety of factors (e.g., dose, exposure time, sex, and life stage). Thus, although the available literature allows, to a certain degree, for extrapolation of results on farmed species, additional research, with a particular focus on commercially important species (e.g., cultured in RAS), should be carried out to fully understand the implications for aquaculture production and, ultimately, global food security.

PMC10000196

Kazutoshi Fujita,Makoto Matsushita,Marco A. De Velasco,Koji Hatano,Takafumi Minami,Norio Nonomura,Hirotsugu Uemura

The Gut-Prostate Axis: A New Perspective of Prostate Cancer Biology through the Gut Microbiome

21-02-2023

gut,microbiome,prostate cancer,castration resistant prostate cancer,microbiota

Simple Summary The gut microbiome plays important roles in the development of several diseases. The gut microbiome is a dynamic system that is affected by several factors, such as dietary habits, and since prostate cancer and diet are closely linked, it is reasonable to hypothesize that a gut microbiome—affected by diet—could regulate prostate cancer far from the gut, thus creating a gut-prostate axis. Gut dysbiosis result in the leakage of gut bacterial metabolites, such as short-chain fatty acids and lipopolysaccharide into the systemic circulation, leading to the prostate cancer growth. Patients with prostate cancer have a distinct gut microbiome. Furthermore, the gut microbiome produces androgen, affecting castration-resistance of prostate cancer. The gut-prostate axis could be a new target for the prevention and management of human prostate cancer. Abstract Obesity and a high-fat diet are risk factors associated with prostate cancer, and lifestyle, especially diet, impacts the gut microbiome. The gut microbiome plays important roles in the development of several diseases, such as Alzheimer’s disease, rheumatoid arthritis, and colon cancer. The analysis of feces from patients with prostate cancer by 16S rRNA sequencing has uncovered various associations between altered gut microbiomes and prostate cancer. Gut dysbiosis caused by the leakage of gut bacterial metabolites, such as short-chain fatty acids and lipopolysaccharide results in prostate cancer growth. Gut microbiota also play a role in the metabolism of androgen which could affect castration-resistant prostate cancer. Moreover, men with high-risk prostate cancer share a specific gut microbiome and treatments such as androgen-deprivation therapy alter the gut microbiome in a manner that favors prostate cancer growth. Thus, implementing interventions aiming to modify lifestyle or altering the gut microbiome with prebiotics or probiotics may curtail the development of prostate cancer. From this perspective, the “Gut–Prostate Axis” plays a fundamental bidirectional role in prostate cancer biology and should be considered when screening and treating prostate cancer patients.

The Gut-Prostate Axis: A New Perspective of Prostate Cancer Biology through the Gut Microbiome The gut microbiome plays important roles in the development of several diseases. The gut microbiome is a dynamic system that is affected by several factors, such as dietary habits, and since prostate cancer and diet are closely linked, it is reasonable to hypothesize that a gut microbiome—affected by diet—could regulate prostate cancer far from the gut, thus creating a gut-prostate axis. Gut dysbiosis result in the leakage of gut bacterial metabolites, such as short-chain fatty acids and lipopolysaccharide into the systemic circulation, leading to the prostate cancer growth. Patients with prostate cancer have a distinct gut microbiome. Furthermore, the gut microbiome produces androgen, affecting castration-resistance of prostate cancer. The gut-prostate axis could be a new target for the prevention and management of human prostate cancer. Obesity and a high-fat diet are risk factors associated with prostate cancer, and lifestyle, especially diet, impacts the gut microbiome. The gut microbiome plays important roles in the development of several diseases, such as Alzheimer’s disease, rheumatoid arthritis, and colon cancer. The analysis of feces from patients with prostate cancer by 16S rRNA sequencing has uncovered various associations between altered gut microbiomes and prostate cancer. Gut dysbiosis caused by the leakage of gut bacterial metabolites, such as short-chain fatty acids and lipopolysaccharide results in prostate cancer growth. Gut microbiota also play a role in the metabolism of androgen which could affect castration-resistant prostate cancer. Moreover, men with high-risk prostate cancer share a specific gut microbiome and treatments such as androgen-deprivation therapy alter the gut microbiome in a manner that favors prostate cancer growth. Thus, implementing interventions aiming to modify lifestyle or altering the gut microbiome with prebiotics or probiotics may curtail the development of prostate cancer. From this perspective, the “Gut–Prostate Axis” plays a fundamental bidirectional role in prostate cancer biology and should be considered when screening and treating prostate cancer patients. The incidence of advanced prostate cancer has been increasing in the USA and Japan, although the incidence of all prostate cancer in the USA has decreased since the recommendation of the US preventive service task force for prostate-specific antigen (PSA) screening in 2012 [1]. Prostate cancer develops in patients in their 50 s and is a slow-growing tumor. Prostate cancer develops due to the mutations in driver genes, but several factors, such as genetic background and lifestyle affect the development of prostate cancer. Overall, the incidence of prostate cancer is high in Western countries compared with that in Asia. Japanese who lived in Hawaii had a higher incidence of prostate cancer compared with Japanese who lived in Japan [2]. Obesity is also associated with the incidence of prostate cancer. A meta-analysis showed that obese men had a higher risk of advanced prostate cancer [3], and dietary habits are one of the major factors affecting prostate cancer. Western-style diets, especially a high-saturated-fat diet, are associated with increased risk of prostate cancer [4]. High-fat diets (HFD) promote local inflammation in prostate tumors which leads to the increased secretion of inflammatory cytokines, such as IL-6 that culminates into increased recruitment and infiltration of myeloid-derived suppressor cells (MDSCs) [5]. Il-6 is a pleiotropic cytokine that in this context, also promotes prostate cancer growth by activating STAT3 signaling. However, the mechanisms as to how consuming a high-fat diet leads to local tumor inflammation remain largely unknown. For some time, the mechanisms that link dietary habits to prostate cancer remained elusive; however, recent advances in technology have allowed us to come closer to unraveling the mystery. Particularly, developments in next generation sequencing have enabled us to probe deeper. For example, we have been able to capture whole profiles of microbes through 16S rRNA sequencing. By having a better understanding of gut microbiota, we are in a better position to discover associations of the gut microbiome with various diseases. The gut microbiome is a dynamic system that is affected by several factors, such as dietary habits, and since prostate cancer and diet are closely linked, it is reasonable to hypothesized that a gut microbiome—affected by diet—could regulate prostate cancer far from the gut, thus creating a gut-prostate axis. We have just begun connecting the dots that exist between the gut microbiome and prostate cancer, and more and more, additional dots are being discovered and linked. In this review, we discuss the current status of the “gut-prostate axis” as regulated by the gut microbiome. Trillions of microorganisms and their genetic material constitute the gut microbiome. The typical gut contains 1013 to 1014 bacteria, and these microorganisms are important as they supply nutrients, such as essential amino acids and vitamins, that cannot be produced by humans [6]. Gut bacteria ferment dietary fiber and produce monosaccharides and short-chain fatty acids (SCFAs), such as butyrate, acetate, propionate, and isopropionate [7]. Humans are then able to utilize absorbed acetate and propionate as substrates for lipid, glucose, and cholesterol metabolism [8,9]. Herbivores have a long gastrointestinal tract and harbor gut microbes that allow them to digest cellulose and other plant materials to produce short-chain fatty acids as an energy source, and produce amino acids from ammonia by fermentation in the gut lumen [10]. The gut microbiota also coevolves with the host [11]. For instance, the giant panda consumes bamboo as an energy source, but the gastrointestinal tract of the giant panda is short and simple. Genetic sequencing showed that giant pandas have all the genes necessary for digesting meat, but not cellulose, which is the main component of bamboo [12]. Metagenomic analysis of gut microbes of giant pandas revealed putative genes, encoding cellulose-digesting enzymes and hemicellulose-digesting enzyme from gut microbes indicating that this adaptation enabled them to use bamboo as an energy source [13]. Gut microbiota is affected by several factors. Genetic background is an innate factor that affects the gut microbiome and this was demonstrated with a study analyzing the gut microbiome from twins which showed that host genetics do indeed influence the composition of the human gut microbiome [14]. A genome-wide association study of the host genome revealed 31 loci that affect the microbiome, and the lactase gene locus showed a significant age-dependent association with the abundance of Bifidobacterium [15]. In addition to innate factors, perinatal factors contribute to the developing gut microbiome. Everyone’s gut microbiome is established early during the neonatal stage. The neonate gut microbiome is affected by the amniotic fluid, maternal lifestyle, and maternal exposure to antibiotics even before birth. After birth, each individual develops a unique gut microbiome based on their sex, race, and lifestyle. Sex differences affect gut microbial composition in mice and humans. Young adult women in Western countries had a higher diversity of gut microbiota than men, but these differences were not found in Chinese individuals [16]. Testosterone also affects the gut microbiome, and the composition of the gut microbiome changes gradually with puberty. In pubertal subjects, the abundance of the genera Adlercreutzia, Ruminococcus, Dorea, Clostridium, and Parabacteroides is associated with the levels of testosterone [17]. Androgen receptor signalling is involved in the maintenance of diversity of gut microbiota; androgen-receptor knock-out mice experienced HFD-induced metabolic syndrome and gut dysbiosis, but intervention of the gut microbiome by antibiotics prevented metabolic syndrome [18]. Lifestyle is one of the main factors affecting gut microbiota. Results from 16S rRNA sequencing of feces has revealed that clusters of individuals with similar lifestyles share similar gut microbiomes. For example, family members share a similar gut microbiome, and obesity is associated with phylum-level changes in gut microbiota [19]. Further evidence of this phenomenon is characterized by differences of the gut microbiome observed between individuals from Western and non-industrialized countries [20]. For instance, the gut microbiome of Japanese is different from that of people in other countries, including Western and even other Asian countries, such as China. The Japanese gut microbiome has a greater abundance of taxa from the Actinobacteria phylum, particularly bacteria from the genus Bifidobacterium, than that in other nations [21]. Interestingly, the gut microbiome also differs among people living in different regions, even within the same country. The gut microbiota of 7009 individuals from 14 districts within one province in China was analyzed, and the individual’s geographic location showed an association with microbiota variation. Furthermore, microbiota-based metabolic disease models developed in one location failed when used elsewhere [22]. Japanese people have a unique dietary culture and habits compared to Western people, and given that individuals generally have a lower BMI and longer lifespans, one can surmise that these factors are related. Some Japanese people consume Western-style diets as well as traditional Japanese food, thus this group of individuals with varied Japanese lifestyles could serve as a good model to analyze the association between lifestyle, gut microbiome, and diseases. Additional factors can influence microbial composition such as exercise and physical activity. For instance, gut microbiomes from athletes also differ from those of normal populations. Athletes had a higher diversity of gut microbiota and significantly higher proportions of the genus Akkermansia compared with individuals with high BMI, indicating that exercise can affect the gut microbiome [23]. It should be noted that gut microbes interact with each other, and the overall picture of the microbiome cannot be determined by changes in single bacterial taxa. For example, obesity reduces SCFA-producing Bifidobacteria, but the total amount of SCFAs in the feces of obese people is increased [24]. This discrepancy suggests that SCFA production will be increased in gut microbiome of obese people by SCFA-producing taxa other than Bifidobacteria. In addition to the identification of the intestinal bacterial taxa, functional analysis of the microbiome and measurement of bacterial metabolites may also be useful in understanding the overall picture of the gut microbiota. Lifestyles affecting prostate cancer risk also change the gut microbiome (Table 1). Obesity, well-known risk factors of prostate cancer, decrease the ratio of Firmicutes to Bacteroidetes [25]. High-fat diets also affect gut microbiome, decreasing Bacteroidetes and increasing Firmicutes and Proteobacteria [9,26]. Dairy products increase prostate cancer risk, although it is still controversial [27]. Dairy products increase Lactobacillus and Bifidobacterium and decrease Bacteroidetes [28,29,30]. Gut dysbiosis impairs gut wall integrity, increases gut permeability, and reduces the expression of tight junction proteins such as zonula occludens (ZO)-1 and occludin, causing a “leaky gut.” A leaky gut results in the translocation of gut metabolites or bacterial components, such as lipopolysaccharide (LPS), into systemic circulation [31,32]. Akkermansia muciniphilia is involved in the maintenance of a healthy gut wall by degrading mucin, and its abundance is inversely associated with several diseases [33,34,35]. SCFAs are major metabolites of gut microbes, and butyrate plays a role in gut barrier function, and immunoregulations [36]. Obese individuals have higher levels of SCFAs in their feces compared to lean individuals [37], and the ratio of Firmicutes to Bacteroidetes is decreased in obese subjects. Bifidobacterium, known to improve the gut mucosal barrier and lower intestinal LPS levels, is reduced in the feces of obese people [25,38]. Interventions to improve dietary habits could change the gut microbiome. A randomized controlled study of obese individuals with metabolic syndrome showed that an energy-restricted Mediterranean diet and increased physical activity changed the gut microbiota. Changes in Lachnospiraceae were positively associated with adherence to the Mediterranean diet [39]. Gut microbes have direct contact with the intestinal wall; consequently, several intestinal diseases are affected by gut microbiota directly or indirectly through the modulation of local immune systems, such as regulatory T cell, dendric cells and CD4+ T cell [40]. In the gut microbiota of IBD patients, the abundance of specific bacteria, such as Enterobacteriaceae and Fusobacteria, are increased, indicating a direct association between gut microbes, local inflammation, and altered local host immunity [41,42]. Fecal microbiota transplantation, defined as the administration of fecal material containing distal gut microbiota from a healthy donor to the gastrointestinal tract of patients with IBD, has been conducted as a remedial treatment [43]. A systematic review showed that this approach may increase the rate of patients achieving clinical remission in ulcerative colitis (RR = 2.03) [43]. Apart from the gut, diseases of the liver and central nervous system are also reportedly affected by the gut microbiome. The liver receives bacterial components and metabolites that are absorbed in the intestinal tract and delivered via portal circulation [44]. Phenyl sulfate, a metabolite derived from intestinal bacteria, acts as an aggravating compound in diabetic kidney disease and contributes to albuminuria [45]. However, inhibition of sodium-glucose cotransporter 1 (SGLT1) in the gut ameliorates renal failure by altering the gut microbiome and reducing phenyl sulfate concentrations [46]. The increased influx of gut microbiota-derived endotoxins in portal circulation promotes TLR-4 expression and is associated with hepatic inflammation and steatosis [47,48]. Gut dysbiosis due to a HFD impairs the barrier of the intestinal wall, leading to the leakage of LPS into the systemic circulation. Systemic inflammation due to endotoxemia is implicated with the pathogenesis of insulin resistance and type 2 diabetes mellitus [49]. Endotoxins and amyloids from gram-negative bacteria can penetrate the blood-brain barrier and induce amyloid β aggregation and neuroinflammation in the central nervous system, suggesting that bacterial molecules and metabolites may be involved in the onset and progression of Alzheimer’s disease [50]. Gut microbiota-derived SCFAs promote neuroinflammation and tau-mediated neurodegeneration in the hippocampus [51]. These gut microbiome-mediated associations between gut and these distant organs are referred to as the “gut-brain axis” and “gut-liver axis” (Figure 1). However, these associations are not limited to these organs and are likely to include other systems. In recent years, it has become evident that gut microbiota affects various types of cancers. Multiple bacterial taxa and their metabolites have contributed to the development and progression of colorectal cancer [52]. Metagenomic and metabolomic studies on feces from participants who underwent colonoscopy showed that the relative abundance of Fusobacterium nucleatum was correlated with cancer progression [53]. Fusobacterium nucleatum has been reported in other studies as a colorectal cancer-associated bacterium that promotes tumor progression in a mouse model of intestinal cancer in a non-inflammatory manner [54]. In the liver, translocated bacterial metabolites and components may be involved in hepatocellular carcinoma (HCC). Obesity-induced hepatic translocation of lipoteichoic acid, a gram-positive intestinal bacterial component, accelerated senescence of hepatic stellate cells, promoting HCC progression through PGE2-mediated suppression of antitumor immunity [55]. It has been suggested that the gut microbiota may influence the development of breast cancer by deconjugating conjugated estrogen excreted in the intestinal tract, and thus allowing the biologically active form to be reabsorbed by the host [56]. In addition, intestinal bacteria can metabolize estrogen-like compounds, such as enterodiol and enterolactone, suggesting that gut microbiota plays a role in breast cancer development [57]. The focus on gut microbiota has not only been directed toward its impact on cancer development and progression, but also on the indirect effects in response to drug therapy, including immune checkpoint inhibitors (ICIs) [58]. In patients with advanced renal cell carcinoma (RCC) treated with anti-PD-L1 therapy, antibiotic use during treatment compared with no use was associated with an increased rate of primary progressive disease (75 vs. 22%, respectively) and shorter progression-free survival (1.9 vs. 7.4 months, respectively HR = 3.1), suggesting that antibiotic-induced dysbiosis reduced the clinical benefit from immune checkpoint inhibitors [59]. Metagenomic data of fecal samples from advanced RCC patients treated with nivolumab showed that the abundance of Akkermancia muciniphila and Bacteroides salyersiae was increased in responders, and that transplantation of these bacteria or feces from responder patients to RCC mouse models rescued responsiveness to anti-PD-1 plus anti-CTLA-4 treatment from mice colonized with the microbiota of non-responder patients [60]. The mechanism by which the gut microbiome influences ICI therapeutic efficacy is not fully understood; however, further studies will lead to innovative methods to enhance ICI treatments. The association between the gut microbiome and prostate cancer has been studied in human samples. In 2018, Golombas et al. examined the gut microbiome in 20 men with either benign prostatic disease or high-risk prostate cancer and reported that Bacteriodes massiliensis was abundant in patients with prostate cancer compared to controls [61]. Sfanos et al. compared the gut microbiome of patients with prostate cancer who received androgen deprivation therapy (ADT) to those of healthy volunteers and found that Akkermansiia muciniphila and Ruminococcaceae were increased in patients treated with ADT [62]. Liss et al. analyzed rectal swabs from 133 men who received prostate needle biopsy and showed that Streptococcus and Bacteroides were increased in patients with prostate cancer. Although 16S rRNA amplicon sequencing alone cannot specifically identify functional genes, it can be used with various computational tools to infer the functional metagenome [63]. Predicted metagenome analysis revealed that the folate and arginine pathways were down-regulated in the gut microbiome of men with prostate cancer therefore implicating these pathways with the pathogenesis of prostate cancer. Bacteroides massiliensis were also found to be increased in the gut microbiome from patients with prostate cancer by another study using a small cohort (8 men with benign prostate hypertrophy and 12 with high-risk prostate cancer) [61]. Matsushita et al. analyzed the gut microbiome of 152 men who underwent prostate biopsy and found that Rikenellaceae, Alistipes, and Lachonospira were increased in patients with high-risk prostate cancer [64]. The predictive value of these bacterial taxa was similar to serum PSA levels for the high-risk prostate cancer group. However, the index comprised from a profile of 18 bacteria taxa identified high-risk prostate cancer cases more precisely. The area under the curve (AUC) of reservoir operating characteristic curve analysis of this index for detecting high-risk prostate cancer was 0.85, while the AUC of serum PSA levels was 0.74. Functional pathway analysis showed that five metabolic pathways (starch and sucrose metabolism, phenylpropanoid biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, cyanoamino acid metabolism, and histidine metabolism) were positively associated with high-risk prostate cancer. The analysis of fecal microbiome of 23 patients with metastatic castration-resistant prostate cancer (CRPC) resistant to enzalutamide prior to treatment with anti-PD-1 showed that the responders have increased levels of Streptococcus salivarius. Interestingly, Akkermansia muciniphila levels were reduced in the fecal samples from responders [65]. The above-mentioned studies suggest that the gut microbiome influences prostate cancer and vice versa. Until recently, the precise mechanisms driving their interactions remained largely undiscovered, however, new findings have shed light on the subject (Figure 2). In 2021, Matsushita et al. reported that SCFAs originating from the gut microbiome promoted the growth of prostate tumors in a transgenic mouse model of prostate cancer [66]. A HFD is considered to be a risk factor for prostate cancer and is implicated in promoting prostate cancer growth by inducing local inflammation [5]. Antibiotics administered to prostate-specific conditional Pten-knockout mice fed a HFD suppressed prostate cancer growth. Interestingly, different types of antibiotics showed different effects on tumor growth—indicating that specific taxa are responsible for this phenomenon. For example, gentamycin suppressed HFD-induced prostate cancer growth, but neomycin did not. Microarray analysis of prostate tumors showed that IGF-1 was significantly down-regulated in HFD-fed Pten-knockout mice treated with antibiotics compared with HFD-fed Pten-knockout mice without antibiotics. Antibiotics also decreased the insulin-like growth factor 1 (IGF-1) levels in serum in wild-type HFD-fed mice. IGF-1 stimulated prostate cancer growth via the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. Short chain fatty acids (SCFAs), such as butyrate, acetate, isobutyrate, and lactate are major metabolites of the gut microbiome, SCFAs play important roles in the regulation of intestinal immune cells with anti-inflammatory effects. SCFAs are also known to suppress colon cancer. Butyrate, the major compound of SCFAs suppress colonic carcinogenesis through this anti-inflammatory effect [67]. On the other hand, it also has a variety of other physiological effects, such as Wnt signal modulation, and promotes colorectal cancer, depending on its concentration [68,69]. However, in the Pten-knockout prostate cancer mouse model, the administration of antibiotics decreased SCFA levels in the gut. SCFAs from gut microbiota are known to stimulate IGF-1 production, leading to bone growth [70]. In the Pten-knockout prostate cancer mouse model, SCFAs also stimulated the production of IGF-1 in prostate tumors and other organs, such as liver, contributing to augmented prostate cancer growth. Notably, IGF-1 expression was upregulated in prostate cancer of severe obese patients compared with non-obese patients. Patients with benign prostatic hyperplasia (BPH) also have higher levels of fecal isobutyric acid and isovaleric compared to healthy individuals, and isobutyric acid, isovaleric acid, and isocproic acid were associated with the occurrence of metabolic syndrome in patients with BPH [71]. Furthermore, the Firmicutes/Bacteroidetes (F/B) ratio was significantly higher in Japanese men with enlarged prostates than in men with normal-size prostates [72]. SCFAs derived the gut microbiota may be a risk factor for prostate cancer and BPH by a similar mechanism. BPH is a very frequent age-related disease [73]. BPH, characterized by hyperplasia of the transition zone, is associated with inflammation, oxidative stress, and other several biological factors [74]. The relationships between different diets and the development of BPH has been discussed for some time [75]. HFD, in which 60% of kcal consists of lipids, promoted BPH in rat models, and activation of ERK1/2 was likely involved in this process [76]. Matsushita et al. reported that HFD also promoted prostate cancer growth via gut microbiome through a similar mechanism, suggesting that the connection between BPH and diet may be also mediated by the gut microbiome [66]. Fat consumption was reported to increase the incidence of BPH in humans. In a 7-year prospective study of 4770 subjects in The Prostate Cancer Prevention Trial, the highest fat intake group had a significantly increased hazard ratio by 31% compared with the lowest group [77]. It is important to note that the consumption of a HFD causes gut dysbiosis which leads to the development of a “leaky gut” by down-regulating tight junction molecules, such as ZO-1 [32,78]. Leaky gut leads to the leakage of bacterial components, such as lipopolysaccharide (LPS) or lipoteichoic acids (LTA), into systemic circulation. Leaked LPS and LTA in turn provoke systemic inflammation which have wide ranging implications including cancer promoting effects [79]. In prostate cancer, LPS activates mast cells via toll-like receptor 4 [78]. HFD-fed Pten-knockout mice also demonstrated upregulated levels of histamine decarboxylase (HDC), which plays a crucial role in histamine production. Fexofenadine, an H1 receptor blocker, suppressed the expression of inflammatory cytokines, such as Il-6, IL-10, IL-4, and IL-17, and reduced infiltration of MDSCs into prostate tumors. Furthermore, fexofenadine suppressed prostate cancer growth in HFD-fed mice. This upregulation in HDC levels is due to leaked LPS from dysbiotic gut microbiota of HFD-fed mice, and thus LPS administration to control diet-fed mice leaded HDC upregulation. In addition, inhibition of LPS suppressed the prostate cancer growth of HFD-fed mice. Like obese HFD-fed mice, severely obese patients with prostate cancer exhibited increased tumor-infiltrating mast cells. Gut dysbiosis could also be associated with drug-resistance in prostate cancer [78]. Antibiotics-induced dysbiosis, characterized by the enrichment of Proteobacteria, resulted in the elevation of tumor LPS due to an increase in gut permeability. Intratumoral elevation of LPS activated the NF-κB-IL6-STAT3 axis, leading to prostate cancer growth and docetaxel-resistance [80]. Androgen and its receptor have a pivotal role in the development of CRPC and these studies provide evidence that shows that the gut microbiome is intricately implicated and are further explored in the next section. ADT has been the gold standard for the treatment of prostate cancer for decades. However, patients eventually develop resistance and progress to CRPC which relies on minute levels of androgens for its growth by amplifying the androgen receptor [81]. Androgens are produced in the testis, adrenal glands, and in prostate tumors by cancer cells, but recent studies suggested that the gut microbiota also plays a role in androgenesis (Figure 3). In the analysis of 31 young Korean men aged 25–65 years using 16SrRNA sequencing, the abundance of Acinetobacter, Dorea, Ruminococcus, and Megamonas correlated significantly with serum testosterone levels [82]. In the analysis of the gut microbiota of 54 Japanese men aged 65 years or older, serum testosterone levels were positively correlated with the abundance of Firmicutes [83]. Several studies have also reported the relationship between the microbiome and polycystic ovarian syndrome (PCOS), a disease caused by high testosterone production in women [84,85]. Testosterone levels increased when feces from male mice were transplanted into female mice, suggesting that specific intestinal bacteria promoted testosterone production. In addition, Firmicutes synthesize testosterone or promotes its reabsorption through unconjugation [86]. Gut microbiota modulate the enterohepatic circulation of androgens, affecting systemic androgen levels. Gut bacteria can also produce androgens from glucocorticoids [86]. ADT also affects the gut microbiome in both mice and humans and promotes the expansion of specific bacteria [87]. ADT plus abiraterone acetate depletes androgen-utilizing and pro-inflammatory Corynebacterium and increases Akkermansia muciniphila in the gut. Predicted metagenomic analysis from 16S rRNA sequencing suggested that patients with abiraterone acetate increased bacterial biosynthesis of vitamin K2, which is known to be an inhibitor of androgen-dependent and-independent tumor growth [88]. Ruminococcus has genes sharing high sequence homology with human CYP17 and Ruminococcus can convert pregnenolone and hydroxypregnenolone in feces to DHEA and testosterone. Patients with CRPC had a higher abundance of Ruminococcus in their feces and promoted prostate cancer growth via the production of androgens in the gut. Abiraterone acetate, a selective inhibitor of CYP17A1, inhibited the bacterial conversion of pregnenolone to DHEA and testosterone. In contrast, FMT with hormone-sensitive microbiota or administration of Prevotella stercorea can decrease androgens levels in CTX mice and delay the onset of CRPC [87]. In this context, androgenesis by gut microbiota should be considered for patients undergoing ADT for metastatic hormone-sensitive prostate cancer or CRPC. Prostate cancer pathobiology is affected not only by genomic mutation, but also by epigenetic modification, namely the acquired regulation of gene expression [89]. Various environmental factors can cause alterations in the epigenome and may be drivers of cancer formation and progression [90]. Aberrant DNA hypermethylation is a prevalent epigenetic modification responsible for the inactivation of tumor suppressor genes in prostate cancer, and GSTP1, a class of Glutathione S-transferases (GSTs), a family of enzymes responsible for processes that protect cells from xenobiotics, was earliest reported to be hypermethylated in human prostate cancer [91]. Similarly, DNA hypermethylation is involved in the expression not only of DNA repair genes but also of the genes involved in cell cycle, apoptosis, and cell adhesion, which have been attempted to be regulated by dietary therapy [92]. The methyl group is extracted from S-adenyl methionine, and bacterial metabolites such as folate and betaine are essential for its synthesis [93]. Certain strains of Lactobacillus and Bifidobacterium used as probiotics have the ability to generate folate, and such strains may enhance prostate cancer risk via DNA hypermethylation [94]. Contrarily, in a gene-based predictive functional analysis of the gut microbiota by Liss et al. the function to synthesize folate was significantly reduced in prostate cancer patients compared to men without cancer [63]. It is possible that the functional analysis is based on gene prediction and does not reflect the actual folate synthesis capacity, but further studies are needed to address this discrepancy. Epigenetic modifications of histones, which give the backbone to chromatin, have been reported in prostate cancer [89]. One type of histone modification is methylation, which may also be influenced by gut microbiota-derived metabolites involved in methyl group donation. The other type, histone acetylation, leads to chromatin loosening, leading to transcription activation. Histone deacetylases (HDACs), which act in transcription inactivation by clearing acetyl groups, are inhibited by short-chain fatty acids (SCFAs) produced by some anaerobic bacteria fermenting dietary fiber [95]. Butyrate, one type of SCFA, at high concentrations, inhibited prostate cancer growth in vitro by altering the expression of cell cycle regulators and AR through the epigenetic histone modification [96]. However, butyrate has contrasting effects on cancer cells depending on its concentration, and we have shown that deficiency of gut microbiota-derived SCFAs rather inhibits prostate cancer growth in vivo [66,97]. These findings suggest that the gut microbiota may also be involved in epigenetic modifications of prostate cancer cells, although these associations have not yet been directly demonstrated. Future studies are needed. In these human gut microbiota analyses, the proportion of several intestinal bacteria seems to change according to the prostate cancer status of the host. Most of the human-reported studies have been conducted in the limited regions of Asia and the United States, although the composition of the gut microbiota varies between regions due to the diversity of lifestyles such as dietary habit. In other to achieve the identification of intestinal bacteria that truly work as promotive or preventive factors of prostate cancer linked to lifestyles, extensive global microbiota research of prostate cancer patients will be necessary. The gut microbiome is greatly influenced by several environmental factors, such as lifestyle. Change in the gut microbiota can be involved in prostate cancer progression through its metabolites and endotoxins. A greater understanding of the molecular mechanisms underlying these bidirectional interactions have allowed us to establish a “gut-prostate-axis”. These interventions could be further incorporated with current treatments as novel strategies for the prevention and management of human prostate cancer.

PMC10000198

Jun Chen,Xuehai Cao,Zhouyin Huang,Xingping Chen,Tiande Zou,Jinming You

Research Progress on Lycopene in Swine and Poultry Nutrition: An Update

28-02-2023

antioxidant,functional feed additive,lycopene,poultry,swine

Simple Summary Lycopene is a natural, red-colored pigment that is found in plants and has particularly robust antioxidant activity among the carotenoids. Plenty of research has highlighted its antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. Emerging evidence has promoted the development of lycopene as a natural antioxidant feed supplement to enhance the health status and performance of pigs and poultry. This review aimed to summarize the latest research advances on lycopene in swine and poultry, to deepen our understanding of its biological functions and practical applications in livestock production. Abstract Oxidative stress and in-feed antibiotics restrictions have accelerated the development of natural, green, safe feed additives for swine and poultry diets. Lycopene has the greatest antioxidant potential among the carotenoids, due to its specific chemical structure. In the past decade, increasing attention has been paid to lycopene as a functional additive for swine and poultry feed. In this review, we systematically summarized the latest research progress on lycopene in swine and poultry nutrition during the past ten years (2013–2022). We primarily focused on the effects of lycopene on productivity, meat and egg quality, antioxidant function, immune function, lipid metabolism, and intestinal physiological functions. The output of this review highlights the crucial foundation of lycopene as a functional feed supplement for animal nutrition.

Research Progress on Lycopene in Swine and Poultry Nutrition: An Update Lycopene is a natural, red-colored pigment that is found in plants and has particularly robust antioxidant activity among the carotenoids. Plenty of research has highlighted its antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. Emerging evidence has promoted the development of lycopene as a natural antioxidant feed supplement to enhance the health status and performance of pigs and poultry. This review aimed to summarize the latest research advances on lycopene in swine and poultry, to deepen our understanding of its biological functions and practical applications in livestock production. Oxidative stress and in-feed antibiotics restrictions have accelerated the development of natural, green, safe feed additives for swine and poultry diets. Lycopene has the greatest antioxidant potential among the carotenoids, due to its specific chemical structure. In the past decade, increasing attention has been paid to lycopene as a functional additive for swine and poultry feed. In this review, we systematically summarized the latest research progress on lycopene in swine and poultry nutrition during the past ten years (2013–2022). We primarily focused on the effects of lycopene on productivity, meat and egg quality, antioxidant function, immune function, lipid metabolism, and intestinal physiological functions. The output of this review highlights the crucial foundation of lycopene as a functional feed supplement for animal nutrition. Oxidative stress severely threatens the productivity and health status of farm animals, which results in huge economic losses for the livestock industry [1]. In animal production, many factors, such as changes in the environment [2], physiological stages [3,4], and exogenous pathogenic toxins (such as mycotoxins) [5], can cause oxidative stress, thus disturbing the redox balance in animal bodies. Oxidative stress refers to the imbalance between antioxidants and pro-oxidants [6]. The excessive production of reactive oxygen species (ROS) and reactive nitrogen radicals (RNS) will cause irreversible damage to cell lipids, proteins, and DNA, thus affecting the physiological functions and production performance of the animals [7]. Antioxidation is defined as the process of antioxidant defense against oxidation in organisms [8]. The antioxidant substances in the body are mainly divided into two categories; one is synthesized by the body itself, and the other is obtained from food [9]. When animals are in special circumstances such as high temperatures, weaning, pregnancy, and so on, the supplementation of exogenous antioxidant substances (such as plant-derived polyphenols and carotenoids) can effectively alleviate the oxidative stress status in animals, reduce oxidative damage, and improve their health and production performance [10,11]. In addition, antibiotic resistance and residues have adversely affected animal production, human health, and environmentally sustainable development [12]. Since the European Union (EU) banned the use of antibiotics as feed additives in animal feed in 2006, researchers have been trying to explore plant-derived feed supplements as safe antibiotic alternatives [13]. After July 2020, China officially entered a new era of in-feed antibiotic bans in livestock production [14]. Therefore, it is urgently necessary to develop natural, green, and safe antibiotic alternatives for the swine and poultry industry. Lycopene (C40H56), a red-colored carotenoid, is a natural pigment found in plants, mainly in fruits and vegetables such as tomatoes, carrots, watermelon, and guava [15]. Lycopene has the greatest antioxidant potential among carotenoids due to its chemical structure [16]. Structurally, it contains 2 non-conjugated double bonds and 11 conjugated double bonds [17]. Lycopene has been listed as a nutrient and food additive in more than 50 countries and is widely used in healthy foods, medicine, cosmetics, agriculture, and other fields [18]. Lycopene is a powerful antioxidant, which is the fundamental basis for its health-promoting effects, including anti-inflammatory, anticancer, and antidiabetic potential [19], cardiovascular-protecting abilities [20], and neurobiological, antihypertensive, and anti-aggregative effects [21]. Interestingly, there is an ever-increasing body of evidence indicating that lycopene could be developed as a functional feed additive for swine and poultry. In detail, lycopene has been reported to improve productivity, meat quality, egg quality, antioxidant, immunity, lipid metabolism, and intestinal physiological functions [22,23,24,25]. The biological functions of lycopene are primarily due to its chemical structure [16]. In this paper, we first briefly introduce the sources, physicochemical properties, digestion and absorption, and biological functions of lycopene. Then, we systematically summarize the latest research progress of lycopene in swine and poultry over the last ten years (2013–2022). Lycopene was first discovered as a red pigment in tomatoes by Millardet in 1876; 27 years later, it was named lycopene by Schunck in reference to the scientific name of tomato (Lycopersicon esculentum) [26]. Lycopene is widely distributed in tomatoes and also in red vegetables and fruits, such as carrots, sweet potatoes, pumpkin, watermelons, apricots, papaya, pink grapefruits, pink guavas, and rosehips [27]. Although lycopene exists in a variety of foods, tomato plays a leading role in lycopene-producing sources because it is the main extraction source, as well as the cheapest raw material for lycopene [28]. It has been reported that the global gross production of tomatoes was 180.8 million tonnes in 2019 [29]. Tomatoes are a rich source of lycopene, accounting for 80% to 90% of all carotenoid food content [27]. The extraction techniques of lycopene from raw materials commonly include conventional methods, microwave- and ultrasound-assisted extraction, supercritical fluid extraction, and enzyme-assisted extraction [30]. Most importantly, biosynthetic methods have been developed for large-scale lycopene production using biotechnology [31]. Microbial fermentation is a typical traditional biotechnology in lycopene production [32]. Additionally, modern biotechnology, including genetic engineering, protein engineering, and metabolic engineering, has also been applied to lycopene production [31]. Lycopene is a form of red-colored carotenoid with the molecular formula of C40H56 (Figure 1) [28]. It is a polyolefin chain composed of 13 double bonds, 11 of which are conjugated into a linear array (red color factor) [27], making it longer than other carotenoids [33]. Owing to the planar symmetry in its structure, lycopene does not have the same activity as vitamin A [34]. Lycopene is a red waxy pigment, which exists in nature in the form of slender needle-like crystals [33]. It is a liposoluble substance that is insoluble in water and easily soluble in benzene, chloroform, and acetone [27]. In nature, lycopene mostly exists in an all-trans configuration, which is relatively stable in terms of thermodynamics. However, at least 50% of its cis isomers are found in the plasma and tissues of humans [33]. The most common forms are 5-cis, 9-cis, 13-cis, and 15-cis isomers, which suggests that cis isomers are more easily absorbed and utilized by both humans and animals [35]. The absorption mode of lycopene is similar to that of lipids, occurring via a passive diffusion pathway [36]. The lycopene in the food matrix is released under the action of gastric acid, bile acid, and enzymes [36]. Upon entering the intestine, lycopene is combined with dietary lipids to form chylomicrons, which are then transported into the mesenteric lymphatic system via diffusion and permeation [37]. Thereafter, the lycopene is finally discharged into portal circulation [36]. This is the main way for lycopene to be absorbed from the gastrointestinal tract [37]. Extrahepatic lipoprotein lipase can partially degrade chyle particles into chyle particle residues. Lycopene and its metabolites are randomly released and transported by low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and are finally distributed to the target tissues [35]. The chemical structure of lycopene will affect its distribution process. Through the circulatory system, it will preferentially accumulate in the testis, adrenal gland, liver, and prostate. This uneven distribution indicates its unique biological role in these tissues [38], such as its regulatory role in hepatic lipid metabolism [39]. The unique double-bond structure of lycopene makes it far better than other carotenoids at scavenging free radicals in humans and animals. The greatest antioxidant carotenoid is lycopene [16], followed by tocopherol, carotene, cryptoxanthin, zeaxanthin, β-carotene, and lutein [27]. Lycopene is a robust antioxidant, which is the fundamental basis for its health-promoting effects [40,41]. Furthermore, there is an ever-increasing body of evidence to show that lycopene has anti-inflammatory, anticancer, and antidiabetic potential [19]. Additionally, lycopene has been demonstrated to exert cardiovascular-protecting effects [20], neurobiological effects, and antihypertensive and anti-aggregative effects [21]. Most recently, lycopene has been developed as an effective feed supplement for swine and poultry due to its potent antioxidant potential and red-colored pigment characteristics. The next section of this review will provide a systematic overview of research progress on lycopene in swine and poultry nutrition during the past decade (2013–2022), mainly focusing on its beneficial effects on production performance, meat quality, egg quality, antioxidant function, immune function, lipid metabolism, and intestinal barrier function. Figure 2 presents an overview diagram of the beneficial effects of lycopene on swine and poultry. During the last ten years, lycopene has garnered extensive research attention in the field of swine and poultry production. Sun et al. [22] reported that 50 mg/kg dietary lycopene supplementation during gestation and lactation improved the reproductive performance of sows, including increased born-alive piglets, weaned piglets, litter birth weight, litter weaning weight, and decreased born-dead piglets. The authors finally concluded that lycopene promoted sow reproductive performance by regulating milk composition, placental immunity, and antioxidant ability [22]. However, it appears that dietary lycopene inclusion does not influence the production performance of farm animals during the finishing period, especially in the case of finishing pigs. For instance, Fachinello et al. pointed out that dietary addition with a variety of lycopene dosages (12.5, 25, 37.5, and 50 mg/kg) did not impact the growth performance of finishing pigs [42]. Likewise, Fachinello et al. indicated that feeding 12.5, 25, 37.5, and 50 mg/kg of lycopene to finishing pigs did not affect their carcass characteristics or relative organ weights [43]. A recent study conducted by Wen et al. also showed no effects on the performance and carcass characteristics of finishing pigs from dietary lycopene supplementation at 100 or 200 mg/kg [23]. In a poultry study, Sun et al. demonstrated that feeding 40 mg/kg lycopene to Xinghua breeding hens for 35 days increased the fertilization rate and hatchability of their eggs [44]. An et al. also found that the dietary addition of 20 mg/kg lycopene or 1.7% tomato paste for 28 days elevated the egg weight and egg production of Hy-line Brown laying hens [45]. The beneficial additive effect of lycopene on productive performance and carcass characteristics was also observed in Japanese quail, according to a study by Al-Jrrah et al. [46]. In addition, Amer et al. noted that dietary supplementation with lycopene at 300 mg/kg increased the relative growth rate of Japanese quail [47]. In a 42-day feeding trial conducted by Wan et al., supplementation with 10, 20, or 30 mg/kg lycopene increased the average daily gain (ADG), feed conversion ratio (FCR), and final body weight of 1-day-old broilers [48]. Similarly, Wan et al. noticed that broilers supplemented with 100 mg/kg lycopene demonstrated greater body weight at day 21 of the feeding trial [49]. Interestingly, Mezbani et al. revealed that the dietary inclusion of lycopene (50, 100, and 200 mg/kg) enhanced growth performance without affecting the carcass performance of Ross 308 broilers, as indicated by an increased ADG, average daily feed intake (ADFI), and decreased FCR [50]. On the contrary, using tomato paste as a lycopene source, Lee et al. observed no effects on the growth performance and relative organ weights of broilers fed diets containing 10 or 20 mg/kg of lycopene, or 17 g/kg of tomato paste [51]. Most importantly, lycopene has been demonstrated to exert growth-promoting effects on farm animals under stressful conditions, such as heat stress, and due to mycotoxin feed contamination [52,53,54,55,56,57]. For example, Sarker et al. proved that the supplementation of 200 mg/kg lycopene for 42 days increased the ADG and decreased the FCR of broilers under the aflatoxin B1 (AFB1) challenge condition [52]. Moreover, the results of Sarker et al. showed that the inclusion of lycopene promoted the growth performance of broilers under the AFB1 challenge, as indicated by increased ADG (day1–21: 100 mg/kg lycopene; day22–42 and day1–42: 200 or 400 mg/kg lycopene) and decreased FCR (day22–42 and day1–42: 200 or 400 mg/kg lycopene) [53]. Under heat stress conditions, the supplementation of 0, 200, and 400 mg/kg lycopene for 42 days has also been confirmed to linearly elevate the growth performance of Ross 308 broilers, as reflected by an increased cumulative feed intake, weight gain, and reduced FCR [54]. It should be noted, however, that the high-dosage supplementation of lycopene (500 mg/kg) has been demonstrated to negatively influence the growth performance of Hubbard broilers, including decreased ADFI and ADG during the first week of the feeding trial [55]. Therefore, in light of the observed results, lycopene (or tomato paste) has great potential to be utilized as a growth-promoting supplement (or feedstuff) for swine and poultry. However, further research is needed to confirm its favorable effects on the production performance of pigs and poultry, using feeding trials with a large population of animals. Along with rapid economic development and livestock production technology improvement, the ever-increasing attention of consumers has been paid to meat and egg quality in place of meat and egg quantity [58,59]. The quality of meat and eggs is a key criterion for consumers when choosing livestock products [60]. In recent years, lycopene has obtained substantial attention as a natural feed supplement intended to improve meat and egg quality in animal production. As reported by Wen et al., the dietary inclusion of lycopene improved the meat quality of finishing pigs, including reduced L* and b* values, elevated a* value, and intramuscular fat and crude protein contents of the longissimus dorsi (LD) muscle [23]. The thawing loss of the longissimus lumborum (LL) muscle was also found to be linearly reduced when finishing pigs were fed diets supplemented with 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene [43]. Besides this, lycopene was demonstrated to promote muscle fiber type transformation in pigs, which is of great significance in determining meat quality [23]. Specifically, the mRNA levels of Cytc, TFAM, TFB1M, CS, COX1, MyHC1, MyHC IIa, MyHC IIx, and TNNI1 were up-regulated, while the mRNA level of MyHC IIb was down-regulated in the LD muscle by lycopene supplementation [23]. In addition, the protein expression of slow MyHC, Cytc, myoglobin, slow-twitch fiber percentage, succinic dehydrogenase (SDH), and malate dehydrogenase (MDH) was increased, while fast MyHC, fast-twitch fiber percentage, and lactate dehydrogenase (LDH) activity were decreased in the LD muscle of finishing pigs fed lycopene diets (100 or 200 mg/kg lycopene) [23]. Importantly, the oxidative stability of pork has been reported to be elevated by dietary lycopene feeding (20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste) [61]. As we know, lipid oxidation negatively influences the color, nutritional value and flavor, and shelf-life of meat [62]. An et al. reported that the oxidative indices in fresh pork belly meats were decreased when finishing pigs were fed lycopene-supplemented diets, including decreased malondialdehyde (MDA) levels and increased lycopene levels [61]. Likewise, Wen et al. noted that dietary lycopene inclusion improved LD muscle antioxidant status, as indicated by increased total superoxide dismutase (T-SOD) and catalase (CAT) activities, decreased MDA level, up-regulated mRNA levels of SOD1, SOD2, CAT, GPX1, GST, GR, and Nrf2, and down-regulated Keap1 mRNA levels [23]. Similar findings were reported by Correia et al., who observed improved oxidative stability of the LL muscle in young pigs fed 5% tomato pomace for 5 weeks [63]. However, An et al. found that fatty acid composition in the fresh belly meat of finishing pigs was unaffected by dietary lycopene supplementation with 20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste in a 28-day feeding trial [61]. Regarding egg quality, the study by Shevchenko et al. showed that supplementing High Line W36 laying hens’ diets with lycopene (20/40/60 mg/kg) for 90 days resulted in improved egg quality, as indicated by an increased carotenoid level and yolk color in fresh eggs or in eggs undergoing a 4 °C and 12 °C storage period [24]. Orhan et al. also indicated that feeding Lohman LSL laying hens with 20 mg/kg lycopene as a purified powder or tomato powder for 84 days improved egg quality, including increased egg weight, yolk color, yolk weight, yolk ratio, yolk lycopene level, and decreased yolk MDA and cholesterol levels [64]. An et al. also found that the dietary addition of 10 or 20 mg/kg lycopene for 28 days increased yolk color and lycopene levels and decreased MDA levels in the eggs of Hy-line Brown laying hens [45]. A previous study by Sun et al. indicated that feeding 40 mg/kg of lycopene to Xinghua breeding hens for 35 days increased the lycopene levels in the serum, eggs, and liver, and an elevated yolk color score [44]. Additionally, Sahin et al. showed that the supplementation of 200 and 400 mg/kg lycopene for 42 days improved the muscle antioxidant status of Ross 308 broilers under heat stress conditions, including a linearly increased lycopene level, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities, decreased MDA level, down-regulated Keap1, and up-regulated Nrf2 protein expression (400 mg/kg lycopene) [54]. Similarly, Lee et al. noticed that dietary supplementation with 10, 20 mg/kg lycopene, or 17 g/kg tomato paste for broilers decreased the thiobarbituric acid-reactive substance (TBARS) value of LDL isolated from broilers at both 2 weeks old and 4 weeks old [51]. However, in laying quails, Hsu et al. suggested that the supplementation of lycopene (6 and 18 mg/kg lycopene as commercial or bacterial lycopene) for 28 days did not affect laying performance or egg quality, or the serum antioxidant status [65]. Therefore, given the current findings, lycopene has huge development value in terms of being utilized as a green feed supplement for meat and egg production, as well as for producing lycopene-enriched meats and eggs as functional foods for humans [66]. The antioxidant potential of lycopene is one of its predominant biological characteristics, as has been proven in swine and poultry by several animal nutritionists. For instance, Sun et al. demonstrated that dietary 50 mg/kg lycopene supplementation during gestation increased total antioxidant capability (T-AOC) and GSH-Px activity and decreased the H2O2 and ROS levels in the placental tissues of sows [22]. Additionally, the mRNA levels of GPX1, GPX4, FABP4, SLPI, ANX1, and APOE in the placenta were also up-regulated by lycopene supplementation at 50 mg/kg [22]. In finishing pigs, the dietary supplementation of 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene linearly reduced the TBARS level and increased the 2,2 diphenyl 1 picrylhydrazyl level in the liver [43]. The gene expression of SOD1 and CAT were also found to be linearly affected by dietary lycopene supplementation (0, 12.5, 25, 37.5, and 50 mg/kg) [42]. Similarly, as reported by Wen et al., the dietary inclusion of lycopene improved antioxidant status, including increased T-AOC, T-SOD, GSH-Px, and CAT activities in the serum and liver, and decreased hepatic MDA levels in finishing pigs [23]. Lycopene was also reported to enhance the antioxidant ability and reduce oxidative damage in piglets. In a study conducted by Meng et al., supplementing 50 mg/kg lycopene to weaned piglets for 28 days increased the activities of CAT in serum and SOD in the jejunum, and decreased H2O2 levels in the serum and jejunum [25]. Lycopene supplementation also up-regulated the mRNA levels of NRF2, SOD2, CAT, GLUT2, GLUT5, CD36, CLDN1, and IL-22, along with the protein levels of NRF2, CD36, and CLDN1, and down-regulated the mRNA and protein levels of KEAP1 [25]. Those results indicate that lycopene could activate the antioxidant signaling pathway (Keap1/Nrf2) and drive the downstream antioxidant gene expression. Using an in vitro model of a pig embryo, Kang et al. noticed that treating the embryo with 0.1 μM lycopene for 6 days increased the blastocyst formation of embryos, the number of total cells, and the trophectoderm [67]. The authors further investigated the underlying mechanisms and finally concluded that the beneficial effects of lycopene on porcine embryos were attributed to its reducing oxidative stress and apoptosis [67]. In an in vitro model with piglet Sertoli cells, lycopene was also demonstrated to alleviate zearalenone-induced oxidative injury via regulating the Nrf2 signaling pathway [68]. In a broiler-feeding trial by Wang et al., supplementing lycopene (especially at 30 mg/kg) to 1-day-old broilers for 42 days markedly elevated the serum antioxidant status, as indicated by increased T-AOC, GSH-Px, and SOD activities, and decreased MDA level on day 21 of the study [48]. Moreover, the hepatic antioxidant status was also improved by lycopene supplementation, as suggested by increased GSH-Px, SOD activities, decreased MDA levels (10, 20, and 30 mg/kg lycopene) at day 21 of the study, and increased GSH-Px activity (30 mg/kg lycopene), decreased MDA level (10, 20, and 30 mg/kg lycopene) at day 42 of the study [48]. Meanwhile, the Nrf2 pathway was activated by lycopene supplementation, which further drove Nrf2-downstream antioxidant gene expression, including SOD2, NQO1, and HO-1 [48]. Feed AFB1 contamination commonly occurs in poultry production and seriously threatens the productivity and health of poultry [69]. It has been confirmed that oxidative stress is one of the key mechanisms of AFB1 toxicity, and the addition of exogenous antioxidants (such as antioxidant polyphenols and carotenoids) in diets can effectively alleviate the adverse effects of AFB1 in poultry [70]. In an experiment by Wan et al., the dietary addition of 100, 200, or 400 mg/kg lycopene all markedly decreased the hepatic activities of cytochrome P450 1A1 (CYP1A1) and cytochrome P450 2A6 (CYP2A6) in broilers fed AFB1-contaminated diets in a 42-day feeding trial [71]. The antioxidant status was also enhanced by lycopene supplementation, as suggested by an increased GSH (200 mg/kg lycopene) level, glutathione s-transferase (100 and 400 mg/kg lycopene), glutamine-cysteine ligase, CAT (100 mg/kg lycopene), GSH-Px activities (100, 200 and 400 mg/kg lycopene), decreased AFB1-8,9-epoxide-DNA and ROS levels (100, 200, and 400 mg/kg lycopene), MDA, 8-hydroxydeoxyguanosine (100, 200 and 400 mg/kg lycopene), 4-hydroxynonenal, and protein carbonyl levels (200 and 400 mg/kg lycopene) [71]. Similarly, Sarker et al. documented the finding that feeding 200 mg/kg lycopene for 42 days to broilers fed AFB1-contaminated feed elevated the hepatic mitochondrial antioxidant status, including greater mGSH, GSH-Px, MnSOD, and ATP levels, along with lower ROS and H2O2 levels, and reduced mitochondrial swelling [52]. Additionally, hepatic mitochondrial function was improved, such as increased complex III and complex V, and an up-regulated mRNA level of MnSOD, Trx2, TrxR2, Prx3, PGC-1α, NRF1, and TFAM [52]. El-Sheshtawy et al. found that the dietary addition of 100 mg/kg lycopene for 10 days in Pekin ducks decreased the hepatic aflatoxin residue and increased hepatic antioxidant status (increased T-AOC, GST, and CAT activities, and decreased MDA content) [72]. Mezbani et al. indicated that the dietary addition of lycopene improved the serum antioxidant status of Ross 308 broilers, which is suggested by an increase in GSH-Px activity, decreased MDA content (100, 200 mg/kg lycopene), and increased CAT activity (50, 100, 200 mg/kg lycopene) [50]. Sahin et al. observed that dietary supplementation with 200 or 400 mg/kg lycopene for 42 days improved the antioxidant status of Ross 308 broilers under heat-stress conditions, including linearly increased serum lycopene content, GSH-Px, and SOD activities, along with decreased MDA content [54]. In laying hens, as observed by Orhan et al., supplementation of 20 mg/kg lycopene as a purified powder or tomato powder for 84 days decreased serum MDA and cholesterol levels in Lohman LSL laying hens [64]. Likewise, An et al. documented that the dietary addition of 10, 20 mg/kg lycopene or 1.7% tomato paste for 28 days all increased hepatic lycopene levels, while supplementation with 20 mg/kg lycopene or 1.7% tomato paste decreased serum MDA levels in Hy-line Brown laying hens [45]. Sun et al. noted that feeding lycopene to Xinghua breeding hens for 35 days increased the antioxidant status, including increased serum SOD (day21, 20, 40, or 80 mg/kg lycopene; day35, 80 mg/kg lycopene), T-AOC (day21, 20, 40, or 80 mg/kg lycopene), GSH/GSSG (day21,28,35, 20, 40, or 80 mg/kg lycopene), GSH-Px (day14, 20, 40, or 80 mg/kg lycopene), and increased hepatic SOD, T-AOC, and GSH/GSSG (day35, 20, 40, or 80 mg/kg lycopene) [44]. Amer et al. observed that dietary 300 mg/kg lycopene supplementation increased SOD activity and decreased MDA levels in both the liver and chest muscle of Japanese quail [47]. Sun et al. evaluated the beneficial effects of lycopene (20, 40, and 80 mg/kg) on breeding hens under lipopolysaccharide challenge conditions and observed higher GSH/GSSG in serum at 24 h post-injection of lipopolysaccharide by lycopene feeding at 20, 40, or 80 mg/kg [73]. Therefore, as a natural antioxidant, lycopene could be developed as a functional supplement for swine and poultry. However, along with in vivo feeding trials, more in vitro or ex vivo studies are required to illuminate the specific mechanism of action of lycopene, which is of great importance for promoting the research and application of lycopene in animal nutrition. Inflammation is reported to be the cost paid by livestock productivity, and it is the basis for allocating nutrient resources between growth and survival in livestock production [74]. Inflammatory reactions are implicated in animal diseases (such as diarrhea and enteritis), resulting in compromised productivity and higher mortality levels in swine and poultry [75]. Lycopene has been demonstrated to have health-promoting effects on farm animals due to its immune-regulatory functions [56]. Sun et al. highlighted that feeding gestating sows with 50 mg/kg lycopene improved their placental immunity status, including increased secretory immunoglobulin A (sIgA), immunoglobulin G (IgG), immunoglobulin M (IgM) levels, and decreased interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) levels [22]. The authors also noticed that the mRNA levels of IL-1β, IL-8, and TNF-α in the placenta were also down-regulated by lycopene supplementation [22]. In a recent study by Liu et al., the dietary addition of 200 mg/kg lycopene for 70 days notably improved the intestinal immune status of finishing pigs, as indicated by decreased IL-1β, TNF-α, and nuclear factor κ-B (NF-κB) levels in the duodenum, down-regulated mRNA levels of IL-1β in the jejunum, and up-regulated interleukin-10 (IL-10) mRNA levels in the duodenum and jejunum [76]. Fachinello et al. investigated the impacts of lycopene on the immune responses of finishing pigs and observed that supplementing 0, 12.5, 25, 37.5, and 50 mg/kg lycopene for 28 days linearly increased the plasma albumin and lymphocyte levels, and linearly and quadratically affected neutrophils and the neutrophil/lymphocyte ratio, and also quadratically affected eosinophils in the blood and anti-bovine serum albumin (BSA) production [77]. The immunomodulatory effects of lycopene have also been reported in poultry. In a study conducted by Shevchenko et al., feeding High Line W36 laying hens with lycopene (20, 40, and 60 mg/kg) for 90 days markedly decreased the leukocytes and erythrocytes, but had no effects on the serum antibody titer of hens vaccinated against Newcastle disease, avian rhinotracheitis, egg drop syndrome, and infectious bronchitis [78]. In a 42-day feeding trial by Alwash et al., supplementing the diet with lycopene increased the blood-packed cell volume, heterophils/lymphocytes, and blood hemoglobin level of Japanese quail under heat-stress conditions [79]. Additionally, Sarker et al. revealed that the supplementation of 200 mg/kg lycopene improved the intestinal immunity of broilers under AFB1 challenge conditions, as suggested by increased IL-10 levels at day 21 and day 42, and decreased the interferon-γ (IFN-γ) levels in jejunum at day 21 of the study [80]. Furthermore, Sun et al. evaluated the beneficial effects of lycopene on breeding hens under lipopolysaccharide challenge conditions and found that feeding lycopene (20, 40, and 80 mg/kg) to hens for 35 days augmented the immune organ indices of the thymus, spleen, and bursal [73]. However, a high supplemental dosage of lycopene could cause side effects in farm animals. As reported by Pozzo et al., the immune organ weight of the spleen and bursa of Fabricius were reduced by lycopene supplementation at up to 500 mg/kg in the diets of Hubbard broilers [55]. Therefore, further studies are urgently needed to determine the optimal supplemental dosage of lycopene for swine and poultry. Further research is also warranted to clarify the underlying mechanism of action in terms of the immunomodulatory effects of lycopene. Lycopene has been demonstrated to exert modulatory effects on lipid metabolism in swine and poultry. Most recently, Meng et al. reported that supplementing 50 mg/kg lycopene to weaned piglets for 28 days increased their serum total cholesterol (TC) level [25]. In finishing pigs, supplementation with 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene linearly affected plasma cholesterol, high-density lipoprotein (HDL) and LDL levels, and the LDL/HDL ratio of pigs [42]. Wen et al. also proved that the dietary inclusion of lycopene at 100 or 200 mg/kg up-regulated the mRNA levels of AMPKα1, AMPKα2, Sirt1, PGC-1α, and up-regulated the protein levels of P-AMPK/AMPK, NRF1, CaMKKβ, Sirt1, PGC-1α in LD muscle of finishing pigs [23]. However, a study by An et al. indicated that serum lipid profiles of finishing pigs were unaffected by dietary supplementation of 20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste in a 28-day feeding trial [61]. The placental protein expressions of APOE, ANX1, SLPI, and FABP4 are up-regulated for sows that are fed diets supplemented with 50 mg/kg lycopene during gestation [22]. In broilers, a study by Wan et al. suggested that the dietary inclusion of lycopene at 100 mg/kg for 42 days decreased the abdominal fat weight and abdominal fat percentage of broilers [49]. These results indicated a lipid-lowering property of lycopene. Concurrently, the plasma concentrations of total triglyceride (TG), TC, low-density lipoprotein cholesterol (LDLC), and the hepatic concentrations of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were reduced in broilers that were fed diets supplemented with 100, 200, or 400 mg/kg of lycopene [49]. Moreover, supplementation of 100, 200, or 400 mg/kg of lycopene all up-regulated the mRNA levels of AMPK-α, and down-regulated the mRNA levels of SREBP-1, FAS, and ACC in the livers of broilers, which are indicative of hepatic lipid metabolism [49]. In an experiment by Wan et al., the dietary addition of 100, 200, or 400 mg/kg lycopene for 42 days decreased the levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum of broilers fed AFB1-contaminated diets [71]. A study by Mezbani et al. also indicated that feeding lycopene to Ross 308 increased their glucose and HDL (100, 200 mg/kg lycopene) and decreased their cholesterol, triglyceride, VLDL (50, 100, 200 mg/kg lycopene) levels, also decreasing the hepatic activities of ALT and ALP (100, 200 mg/kg lycopene) [50]. Additionally, Lee et al. revealed that supplementing the diet of broilers with lycopene or 17 g/kg tomato paste decreased plasma TG (10, 20 mg/kg lycopene) and LDL cholesterol (20 mg/kg lycopene) levels at 2 weeks old, and increased the lycopene level in the plasma and liver (10, 20 mg/kg lycopene, 17 g/kg tomato paste) at 4 weeks old [51]. Similar results were found in ducks; the supplementation of 100 mg/kg lycopene for 10 days decreased ALT, AST, gamma-glutamyl transferase (γ-GT), alkaline phosphatase (ALP), and uric acid levels, and increased albumin and total protein levels in the serum of Pekin ducks [72]. However, Pozzo et al. claimed that feeding a high level of lycopene (500 mg/kg) to Hubbard broilers for 35 days decreased the total protein, gamma globulin, albumin, and alpha globulin levels [55]. The regulatory functions of lycopene on fat deposition and lipid metabolism have also been reported in laying and breeding hens. Shevchenko et al. found that feeding High Line W36 laying hens with lycopene (20/40/60 mg/kg) for 90 days markedly increased the serum levels of glucose, creatinine, and ALT, and decreased the serum levels of cholesterol, AST, alkaline phosphatase at day 31 of the study; increased serum levels of glucose, cholesterol, and ALT, and decreased serum level of AST were recorded at day 61 of the study, and increased serum levels of glucose, creatinine, and cholesterol were recorded at day 91 of the study [24]. Additionally, Orhan et al. fed Lohman LSL laying hens with 20 mg/kg lycopene as a purified powder or tomato powder for 84 days and found that lycopene decreased the protein expression of intestinal NPC1L1, MTP, ACAT2, hepatic SREBP1c, ACLY, and LXRα, and increased the protein expression of hepatic ABCG5 and ABCG8 [64]. In a study with quails, Hsu et al. noticed that feeding laying quail with lycopene for 28 days decreased the yolk triglyceride level (6, 18 mg/kg commercial lycopene; 18 mg/kg bacterial lycopene), serum triglyceride level (18 mg/kg bacterial lycopene), and serum total lipid level (6 and 18 mg/kg commercial lycopene) [65]. Under heat-stress conditions, Japanese quail that were fed lycopene diets for 42 days had increased blood glucose and decreased triglyceride levels (with 150, 200, 250, and 300 mg/kg lycopene), increased cholesterol (with 250 and 300 mg/kg lycopene), total protein, and globulin levels (300 mg/kg lycopene) [79]. In contrast, An et al. did not observe alterations in the serum lipid profile of Hy-line Brown laying hens fed 10 or 20 mg/kg lycopene or 1.7% tomato paste diets for 28 days [45]. In breeding hens, Sun et al. highlighted that supplementing lycopene to Xinghua breeding hens for 35 days decreased the total cholesterol (TCHO) levels (day7,35, 20, 40, and 80 mg/kg lycopene), increased high-density lipoprotein cholesterol (HDLC) (day35, 20, 40, 80 mg/kg lycopene), and T3 level (day14, 20 mg/kg lycopene) in the serum of hens [44]. Tian et al. demonstrated that feeding lycopene (20, 40, and 80 mg/kg) to Xinghua breeding hens for 35 days regulated the gene expression of fat metabolism. Specifically, lycopene up-regulated the hepatic mRNA levels of PPARα, RARα, PGCα (40 and 80 mg/kg lycopene), RXRα (20, 40 and 80 mg/kg lycopene), and down-regulated mRNA levels of FABP10 (40, 80 mg/kg lycopene), FABP1 (20, 40, and 80 mg/kg lycopene), and FATP4 (40 mg/kg lycopene). In the duodenum, the RARα mRNA level was up-regulated by the supplementation of 40 or 80 mg/kg lycopene. In the jejunum, lycopene up-regulated the mRNA levels of PPAPγ, RXRγ (40 and 80 mg/kg lycopene), and RXRα (20 and 40 mg/kg lycopene), and down-regulated the mRNA levels of FATP4 (40 and 80 mg/kg lycopene) [81]. Sun et al. evaluated the beneficial effects of lycopene on breeding hens under lipopolysaccharide challenge conditions and found that lycopene (at 20, 40, and 80 mg/kg) decreased the levels of TCHO, LDLC, and blood urea nitrogen (BUN), and increased HDLC and T3 levels in serum [73]. Therefore, lipid metabolism regulation by lycopene could partially explain the improvement of meat quality, egg quality, and health promotion in swine and poultry. Given the lowering-lipid ability of lycopene, it also has great potential for development as a functional additive to counteract obesity and diabetes in humans. The intestinal tract is the main site of digestion and the absorption of feed nutrients in farm animals and is also an indispensable line of defense against exogenous pathogenic microorganisms and their toxins [82,83,84]. The maintenance of normal intestinal physiological functions and gut homeostasis is a prerequisite for productivity efficiency and well-being in swine and poultry [85,86]. Lycopene has been confirmed to improve the intestinal morphology of pigs, which is indicative of gut health. Most recently, Meng et al. reported that feeding 50 mg/kg lycopene to weaned piglets for 28 days increased villus height (VH) and the ratio of VH to the crypt depth (CD) of the jejunum [25]. The jejunum’s digestive enzyme activities were also increased by lycopene supplementation in piglets, as indicated by elevated lipase, sucrase, maltase, and lactase activities [25]. Consistently, Liu et al. also investigated the beneficial effects of lycopene supplementation on the intestinal morphology of finishing pigs and found that the supplementation of 200 mg/kg lycopene for 70 days significantly augmented the ratio of VH/CD of the jejunum in finishing pigs [76]. In addition, the authors also noticed that the addition of 200 mg/kg lycopene notably up-regulated the mRNA and protein expression of Claudin-1 in the jejunum of pigs, which is indicative of intestinal integrity enhancement via lycopene feeding [76]. The regulatory effects of lycopene on intestinal physiological functions may be attributed to its antioxidant properties. As reported by Liu et al., the supplementation of 100 or 200 mg/kg lycopene for 70 days significantly increased CAT activity in the jejunum and decreased MDA levels in the duodenum of finishing pigs, which suggested that lycopene supplementation promoted intestinal antioxidant capacity [76]. Similar results have been reported in poultry studies. Sarker et al. reported that dietary supplementation with lycopene (at 100, 200, or 400 mg/kg) improved the intestinal morphology of broilers undergoing an AFB1 challenge [53]. The authors also observed elevated digestive enzyme activities in the small intestine, including duodenum amylase and lipase activities (day21: 200, 400 mg/kg lycopene; day42: 200 mg/kg lycopene), and jejunum amylase and lipase activities (day42: 200, 400 mg/kg lycopene), as well as ileum amylase activity (day42: 200 mg/kg lycopene) [53]. According to the findings of Sarker et al., supplementation with 200 mg/kg lycopene enhanced the intestinal integrity of broilers under AFB1 challenge conditions, as suggested by reduced serum diamine oxidase concentrations, with up-regulated jejunum ZO-1 and Claudin-1 mRNA levels at day 42 of the experiment [80]. Additionally, Al-Jrrah et al. noted that feeding Japanese quail with lycopene improved their gastrointestinal and organ development [46]. Similarly to the results in swine, Sarker et al. found that supplementing feed with 200 mg/kg lycopene improved the intestinal antioxidant of broilers under AFB1 challenge conditions, as suggested by decreased jejunum H2O2 (day42) and MDA levels (day21, day42), along with increased GSH, GST, and GR levels, as well as up-regulated mRNA levels of Nrf2, HO-1, Cu/ZnSOD, MnSOD, and CAT (day42) [80]. Hence, the maintenance of intestinal physiological functions is another crucial biological function of lycopene for swine and poultry. A summary of lycopene as a functional feed additive for swine, meat-producing, and egg-producing poultry is presented in Table 1, Table 2 and Table 3, respectively. Lycopene is widely considered an effective antioxidant that can capture oxygen free radicals, regulate the cellular expression of related transcription factors, enhance the activities of antioxidant enzymes, and protect cells from free radical damage. As a natural pigment and potent antioxidant, lycopene can be developed as a functional feed supplement for swine and poultry. Several pieces of research have revealed that lycopene has great potential to improve productivity, meat quality, and egg quality, as well as antioxidant activity, immune function, lipid metabolism, and intestinal physiological functions. In view of the current knowledge, lycopene could be supplemented as lycopene products (including commercial lycopene and tomato paste) at 10–400 mg/kg in swine and poultry diets, while a higher supplemental dosage could adversely affect the productivity and health of animals. However, the research on lycopene to alleviate oxidative stress and promote productivity is still in the initial stages, and the research results have not been widely popularized. Therefore, future research directions should further explore the additive effects of lycopene on swine and poultry at different physiological phases and different rearing environments, and, finally, establish the optimum supplemental dosage for the productivity and health of swine and poultry. More importantly, further research is warranted to elaborate the specific mechanism of action of lycopene, which is of great significance in promoting the research and application of lycopene in animal nutrition, or even in human nutrition.

PMC10000201

Yiyue Ren,Zhen Song,Jens Rieser,Jörg Ackermann,Ina Koch,Xingyu Lv,Tong Ji,Xiujun Cai

USP15 Represses Hepatocellular Carcinoma Progression by Regulation of Pathways of Cell Proliferation and Cell Migration: A System Biology Analysis

21-02-2023

USP15,HCC,pathway analysis,system biology,cell behavior

Simple Summary Expression levels of the protein USP15 correlated with slow proliferation and slow migration of HCC cells. In HCC patients, high expression of USP15 in tumor tissue was associated with significantly decreased risk for mortality and cancer relapse. Overexpression of USP15 led to reduced tumor growth in a mouse model. USP15 regulated indirectly tumor-related proteins through interactions mediated by other proteins. The functions “cell migration” and “cell proliferation” were significantly enriched in a network of pathway hierarchies that we found relevant for the regulatory role of USP15 in HCC. Six cluster of pathways described the link between experimental observed phenotypes and expression levels of USP15. Abstract Background: Hepatocellular carcinoma (HCC) leads to 600,000 people’s deaths every year. The protein ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a ubiquitin-specific protease. The role of USP15 in HCC is still unclear. Method: We studied the function of USP15 in HCC from the viewpoint of systems biology and investigated possible implications using experimental methods, such as real-time polymerase chain reaction (qPCR), Western blotting, clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS). We investigated tissues samples of 102 patients who underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). Tissue samples were immunochemically stained; a trained pathologist then scored the tissue by visual inspection, and we compared the survival data of two groups of patients by means of Kaplan–Meier curves. We applied assays for cell migration, cell growth, and wound healing. We studied tumor formation in a mouse model. Results: HCC patients (n = 26) with high expression of USP15 had a higher survival rate than patients (n = 76) with low expression. We confirmed a suppressive role of USP15 in HCC using in vitro and in vivo tests. Based on publicly available data, we constructed a PPI network in which 143 genes were related to USP15 (HCC genes). We combined the 143 HCC genes with results of an experimental investigation to identify 225 pathways that may be related simultaneously to USP15 and HCC (tumor pathways). We found the 225 pathways enriched in the functional groups of cell proliferation and cell migration. The 225 pathways determined six clusters of pathways in which terms such as signal transduction, cell cycle, gene expression, and DNA repair related the expression of USP15 to tumorigenesis. Conclusion: USP15 may suppress tumorigenesis of HCC by regulating pathway clusters of signal transduction for gene expression, cell cycle, and DNA repair. For the first time, the tumorigenesis of HCC is studied from the viewpoint of the pathway cluster.

USP15 Represses Hepatocellular Carcinoma Progression by Regulation of Pathways of Cell Proliferation and Cell Migration: A System Biology Analysis Expression levels of the protein USP15 correlated with slow proliferation and slow migration of HCC cells. In HCC patients, high expression of USP15 in tumor tissue was associated with significantly decreased risk for mortality and cancer relapse. Overexpression of USP15 led to reduced tumor growth in a mouse model. USP15 regulated indirectly tumor-related proteins through interactions mediated by other proteins. The functions “cell migration” and “cell proliferation” were significantly enriched in a network of pathway hierarchies that we found relevant for the regulatory role of USP15 in HCC. Six cluster of pathways described the link between experimental observed phenotypes and expression levels of USP15. Background: Hepatocellular carcinoma (HCC) leads to 600,000 people’s deaths every year. The protein ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a ubiquitin-specific protease. The role of USP15 in HCC is still unclear. Method: We studied the function of USP15 in HCC from the viewpoint of systems biology and investigated possible implications using experimental methods, such as real-time polymerase chain reaction (qPCR), Western blotting, clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS). We investigated tissues samples of 102 patients who underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). Tissue samples were immunochemically stained; a trained pathologist then scored the tissue by visual inspection, and we compared the survival data of two groups of patients by means of Kaplan–Meier curves. We applied assays for cell migration, cell growth, and wound healing. We studied tumor formation in a mouse model. Results: HCC patients (n = 26) with high expression of USP15 had a higher survival rate than patients (n = 76) with low expression. We confirmed a suppressive role of USP15 in HCC using in vitro and in vivo tests. Based on publicly available data, we constructed a PPI network in which 143 genes were related to USP15 (HCC genes). We combined the 143 HCC genes with results of an experimental investigation to identify 225 pathways that may be related simultaneously to USP15 and HCC (tumor pathways). We found the 225 pathways enriched in the functional groups of cell proliferation and cell migration. The 225 pathways determined six clusters of pathways in which terms such as signal transduction, cell cycle, gene expression, and DNA repair related the expression of USP15 to tumorigenesis. Conclusion: USP15 may suppress tumorigenesis of HCC by regulating pathway clusters of signal transduction for gene expression, cell cycle, and DNA repair. For the first time, the tumorigenesis of HCC is studied from the viewpoint of the pathway cluster. Hepatocellular carcinoma (HCC) is one of the most common cancer types worldwide. The development of treatment methods and research on relevant mechanisms is an important topic in current cancer research [1]. HCC accounts for the majority of cases of primary liver cancer, and every year, more than 600,000 people die of HCC [2,3]. In the focus of current molecular cancer therapies have been protein kinases that mediate antitumor activity primarily through abnormal expression and tumor-dependent or unregulated inactivation of target enzymes. Efforts have increased the number of treatment options [4]. Sorafenib (FDA 2007) and Lenvatinib (FDA 2018) are clinical first-line drugs in the treatment strategy of liver cancer. Post-translational modification, such as ubiquitination, plays an important role in tumorigenesis [5,6,7,8]. Ubiquitination is related to proteasome degradation [9]. Tagging genes such as p27, p53, or NF-κB, ubiquitin (Ub) influences pathways that play an important role in tumorigenesis [5,6,7,8]. Ubiquitin-specific proteases (USPs) remove ubiquitin from proteins and hence may regulate tumor-related pathways [10,11]. Its simple chemical structure makes ubiquitin a preferable target for anticancer therapy [12]. The adjustment of the balance between ubiquitination and deubiquitination may lead to lesser side effects than targeting the large proteasome complex with drugs such as Bortezomib [13]. Ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a member of the family of ubiquitin-specific proteases. USP15 shows high similarity with the proto-oncogene USP4, which cleaves ubiquitin [14]. USP15 was identified in 1999 and its role in tumorigenesis has been reported recently. A relationship between USP15 and cancer has been proposed by the observation of its stabilization function on the TGF-β receptor in glioblastoma [15]. USP15 has been shown to be elevated in breast and ovarian cancer [15]. USP15 has been shown to repress apoptosis in melanoma and colorectal cancer cell lines through stabilizing the E3 ubiquitin protein ligase MDM2 [16]. USP15 has been shown to suppress glioblastoma cell growth through stabilizing the E3 ubiquitin protein ligase HECTD1 [17]. USP15 has been shown to regulate DNA homologous recombination repair, which has been related to tumorigenesis in the breast cancer cell line MCF7 and human osteosarcoma cell line U2OS [18]. USP15 may be expected to regulate ubiquitination pathways of proteins such as TGF-β, MDM2-p53, Keap1/Nrf2, and Wnt/β-catenin. Ubiquitination pathways may play an important role in HCC. USP15 may regulate tumorigenesis in HCC by multiple pathways. The role of USP15 in HCC is, however, unknown. USP15 has been shown to act as an oncogene or as a tumor-suppressing gene [15,16,17,18]. Systems biology applies network analysis and data integration to explore biomedicine questions. The holistic view of systems biology may help to unravel the complex function of USP15 in the progression of HCC. We applied algorithms of systems biology and experimental methods to investigate the function and mechanism of USP15 in HCC. We performed a tumor-normal comparison with the database of UCSC Xena (https://xena.ucsc.edu/#analysis, accessed on 5 May 2022) [19] and the tool Gene Expression Profiling Interactive Analysis 2 (GEPIA2, http://gepia2.cancer-pku.cn/#index, accessed on 7 March 2020) [20]. The Xena provided data sources of the Genotype-Tissue Expression Project (GTEx) of The Cancer Genome Atlas (TCGA). We chose the publicly available dataset of liver hepatocellular carcinoma (LIHC) with 369 primary tumor tissue samples (no recurrent tumor) from TCGA and 160 samples from GTEx (dataset: TCGA TARGET GTEx). Boxplot analysis for USP15 expression of RNA-Seq by Expectation–Maximization (RESM) was applied in log scale (dataset: gene expression RNAseq-RSEM norm_count), see Figure 1A. With GEPIA2, we considered mRNA levels of USP15. We split the 369 tissue samples (no recurrent tumor) into two equal-sized groups; one with high expression of USP15 and a second group with low expression of USP15. Before splitting, we normalized the mRNA level of each tissue to the corresponding mRNA level of the control glyceraldehyde 3-phosphate dehydrogenase (GADPH). We used the median value of the 369 normalized USP levels as the threshold for the split between the two groups. We compared the Kaplan–Meier plots of the two groups, see Figure 1B. To compute the hazard ratio, we applied the Cox Proportional-Hazards Model. We chose a 95% confidence interval (CI). We applied the log-rank test to compare two Kaplan–Meier curves. We investigated tissues samples of 102 patients that were diagnosed with HCC and underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). For detailed information on the clinical and pathological characteristics of the patient cohort, we refer to Table 1. We collected a cancerous tissue sample for each of the 102 patient samples. As a control, we took 27 samples from non-cancerous tissue adjacent to the tumor. All specimens were immediately immobilized in formalin and embedded in paraffin block for subsequent pathological testing. We used ten pairs of fresh frozen tissue specimens from the hospital tissue bank of SRRSH for Western blot analysis. We evaluated tumor stages according to the American Joint Committee on Cancer (AJCC) staging system and defined clinical stages according to the Barcelona Clinic Liver Cancer (BCLC) staging system. This study was approved by the Medical Ethics Committee of SRRSH with the number: 20190211-90, and all patients were informed. All the procedures conformed with the Declaration of Helsinki [21]. Cell lines HCCLM3, SKhep-1, HA22T, HepG2, Huh1, Huh7, hep3B, QGY-7703, SMMC-7721, Bel-7404, SNU-387, SNU-449, SNU-423, and SNU-745 were provided by the Cang laboratory at Zhejiang University, Hangzhou, China. Cell lines HCCLM3, SKhep-1, HA22T, HepG2, Huh1, Huh7, hep3B, SMMC-7721, Bel-7404, and LO-2 were grown in DMEM (Gibco, Waltham, MA, USA, cat#C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Cellmax, Beijing, China cat#SA102.02). Cell lines SNU-387, SNU-449, SNU-423, SNU-745, and QGY-7703 were grown in RPMI-1640 (Gibco, cat# C11975500BT) supplemented with 10% FBS (Cellmax, cat#SA102.02). All cells were maintained in an atmosphere of 5% CO2 in a humidified 37 °C incubator. We designed the single-guide RNA (sgRNA), using the online sgRNA design website (http://crispr-era.stanford.edu/, accessed on 12 November 2017). The sgRNA was cloned into the lentiCRISPRv2 plasmid (Addgene, Watertown, MA, USA cat#52961, standard protocol). Full-length USP15 cDNA was kindly provided by the Cang laboratory (Zhejiang University). The cDNA was amplified and cloned into the expression vector p3xFlag-CMV-7.1 (Sigma, St. Louis, MO, USA, cat#E4026) using DNA restriction enzymes XbaI and BamHI. We cloned the full-length cDNA of USP15 into the plasmid pXF4H [23] at the restriction sites EcoRI and BamHI; pXF4H was provided by the Cang laboratory. For the sequences of the primers, we refer to Table 2. Lentivirus expressing sgRNA was produced by co-transfecting cells HEK293T with a mixture of plasmid lentiCRISPRv2-sgUSP15 (or plasmid lentiCRISPRv2-sgControl), the Gag-Pol packaging plasmid psPAX2 (Addgene, cat#12260), and the envelope plasmid B19/VSVG (Addgene, cat#88865) at a 4:3:2 ratio. We used transfection reagent Lipofectamine 2000 (lipo2000, Thermo Fisher Scientific, Waltham, MA, USA, cat#11668019). Virus particles were harvested 48 h after transfection. Lentivirus-infected cells were screened with puromycin (Thermo Fisher Scientific, cat#A1113802: Huh1,1µg/µL; HA22T, 1µg/µL). RNA was purified from liver tissue samples using Trizol (Thermo Fisher Scientific cat#204211) according to the manufacturer’s protocol. The reverse transcript was performed using PrimeScript RT Master Mix kit (Takara, Tokyo, Japan, cat#RR037B). Real-time PCR was carried out in SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA, cat#1725270) with ABI PRISM 7500 Sequence Detection System. The USP15 primer sequences were as follows: forward, 5’-CTG CTC AAA ACC TCG CTC C-3’ and reverse, 5’-CAA TGG GTC CAG GAT ACA CA-3’. The GAPDH primer sequences were as follows: forward, 5’-AGGTCGGTGTGAACGGATTTG-3’ and reverse, 5’-TGTAGACCATGTAGTTGAGGTCA-3’. Each measurement was performed in triplicate, and results were normalized to the expression of the internal reference gene GAPDH. For protein extraction, tumor tissues or cell pellets were homogenized in the buffer of RIPA (Sigma, cat#R0278), which contained a cocktail of protease inhibitors (Roche, Basel, Switzerland, #4693116001). After incubation on ice for 30 minutes, we centrifuged for 15 minutes at 4 °C at 12,000× g. Protein concentration was quantified using the kit BCA protein assay (Thermo Fisher Scientific, cat#A53227). Equal amounts of protein were separated discontinuously on a 4–12% SDS–PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). Non-specific binding sites on the membranes were blocked for 1 h with 5% non-fat milk (BD, Franklin Lakes, NJ, USA, cat#232100). After blocking, membranes were incubated overnight at 4 °C with primary antibodies, washed three times with TBS/T, and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactions were visualized using Clarity Western ECL substrate (Bio-Rad, cat#VL001), and the blots were imaged using a luminescent image analyzer (Fujifilm, Tokyo, Japan). The following antibodies were used: anti-USP15 (Santa Cruz, Santa Cruz, CA, USA, cat#sc-100629), anti-β-catenin (Cell Signaling Technology, Danvers, MA, USA, cat#8480), anti-Phospho-Nrf2 (Ser40) (Thermo Fisher Scientific, cat#PA5-67520), anti-Phospho-Rb1 (Ser780) (Cell Signaling Technology, cat#9307), anti-IκBα (Cell Signaling Technology, cat#4814), anti-N-cadherin (Cell Signaling Technology, cat#13116), anti-Phospho-Smad2 (Ser465/467) (Cell Signaling Technology, cat#3108), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, cat#4370), anti-Phospho-AKT (Ser473) (Cell Signaling Technology, cat#4060), anti-C-myc (Cell Signaling Technology, cat#18583), anti-SQSTM1 (P62) (Abcam, Cambridge, UK, cat#ab56416), anti-β-actin (Cell Signaling Technology, cat#3700), and anti-GAPDH (Abcam, cat#ab181602). Paraffin-embedded sections were provided by the department of pathology of the SRRSH. The tissues were paraffin-embedded using a Tissue-Tek automated processor (Sakura) and cut into 3 μm tissue sections. The tissue sections were deparaffinized and rehydrated. For antigen retrieval, the sections were immersed in 10 mM citrate buffer (pH 6.0) and boiled for 10 min in a microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Non-specific binding sites were blocked with 5% normal goat serum for 30 min. The sections were incubated with an antibody against USP15 (1:100, Santa Cruz, cat#sc-100629) overnight at 4 °C. The sections were incubated with the secondary antibody, and the expression of USP15 in the tissues was observed via microscopy after DAB staining and hematoxylin staining. By visual inspection, two trained pathologists assigned a score—strong, moderate, weak, or negative—to an immunochemically stained tissue. All decisions were made blinded to the clinicopathologic characteristics. Figure S1 in the supplemental material shows a reference image for each of four staining scores. If the decision was not unanimous, a third pathologist repeated the evaluation and made an independent decision. We denoted tissue with the scores strong or moderate as tissue with high expression of USP15. We denoted tissue with the scores weak or negative as tissue with low expression of USP15. For the comparison of the scores of two patient groups, we replaced the scores negative, weak, moderate, and strong by the numbers 1, 2, 3, and 4, respectively, and applied the Mann–Whitney U test. All tissue slides are accessible on reasonable request. The cell lines Huh1 and HA22T cells with USP15-stable overexpression and/or knockout (200 μL FBS-free DMEM medium suspension, 1 × 105/mlHA22T or HUH1) were sucked into the upper chamber of Transwell (PC membrane with 8.0-μm pore size). After 24 h of incubation, the chambers were washed with PBS (pH 7.4) three times to remove the cells in the upper chamber. The cells were fixed with 4% paraformaldehyde for 15 min then stained with crystal violet (0.01% in ethanol) for 25 min followed by washing three times. The cells were counted in an inverted microscope, and five visions were randomly taken at ×200. The average number of cells was considered. Wound healing assay was carried out by scratching a 6-well dish with a 10-μL pipette tip when the dish was at 80% confluences (including SMMC-7721vec/usp15 overexpression and QGY-7703vec/usp15 overexpression). The widths of the scratches were evaluated at time points 0, 6, 12, 24, and 30 h after scratching. We seeded in round numbers 1 × 103 cells per well into 96-well plates. We added cell counting kit CCK8 (5 mg/ml, YEASEN, cat#40203ES60) to the wells. We measured absorbance at wavelength 450 nm. BALB/c nude mice (n = 10, 4 weeks of age, female) were obtained from ZhiYuan biological company (Hangzhou, China). The nude mice were housed in a pathogen-free facility under controlled temperature (22 ± 2 °C) and lighting (12-h light/dark cycle) conditions. We used two human liver cell lines, Huh7-PLVX and Huh7-PLVX-USP15. Huh7-PLVX was the Huh7 cell line transfected with an empty lentiviral expression vector (PLVX). We used cell line Huh7-PLVX as a control. The second cell line, Huh7-PLVX-USP15, was the Huh7 cell line transfected with a lentiviral expression vector to overexpress USP15. We compared the two cell lines Huh7-PLVX-USP15 and cell line Huh7-PLVX to evaluate the effect of high expression of USP15. Both of the cell lines were suspended in sterilized PBS. Mice were randomized and subcutaneously injected with either cell line Huh7-PLVX or cell line Huh7-PLVX-USP15 (five mice per group, per mouse: 5 × 106 cells suspended in 100 µL of PBS,). After 25 days, the mice were euthanized, and the tumors were excised. The dimensions of the implanted tumors were measured with a vernier caliper. The tumor volume was computed by the formula: volume (cm3) = 0.5 × length (cm) × width2 (cm2). For further analysis, e.g., immunochemical staining, the tumors were embedded in paraffin. All animal experiments were performed under the guidelines reviewed by the Animal Ethics Committee of the Biological Resource Centre of the Agency for Science, Technology and Research at the Sir Run Run Shaw Hospital. Interaction partners of USP15 (layer 1 proteins) were searched in the IntAct database (https://www.ebi.ac.uk/intact/, accessed on 15 March 2020) [24] with species restriction to human. We found 106 interaction partners including USP15 (layer 1 proteins). We applied a breadth-first search (BFS) strategy to expand the set of layer 1 proteins. We found 6175 interactions partners (layer 2 proteins) for the layer 1 proteins (IntaAct, human). For visualization of interaction networks, we chose the circle layout of Cytoscape 3.8 [25]. We constructed sub-networks to describe the interaction between USP15 and given small sets of proteins, called target genes. The sub-network had a node for USP15, each target gene, and each of their interaction partners. Each edges denoted an interaction reported in IntAct for human. We downloaded the list of genes of HCC from the cBioPortal for Cancer Genomics (Dataset: HCC, TCGA, PanCancer Atlas) [26,27]. We selected genes with a mutation rate > 1%. The HCCgenes were checked to be registered in the OncoKB database [24], see Table S1 for a list of the 295 identified genes. We determined a set of 143 genes that simultaneously were gene of HCC and interaction partners of USP15 (layer 1 protein or layer 2 protein). We denoted the 143 genes that interact with USP15 as the set of HCC-USP15 genes. For a complete list, we refer to Table S2 in the supplemental material. The human hepatoma cell lines HA22T sg-control and knockout HA22T sgUSP15 were treated with 1 mL of TRIzol reagent (Thermo Fisher Scientific, cat#204211) per 107 cells and stored at −80 °C and afterwards sent in three replicates to Hangzhou Kaitai Biotechnology Co., Ltd. (Hangzhou, China) to perform mRNA extraction and sequencing. The tool DESeq2 was applied to estimate log-fold changes based on mean counts of triple replicates and false discovery rates (FDR) [28]. The thresholds for the selection of significant genes were set to FDR < 0.1 and log2 fold changes Log2FC > 1. We downloaded the relation of pathway hierarchies from Reactome (https://reactome.org/download/current/ReactomePathwaysRelation.txt, accessed on 20 July 2020) [29]. Only human pathways were considered. A network represented each pathway hierarchy as a node and each relation between two pathway hierarchies as an arc. The network had 2423 nodes and 2424 directed edges. We imported the network in Cytoscape 3.8 [25,29,30]. For the mapping, we extracted a list of Uniprot IDs for the genes from the database Uniprot (https://www.uniprot.org/uploadlists/, accessed on 28 July 2020) [31] and downloaded the mapping list “UniProt2Reactome_All_Levels” from Reactome (https://reactome.org/download/current/UniProt2Reactome.txt, accessed on 28 July 2020) [29]. We selected only the human entries in the mapping list to generate the file “UniProt2Reactome_human.txt”. We filtered the Uniprot IDs of the genes in the human mapping list and generated a relevant mapping list “pathway_matching_all.txt”. We imported the file “pathway_matching_all.txt” into the network of pathway hierarchies in Cytoscape. For the enrichment analysis, we mapped all the genes extracted from the file “UniProt2Reactome_All_Levels.txt” to the network of pathway hierarchies, applying our Python script for computing the fraction of genes in each pathway. The complete network of human pathway hierarchies was analyzed using the tool CentiScape [32]. We identified 28 source nodes with an in-degree of zero to decompose the network into the 28 sub-networks of its connected components. Each sub-network was associated with an individual source node. The sizes of the sub-networks ranged from 606 nodes for the hierarchy “diseases” to only 4 nodes for the hierarchy “circadian clock”. For statistical testing, we applied the tools GraphPad Prism 7 (San Diego, CA, USA), SPSS (V25.0, Chicago, IL, USA), and Microsoft Excel (Office 2021, Redmond, WA, USA). If not indicated otherwise, we give numbers in the form of mean ± standard error. We applied Fisher’s exact test for analysis of contingency tables, i.e., to evaluate possible biases in the patient cohort, see Table 1. We applied the Mann–Whitney U test to compare the USP15 levels in tumor and non-tumor tissue in 102 patients. To compare the survival distribution of groups of patients, we produced Kaplan–Meier plots and applied the log-rank test (Mantel–Cox test). For enrichment analysis, we applied the hypergeometric test. We denoted p-values p <0.05 as significant. In graphical representation, we represented significance values as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. We inspected publicly available data in the Xena database and gene expression profiling interactive analysis tool (GEPIA2) [33]. We chose the publicly available dataset of liver hepatocellular carcinoma (LIHC) with 369 tissue samples from TCGA and 160 samples from GTEx for tumor. The USP15 expression levels in tissue samples of liver cancer were significantly lower than those in normal tissue (p < 0.0001, Mann–Whitney U test, Figure 1A). We split the patients into two equal-sized groups, one with high expression of USP15 and a second group with low expression of USP15. The Kaplan–Meier plot of the survival data demonstrated a significantly higher survival rate for patients (expression levels above median) with high expression of USP15 than for patients (expression levels below median) with low expression (Figure 1B, p = 0.018, log-rank test). We retrospectively inspected tissue samples from 102 patients with a diagnosis of HCC at Sir Run Run Shaw Hospital (SRRSH). Information on the cohort of patients is listed in Table 1. Tumor and non-tumor tissue samples were immunochemically stained for USP15 (Figure 1C). By visual inspection, we classified tissue samples according to the degree of staining into four levels: negative, weak, moderate, and strong (Figure S1). A total of 59.2% of non-tumor tissue samples were assigned to strong or moderate staining. In contrast, only 25.4% of tumor samples were assigned to strong or moderate staining. (Table S4). The staining of tissue samples differed significantly in tumor samples and non-tumor samples (p < 0.0001, Mann–Whitney U test, Figure 1D). In accordance with our findings for the publicly available dataset LIHC, USP15 expression tended to be reduced in the tumor tissues of our patient cohort. We split the 102 patients in two groups. Patients with tumor tissue of negative or weak staining were assigned to the group low expression of USP15 (n = 76). Patients with tumor tissue of moderate or strong staining were assigned to a second group, high expression of USP15 (n = 26). We compared the survival data of the two groups by means of Kaplan–Meier plots. High expression of USP15 was associated with significantly decreased risk for mortality: hazard ratio 0.379, p = 0.0105 (log-rank test, Figure 1E). We compared data of tumor-free time periods by means of Kaplan–Meier plots. High expression of USP15 was associated with significantly decreased risk for cancer relapse: hazard ratio 0.420, p = 0.0089 (log-rank test, Figure 1F). The correlation of high survival rate and reduced cancer relapse with high expression of USP15 might suggest a role of USP15 as a tumor suppressor. We compared the protein level of USP15 in the HCC cell lines Huh1, HA22T, and Huh7 with the protein level of USP15 in cell lines LM3, PLC/PRF/5, HLF, and Hep-G2. To measure the level of USP15, we applied Western blotting. For each cell line, we normalized the luminescence of a USP15 antibody by luminescence of a GAPDH antibody. We found the highest normalized expression levels of USP15 for HCC cell lines Huh1, HA22T, and Huh7 (Figure 2A,B; the uncropped Western blots are shown in Figure S7). This finding supported our decision to choose HA22T and Huh1 cell lines to be used as a cell model for researching USP15. We transfected USP15 to cell lines Huh1 and HA22T to overexpress USP15. Overexpressing USP15 in cells Huh1 (Huh1-P-F-USP15) had a reduced proliferation index of, in round numbers, 50% of the control (p values at five time points all less than 0.0001, two-tailed t-test, Figure 2C). Overexpressing USP15 in cells HA22T (HA22T-P-F-USP15) had a slightly decreased proliferation index compared to the control and the proliferation was reduced only 2.86% (p values at 48 h, 60 h and 72 h are less than 0.05, two-tailed t-test, Figure 2D). We applied a migration assay (Transwell) to investigate the motility of the overexpressing cell lines. USP15 overexpressing cells Huh1 (Huh1-P-F-USP15) had a reduced migration rate of 45% of the migration rate of the control (p < 0.01, two-tailed t-test, Figure 2E,F). On USP15 overexpressing cells HA22T (HA22T-P-F-USP15), the migration rate was nearly completely inhibited compared to the control c (p < 0.0001, two-tailed t-test, Figure 2G,H). We transfected cell lines QGY-7703 and SMMC-7721 to overexpress USP15. We applied wound healing assays to evaluate the effect of overexpression of USP15. Overexpressing cells, QGY-7703-PXF4H-USP15 and SMMC-7721-PXF4H-USP15, had a lower movement rate than their controls (two-tailed t-test, Figures S2 and S3). The USP15 overexpression efficiency in the two cell lines is shown in Figure S4. We also use flow cytometry to detect whether the level of USP15 influences the apoptosis rate of the cell. However, in Huh1 and HA22T cell lines, overexpression or knockout USP15 does not change the apoptosis rate of the two cell lines (Figure S5), which indicated that a high level of USP15 represses the proliferation of these cell lines. We tested whether knockout had reverse effects compared to overexpression of USP15. Hepatocellular cell lines Huh1 and HA22T were modified with CRISPR/Cas9 to knock out expression of USP15. The reduced expression of USP15 was tested for knockout cell lines Huh1-sgUSP15 and HA22T-sgUSP15 using Western blotting (Figure 2I,M). Increased proliferation index was observed for knockout cell lines Huh1-sgUSP15 Figure 2J) and HA22T-sgUSP15 (p value at 72 h less than 0.05, two-tailed t-test, Figure 2N). Migration assays showed increased motility knockout cell lines Huh1-sgUSP15 (p < 0.01, two-tailed t-test, Figure 2K,L) and HA22T-sgUSP15 (p < 0.01, two-tailed t-test, Figure 2O,P). To investigate a role of USP15 as a possible tumor suppressor, we subcutaneously injected Huh7 cells into BALB/c nude mice. Five mice, the control group, were injected with cells of control cell line, Huh7-PLVX. Huh7-PLVX were Huh7 cells that were transfected by an empty vector. A second group of five mice, OE group, were injected with cells of cell line Huh7-PLVX-USP15. Huh7-PLVX-USP15 were Huh7 cells that were transfected with a lentiviral expression vector to overexpress USP15. Tumors were excised after 25 days (Figure 2Q). We confirmed a high expression of USP15 by immunostaining of tumor tissue (Figure 2R). We found the volume of tumor significantly larger in the OE group (p = 0.0079, two-tailed t-test) (Figure 2S), and the weight of tumor was also significantly larger in the OE group (two-tailed t-test) (Figure 2T). We selected the proteins from the IntAct database [34] that interact with USP15 (layer 1 proteins). To the set of 105 interaction partners of USP15, we added their interaction partners (layer 2) (Figure 3A). For the set of proteins, we constructed the protein–protein interaction (PPI) network. We deleted all non-human genes and compounds. The resulting network had 105 proteins in layer 1 (Figure 3B) and 6175 proteins in layer 2 (Figure 3C). The PPI network had 17,439 edges. In the following, we denote the network as the human USP15 PPI network. With the searching method shown in Figure 3D, we chose a set of the ten genes (Figure 3E) to see the relations of them to USP15. The ten genes using two conditions: (1) the gene was known to be tumor-related and (2) the availability of an antibody enabled an experimental investigation by Western blotting in our lab. Eight of the ten proteins were members of the human USP15 PPI network (Figure 3F,G). We applied Western blotting to compare gene expression and phosphorylation levels in cell lines Huh1-P-F_USP15 (OE, overexpressing USP15) and Huh1-sgUSP15 (KO, knockout USP15) versus their control cell lines Huh1-PLVX and Huh1-sgControl, respectively, see Material and Methods. We assumed proteins to be regulated by USP15 if overexpression and knockout had reverse effects. We found the eight members of the human USP15 PPI network regulated by USP15 (Figure 4A–I). Also regulated by USP15 was Nrf2, which was not part of the human USP15 PPI network. Nrf2 was, however, an interaction partner of layer 2 proteins. We also tested the level of some downstream proteins of USP15 in the tumor samples from the mouse model. The level of beta-catenin increased, whereas pAKT and pERK decreased (Figure 4J,K). These proteins level changes revealed the mechanism by which USP15 represses tumor growth. We constructed a network of all human pathway hierarchies, see Material and Methods. Nodes and arcs represented pathway hierarchies and hierarchy relations, respectively. The initial network had 2423 nodes and 2424 arcs. Motivated by the experimental evidence of a regulation by USP15 for eight exemplary members of the human USP15 PPI network, we chose all liver cancer genes with a mutation rate of over 1% (295 genes, Table S1) in the database cBioPortal for Cancer Genomics (dataset: HCC, TCGA, PanCancer Atlas) [26,27]. We identified 143 of the liver cancer genes as members of the human USP PPI network (HCC-USP15 genes, Table S2, Figure 5A). We mapped the 143 HCC-USP15 genes to the network of human pathway hierarchies. The number of HCC-USP15 genes per node varied between and (median 2, mean 3.56 ± 2.82). Using NGS, we measured log-fold change in gene expression after a knockout of USP15 in cell line HA22T (Figure 5B). We found 1.4% (n = 203) and 2.0% (n = 278) of the 14,099 identified proteins up-regulated and down-regulated, respectively. We mapped the set of 481 differentially expressed genes to the network of human pathway hierarchies. The number of differentially expressed genes per node varied between and (median, 1; mean, 2.55 ± 1.87). To compute an upper bound for the number of genes, we mapped a list of all human genes to the network of human pathway hierarchies. The total number of genes that may be mapped to a node varied between and (median, 17; mean, 56.37 ± 63.63). We ranked the nodes by the score: A pathway hierarchy with all associated genes being differentially expressed and simultaneously, being an HCC-USP15 gene, e.g., with , would reach the theoretical upper bound of s = 104. The score, s, was intended to quantify the specificity of a node for the 624 genes that may contribute to the regulatory role of USP15 in HCC. The values of s varied between 0 and 625 (median, 0; mean, 7.91 ± 8.49; Table S3). For a color-coded representation of the scores, we refer to Figure 5C, and Figure S6 is the original network which is used as the negative control. We selected a subset of 225 nodes (9.2%) with score values above s = 20. Note that a node with a small fraction of differentially expressed genes and a small fraction of HCC genes, such as , would get a score below 20. Figure S6 was used as blank figure. We denoted the sub-network for the 225 “high score” pathway hierarchies as HCC-USP15 pathways (Figure 6A). We downloaded 163, 75, and 143 pathway hierarchies (database: Reactome) that were associated with cell proliferation, cell migration, and lipid processes, respectively. We tested the hypothesis of a random selection of pathway hierarchies for network HCC-USP15 pathways. Since HCC-USP15 pathways had 225 nodes, we would expect to find mean numbers of , , and pathway hierarchies associated with cell proliferation, cell migration, and lipid processes, respectively. In the network HCC-USP15 pathways, we found an enrichment of 35 pathways (expected mean 15.14, Table S5-proliferation) associated with cell proliferation (p = 0.0077, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with cell proliferation, we refer to Figure 6B. We found an enrichment of 13 pathways (expected mean 6.96 Table S5-migration) associated with cell migration (p = 2.5 × 10−7, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with cell proliferation, we refer to Figure 6C. We found no enrichment of pathways associated with lipid processes. Only seven pathways (expected mean 13.28, Table S5-lipid processes) were associated with lipid processes (p = 0.035, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with lipid processes, we refer to Figure 6D. The complete network of human pathway hierarchies clustered into 28 connected components, so-called clusters (Table 3). We mapped the 225 “high score” pathway hierarchies, i.e., HCC-USP15 pathways, to the 28 clusters. HCC-USP15 pathways were significantly enriched (hypergeometric test) in 8 of the 28 pathway clusters. We assigned six of the enriched clusters to cancer pathways (Figure 7A). For a discussion of cancer-related pathways, we refer to Hanahan and Weinberg [35]. The six tumor-related clusters were “Cellular responses to external stimuli” (33.33%, 0.000165321, Figure 7B), “Cell Cycle” (19.20%, p = 0.000122438, Figure 7C), “DNA repair” (34.43%, p = 6.07738E-09, Figure 7D), “Gene expression” (14.40%, p = 0.0192962, Figure 7E), “Chromatin organization” (28.57%, p = 0.0208616, Figure 7F), and “Signal transduction” (11.46%, p = 0.0479025, Figure 7G). The two clusters “Reproduction” (p = 0.00029321) and “Circadian Clock” (p = 0.00294656) were enriched but not denoted as cancer-related. Liver cancer is one of the most common cancers in China due to high numbers of infections with the hepatitis B virus, and the mechanism behind liver cancer is still insufficiently understood [36,37]. Hepatocellular carcinoma (HCC) covers the majority of cases of primary liver cancer. Previous research has indicated that USP15 plays a role in tumor development, but its regulatory function as an oncogene or tumor-suppressing gene has never been demonstrated. We investigated the function of USP15 in HCC through a synergetic application of experimental methods and system biology methods. In a publicly available dataset (LIHC, GEPIA2) of HCC patients, we found the level of USP15 expression reduced in tumor tissue and associated with high survival rate. Additionally, in our own cohort of 102 patients, we found expression of USP15 reduced in tumor tissue. Within our cohort, a high level of USP15 expression was associated with significantly decreased risk for mortality and cancer relapse. However, the mortality rate decreased sharply in the high-USP15 group at late HCC phase in the database and our clinical data. This would indicate that the tumor suppression role of USP15 only happened in the early phase of HCC, and in the end phase, the tumor suppression role disappeared. The results motivated a more detailed investigation of the function of USP15 and its potential role as a tumor-suppressing gene in liver cancer. We investigated the phenotype of overexpression and knockout of USP15. Overexpression of USP15 in HCC cell lines correlated with slow proliferation and slow migration. Knockout of USP15 in HCC cell lines correlated with fast proliferation and fast migration. In a mouse model, overexpression of USP15 correlated with slow tumor growth. The experimental results suggested a preferable role of USP15 in HCC, presumably via a suppression of cell proliferation and motility. To explore the relevant function of USP15, we constructed a PPI network. A set 105 interaction partners of USP15 contained two liver cancer genes, LRRK and NOTCH1. Ubiquitination of one of the 105 interaction partners may affect a larger set of 6175 proteins (layer 2 proteins). To illustrate, we selected eight layer 2 proteins that were known to be related to cancer. In a cell line, overexpression and knockout had effects on the expression of each of these eight proteins. Tumor-related proteins turned out to be regulated by USP15 via indirect interactions mediated by other proteins. Via indirect interactions, USP15 may be able to regulate a rather high number of proteins. From an investigation of a small set of only eight proteins, we were not able to identify the net effect on tumor progression. We found, for example, expressions of oncogene AKT and the tumor-suppressor gene Rb1 negatively correlated with the level of USP15 expression. A similar regulation of oncogene and tumor-suppressor gene may be interpreted as contradictory effects and their concerted effect on tumor progression is difficult to estimate. Metastasis is a complex process that involves a cascade of multiple steps for the successful establishment of clinically impactful metastases. Metastasis involves multiple steps, including epithelial-mesenchymal transition (EMT), migration, matrix degradation, invasion into lymph vascular tissue, extravasation, adhesion, and mesenchymal-epithelial transition (MET) [20]. EMT involves disassembly of cell–cell junctions, actin cytoskeleton reorganization, and increased cell motility and invasion, as characterized by down-regulation and translocation of beta-catenin from the cell membrane to the nucleus and up-regulation of mesenchymal molecular markers such as vimentin, fibronectin and N-cadherin [38]. We found that N-cadherin was not regulated by USP15. USP15 may have no effect on EMT in metastasis. USP15 may, however, regulate a high number of genes that may be relevant for HCC. In our PPI network of USP15, we identified nHCC = 143 known liver cancer genes (mutation rate > 1%). To identify a large set of proteins that may be regulated by USP15, we applied NGS experiments and determined log-fold changes after a knockout of USP in a cell line (Ha22T). We found ndiff = 478 proteins differentially expressed. The investigation of the regulatory effect of USP15 on single individual proteins may not lead to a conclusive picture of its role in HCC. We decided to explore further the function of USP15 not in terms of regulatory effects on individual proteins but in terms of regulatory effects on pathway hierarchies. We scored the 2443 human pathway hierarchies and selected 225 pathway hierarchies that were simultaneously relevant for liver cancer genes and differentially expressed proteins. Note that USP15 may contribute to multiple cellular functions, of which a majority may be irrelevant for tumor progression in HCC. We found functions “cell proliferation” and “cell migration” significantly enriched in the 225 pathway hierarchies that were predicted to be relevant for the role of USP15 in HCC. Note that the relevance of the functions “cell proliferation” and “cell migration” was already indicated by our cell line experiments. Compared to the gene regulation of individual proteins, pathway hierarchies may offer a higher level of viewpoint that can be helpful to understanding the regulation of tumorigenesis. We found the 255 relevant pathway hierarchies enriched in 8 of the 28 clusters, i.e., connected components, of the network of all human pathway hierarchies. Six of the clusters can easily be related to cancer, see Table 3 and Figure 7. Prominent examples were the clusters for signal transduction and gene expression. The cluster signal transduction contained 69 out of 163 proliferation pathways and 35 out of 75 migration pathways, see Table S5 in the supplemental material. The cluster gene expression contained 22 proliferation pathways. The enrichment of these two clusters indicated that USP15 may regulate the two clusters’ signal transduction and gene expression. By such a regulation, USP15 may influence cell proliferation and cell migration in tumorigenesis of HCC. The cluster cell cycle contains cell cycling checkpoints which play an important role in cancer [35]. The cluster DNA repair can be related to cancer progression because the accelerated replication of cancer cells induces so-called replication stress [39]. Replication stress in combination with an impaired DNA repair has been associated with cellular senescence and barriers to malignant progression [40,41]. The role of ubiquitination in DNA damage response has been previously reported by Ramadan and Dikic [17]. Peng et al. reported the involvement of USP15 in regulation of DNA-homologous recombination repair [18]. In particular, Peng et al. demonstrated the recruitment of USP15 to DNA double-strand breaks (DSBs) by the mediator of DNA damage checkpoint protein 1 (MDC1). Recruited to DSBs, USP15 can be deubiquitinated and demobilize a heterodimer of the BRCA1-associated RING domain protein 1 (BARD1) and the BRCA1 C Terminus (BRCT). Note that binding of heterodimer BARD1/BRCA1 to the damaged DNA prevents transcription and restores genetic stability [42]. The independent findings by other groups of a regulatory role of USP15 in DNA repair supports the meaningfulness of our approach. The cluster cellular responses to external stimuli included pathways such as cellular response to heat stress, heat shock factor 1 (HSF1)-dependent transactivation, regulation of HSF1-mediated heat shock response, and HSF1 activation and attenuation phase, see Figure S7B in the supplemental material. HSF1 has been reported to function as a negative regulator of non-homologous end joining (NHEJ) in DNA repair activity [43]. Note that abnormal NHEJ plays an important role in tumorigenesis [44]. HSF1 has been reported to be involved in stress-induced cancer cell proliferation via IER5 [45]. The cluster cellular responses to external stimuli included additional pathways that are cancer-related, such as cellular senescence, DNA damage/telomere stress-induced senescence, and formation of senescence-associated heterochromatin foci, see Figure S7B in the supplemental material. Among the involved proteins were tumor protein P53 (TP53) and retinoblastoma protein (RB1), both of which may act as transcription factors or as a tumor suppressor. TP53 can bind to the promoter of cyclin-dependent kinase inhibitor 1 (CDKN1A) and may trigger transcription [46]. CDKN1A inhibits the activity of cell division protein kinase 2 (CDK2) and leads to an arrest of cell cycle at G1/S phase [47]. In chromatin organization, USP15-related cancer genes enriched in PKMTs and RMTs. Lysine methyltransferases (KMTs) and arginine methyltransferases (RMTs) regulate the Methylation levels of histone by adding methyl groups to histone, which play roles in regulating digestive cancer, including liver cancer [48]. H3K9me2 in the promoter region of RARRES3 gene suppresses its transcription and promotes cancer cell migration in HCC [49]. H3K27me3 enriched in the Kruppel-like factor 2 (KLF2) promoter leads to the tumor cell population in GC and CRC [50]. The decreased H3K27me3 in the promoter of SLUG activates the transcription and promotes migration and invasion in HCC [51]. All this evidence clearly showed that chromatin organization strongly relates to HCC and indicated that USP15 regulates HCC via histone methylation level. To describe phenotypes, we found pathway hierarchies more suitable than a single gene or a small set of genes. Pathway clusters offered a coarser-grained—but still meaningful—viewpoint on regulation. Pathway clusters could be linked phenotypes and helped to understand the relations between USP15 and phenotypes of its regulatory function in HCC. We found high level of USP15 expression to be significantly correlated with high survival rate in HCC patients. Relevant cellular phenotypes were slow proliferation and slow migration. We confirmed the role of USP15 as a tumor suppressor in a mouse model. To link the experimentally observed phenotypes to molecular function of USP15 we constructed a protein–protein interaction network. We found the regulatory function of USP15 to be mediated by indirect interactions. Indirect interactions may enable USP15 to regulate thousands of other proteins. The phenotype of complex regulation is hard to predict. We proposed and tested a theoretical approach based on pathway hierarchies. The viewpoint of pathway hierarchies offered a meaningful coarse-grained viewpoint on the regulatory function of USP15. We were able to link our experimentally observed phenotypes to clusters of pathways. Furthermore, in other applications, clusters of pathways may be an appropriate viewpoint to understand the regulatory function of proteins and may offer a starting point to select molecular mechanisms for further experimental investigation.

PMC10000210

Yunfa Xie,Zhiying Chen,Yanling Wang,Xiayun Peng,Ni Feng,Xiaoye Wang,Yinsheng Tang,Xun Li,Chunrong Xu,Chuanhuo Hu

Supplementation of Schisandrin B in Semen Extender Improves Quality and Oxidation Resistance of Boar Spermatozoa Stored at 4 °C

25-02-2023

schisandrin B,sperm quality,boar,oxidative stress

Simple Summary Sch B is the active component extracted from Sch B Fructus. It shows reproductive protection and spermatogenesis effects in animals. As far as we know, no research has been conducted with Sch B on boar semen preservation at 4 °C. Therefore, the current research aimed to explore the effects of Sch B on quality, oxidation resistance, and decapacitation of boar spermatozoa at 4 °C. The results showed that the positive effects of 10 μmol/L Sch B were obvious for boar semen preservation at 4 °C. Abstract During cold storage, boar spermatozoa undergo oxidative stress, which can impair sperm function and fertilizing capacity. The objective of the present study was to assess the effects of Schisandrin B (Sch B) in semen extenders on the quality of boar semen stored at hypothermia. Semen was collected from twelve Duroc boars and diluted in extenders supplemented with different concentrations of Sch B (0 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L). Here, we demonstrated that 10 μmol/L Sch B provided the best effects on motility, plasma membrane integrity, acrosome integrity, sperm normality rate, average movement velocity, wobbility, mitochondrial membrane potential (MMP), and DNA integrity of sperm. The results of Sch B effects on antioxidant factors in boar sperm showed that Sch B significantly elevated the total antioxidant capacity (T-AOC) and markedly decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) content of sperm. The expression of catalase (CAT) and superoxide dismutase (SOD) mRNA was increased, while the expression of glutathione peroxidase (GPx) mRNA demonstrated no change compared to non-treated boar sperm. Compared to the non-treated group, Sch B triggered a decrease in Ca2+/protein kinase A (PKA) and lactic acid content in boar sperm. Similarly, Sch B led to a statistically higher quantitative expression of AWN mRNA and a lower quantitative expression of porcine seminal protein I (PSP-I) and porcine seminal protein II (PSP-II) mRNA. In a further reverse validation test, no significant difference was observed in any of the parameters, including adhesion protein mRNA, calcium content, lactic acid content, PKA, and protein kinase G (PKG) activity after sperm capacitation. In conclusion, the current study indicates the efficient use of Sch B with a 10 μmol/L concentration in the treatment of boar sperm through its anti-apoptosis, antioxidative, and decapacitative mechanisms, suggesting that Sch B is a novel candidate for improving antioxidation and decapacitation factors in sperm in liquid at 4 °C.

Supplementation of Schisandrin B in Semen Extender Improves Quality and Oxidation Resistance of Boar Spermatozoa Stored at 4 °C Sch B is the active component extracted from Sch B Fructus. It shows reproductive protection and spermatogenesis effects in animals. As far as we know, no research has been conducted with Sch B on boar semen preservation at 4 °C. Therefore, the current research aimed to explore the effects of Sch B on quality, oxidation resistance, and decapacitation of boar spermatozoa at 4 °C. The results showed that the positive effects of 10 μmol/L Sch B were obvious for boar semen preservation at 4 °C. During cold storage, boar spermatozoa undergo oxidative stress, which can impair sperm function and fertilizing capacity. The objective of the present study was to assess the effects of Schisandrin B (Sch B) in semen extenders on the quality of boar semen stored at hypothermia. Semen was collected from twelve Duroc boars and diluted in extenders supplemented with different concentrations of Sch B (0 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L). Here, we demonstrated that 10 μmol/L Sch B provided the best effects on motility, plasma membrane integrity, acrosome integrity, sperm normality rate, average movement velocity, wobbility, mitochondrial membrane potential (MMP), and DNA integrity of sperm. The results of Sch B effects on antioxidant factors in boar sperm showed that Sch B significantly elevated the total antioxidant capacity (T-AOC) and markedly decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) content of sperm. The expression of catalase (CAT) and superoxide dismutase (SOD) mRNA was increased, while the expression of glutathione peroxidase (GPx) mRNA demonstrated no change compared to non-treated boar sperm. Compared to the non-treated group, Sch B triggered a decrease in Ca2+/protein kinase A (PKA) and lactic acid content in boar sperm. Similarly, Sch B led to a statistically higher quantitative expression of AWN mRNA and a lower quantitative expression of porcine seminal protein I (PSP-I) and porcine seminal protein II (PSP-II) mRNA. In a further reverse validation test, no significant difference was observed in any of the parameters, including adhesion protein mRNA, calcium content, lactic acid content, PKA, and protein kinase G (PKG) activity after sperm capacitation. In conclusion, the current study indicates the efficient use of Sch B with a 10 μmol/L concentration in the treatment of boar sperm through its anti-apoptosis, antioxidative, and decapacitative mechanisms, suggesting that Sch B is a novel candidate for improving antioxidation and decapacitation factors in sperm in liquid at 4 °C. At present, the preservation of pig semen mainly includes chill storage (4 °C) and ultra-cryopreservation (−196 °C). Cryopreservation results in the low fertility of pig semen, which has a serious impact on research in the boar breeding industry. The pregnancy rate and litter size are similar at 4 °C fluid storage compared with natural mating. Therefore, liquid storage at 4 °C is an effective method to preserve boar semen. In mammals, boar sperm is susceptible to temperature due to its special membrane structure. Especially under low-temperature conditions, the sperm membrane structure is easily destroyed, resulting in ultrastructural changes in the acrosome and plasma membrane, including deterioration of sperm motility, viability, and DNA integrity, resulting in reduced sperm quality and thus reduced sperm longevity and fertility. Even if appropriate chilling and cryopreservation protocols have already been developed for many species, as shown in bull [1,2], ram [3,4], goat [5], and mouse [6] studies. There are limited data on the successful freezing, liquid storage, and cryopreservation of boar sperm. The cryopreservation of boar semen is still in the research and improvement stage; it is more commonly used in the preservation of sperm of threatened species and has not been widely used, which makes the cryopreservation research of boar semen of very important practical significance. Oxidative stress is an important factor affecting sperm quality during the liquid storage of boar semen in vitro. Adding various protective components to the diluent is an effective method to maintain the survival and function of sperm. Schisandrin B (Sch B) is mainly derived from Schisandra chinensis (Turcz) Baillon and has the highest content in Schisandra chinensis. Sch B, C23H28O6 with a molecular weight of 400.6, is a natural antioxidant that not only enhances the antioxidant status of cells but also has been found to prevent the structural and functional degradation of cell mitochondria, both of which are critical to cell survival. Experimental investigations have demonstrated that Sch B has multiple pharmacological properties, including anti-oxidation, anti-inflammation [7], anti-apoptosis [8], and anti-tumor [9] effects. Meanwhile, research suggests that Sch B may be a potential therapeutic agent against anxiety associated with oxidative stress through reduced malondialdehyde (MDA) levels, production of reactive oxygen species (ROS), and neuronal damage [10]. In terms of reproductive function, Sch B has spermatogenic effects and can repair damaged seminiferous tubules and spermatogenic cells in the testis, providing a novel way to treat male infertility [11]. Based on its antioxidant and reproductive function protection properties, we speculate that Sch B could be used for sperm protection during liquid preservation of boar semen at 4 °C. To date, the antioxidative effects of Sch B in the preservation of boar sperm and the mechanism of protective effects by Sch B have not been determined. In this context, Sch B is used for the first time in this study to explore its effects on boar sperm liquid preservation, revealing the underlying mechanism(s) of Sch B in sperm quality parameters, antioxidant capacity, and sperm fertilization ability during liquid preservation of boar semen at 4 °C. We further evaluated the effects of Sch B on sperm capacitation parameters before and after capacitation. Unless otherwise stated, all chemicals used in this study were purchased from Solarbi (Beijing, China). Sch B was obtained from Victory Biological Technology (Cat. No. 61281-37-6, Sichuan Province, China). Mixed semen from 12 mature Duroc boars aged 2–4 years was collected through the hand-gloved technique. Boars were fed at the Keda Livestock and Poultry Improvement Co., Ltd. (Guangxi Province, China). An entire ejaculation was collected twice a week. Only ejaculates that satisfied the sperm quality requirements to produce commercial semen AI-doses (namely, sperm concentration >200 × 106 sperm/mL, sperm motility >70%, and morphologically normal sperm >75%) were included in the study. For seminal plasma harvesting, entire ejaculates were centrifuged twice (1500× g for 10 min at room temperature (Rotofix 32A, Hettich Centrifuge UK, Newport Pagnell, Buckinghamshire, UK)). In this study, three experiments were carried out. Boar semen samples were diluted in the extender (glucose 3 g, sucrose 4 g, and egg yolk 20 mL in double-distilled water and volume made up to 100 mL) to a concentration of 80 million sperm/mL. Sch B was added to yield six different final concentrations: 0, 2.5, 5, 10, 20, and 40 μmol/L. The extended sperm were incubated at 4 °C for 24 h. The sperm motility, abnormal sperm morphology, average movement velocity, wobbility (wobbility is the measure by which the sperm head wobbles along the average spatial path of its actual trajectory), acrosome integrity, plasma membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were assessed. Each experiment was repeated three times. In experiment 2, we measured sperm superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) mRNA expression, total antioxidant capacity (T-AOC) ability, ROS content, MDA content, Ca2+ content, lactic acid content, the mRNA expression of AWM, AQN1, AQN3, porcine seminal protein I (PSP-I), porcine seminal protein II (PSP-II), protein kinase A(PKA), and protein kinase G (PKG) activity before capacitation. Each experiment was repeated three times. We measured Ca2+ content, lactic acid content, the mRNA expression of AWM, AQN1, AQN3, PSP-I, PSP-II, PKA, and PKG activity after sperm capacitation. Each experiment was repeated three times. After rewarming, 1 mL sperm was centrifuged at a speed of 900× g/min in a cryogenic centrifuge at 4 °C for 5 min, washed with PBS, repeated 3 times, and then suspended with PBS to adjust the sperm density to 1 × 106~2 × 106 sperm/mL. Then, 20 μL of the sperm resuspended in PBS was applied to a slide, covered with a coverslip, and the sperm motility, abnormal sperm morphology, average movement velocity, and wobbility were examined by CASA (CEROSII, IMV, Shanghai, China) after low-temperature preservation. A sample of 1 mL of rewarmed sperm was taken and centrifuged with phosphate-buffered saline (PBS) at 900× g/min in a low-temperature centrifuge at 4 °C for 5 min. This was repeated three times and added to the pre-configured hypotonic solution. The sperm density was adjusted to 1 × 106~2 × 106 sperm /mL, and incubated at 37 °C for 30 min. Then, 15 μL of the mixture was put on a slide, covered with a cover slide, and the tail bending rate of sperm was observed under a microscope at 400 times. At least 200 sperm were counted, and this was repeated 3 times. After sperm rewarming, the sperm density was adjusted to 1 × 106~2 × 106 sperm /mL, and the semen was washed by PBS centrifugation 3 times. The sperm were fixed with formalin and sodium citrate in the EP tube. The fixed sperm 2000× g/min was centrifuged for 5 min and then the supernatant was discarded. The sperm were washed once with PBS. Then, 100 μL of PBS was added to the sperm to make a suspension. A 10 μL mixed smear was taken and dried naturally. The air-dried slides were stained in a staining jar with Coomassie brilliant blue, and then the staining solution was slowly rinsed off with water and placed on a staining rack to dry naturally. The acrosome integrity was observed under the microscope (acrosomes in intact sperm were stained with Coomassie brilliant blue to a smooth and uniform blue surface). The experiment was repeated 3 times. The operation was carried out under dark conditions according to the instructions of the AO fluorescent staining kit (Cat. DA0037, Leagene, Beijing, China). After staining, the same samples were examined under a fluorescence microscope (the stained yellow-green fluorescence was the nucleus of normal sperm, and the green granules were the nucleus of DNA damage). At least 200 spermatozoa were counted per smear. The experiment was repeated 3 times. The sperm density was adjusted to (1–2) × 106 sperm /mL. The sperm were rewashed with PBS by centrifugation (900× g; 5 min), and the supernatant was discarded. The operation was performed according to the MMP detection kit (JC-1) (Cat. CA1310, Solarbio, Beijing, China), and the fluorescence values of each group were detected by a multi-functional microplate tester. The experiment was repeated 3 times. After being rewarmed, the sperm density was adjusted to (1–2) × 106 sperm /mL. The semen was centrifuged 3 times with PBS. Oxidant–antioxidant parameters (MDA (Cat. BC0025, Solarbio, Beijing, China), ROS (Cat. S0033S, Beyotime, Shanghai, China), and sperm T-AOC (Cat. BC1310, Solarbio, Beijing, China)) were identified in fresh, Sch B-treated, and non-treated sperm. Sperm MDA (lipid peroxidation marker) concentration was detected by a fluorescence enzyme labeler according to the manufacturer’s instructions, following the Tecan protocol. The absorbance was determined at 450 nm, 532 nm, and 600 nm. MDA measurements were presented in nmol/g protein. In addition, the activity of ROS enzymes and sperm T-AOC was measured in boar sperm, as previously analyzed. The experiment was repeated 3 times. ROS: relative fluorescence unit (RFU); MDA: nmol/mg; T-AOC: μmol/mg. The total RNA was extracted using the TRIzol reagent (Vazyme, Nanjing, China) for the boar sperm. The first-strand cDNA was synthesized from the total RNA using a reverse transcription kit (Cat. R211-02, Vazyme, Nanjing, China). Amplification reactions were conducted in triplicate using gene-specific primers designed from the clone sequences shown in Table 1. The PCR products in each cycle were monitored using a fluorescence quantitative PCR instrument (PRISMR 7500, ABI, Los Angeles, CA, USA). The data were analyzed using the comparison Ct (2−ΔΔCt) method and were expressed as fold changes relative to the respective controls. Each sample was analyzed in triplicate. The experiment was repeated 3 times. Boar sperm was taken from each group after being cryopreserved and centrifuged with PBS 3 times (4 °C, 900 g/min, 5 min); then, the sperm density was adjusted to 2 × 106/mL and centrifuged with 1/5 (4 °C, 900× g/min, 5 min). After the supernatant was abandoned, 300 μL PBS was added to the reinserted sperm. A 20 μL 0.1 mmol·L−1 Ca2+ fluorescent probe Fluo-3 (Cat. IF0150, Solarbio, Beijing, China) was added into the probe, which was fully blown with a pipette gun, and then incubated in a constant temperature incubator at 37 °C for 30 min. After centrifugation for 5 min (1500× g/min), the supernatant was abandoned and resuspended in 500 μL PBS. Then, the 200 μL sperm suspension was placed in a 96-well plate, and the fluorescence values of each experimental group were detected by a multifunctional enzyme marker [12]. The experiment was repeated 3 times. Ca2+: relative fluorescence unit (RFU). Lactic acid content was evaluated by using the lactic acid content assay kit (Cat. No. BC2230, Solarbio, Beijing, China). Samples were centrifuged at 3000× g for 5 min and the sperm pellets were re-suspended to a final concentration of 1 × 106 cells in 0.5 mL of PBS. After ultrasonic fracturing in an ice bath, the samples were centrifuged at 8000× g/min for 10 min at 4 °C, and the supernatant was taken to measure the protein concentration and placed on ice until use. Then, 0.02 mL of distilled water, a standard tube, and the sample were added to the blank tube, standard tube, and determination tube, respectively, and then 1 mL enzyme working solution and 0.2 mL chromogenic agent were added, respectively, to mix evenly and react accurately at 37 °C for 10 min. Finally, 2 mL terminating solution was added. We mixed this evenly in a colorimetric dish with a 1 cm optical diameter and measured the absorbance of each tube at 530 nm [13]. The experiment was repeated 3 times. Lactic acid: nmol/g. Both PKA and PKG were identified in boar sperm using commercially available ELISA kits for PKA (Cat. MM-77570O1, Meimian, Jiangsu, China) and for PKG (Cat. MM-77540O1, Meimian, Jiangsu, China). We established the standard curve according to the kit’s instructions. Each test was performed in triplicate. As described in a previous study [14], after sperm collection, samples were centrifuged with PBS (4 °C, 900× g/min, 5 min) 3 times to wash away impurities, and then the sperm density was adjusted to 1 × 106~2 × 106 sperm/mL with Biggers–Whitten–Whittingham (BWW) capacitive medium, which was placed in a 5% CO2 cell incubator and incubated at 37 °C for 2 h. Data were analyzed using one-way ANOVA (version 13.0, SPSS Inc., Chicago, IL, USA). When a significant difference (p < 0.05) was observed among treatments, Tukey’s studentized range test was used for post-test comparisons. Percent motility was arcsine-square root transformed, and the means of three straws per treatment were used for analysis. Values presented are mean ± SD. The effects of Sch B on motility, abnormal sperm morphology, average movement velocity, and wobbility at 4 °C were initially investigated in this sperm quality parameters analysis. Sperm motility is shown in Figure 1A. Sperm motility in 10 Sch B was significantly higher (p < 0.01), while 40 Sch B was markedly decreased (p < 0.001) compared to the 0 Sch B group. There was no significant difference between the 0 Sch B group and the Sch B-treated group (2.5, 5, and 20 Sch B) (p > 0.05). The differences in abnormal sperm morphology are shown in Figure 1B. Compared to 0 Sch B, 5 Sch B and 10 Sch B significantly decreased the abnormal sperm morphology level in a dose-dependent manner. On the contrary, 40 Sch B significantly increased the abnormal sperm morphology level compared with 0 Sch B. The effects of hypothermic injury on the average movement velocity of boar spermatozoa are shown in Figure 1C. There was no significant difference in the average movement velocity (p > 0.05) in 2.5 Sch B and 20 Sch B compared with 0 Sch B. The average movement velocity in 10 Sch B and 5 Sch B was significantly higher than 0 Sch B (p < 0.001 and p < 0.01, respectively) on the hypothermic liquid preservation. A significantly lower average movement velocity was observed in 40 Sch B than in 0 Sch B (p < 0.01) The results of sperm wobbility after chilling are shown in Figure 1D. Our results showed that the addition of 10 μmol/L Sch B to the base solution can significantly improve the wobbility compared to 0 Sch B (p < 0.05), but there was no significant difference between the other Sch B-treated groups (2.5, 5, 20, and 40 Sch B) and 0 Sch B (p > 0.05). After treatment with Coomassie bright blue, the acrosomal reaction rate of sperm was detected, as shown in Figure 2A. All Sch B-treated groups significantly (p < 0.01) enhanced plasma membrane integrity, except for 40 Sch B. When the Sch B concentration exceeded 10 μmol/L, the sperm acrosome integrity showed a decreasing trend with increasing concentration. The percentage of plasma membrane integrity of sperm in the hypothermia treatment groups is presented in Figure 2B. The plasma membrane integrity in 2.5, 5, and 10 Sch B was significantly higher than in 0 Sch B (p < 0.01). It is worth noting that the plasma membrane integrity of boar sperm was significantly (p < 0.05) lower when the concentration of Sch B was 40 μmol/L. The sperm DNA damage rate from representative semen samples of all six groups at the chilled–thawed stage is presented in Figure 3A. The DNA damage rate in 5, 10, and 20 Sch B was significantly lower than in 0 Sch B (p < 0.01). The difference between 0 Sch B and 2.5 Sch B was not significant (p > 0.05). When the concentration of Sch B was 40 μmol/L, the DNA damage rate showed an increasing trend. Sperm MMP at the chilled-thawed stage are presented in Figure 3B. The percentage of sperm with MMP was higher (p < 0.01) in 10 Sch B than in 0 Sch B, while no significant difference was observed between 20 Sch B, 40 Sch B, and 0 Sch B after chilling (p < 0.05). The MMP was significantly lower in the 2.5 and 5 Sch B than in 0 Sch B (p < 0.01). The expression level of SOD and CAT was significantly increased in sperm with Sch B compared to 0 Sch B (p < 0.05, p < 0.01). The 0 Sch B showed a significant decrease (p < 0.001) in the mRNA level of sperm SOD and CAT compared to the fresh sperm (Figure 4). The differences in the quantitative expression of GPx mRNA were not significant in the fresh and Sch B-treated sperm compared to the non-treated sperm (p > 0.05). The 0 Sch B samples had significantly higher activity in sperm ROS compared to fresh sperm (p < 0.001; Figure 5A). The Sch B-treated samples revealed lower activity in sperm ROS (p < 0.01) compared to the 0 Sch B samples. When compared to fresh, 0 Sch B had a significantly lower T-AOC level (p < 0.001, Figure 5B). Compared to 0 Sch B, the Sch B-treated sperm had a significant increase (p < 0.001) in the T-AOC level. Compared to fresh, the 0 Sch B sperm had a significant increase in the sperm lipid peroxidation marker MDA (p < 0.001, Figure 5C), whereas the Sch B-treated sperm had a lower MDA content than the 0 Sch B sperm (p < 0.001). Compared to fresh, 0 Sch B had significantly higher sperm Ca2+ and lactic acid contents (p < 0.01 and p < 0.001, respectively). Compared to 0 Sch B, Sch B-treated samples revealed significantly lower sperm Ca2+ and lactic acid contents (p < 0.05 and p < 0.01, respectively, Figure 6). The mRNA level of AWM was significantly reduced in 0 Sch B sperm compared to fresh (p < 0.001). However, boar sperm treated with Sch B at a dose of 10 μmol/L showed a significant increase (p < 0.01) in the mRNA level of AWM compared to 0 Sch B (Figure 7). The mRNA level of AQN1 was identified in fresh, Sch B-treated, and non-treated sperm. Both the non-treated and Sch B-treated sperm showed a significant reduction in the level of AQN1 mRNA compared to the fresh samples (p < 0.001) (Figure 7). No significant difference was observed between the non-treated and Sch B-treated samples (p ≥ 0.05). The mRNA levels of AQN3 in both the 0 Sch B and Sch B-treated sperm showed significant decreases compared to the fresh sperm (p < 0.01). AQN3 mRNA levels between the 0 Sch B and Sch B-treated sperm showed no significant differences (p ≥ 0.05). Quantitative expression of both PSP-I and PSP-II mRNA (Figure 7) showed significant increases in 0 Sch B compared to the fresh sperm (p < 0.001 and p < 0.01, respectively). When sperm were treated with Sch B at a dose of 10 μmol/L, a significant decrease in the quantitative expression of both PSP-I and PSP-II mRNA was identified compared to 0 Sch B (p < 0.01 and p < 0.05, respectively). PKA was increased in sperm without any additives compared to fresh (p < 0.05). However, boar sperm treated with 10 μmol/L Sch B showed a decrease in the PKA level compared to 0 Sch B as measured by ELISA immunoassay techniques (Figure 8A). Compared to the fresh, the 0 Sch B and 10 Sch B sperm both had no significant differences in PKG levels. PKG level between the 0 Sch B and Sch B-treated sperm showed no significant differences (p ≥ 0.05, Figure 8B). Compared to 0 Sch B, the capacitation groups demonstrated significantly higher (p < 0.01) sperm Ca2+ and lactic acid contents. The Ca2+ and lactic acid contents in the 10 Sch B sample were not significantly different compared with the 0 Sch B capacitation sample (p > 0.05, Figure 9). mRNA levels of both AWM and AQN1 were significantly reduced in the capacitation groups compared to 0 Sch B (p < 0.01). However, 10 Sch B showed no significant differences (p ≥ 0.05) in the AWM and AQN1 mRNA levels compared to 0 Sch B capacitation as measured by quantitative PCR (Figure 10). The mRNA levels of AQN3 in 0 Sch B capacitation and 10 Sch B showed no significant differences compared to 0 Sch B, as did 10 Sch B compared to 0 Sch B capacitation (p ≥ 0.05). Quantitative expression of both PSP-I and PSP-II mRNA (Figure 10) was significantly increased in 0 Sch B capacitation compared to 0 Sch B (p < 0.01 and p < 0.001, respectively). The 10 Sch B sample did not show significant differences with regard to the parameters of the two genes compared to the 0 Sch B capacitation sample. The results of Sch B affecting sperm PKA and PKG levels of capacitated boar spermatozoa are shown in Figure 11. After 24 h of liquid storage, the sperm PKA levels of 0 Sch B capacitation and 10 Sch B were higher than 0 Sch B (p < 0.05), and the sperm PKA levels were not significantly different between 0 Sch B capacitation and 10 Sch B. Sperm PKG levels are shown in Figure 11. The PKG level in 0 Sch B was not significantly different compared with 0 Sch B capacitation (p > 0.05), and 0 Sch B capacitation showed no significant difference compared to 10 Sch B (p > 0.05). Sch B, isolated from Schisandra chinensis, has been documented to possess diversified pharmacokinetic properties, among them neurogenesis [15], anti-inflammatory [16], anti-tumor [17], and liver-protective [18] effects. The present study validated the effect of Sch B on male infertility [11], suggesting that Sch B is a potential natural active ingredient for reproduction protection. A previous study determined that oral administration of Sch B at 2 weeks not only increased the sperm number and average number of births but also increased sperm motility parameters in mice. These results suggest that Sch B may have positive effects on reproduction and improve sperm quality. Our present sperm quality parameters show that Sch B triggered an increase in the motility, average movement velocity, wobbility, plasma membrane integrity, and acrosome integrity of boar sperm, while decreasing abnormal sperm morphology, indicating that Sch B could directly act on sperm to enhance its motility. Notably, the plasma membrane integrity and acrosome integrity were increased after treatment with Sch B. Suggesting that Sch B has potential improvement functions on sperm quality parameters equally in in vitro models and at the cellular level, a previous study demonstrated that Sch B can modulate DNA damage through inhibition of MAPK/p53 signaling [19]. Our results are consistent with previous research on rodents in that administration with Sch B can decrease DNA damage of boar sperm in vitro. Subsequently, the results of the MMP changes showed that the MMP increase may be responsible for the decreased apoptosis and increased quality in Sch B-treated sperm. Our results correspond with a previous study in that the mitochondrial functional and structural integrity were improved by long-term Sch B treatment in rat kidneys [20]. These data corroborate the previous suggestion that Sch B increased the quality parameters by causing changes in anti-apoptotic ability, DNA damage reduction, and increased MMP. Sch B in concentrations of 0–10 μmol/L had a dose-dependent positive effect on sperm characteristics, while the higher levels of Sch B supplementation (20 and 40 μmol/L) had either a negative or no effect on sperm characteristics. Accordingly, we selected 10 μmol/L Sch B as the optimal concentration for a follow-up study. Oxidative stress is a leading factor contributing to the poor overall quality of spermatozoa after the freezing and thawing process since the spermatozoa are rich in polyunsaturated fatty acids and are an easy target for ROS, which results in lipid peroxidation of the spermatozoan plasma membrane [21]. It has been well established in sperm that ROS content is inversely correlated with sperm motility [11]. Meanwhile, MDA is also involved in lipid peroxidation, which is an important indicator of cellular oxidative damage. The results of our analysis showed that the MDA and ROS contents were notably down-regulated and that the expression levels of GPx had no significance, accompanied by a significantly improved SOD and CAT mRNA expression and T-AOC levels after Sch B treatment in sperm, which is consistent with other studies performed in rodents, except for GPx [22,23]. Supplementation of extenders with CAT, SOD, and GPx can preserve sperm motility and DNA integrity, thereby improving cooled storage, especially with poor semen quality [24,25]. These results suggest that Sch B may improve sperm quality by increasing the antioxidant capacity of sperm. Donà et al. reported that spermatozoon ROS content directly influences the levels and locations of tyrosine phosphorylation and then enables the spermatozoa to undergo an acrosome reaction [26]. In boars, the majority of seminal plasma proteins belong to the sperm-adhesin family, a group of (glyco) proteins built by a single CUB domain architecture [27]. Previous research has shown that the addition of seminal plasma attenuates in vitro and cooling-induced capacitation-like changes in boar spermatozoa [28]. Premature capacitation-like changes lead to spontaneous acrosome reactions and cell death, with a subsequent decrease in sperm fertilizing ability. The sperm adhesion protein gene family plays an important role in the processes of spermatogenesis, ejaculation, and sperm–egg binding. AQN-1, AQN-3, AWN, PSP-I, and PSP-II adhesion protein genes are the main members of the sperm adhesion protein gene family. The AQN-1 and AWN proteins are involved in the formation of the acrosomal reaction inhibition receptor complex before sperm-egg binding and premature sperm capacitation can be prevented by them [29]. AWN and AQN-3 also mediate sperm binding to the pellucida during fertilization [30]. It could be argued that the PSP-I/PSP-II heterodimer is responsible for the stabilization of the sperm membrane, thus counteracting the destabilizing phenomena that lead to membrane destabilization, impaired acrosome integrity, and reduced cell viability. Several studies have demonstrated a significantly higher expression of both genes in low-litter size boar spermatozoa than in high-litter size spermatozoa [31,32]. In addition, in another study, it was reported that the expression of PSP-I protein in seminal plasma was negatively correlated with the motility and morphology of boar sperm [33] and litter size [34]. Our results showed that Sch B could significantly increase the expression of AWN, whereas it reduced the expression of and decreased PSP-I and PSP-II in the boar sperm. However, the results of the expression of AQN-1 and AQN-3 were not significant. This is the first evidence revealing that the molecular mechanism of Sch B is involved in sperm capacitation by regulating the expression of the sperm adhesion protein gene family, suggesting that adding Sch B to a semen extender can improve sperm quality in the cryogenic liquid state by reducing capacitation. Ca2+ signaling is of particular significance in sperm, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis, and acrosome reaction). Previous studies have indicated that the increase in Ca2+ is related to sperm death. An excessive Ca2+ level affects capacitation during the fertilization of sperm, which leads to sperm death [35,36]. Fluorometric recordings of sperm loaded with fluo3-AM revealed that low-temperature preservation evoked elevations of intracellular Ca2+. Therefore, the high Ca2+ concentration during the cryopreservation of sperm is an important reason for its quality decline. In contrast, when boar spermatozoa were treated with Sch B in our study, decreases in the spermatozoa’s membrane instability and calcium concentration were observed. It has been reported that lactic acid is a sperm motility inactivation factor [37]. Carr concluded that the low pH of the cauda epididymal fluid from bulls and dogs, plus the presence of lactate, is sufficient to cause inhibition of sperm motility [38]. In our study, we found that the lactate content of sperm decreased after the addition of Sch B, which explained the possible factor of the previously detected increase in sperm motility, and we speculated that lactate content might be an important factor affecting sperm capacitation. Sperm-capacitated processes shorten their lifespan and result in premature death [20]. Ejaculated spermatozoa are unable to fertilize the oocyte; on entry to the female reproductive tract, maturational changes occur in sperm that render them competent for fertilization in a process known as capacitation [39,40]. Even though the molecular basis of this process remains unclear, it correlates with a series of cellular and biochemical changes, including cholesterol efflux from the sperm plasma membrane [41], ion influx, membrane hyperpolarization [42,43], cAMP production [44], kinase activation and protein phosphorylation [45]. Enzymes such as members of the PKA are directly regulated by cAMP [44]. Thus, this nucleotide orchestrates several downstream events with critical outcomes in cell physiology. Activation of PKA enzymes is essential for capacitation, and thus, cAMP levels are tightly regulated during this process. cAMP, an important second messenger, activates PKA, leading to potassium channel opening and calcium channel closing, which can decrease the concentration of calcium in the cytoplasm. Previous results suggest that [46] Sch B was able to significantly improve erectile dysfunction in rats with nerve injury through the cAMP-PKA pathways. Similar results were found in our present capacitation investigation in that Sch B triggered an increase in the PKA level, resulting in a Ca2+ concentration decrease. PKG inhibitors have been reported to inhibit sperm, hyperactivation, and the acrosome reaction. It has also been reported that increased PKG in the cGMP/PKG signaling pathway can increase Ca2+ and tyrosine-phosphorylated proteins, promote hyperactivation, and induce the acrosome reaction, which ultimately facilitate sperm capacitation [47]. Our study showed that adding Sch B had no effect on the level of PKG in boar sperm. We believe that adding Sch B in a semen extender can regulate sperm Ca2+ concentration through the cAMP-PKA pathway, which leads to the decrease of sperm number with capacitation. These findings suggest that Sch B was identified as a primary candidate for a decapacitation factor. Since our data showed that Sch B could affect the capacitation state of sperm by regulating sperm adhesion proteins and the CAMP-PKA pathway, we therefore hypothesized that Sch B may be involved in decapacitation. To test this hypothesis, we further assessed Ca2+ influx, lactic acid content, the quantitative expression of sperm adhesion protein gene family mRNAs, and the PKA/PKG activity of boar spermatozoa after capacitation. However, no significant difference (p > 0.05) was observed in any of the capacitation-related parameters (Ca2+ and lactic acid content, expression of sperm adhesion protein gene family, PKA and PKG levels) evaluated in samples with Sch B compared to 0 Sch B. Current studies on sperm decapacitation factors have shown that they can prevent premature capacitation and the acrosome reaction but have no decapacitation effect on sperm that has been capacitated [48,49]. In other words, Sch B does not reverse capacitation when spermatozoa are highly capacitated. Therefore, we suspected that Sch B acted only on non-capacitated sperm, as it did not reverse capacitated sperm. This selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated sperm. Collectively, our study based upon sperm quality parameters, antioxidant capacity, and sperm fertilization ability during liquid preservation of boar semen at 4 °C significantly supports the effect of Sch B at a dose of 10 μmol/L as a semen storage protectant. This was evidenced by downregulation of Ca2+, reduced PKA, and regulated sperm adhesin family, reduced amounts of capacitated sperm, elevated antioxidative levels (SOD, CAT, and T-AOC increased, ROS, and MDA decreased), lowered proapoptotic properties of DNA damage, increased MMP, and, ultimately, the protected quality of boar sperm. Based on the above results, Sch B was identified as a primary candidate for an antioxidant and decapacitation factor in sperm in liquid at 4 °C. In conclusion, our findings demonstrated that 10 μmol/L is the optimal concentration of Sch B supplement in boar semen extender. Sch B enhanced sperm quality by increasing oxidation resistance and reducing the capacitation-like changes in spermatozoa stored at 4 °C. However, it did not have reversing effects on the capacitation status of capacitated spermatozoa.

PMC10000225

Cristián A. Valenzuela,Marco Azúa,Claudio A. Álvarez,Paulina Schmitt,Nicolás Ojeda,Luis Mercado

Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis

28-02-2023

autophagy,skeletal muscle,fish,immune response,Piscirickettsia salmonis

Simple Summary In mammals, autophagy plays a fundamental role in the defense against intracellular pathogens; however, in fish, this noncanonical function has not been described. In this context, it was proposed to study whether autophagy was modulated/activated in muscle cells challenged with a bacterial pathogen. Muscle cell cultures were performed and challenged with Piscirickettsia salmonis, the main threat to the salmon industry. Genes associated with immune response and autophagy were evaluated. In addition, the protein content of the LC3-II-specific marker of the autophagic process was evaluated via Western blot. Additionally, genes associated with vesicular traffic and endocytosis were evaluated, finding that P. salmonis promotes these processes. The results show a concomitant modulation of the genes associated with the immune response, vesicular trafficking, and autophagy, suggesting an early intracellular response by the muscle cell against this bacterium. Due to the necessity of seeking and discovering new alternatives and strategies to fight intracellular pathogens in the salmon industry, a better understanding of how autophagy participates in immune system responses may lead to the development of new technologies that allow for the effective control of intracellular pathogens, improving animal welfare and contributing to the sustainability of the global fish industry. Abstract Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1β, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.

Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis In mammals, autophagy plays a fundamental role in the defense against intracellular pathogens; however, in fish, this noncanonical function has not been described. In this context, it was proposed to study whether autophagy was modulated/activated in muscle cells challenged with a bacterial pathogen. Muscle cell cultures were performed and challenged with Piscirickettsia salmonis, the main threat to the salmon industry. Genes associated with immune response and autophagy were evaluated. In addition, the protein content of the LC3-II-specific marker of the autophagic process was evaluated via Western blot. Additionally, genes associated with vesicular traffic and endocytosis were evaluated, finding that P. salmonis promotes these processes. The results show a concomitant modulation of the genes associated with the immune response, vesicular trafficking, and autophagy, suggesting an early intracellular response by the muscle cell against this bacterium. Due to the necessity of seeking and discovering new alternatives and strategies to fight intracellular pathogens in the salmon industry, a better understanding of how autophagy participates in immune system responses may lead to the development of new technologies that allow for the effective control of intracellular pathogens, improving animal welfare and contributing to the sustainability of the global fish industry. Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1β, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes. Autophagy is a highly conserved catabolic process essential for cellular homeostasis, degrading cytosolic macromolecules and damaged organelles to guarantee energy supply and nutrient availability for the cell. Furthermore, autophagy can modulate innate and adaptative immunity, contributing to the clearance of pathogens from the cell [1]. Under normal conditions, autophagy occurs at low levels in almost all cells, but is strongly induced under nutrient deprivation, amino acid starvation, cellular stress, cytokine exposure and pathogen infections [2,3]. Additionally, autophagy can act through a noncanonical pathway in antigen processing and presentation for MHC-I and MHC-II, restricting intracellular pathogens and regulating adaptive immunity [4]. At the molecular level, the autophagic pathway involves the concerted action of evolutionarily conserved ATG gene products involved in the initiation (atg9, atg13 and becn1), autophagosome formation (atg5, atg12 and atg16), the elongation of autophagic processes (atg7 and atg4), and autophagosome closure (gabarap and lc3) [5]. Autophagy is triggered by activating a regulatory protein complex that recruits microtubule-associated light chain protein 3 (LC3) to the nascent autophagosome [6]. The conversion of LC3 into its active form, LC3-II, through conjugation to phosphatidylethanolamine (PE), allows for the transformation of the membrane into a closed double-membrane system [7]. Lastly, the autophagosome binds to the endosomal or lysosomal compartment, forming the autolysosome where organelles and macromolecules are degraded by a pool of proteolytic enzymes [8]. In mammals, autophagic activation is associated with immune signals and molecular pathways, involving toll-like receptor (TLR) activation through different pathogen-associated molecular patterns (PAMPs) [9]. For instance, autophagy activated by the endosomal TLR signaling pathway in macrophages plays a key role as a mechanism for Leishmania major infection resistance [10]. Mammalian skeletal muscle, a nonclassical immune tissue, possesses several membrane-bound TLRs with the ability to detect different PAMPs, triggering signaling pathways to activate autophagy [11]. These findings associate TLR signaling and autophagy, providing a potential molecular mechanism for the induction of autophagy in response to pathogen invasion. Therefore, the ability of TLR ligands to stimulate autophagy may be used to treat intracellular pathogens in mammals [12]. Moreover, several proinflammatory cytokines, such as IFN-γ, TNF-α, IL-1, IL-2, IL-6, and TGF-β induce autophagy, while anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 display an inhibitory effect [13]. All these data suggest a close relation between the immune response and autophagy in mammalian skeletal muscle. However, this interaction is unknown in fish, and only a few reports related to autophagy exist. Nevertheless, due to the direct relation of autophagy and growth, a key parameter for fish-industry productivity, autophagic markers have been studied under different diets and nutritional trials. Results showed a direct association between fish’s energy requirement and the activation of autophagy [14]. For instance, the autophagic flux and the overexpression of autophagic genes atg4, atg9, atg12, lc3, gabarap and becn1 was observed in vitro in epithelial gill cell line RT-gill-W1 of rainbow trout under nutrient restriction, and in the muscle of trout challenged with Flavobacterium psychrophilum [15,16]. Nevertheless, the modulation of autophagy in fish muscle cells during an immune-like response against an intracellular pathogen has not been elucidated. This study aims to describe and characterize autophagic modulation through the immune response of fish muscle cells after a challenge with a critical bacterial pathogen for the salmon industry. Through the immune challenge of primary cultures of muscle cells with Piscirickettsia salmonis, a fastidious intracellular pathogen, we revealed the transcript upregulation and protein production of several immune markers and genes involved in autophagy. Results obtained in the present work give new insight into the role of autophagy in the immune response against bacterial pathogens in muscle cells. The present research was approved by the bioethics committee of Pontificia Universidad Católica de Valparaíso (PUCV) BIOEPUCV-B 334-2020 and the Agencia Nacional de Investigación y Desarrollo (ANID) of the Chilean government, and adhered to all animal welfare procedures. In total, 24 rainbow trout (Oncorhynchus mykiss) with body weight of 10 ± 1.7 g and body length of 10 ± 1.5 cm were obtained from the Río Blanco Aquaculture Center (Valparaíso, Chile). These fish were kept in 3 tanks at a density of 8 fish per 25-L tank, using 1 tank per trial. Fish were maintained at a temperature of 16 °C, fed daily with commercial feed, and provided with a photoperiod of 13 h of light and 11 h of darkness. Muscular cells were prepared from 6-8 g of the skeletal muscle of four rainbow trout as previously reported [17]. Briefly, the dorsal white muscle was collected and disintegrated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 9 mM NaHCO3, 20 mM HEPES, 10% horse serum at pH 7.4, 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma, St. Luis, MO, USA). After mechanical dissociation, the muscle was washed with DMEM and then digested with 0.2% collagenase solution in DMEM for 1 h at 18 °C. The suspension was centrifuged at 500 g for 5 min at 15 °C, and the resulting pellet was digested with a 0.1% trypsin solution in DMEM for 30 min at 18 °C. Trypsin was deactivated with complete medium (DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at pH 7.4), the suspension was filtered and centrifuged at 5000× g for 10 min at 18 °C, and 5 mL of DMEM was lastly added to the resulting pellet. The cell suspension was collected to count cells and evaluate viability with Trypan Blue 0.2%. Cells were seeded at a density of 5 × 105 per well in plates previously treated with poly-L-lysine (2 μg/cm2) and laminin (20 μg/mL). The cells were incubated at 18 °C for 14 days, 7 days in a proliferation medium containing DMEM, 9 mM NaHCO3, 20 mM HEPES, 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at pH 7.4, and 7 days in a differentiation medium composed of DMEM (9 mM NaHCO3, 20 mM HEPES, 100 U/mL penicillin, and 10 mg/mL streptomycin). This procedure was repeated three times (n = 3) per experimental trial described in the next sections. A P. salmonis field isolate, Psal-104a, was obtained from the Chilean National Piscirickettsia salmonis Strain Collection at Pontificia Universidad Católica de Valparaíso (PUCV). The bacteria were cultured in basal medium (BM) broth composed of yeast extract (Merck, St. Louis, USA) 2.0 g L−1, peptone from meat (peptic digested, Merck) 2.0 g L−1, (NH4)2SO4 1.32 g L−1, MgSO4·7H2O 0.1 g L−1, K2HPO4 6.3 g L−1, NaCl 9.0 g L−1, CaCl2·2H2O 0.08 g L−1, FeSO4·7H2O 0.02 g L−1, and glucose 5 g L−1 at 18 °C at 100 rpm. Exponentially growing P. salmonis (O.D. 600 = 0.3) was recovered via centrifugation at 5000× g, washed three times with saline-buffered solution (SBS: 0.15 M NaCl, 7.3 mM KH2PO4, 11.5 mM K2HPO4, pH 6.0) supplemented with 4% lactose, and resuspended in DMEM containing 9 mM NaHCO3, 20 mM HEPES, at pH 7.4. Bacterial cells were counted using a Petroff–Hausser chamber. Muscular cells were infected at a multiplicity of infection (MOI) of 10 for 4, 6, 8, and, 24 h. After the experimental times, the pathogen was removed and then sampled. As a positive control for autophagic activation, muscle cells were treated with rapamycin (50 nm), and DMEM was used as a vehicle for control, sampled at the beginning of the experiment (0 hpi). The total RNA from the primary culture of skeletal muscle was extracted according to the manufacturer’s guidelines for E.Z.N.A total RNA Kit I (Omega Bio-tek, Norcross, GA, USA), and quantified with NanoDrop LITE spectrophotometer (Thermo Scientific, Rockford, IL, USA). Residual genomic DNA was removed, and 1 µg of RNA was used for cDNA synthesis via RevertAid First strain cDNA Synthesis Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Real-time quantitative PCR (qPCR) assays were performed in a AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA) with 15 µL volume reactions, of which 1 µL was cDNA (a 2-fold dilution), 0.2 μl of each primer (250 nM), and 8.8 µL of Takyon master mix (Eurogentec, Seraing, Belgium). The primers used in this study were designed and validated in our laboratory, and the sequence and efficiency of each primer are listed in Supplementary Table S1. Thermal cycling conditions were as follows: initial activation 95 °C for 10 min, 40 cycles of 30 s denaturation at 95 °C, 30 s annealing at 60 °C, and 30 s elongation at 72 °C, and each sample was loaded in duplicate. Relative expression analysis was conducted using geNorm software (https://genorm.cmgg.be/ (accessed on 30 March 2021)), and the results are expressed as fold changes using EF-1α and b-actin as housekeeping reference genes. Total protein was extracted from primary culture cells in 150 µL Piercetm RIPA buffer (Thermo) supplemented with 8 mM EDTA, PMSF 1 mM, and a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA), centrifuged at 12,000× g, and solubilized at 4 °C. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Hanover Park, IL, USA). The total protein (10 µg) was loaded into each line, separated with 18% SDS-PAGE, transferred to a nitrocellulose membrane, and blocked for 1 h at 37 °C in Tris-buffered saline (TBS-Tween) with 3% BSA. Primary antibody incubations (LC3A/B (D3U4C) XP® Rabbit mAb #12741, Actine (INVITROGEN), and ant-Rab7a, diluted 1:1000, 1:2000 and 1:500, respectively) were performed overnight at 4 °C. The anti-Rab7a polyclonal antibody was developed in our lab. Briefly, a recombinant Rab7a was used for specific antibody production (Supplementary Figure S1). CF-1 mice (5 weeks old) were subcutaneously injected at 1, 14, and 28 days with 30 μg of peptide diluted 1:1 in FIS, as a T helper cell activator, and in 1:1 Freund’s adjuvant (Thermo Scientific). The antiserum was collected on Day 42 and centrifuged at 700 g for 10 min, and the supernatant was stored at −20 °C. Antibody validation was determined via Western blot (Supplementary Figure S1). Membranes were washed with 1X TBS and incubated for 1 h at room temperature with the appropriate secondary antibody. After washing, the membranes were visualized using commercial kit WESTAR SUPERNOVA from Cyanagen. Then, the membrane was visualized with the ChemiDoc Imaging Systems Bio-Rad system (Bio-Rad Laboratories, Hercules, CA, USA). Digitized images were used for the densitometric analyses of the bands in Image J software(National Institutes of Health, Bethesda, Maryland, USA). Differences are expressed as fold changes in protein content. Western blots were performed for all individual samples per experimental treatment; however, only one representative blot film is shown in the figures (Supplementary Figures S2–S4). Adherent cells were washed with 1X PBS and fixed with 4% paraformaldehyde (PFA) (in 1x PBS) for 10 min and permeabilized/blocked with 3% BSA with 0.3% Triton in 1X TBS for 30 min, and the quenching solution (50 mM ammonium chloride) was added for 10 min. Then, the cells were incubated overnight with the anti-LC3 antibody diluted at 1:100 (LC3A/B (D3U4C) XP® Rabbit mAb #12741 in 1% BSA at room temperature. Samples were washed with 1X PBS and 0.02% TBST, and incubated with a commercial secondary antibody (1:750) (Invitrogen, Thermo Scientific) for 90 min at room temperature. Nuclear staining was performed with DAPI (Vector Laboratories) for conventional fluorescence microscopy, following the manufacturer’s instructions. For image capture, the samples were placed in a Leica CTR5000fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Western blots were analyzed using the LC3-II/actin and Rab7a/actin ratios. All generated data were evaluated with one-way ANOVA and Student’s t test. Data are expressed as the mean (n = 3 per treatment) ± standard error of the mean (SEM), and p < 0.05 was considered statistically significant. All analyses were performed using general linear models on Graph Prism 7.0 software (GraphPad Company, Boston, MA., USA). To understand and characterize the response of muscle cells against bacterial pathogens, rainbow-trout skeletal muscle cells were challenged with P. salmonis for 6, 8, and 24 h. Next, immune molecular markers tlr3, tlr9, il-1β, tnfα, il-8, hepcidin, mhc-I and mhc-II were measured with RT-qPCR, and results showed a concomitant overexpression of toll-like receptors 3 and 9 after 6 h of challenge (Figure 1A). Proinflammatory cytokines il-1β, tnfα and il-8 were modulated compared to the noninduced controls, where tnfα and il-8 were upregulated significantly after 6 and 8 h after the challenge, respectively (Figure 1B,C). Antimicrobial peptide hepcidin increased after 6, 8, and 24 h after the challenge, but no statistical significance was observed (Figure 1C). No changes were observed in the mRNA levels of mhc-I and mhc-II during the entire trial (Figure 1D). To describe the modulation of autophagy in muscle cells after bacterial challenge, autophagic markers were analyzed via RT-qPCR, Western blot, and immunofluorescence. Rapamycin was used as the positive control of autophagic activation. Autophagic genes associated with the induction of this process were upregulated, particularly becn1, of which the mRNA level was significantly increased after 6 and 24 h of the challenge (Figure 2A). In the case of autophagosome formation genes, the overexpression of atg5 was observed after 6 h of treatment, while the transcript level of atg12 was downregulated after 8 h (Figure 2B). The mRNA levels of atg4, an elongation of an autophagosome molecular marker, was downregulated after 8 h of challenge (Figure 2C). Lastly, the transcript level of lc3 was significantly downregulated after 8 h of challenge (Figure 2D). At the protein level, protein autophagic marker LC3-I/LC3-II was evaluated via immunofluorescence and Western blot. Agreeing with the gene expression of autophagic induction gene analysis, the protein content of LC3 showed an increase in fluorescence signal in the muscular cells challenged with P. salmonis compared with the control group at 6 h after challenge (Figure 3A). An increase in the LC3-II protein content was observed through Western blot analysis after 6 h of stimuli (Figure 3B). In addition, the mRNA levels of small GTPase proteins rab5a and rab7a were upregulated at 6 h after challenge, while the transcript level of rab11a was increased after 24 h (Figure 3C). Lastly, the protein content of Rab7 showed an increasing trend through Western blot (Figure 3D). In mammals, the ability of muscles to respond and participate in the immune response against pathogens and in autoimmune events is well-characterized [18]. This ability is represented by the immunocompetent attributes of muscle cells, such as the expression of costimulatory molecules, antigen-presenting machinery, proinflammatory cytokines, and pathogen recognition receptors [19,20,21,22,23]. Moreover, the interaction between muscle cells and resident immune cells is an essential step for wound healing and damage regeneration in this tissue [24,25]. In fish, the skeletal muscle appears as an active immunological organ, where immune reactions occur after an in vivo challenge with bacterial pathogens [26,27]. However, the ability of a muscle cell to express classical immune-like molecules by itself is still under study, and only few reports address how this type of cells may respond against PAMPs and pathogens [28,29]. For example, rainbow-trout myotubes responded against Piscirickettsia salmonis through the upregulation of tlr1, tlr22, and il-8 at 8 h after a challenge [17]. In the present study, the immune response against P. salmonis generated a concomitant overexpression of tnfα, tlr3, and tlr9 after 6 h of challenge. Toll-like receptors 3 and 9 are associated with an intracellular/antiviral response where these receptors can detect double-strand RNA and CpG DNA, respectively [30]. Reports in mammals showed how these receptors can be involved in diminishing the growth of intracellular bacterium Salmonella typhimurium [31]. Salmon head kidney cells (SHK-1) respond to an infection with P. salmonis through the overexpression of tlr1 and tlr5s [32]. Furthermore, P. salmonis could induce a flagellin-dependent TLR5 activation in Atlantic salmon, which resulted in the upregulation of proinflammatory genes [33]. The overexpression of intracellular receptors suggests that intracellular defense mechanisms could be activated in response to P. salmonis infections. Despite the overexpression of markers associated with an intracellular response, the RNA levels of molecular markers DotA and DotB of P. salmonis were not detected via RT-qPCR (data not shown), which could indicate that, even though the bacterium does not enter the cell, the intracellular defense mechanisms are stimulated. On the other hand, proinflammatory cytokine tnfα represents an early response of muscular cells against bacterial pathogens. Indeed, an increase in NF-κB and TNFα at 48 h after a challenge against Vibrio anguillarum was observed on fish muscle [34]. Additionally, at 8 h after a challenge, the mRNA level of il-8 was upregulated. In higher vertebrates, TNFα induces the expression of IL-8 [35]. This kind of response may be associated with the need for muscle cells to recruit immune cells to the site of infection, since IL-8 is a chemoattractant for leukocytes [36]. These results contribute to existing data confirming that the muscle cell responds to a bacterial challenge by itself. However, communication with the immune resident’s cells is needed to deploy an effective and robust response. In mammals, the participation of autophagy in the immune response is well-characterized, where autophagy-related gene (ATG) proteins mediate direct pathogen degradation, inflammation restriction, antigen presentation on MHC molecules and survival of memory lymphocyte populations [37]. This process has mainly been studied for its role in the internal quality control of the cell, and for its implication in metabolism and cancer. However, autophagy can participate through a noncanonical pathway to respond against other dangerous stimuli, such as pathogen infection [38]. In fish, the modulation of this process in a pathological context is unclear, and only a few reports show how autophagy is modulated by nutrient restriction and pharmacological treatments [14,39]. Transgenic zebrafish and zebrafish cell lines were used as research tools on the autophagic regulation process, highlighting its role on the protection against pathogen infection, development, and lipid degradation [14]. Autophagy was also linked to development and extracellular matrix formation in zebrafish [40]. Recently, the modulation of autophagic genes atg4, becn1, atg7, lc3, atg12, and gabarap has been evaluated in rainbow-trout liver and muscle under different functional treatments, fasting, and a posteriori F. psychrophilum infection, suggesting a possible role of this process in the resistance against pathogens [16]. In the present work, autophagic genes were up- and downregulated in a time-dependent and coordinated way. The upregulation of becn1 was observed at 6 h after challenge; becn1 is a protein that facilitates the de novo formation of the phagophore, and is critical for the autophagic initiation in embryogenesis and antiviral responses in fish [41,42]. The next step in autophagy is autophagosome formation, where molecules atg5 and atg12 are directly involved [43]. We observed the upregulation of atg5 at 6 h after a challenge with the subsequent downregulation of atg12 after 8 h. Both genes are associated with intracellular immune response against viruses in fish, where atg5 plays crucial roles in viral replication via promoting autophagy in orange-spotted grouper [44], while atg12 can induce both autophagy and the Type I IFN response in large yellow croaker [45]. These two molecules are regulated by RAB5, a small GTPase, which facilitates the early formation of the autophagosome [46]. Six hours after a challenge with P. salmonis, the mRNA levels of rab5a and rab7 increased significantly, suggesting the activation of vesicle traffic in response to the bacteria. In mammals, Rab7a is associated with the endocytic pathway [47]; in fish, this molecule is related with an antiviral response [48], suggesting the activation of different intracellular mechanisms in response to P. salmonis. Lastly, the genes involved in the elongation and closure of the autophagosome were downregulated after 8 h after challenge, particularly atg4 and lc3. Altogether, the present data suggest a tight relation between autophagy and intracellular innate immune response, and are consistent with the used intracellular bacterial pathogen and the evaluated immune markers in our study. A crosstalk between the immune response and autophagy has been reported in mammals. For example, TLRs were directly involved with autophagic activation after TLR ligand stimulations with poly I:C and LPS, among other PAMPs [9]. In addition, the stimulation of TLR7 with different ligands activated autophagy, eliminating intracellular microorganisms even when the target pathogen had not been canonically associated with TLR7 signaling [12]. In this context, the recognized ability of TLR ligands to stimulate autophagy was proposed as a prophylactic alternative to treat intracellular pathogens [12]. In the present study, tlr3, tlr9, tnfα and autophagic genes becn1 and atg5 were upregulated at the same experimental time (6 h after challenge), suggesting a relation between the autophagy and immune response of muscle cells against P. salmonis. This relation was confirmed in mammals where TNFα and other cytokines could induce autophagy [13]. Lastly, to corroborate the activation of autophagy in rainbow-trout muscle cells, we assessed the protein level of LC3-II, a classical protein marker for autophagy. Finding higher protein content of LC3-II in the treated cells added to the presented overall results and the background information, suggesting that the muscle cell deploys an immunelike response where autophagy may play an important role for the clearance of the pathogen. Deeper functional analysis and characterization are required to corroborate the dialogue and regulation between the two essential cellular functions. The skeletal muscle of fish is a fundamental energy source for the organism and a recurrent site of infection where immunelike responses against different bacterial pathogens occur. The present study described for the first time the modulation of the immune response and its possible connection with autophagy in rainbow-trout muscle cells. The challenge of trout muscle cells with P. salmonis, an intracellular pathogen, triggered an immune response concomitant to the activation of the autophagic process. Results suggest an early response by the muscle cells against the bacteria just after only 6 h of challenge, where both immune and autophagic markers were upregulated. Considering the obtained data and the available research information, two important questions arise regarding the possible implication of P. salmonis challenge in fish muscle: (1) is autophagy acting as an intracellular immune mechanism to fight this pathogen or is the bacterium using this mechanism to enter the cell and evade the immune response? These questions are relevant and represent a new line of research that must be answered. Due to the necessity of seeking and discovering new alternatives and strategies to fight intracellular pathogens in the salmon industry, a better understanding of how autophagy participates in immune system responses may lead to the development of new technologies that allow for the effective control of intracellular pathogens, improving animal welfare and contributing to the sustainability of the global fish industry.

PMC10000232

Vincenzo Quagliariello,Irma Bisceglia,Massimiliano Berretta,Martina Iovine,Maria Laura Canale,Carlo Maurea,Vienna Giordano,Andrea Paccone,Alessandro Inno,Nicola Maurea

PCSK9 Inhibitors in Cancer Patients Treated with Immune-Checkpoint Inhibitors to Reduce Cardiovascular Events: New Frontiers in Cardioncology

22-02-2023

cardio-oncology,cardioprotection,cancer,immune-checkpoint inhibitors,atherosclerosis,cholesterol,inflammation

Simple Summary Atherosclerosis is a critical cardiovascular disease associated with the use of immune checkpoint inhibitors (ICIs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key orchestrator of atherosclerotic process and it is also involved in cancer progression and immune-resistance. In this context, data from recent meta-analysis and cardiovascular outcome trials associate PCSK9 levels to reduced ICIs-related cancer responsiveness and to high risk of atherosclerotic cardiovascular diseases. This review summarizes the pleiotropic effects of PCSK9 in heart failure, atherosclerosis, and cancer immune recognition, and outlines its ability to represent a new pharmacological target in patients who develop ICIs-related atherosclerosis to reduce cardiovascular mortality and to improve overall survival. Abstract Cancer patients treated with immune checkpoint inhibitors (ICIs) are exposed to a high risk of atherosclerosis and cardiometabolic diseases due to systemic inflammatory conditions and immune-related atheroma destabilization. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key protein involved in metabolism of low-density lipoprotein (LDL) cholesterol. PCSK9 blocking agents are clinically available and involve monoclonal antibodies, and SiRNA reduces LDL levels in high-risk patients and atherosclerotic cardiovascular disease events in multiple patient cohorts. Moreover, PCSK9 induces peripheral immune tolerance (inhibition of cancer cell- immune recognition), reduces cardiac mitochondrial metabolism, and enhances cancer cell survival. The present review summarizes the potential benefits of PCSK9 inhibition through selective blocking antibodies and siRNA in patients with cancer, especially in those treated with ICIs therapies, in order to reduce atherosclerotic cardiovascular events and potentially improve ICIs-related anticancer functions.

PCSK9 Inhibitors in Cancer Patients Treated with Immune-Checkpoint Inhibitors to Reduce Cardiovascular Events: New Frontiers in Cardioncology Atherosclerosis is a critical cardiovascular disease associated with the use of immune checkpoint inhibitors (ICIs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key orchestrator of atherosclerotic process and it is also involved in cancer progression and immune-resistance. In this context, data from recent meta-analysis and cardiovascular outcome trials associate PCSK9 levels to reduced ICIs-related cancer responsiveness and to high risk of atherosclerotic cardiovascular diseases. This review summarizes the pleiotropic effects of PCSK9 in heart failure, atherosclerosis, and cancer immune recognition, and outlines its ability to represent a new pharmacological target in patients who develop ICIs-related atherosclerosis to reduce cardiovascular mortality and to improve overall survival. Cancer patients treated with immune checkpoint inhibitors (ICIs) are exposed to a high risk of atherosclerosis and cardiometabolic diseases due to systemic inflammatory conditions and immune-related atheroma destabilization. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key protein involved in metabolism of low-density lipoprotein (LDL) cholesterol. PCSK9 blocking agents are clinically available and involve monoclonal antibodies, and SiRNA reduces LDL levels in high-risk patients and atherosclerotic cardiovascular disease events in multiple patient cohorts. Moreover, PCSK9 induces peripheral immune tolerance (inhibition of cancer cell- immune recognition), reduces cardiac mitochondrial metabolism, and enhances cancer cell survival. The present review summarizes the potential benefits of PCSK9 inhibition through selective blocking antibodies and siRNA in patients with cancer, especially in those treated with ICIs therapies, in order to reduce atherosclerotic cardiovascular events and potentially improve ICIs-related anticancer functions. Immune checkpoint inhibitors (ICIs) are increasingly used in oncology to treat multiple malignancies, including melanoma, non-small cell lung cancer, metastatic breast cancer, and others [1,2]. In most cases, ICIs are monoclonal antibody antagonists of programmed death-ligand 1 (PDL-1) or programmed cell death protein 1 (PD-1) or cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) which are the main drivers of peripheral immune tolerance, even towards tumors [3,4]. Briefly, ICIs antagonize the inhibition of lymphocyte uptake against tumors, resulting in a lymphocyte-mediated anticancer effect [4]. Recent trials associate ICIs with radiotherapy [5], standard chemotherapy (anthracyclines or platinum-based anticancer drugs) [6,7], targeted therapies (HER-2 blocking agents, TKi and others) [8], and combination therapies (i.e., PD-1 and CTLA-4 blocking agents) [9,10]. In brief, combination therapies involving ICIs and standard chemotherapies increase lymphocytic infiltration in neoplastic tissue in a pro-inflammatory microenvironment, which make CD56+ and CD3− large granular lymphocytes more reactive against tumor cells [11,12]. However, ICIs therapies are associated with a broad spectrum of endocrine diseases and, albeit, relatively rare cardiovascular side effects [13,14], including myocarditis [15], vasculitis [16], inflammatory endocrinopathies [17], mucositis [18], and arthritis [19]. The main mechanisms of cardiotoxicity are not deeply understood, but NLRP-3/IL-1overexpression, My-D88/TLR4, and cytokine-mediated pathways are still considered to be key orchestrators [20,21] in ICIs-mediated side effects in preclinical and clinical models [21]. Very recently, atherosclerosis has emerged as a new considerable ICI-s mediated side effect in cancer patients [22,23]. Briefly, exposure to PD-1 or PDL-1 or CTLA-4 blocking agents increases VCAM-1 and ICAM-1 expression in luminal membrane of vascular endotheliocytes, thus stimulating IL-1, IL-6, and TNF-α levels associated with instability of the atherosclerotic plaque. Interleukins 1 and 6 and TNF-α induce LDL uptake and their oxidation to OX-LDL in endothelial cells [24,25,26] (Figure 1). A new key driver of the atherosclerosis process is proprotein convertase subtilisin/kexin type 9 (PCSK9) [27]. Briefly, PCSK9 is a protein with key roles in hepatic low density lipoprotein (LDL) homeostasis, reducing the LDL-receptor density in hepatocytes [28]. When PCSK9 binds LDL receptors, it prevents the correct recycling on the cell membrane after the natural bond with LDL particles. This effect increases plasma LDL levels and its associated cardiovascular risk [29]. PCSK9 inhibitors (antibody-based blocking agents and miRNAs) are currently used in clinical practice to reduce LDL levels in high risk cardiovascular patients, intolerant to statins, in order to reduce cardiac risk [30]. However, extrahepatic functions of PCSK9 are increasingly studied in the last three years, involving cardiomyocyte [31], endotheliocyte [32], macrophage, and cancer cell metabolism [33], shedding light on possible therapeutic uses of PCSK9 inhibitors to new off-label clinical applications in cardio-oncology. This review assesses the current knowledge about how PCSK9 interacts with the immune environment in cancer tissue and how PCSK9 inhibitors could be beneficial in patients treated with ICIs in primary prevention of atherosclerosis. First, we describe how PCSK9 is involved in cancer progression and immune escape. Then, we review current knowledge on how PCSK9 inhibitors reduce atherosclerosis initiation and progression in patients without cancer and the main molecular pathways involved. The use of ICIs can change the peripheral immune tolerance in different tissues, exposing patients to neuro-inflammatory diseases, visceral obesity, atherosclerosis, and leptin resistance [34]. Preclinical models with deactivating mutations of PD-1 or PDL-1 or CTLA-4 genes are more exposed to atherosclerotic plaques characterized by high levels of VCAL-1, ICAM-1, galectine-3, oxLDL, high macrophage density, and pro-inflammatory cytokines [35,36]. In brief, the lack of these immune repressor proteins increases atherogenic phenotype mediated by a high uptake of CD3+/CD4+ lymphocytes and CD3+/CD8+ lymphocytes in atherosclerotic plaque [37]. These findings led to the hypothesis that ICIs could increase the risk of atherosclerotic cardiovascular diseases (ASCD) in cancer patients [38] (Figure 1). Recent clinical studies have confirmed this hypothesis: patients treated with ICIs for two years have a three times higher risk of developing atherosclerosis compared to other therapies [39]. There are additional observational studies associated with the high risk of unstable atherogenic plaques in cancer patients treated with ICIs compared to the general population [40]. However, more in-depth studies should be performed to clarify if combinatorial anti-PD-1 and CTLA-4 therapies could affect the ASCD risk more than a monotherapy regimen. A close correlation between atherosclerosis and PCSK9 has also been observed [41]. In a recent meta-analysis [42], eleven studies in patients with CVD were analyzed. Notably, patients with established CVD and high PCSK9 levels had a 52% higher risk of future total cardiovascular events than those with low PCSK9 concentrations. Patients with high levels of PCSK9 experienced more than 20% of cardiovascular events compared to low levels [43]. Mechanistically, PCSK9 plays a key role in platelet aggregation and adherence to endothelial cells, endothelial dysfunctions, and atherosclerosis [44]. Genetic studies associated loss of function mutation in PCSK9 gene with high LDL levels and high rates of heart failure [45]. Other studies associated high serum PCSK9 levels to a more necrotic core of coronary atherosclerosis independently from LDL levels [46]. These data indicate extra hepatic roles of PCSK9 that do not involve cholesterol homeostasis. However, Vlachopoulos et al. do not associate PCSK9 levels to cardiovascular events in high-risk patients; they only do so in the general population [47]. Additionally, other research groups associated high PCSK9 levels to patients with stable CVD, but not in patients with ACS [48]. However, there are several other associations between PCSK9 and cardiovascular diseases. First, during myocardial infarction, PCSK9 serum levels are upregulated due to pro-inflammatory processes [49]. Moreover, other studies demonstrated that circulating PCSK9 levels rapidly rise after the initiation of statin therapy [50], and there is a sustained increase throughout statin use. Furthermore, it has recently been observed that PCSK9 could reduce the efficacy of statin treatment in patients with high cardiovascular risk, by reducing the LDL receptor density on hepatocytes [51]. Furthermore, a recent gender study concluded that white people and Asian people have variants in the PCSK9 gene that may or may not be associated with different LDL concentrations [52]. The main cardiovascular outcomes trials, which will be discussed in the next paragraph, called Evaluation of Cardiovascular Outcomes After an ACS During Treatment With Alirocumab (ODYSSEY) [53] and Further CV Outcomes Research with PCSK9 Inhibition In subjects With Elevated Risk (FOURIER) [54], analyzed cardiovascular benefits of PCSK9 inhibitors in non-cancer patients, and recruited mostly only white patients. Being a minority in the makeup made up of ascitic patients, it is probable that the effects of PCSK9 inhibitors in white people are not the same due to variations in the genetic profile of different races [54]. The PCSK9 gene is located on chromosome 1p32.3 that is expressed in several organs, including the liver, kidneys, small intestine, heart, and cancer cells [55]. The encoded protein is of 692 aminoacids, characterized by three domains: signal domain, pro-domain, and V domain (Figure 2). Interestingly, the key process for maturation and secretion of active PCSK9 is secondary to the cleavage of pro-domain in the S 38 and P47 region [56]. The first clinical evidence on the association between PCSK9 and CV events are based on loss of function or gain of function in PCSK9 gene: patients with gain of function mutations of PCSK9 gene experienced high levels of serum LDL-C, a reduction in LDL receptor levels on hepatocytes (by more than 35%), and premature cardiovascular events compared to non-mutated patients [57]. Contrary, loss of function mutations are associated with low serum levels of PCSK9 and more than 40% reduction of LDL-C with consequent risk reduction in incidences of ischemic heart diseases [57]. PCSK9 is also expressed in cardiomyocytes [58]. Very recent studies described PCSK9 functions in cardiomyocyte autophagy, pyroptosis, ferroptosis, and apoptosis [58]. Detrimental events such as hypoxia, acute inflammation, and heart failure can induce different types of cardiomyocyte cell death through PCSK9 overexpression [59]. Specifically, high cholesterol and insulin levels induce overexpression of PCSK9, which activates DRP-1 in mitochondria triggering autophagy [60]. Moreover, PCSK9 exacerbates mitochondrial ROS production, resulting in the activation of the LKB1-AMPK pathway [61]. Moreover, in cases of OX-LDL intake in the cardiomyocyte, PCSK9 overexpression causes mitochondrial DNA damage, resulting in the activation of NLRP3 inflammasome/Caspase-1/Interleukin-1 pathway [62]. This process activates cardiomyocyte pyroptosis. Increased serum levels of IL-1 and IL-6 directly result in PCSK9 overexpression, which also induces cardiomyocyte apoptosis via caspase-9 and 3 [63]. Another biochemical mechanism of PCSK9 cardiac and endothelial toxicity is mediated by lipid peroxides [64]. Lipid peroxidation, which can be induced by acute inflammation, smoking, and some chemotherapeutic drugs such as anthracyclines, leads to the formation of MDA and 4-HNA by the Fenton reaction [65,66]. PCSK9 activates the Fenton reaction, whose gene expression is activated by the over-intake of Fe3+ in cardiomyocytes and endotheliocytes [66]. Furthermore, intracellular overexpression of PCSK9 results in decreased FFA uptake and utilization through a specific mechanism: PCSK9 competes with FFAs for binding to the CD36/FAT receptor and reduces their membrane recycling, resulting in an increase in systemic FFA levels and a significant reduction in fatty acid beta oxidation and the Krebs cycle [67]; this is the primary driver of atherosclerosis and cardiomyocyte injury processes. Moreover, it is conceivable that PCSK9 has a role in heart failure, hypertrophy, and cardiac fibrosis. These effects could be mediated by TNF-a and some chemokines; in fact, TNF-a upregulates PCSK9 gene expression via Peroxisome proliferator-activated receptors (PPARs) and PPAR gamma coactivator-1a (PGC1) pathways [68]. Current guidelines of the American College of Cardiology and American Heart Association recommend the addition of non-statin cholesterol-lowering therapies for patients at very high risk of major adverse cardiovascular events (MACE) when LDL-C levels remain ≥ 70 mg/dL [69]. About half of coronary heart disease patients on moderate- or high-dose statin therapies reduce LDL-C less than 70 mg/dL [70]. In this category of patients, there could be an additional clinical benefit deriving from the addition or substitution with other cholesterol-lowering agents, such as PCSK9 inhibitors, and this is a point of discussion in cardiology. Some randomized clinical trials have demonstrated how the administration of PCSK9 inhibitors, evolocumab, alirocumab, bococizumab, or inclisirian, as monotherapy or in combination with statins can reduce systemic levels of atherogenic and pro-inflammatory lipoproteins [71,72]. Notably, PCSK9i significantly reduces LDL-C and up to 25% lipoprotein a (Lpa) [73]. Effects on LPa levels are of particular interest in cardiology, considering its well established strong atherogenic, pro-inflammatory, and pro-thrombotic effects [74]. A recent trial concluded that among patients with advanced stable coronary artery disease, Lp(a) is associated with accelerated progression of coronary low-attenuation plaque (necrotic core) [75]. This may explain the association between Lp(a) and the high residual risk of myocardial infarction, providing support for Lp(a) as a treatment target in atherosclerosis [76]. The most known CVOT including PCSK9i are the FOURIER and ODYSSEY Outcomes trials. Patients with established cardiovascular disease or acute coronary syndrome (ACS) and elevated levels of LDL-C, non–high-density lipoprotein cholesterol, or apolipoprotein B were enrolled in these studies [77]. Patients treated with PCSK9i reduced MACE, coronary heart disease, peripheral artery disease, and venous thromboembolic events. Some of these beneficial effects were also associated with LPa reductions [78]. A brief description of the clinical benefits of each PCSK9i is provided below and summarized in Table 1: - Evolocumab Patients treated with evolocumab halved LDL cholesterol values [79]. Furthermore, a recent meta-analysis showed that patients treated with evolocumab also had a slight but significant increase in HDL cholesterol; on the other hand, total cholesterol levels decreased by about 35–37% but with great heterogeneity of age and sex [80]. Evolocumab was not effective in reducing triglyceride levels. Regarding adverse cardiac events, a recent meta-analysis showed that evolocumab reduced myocardial infarction by 27% and stroke by approximately 21% [81]. A further interesting finding concerns unstable angina requiring revascularization which was reduced by 16% in patients treated with evolocumab vs. placebo. A further recent meta-analysis showed that evolocumab treatment reduces composite or CV death, myocardial infarction, stroke, and unstable angina by 15%. Moreover, in HUYGENS (High-Resolution Assessment of Coronary Plaques in a Global Evolocumab Randomized Study) study [82], evolocumab administration stabilized coronary plaque, resulting in regression of atheroma volume in patients with ACS. - Alirocumab Alirocumab is an additional PCSK9 inhibitor that has been extensively studied in cardiovascular outcome trials [83]. A recent meta-analysis showed that patients treated with alorocumab increased HDL cholesterol levels by approximately 5% [84]. Similarly to evolocumab, alirocumab reduces total cholesterol levels without affecting triglyceride levels. Furthermore, alirocumab, similarly to evolocumab, reduced composite of MI, stroke, unstable angina, and CV death. In a biweekly administered regimen, Alirocumab lower LDL-C by 45 to 60% depending on the applied dose (75 mg vs. 150 mg) and by ~ 50% while given monthly in a 300-mg dose [85]. Moreover, we now have evidence that evolocumab and alirocumab treatment, on top of statins, in patients with ACS modifies coronary plaque properties, leading not only to a significant thickening of the fibrous cap, thereby stabilizing it, but also resulting in regression of atheroma volume in PACMAN-AMI (Effects of the PCSK9 Antibody Alirocumab on Coronary Atherosclerosis in Patients With Acute Myocardial Infarction) study [86]. - Bococizumab Bococizumab is an additional PCSK9 blocking monoclonal antibody which, unlike the other antibodies, has shown beneficial effects on MACE only in high-risk patients [87]. Two large studies called SPIRE I [88] and II [89] enrolled more than 27,000 low-risk and high-risk patients (LDL 70 and 100 mg/dl, respectively) treated with bococizumab (150mg) twice a week. In SPIRE I [88], bococizumab showed no significant effects on composite of myocardial infarction, stroke, hospitalization for unstable angina requiring urgent revascularization, and cardiovascular death. IN SPIRE II, [89] however, bococizumab reduced the primary endpoint of the SPIRE studies by 11%. However, a serious problem occurred in these patients, namely the production of antibodies to bococizumab, which resulted in very frequent injection site adverse reactions [90]. - Inclisiran Inclisiran is a novel, small, interfering RNA aimed to target PCSK9 [91]. In detail, the molecule is a modified double-stranded RNA conjugated to triantennary N-acetylgalactosamine (GalNAc). Liver cells are rich in the asialoglycoprotein receptor that binds to GalNAc, therefore, after subcutaneous administration, inclisiran accumulates significantly in the liver [92]. Inclisiran lowered PCSK9 and LDL-C levels in a dose-dependent manner [93]. After 2 doses of inclisiran, LDL-C was reduced by up to 53% at day 180 [94]. Three large, randomized trials evaluated the cardiovascular efficacy of incisiran; these were called ORION 9–10 and 11 trials [95,96]. In the ORION-9 trial, HeFH patients halved LDL cholesterol after approximately 500 days of treatment with inclisirian. In ORION 10 and 11, LDL cholesterol levels were reduced by approximately 52 and 49% over 500 days of treatment with inclisirian. Furthermore, in all trials, inclisirian also significantly reduced levels of total cholesterol, apolipoprotein B, triglycerides, and lipoprotein (a), thereby reducing many cardiovascular risk factors [97]. Another cardiovascular outcome trial, currently underway, called ORION-4 (NCT03705234) [98], recruited 15,000 patients with ASCVD treated with inclisirian (300 mg) administered as a SC injection at randomization, (3 months and then every 6 months) or placebo aimed to evaluate any reductions in coronary heart disease (CHD) death; these were myocardial infarction, fatal or non-fatal ischemic stroke, or urgent coronary revascularization procedures [98]. Inhibitors of PCSK9 have been approved for the treatment of atherosclerotic cardiovascular diseases associated with hypercholesterolemia, however, a central role on immune tolerance in oncology has recently been investigated [99]. Mechanistically, PCSK9 inhibits the recycling of major histocompatibility complex type I (MHCI) to the cell surface by promoting MHC I degradation (Figure 3) [100]. The inhibition of PCSK9 increases the expression of MHC I on the tumor cell surface, promoting intratumoral infiltration of cytotoxic lymphocytes [101]. To be more detailed, PCSK9 directly interacts with amyloid precursor-like protein 2 (APLP2) which literally bridges MHC-I towards lysosomal degradation [102]. All of this blocks MHC-I recycling by inducing peripheral immune tolerance. The same mechanism of promotion of lysosomal degradation by MHC-1 occurs for CD81, CD36, and the LDL receptor [103]. Consequently, the use of PCSK9i can didactically create peripheral immune tolerance against tumor cells by optimizing recognition by T lymphocytes [104]. Furthermore, the reduction of plasma levels of total cholesterol would lead to the inhibition of the ACAT-1 enzyme (deputy for cholesterol esterification) with further enhancement of the antitumor immune response [105]. These data reveal that PCSK9 may be a crucial regulator for cancer immunotherapy. Inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase are the most important and studied pharmacological treatments aimed to decrease total cholesterol and LDL [106]. For every 38.6 mg/dl reduction in LDL-C, ASCVD events are reduced by 21% after 1 year of treatment with moderate- or high-intensity statin therapy. Recent RCTs have demonstrated that statin therapy combined with either ezetimibe or PCSK9 inhibitors reduces ASCVD events in high-risk populations [107]. As described in introduction, treatment with ICIs reduce cancer-induced immune tolerance, providing significant improvements in survival and prognosis of cancer patients at different stages of the disease [108]. PCSK9 is a new key player of cancer immune tolerance [109,110,111]. Recently, it was demonstrated that PCSK9 regulates proliferation and apoptosis in human cancer cells [112]. For example, in neuroglioma and NSCLC, knockdown of PCSK9 gene activates cancer apoptosis through caspase-3 and XIAP/p-Akt pathways [113]. Another preclinical study in melanoma-bearing mice concluded that PCSK9 gene silencing significantly increases the response to ICIs [114,115]. PCSK9 is also expressed and released by cancer cells [116,117]. It was also reported that lowering blood cholesterol levels could boost cancer immunotherapy based on adoptive T cells [118]. Cholesterol regulates the recycling of MHC I in cell membrane [119]. PCSK9 blocking agents reduce systemic levels of total cholesterol and LDL, affecting the risk of atherosclerosis but also reducing the supply of cholesterol by cancer cells [120,121] through the enhancement of apolipoprotein E receptor, CD36, β-secretase 1, and others [122,123]. A study on colon cancer [124] reveals that PCSK9 expression is upregulated in tumor cells compared with non-tumor cells and correlates with the degree of tumor invasiveness. Downregulation of the PCSK9 gene reduces colonic epithelial-to-mesenchymal transition, n-cadherin, and type 9 metalloproteases via the PI3K/AKT pathway. Furthermore, PCSK9 regulates the polarization of colonic peritumor macrophages, resulting in a pro-inflammatory and pro-metastatic phenotype [124]. PCSK9 blocking agents could therefore be studied in patients with metastatic colon cancer. An association study of 14 lobular or ductal breast cancer patients found that systemic levels of PCSK9 are significantly higher in the advanced stage (stage III) than in patients with less aggressive disease stages and in benign lesions (p < 0.05). However, this study involved only a few dozen patients, so studies on a larger cohort of breast cancer patients should be conducted soon [125]. A study of colon-bearing mice treated with PD-1 blocking agents found that treatment with immunotherapy increased tissue expression and systemic levels of PCSK9, CD36, and TGF-β [126]. Combination of PCSK9 and PD-1 blocking agents in these models significantly increased the antitumor efficacy with strong synergism compared to monotherapies [126]. The use of PCSK9 blocking agents increased the levels of pro-inflammatory and immune-stimulant cytokines, of intra-tumoral CD3+CD8+ lymphocytes, reducing CD4+, FOXP3+, CD25+ lymphocyte density. In liver cancer models, researchers [127] demonstrated that PCSK9 is involved in resistance to VEGFR, PDGFR, and RAF kinases inhibitor sorafenib by direct inhibition of phosphatase and tensin homolog (PTEN) and consequent upregulation of the AKT pathway. In another study in colorectal cancel model, [128,129] authors founded that PCSK9 induces oncogenesis in APC/KRAS mutant models of colon cancer and that systemic PCSK9 levels correlate with reduced survival in this patient cohort. PCSK9 blocking agents, especially when combined with statins, inhibit the neoplastic growth of APC/KRAS mutant colon cancer xenograft models by suppressing the KRAS/MEK/ERK pathway. Thus, the investigators conclude that PCSK9 inhibition may be a valid adjuvant therapy for APC/KRAS mutant colorectal cancer by suppression of the KRAS/MEK/ERK oncogenic pathway. A recent review [130] summarizes that PCSK9 blocking agents are able to reduce prostate, lung, colon, and glioblastoma cancer cell growth through the induction of apoptosis, pyroptosis, and necrosis. Moreover, PCSK9 gene silencing reduces liver metastasis in melanoma-bearing mice through induction of intra-tumoral CD3+CD8+ lymphocytes levels [131]. In another recent meta-analysis, patients with PCSK9 loss of function mutation have a lower odds of prostate cancer compared to non-mutant PCSK9 subjects [132]. Another study on ovarian cancer models [133], evidenced that PCSK9 is also upregulated in ovarian cancer cells and correlated with tumor invasiveness by direct stimulation of ERK/MEK pathways. The use of PCSK9 blocking monoclonal antibodies or SiRNAs suppresses the growth of ovarian cancer cells by reducing AKT phosphorylation by reducing endogenous lipogenesis in these cells [133]. A clinical study of non-small cell lung cancer patients has shown that low systemic levels of PCSK9 (<95 ng/mL) predict better responsiveness to the Programmed Death-1 (PD-1) Inhibitor Nivolumab and have better overall survival than patients with higher blood levels (>120 ng/mL) [134]. In a recent biochemical study [135], an aptamer PL1 and Pcsk9 siRNA were able to potentiate anti PD-1/PD-L1 therapies in human colorectal cancer cells through enhancement of IFN-γ and Granzyme B expression. A preclinical research study concluded that PCSK9 inhibition is able to increase major histocompatibility protein class I (MHC-I) membrane density on different cancer cells through the inhibition MHC-I lysosomal degradation, thereby increasing the concentration of intratumoral cytotoxic CD3+ CD8+ lymphocytes [136]. Immune-related adverse events (irAEs) seen in cancer patients treated with ICIs includes myocarditis, myositis, myasthenia gravis–like syndrome, endocrinophaties, and visceral obesity [137,138]. Moreover, immune-related atherosclerotic vascular events (irAVEs) are also still associated with ICIs therapy in preclinical and clinical trials. In a recently published retrospective study, ICIs therapies increased atherosclerotic plaques volume through the involvement of immune-related inflammation pathways. Other ongoing prospective studies (NCT04586894, NCT03709771, NCT04115410) are aimed to demonstrate if ICIs therapies could increase AVEs events and potential mechanisms involved [139,140,141]. However, in cardio-oncology, the reduction of several cardiovascular and cancer risk factors should be strictly considered. Among the best known cardio-oncological risk factors, we have the reduction of hyperglycemia, hypercholesterolemia, especially OX-LDL, triglycerides, homocysteine, insulin levels, hs-CRP, visceral fat, and smoking. Based on this, patients should be encouraged to follow a diet that complies with the European anti-cancer code and the WCRF directives, a non-sedentary lifestyle where possible (which is difficult especially in metastatic patients), and avoid smoking. However, PCSK9i-based drugs could be key cardioprotective strategies to reduce ASCVD in cancer patients treated with ICIs. Based on this, it is conceivable that a target population that could benefit from PCSK9 therapy are cancer patients treated with ICIs therapies, especially those with confirmed ASCVD. To this end, therefore, the benefits would be multiple: 1. enhancement of immunotherapy efficacy; 2. inhibition of mechanisms of resistance to apoptosis; 3. reduction of the risk of destabilization of stenotic plaque; 4. reduction of the risk of atherosclerosis induced by ICIs. To date, no studies have evaluated if PCSK9 blocking agents could reduce ASCVD in cancer patients treated with ICIs. A potential clinical trial could therefore include patients treated with ICIs in monotherapy or in combination with, for example, Ipilimumab, Nivolumab, Pembrolizumab, Durvalumab, Avelumab, or Atezolizumab. Inhibition of PCSK9 has emerged as a novel therapy to treat hypercholesterolemia and related cardiovascular diseases. Recent preclinical and clinical evidence supports the anticancer and immune-stimulating properties of PCSK9 inhibition therapy through the inhibition of peritumoral peripheral immune tolerance and reduction of cytokines involved in cancer cell survival. Cancer patients treated with ICIs in monotherapy or in an association therapy regimen have a three times greater risk of developing atherosclerotic plaques than patients not treated with ICIs and represent a population category that requires close pharmacological monitoring of atherosclerotic risk factors, including systemic inflammation, LDL, and OX-LDL levels. Cancer patients who develop ICIs-related ASCVD could benefit from PCSK9 inhibition therapy in order to reduce atherosclerotic events, cardiovascular mortality, and improve overall survival. Therefore, cardioprotective properties of PCSK9 inhibitors should be urgently explored in randomized clinical trials in patients with cancer at high risk of ASCVD.

PMC10000233

Chiara Benvenuti,Paola Tiberio,Mariangela Gaudio,Flavia Jacobs,Giuseppe Saltalamacchia,Sebastiano Pindilli,Alberto Zambelli,Armando Santoro,Rita De Sanctis

Potential Role of Circulating miRNAs for Breast Cancer Management in the Neoadjuvant Setting: A Road to Pave

23-02-2023

microRNAs,circulating miRNAs,breast cancer,neoadjuvant chemotherapy,pathological complete response

Simple Summary In breast cancer management, neoadjuvant chemotherapy is well established as therapeutic choice for selected high-risk early or locally advanced breast cancer. However, besides there being few clinical genomic classifiers, there is no technology that can predict for certain whether breast cancer patients will benefit from neoadjuvant chemotherapy in terms of pathological complete response and disease-free survival. The analysis of miRNAs from biological fluids at the beginning of therapy is simple and may aid in the identification of patients who will receive the greatest benefit. On the other hand, monitoring circulating miRNA levels during treatment could allow the early identification of patients who will not benefit from it (avoiding unnecessary treatments and related side effects). Therefore, there is an urgent clinical need for non-invasive biomarkers in the neoadjuvant setting, and circulating miRNAs could theoretically meet this need, but there is still a long way to go until their use in clinical practice can be established. Abstract Recently, circulating microRNAs (miRNAs) have emerged as potential non-invasive biomarkers for breast cancer (BC) management. In the context of BC patients undergoing neoadjuvant chemotherapy (NAC), the possibility of obtaining repeated, non-invasive biological samples from patients before, during, and after treatment is incredibly convenient and provides the opportunity to investigate circulating miRNAs as diagnostic, predictive, and prognostic tools. The present review aims to summarize major findings in this setting, thus highlighting their potential applicability in daily clinical practice and their possible limitations. In all the contexts (diagnostic, predictive, and prognostic), circulating miR-21-5p and miR-34a-5p have emerged as the most promising non-invasive biomarkers for BC patients undergoing NAC. Specifically, their high baseline level could discriminate between BC patients and healthy controls. On the other hand, in predictive and prognostic investigations, low circulating miR-21-5p and miR-34a-5p levels may identify patients with better outcomes, in terms of both treatment response and invasive disease-free survival. However, the findings in this field have been very heterogeneous. Indeed, pre-analytical and analytical variables, as well as factors related to patients, may explain the inconsistency among different study results. Thus, further clinical trials, with more precise patient inclusion criteria and more standardized methodological approaches, are definitely needed to better define the potential role of these promising non-invasive biomarkers.

Potential Role of Circulating miRNAs for Breast Cancer Management in the Neoadjuvant Setting: A Road to Pave In breast cancer management, neoadjuvant chemotherapy is well established as therapeutic choice for selected high-risk early or locally advanced breast cancer. However, besides there being few clinical genomic classifiers, there is no technology that can predict for certain whether breast cancer patients will benefit from neoadjuvant chemotherapy in terms of pathological complete response and disease-free survival. The analysis of miRNAs from biological fluids at the beginning of therapy is simple and may aid in the identification of patients who will receive the greatest benefit. On the other hand, monitoring circulating miRNA levels during treatment could allow the early identification of patients who will not benefit from it (avoiding unnecessary treatments and related side effects). Therefore, there is an urgent clinical need for non-invasive biomarkers in the neoadjuvant setting, and circulating miRNAs could theoretically meet this need, but there is still a long way to go until their use in clinical practice can be established. Recently, circulating microRNAs (miRNAs) have emerged as potential non-invasive biomarkers for breast cancer (BC) management. In the context of BC patients undergoing neoadjuvant chemotherapy (NAC), the possibility of obtaining repeated, non-invasive biological samples from patients before, during, and after treatment is incredibly convenient and provides the opportunity to investigate circulating miRNAs as diagnostic, predictive, and prognostic tools. The present review aims to summarize major findings in this setting, thus highlighting their potential applicability in daily clinical practice and their possible limitations. In all the contexts (diagnostic, predictive, and prognostic), circulating miR-21-5p and miR-34a-5p have emerged as the most promising non-invasive biomarkers for BC patients undergoing NAC. Specifically, their high baseline level could discriminate between BC patients and healthy controls. On the other hand, in predictive and prognostic investigations, low circulating miR-21-5p and miR-34a-5p levels may identify patients with better outcomes, in terms of both treatment response and invasive disease-free survival. However, the findings in this field have been very heterogeneous. Indeed, pre-analytical and analytical variables, as well as factors related to patients, may explain the inconsistency among different study results. Thus, further clinical trials, with more precise patient inclusion criteria and more standardized methodological approaches, are definitely needed to better define the potential role of these promising non-invasive biomarkers. Breast cancer (BC) is the most prevalent neoplasm and the leading cause of cancer-related mortality among women globally, although improvements in early diagnostic procedures and advances in treatments have contributed to decreasing the mortality rate over time [1,2,3]. Pathological variables such as tumor grade (G), TNM stage, proliferation index (Ki67), hormone receptor (HR) status, human epidermal growth factor receptor 2 (HER2) expression, and molecular subtype are well-known features that strongly influence patients’ prognoses. Together with clinical (e.g., menopausal status, age, and performance status) and biological (e.g., genomic signature) parameters, these pathological features are essential for the identification of the best-tailored treatment. In BC management, neoadjuvant chemotherapy (NAC) is a well-established therapeutic choice for selected high-risk early and locally advanced BC [4]. However, before starting treatment, the individual response to NAC and the subsequent long-term prognosis has been almost unpredictable to date. Likewise, an early identification of patients with poor tumor response could be crucial for avoiding unnecessary treatments and related side effects. For this reason, there is an urgent clinical need for non-invasive prognostic/predictive biomarkers in the neoadjuvant setting. A biomarker is considered prognostic if it provides information about outcomes regardless of treatment, reflecting the underlying intrinsic behavior of the disease, whereas a biomarker is considered predictive if treatment outcomes are different for biomarker-positive patients compared to biomarker-negative patients [5]. MicroRNAs (miRNAs) are highly conserved small noncoding RNAs formed by approximately 22 nucleotides which play key roles in gene regulatory networks. MiRNA biogenesis is a complex process involving several enzymes that first generate a primary miRNA, then a precursor miRNA (pre-miRNA) and finally a miRNA duplex composed of two strands: the 5p and the 3p (generating from the 5′ end and 3′ end of the pre-miRNA, respectively) [6]. One of the strands (the guide strand) is then loaded into the Argonaute (AGO) family of proteins, generating the miRNA-induced silencing complex (miRISC) that is able to bind transcripts, thus leading to mRNA degradation, translational suppression, or, as has been more recently discovered, an increase in the translation of target mRNA [6,7]. The other strand (the passenger strand) was initially thought to be degraded; however, it has been demonstrated that both strands can be incorporated into the miRISC complex depending on tissue or cell type [8]. The miRBase database (a searchable database of published miRNA sequences and annotations) provides information on stem-loop and mature miRNA sequences, including which is the guide strand (for which the miRBase alias is the miRNA name without any specification: miR) and which is the passenger ones (also named star strand; miRBase alias: miR*). Thus, the lack of specific information about the miRNA strand under investigation in a research paper means that the authors refer to the miRBase alias used for the guide strand. However, for a few miRNAs (e.g., miR-718, -4516, -422a, -484), it has not yet been defined in the miRBase which is the guide strand. The discovery that miRNAs are differently expressed in normal tissue and several tumor types, including BC [9], has generated a great deal of interest in the scientific community. To date, miRNA signatures from normal tissues, cancer tissues, and metastases have been used to classify different types of cancer and have been shown to represent potential biomarkers for diagnosis, prognosis, and treatment response [10,11]. In addition, different studies have shown that miRNAs can be released from cells and enter circulation [9,10,11,12,13,14]. In fact, it has been demonstrated that miRNAs can be found in circulation as a passive consequence of apoptotic and necrotic cell death, or due to active secretion through regulated cellular processes [6,12,13,14,15,16,17]. As a result, these molecules have also been found in several human bodily fluids (including blood, serum, plasma, urine, saliva, seminal fluid, and pleural effusion) [18], in a stable form protected from endogenous RNAses (i.e., in vesicles such as exosomes, microvesicles, and apoptotic bodies, or associated with proteins such as AGO2), thus making circulating miRNA levels well suited for non-invasive analysis in patient samples [19,20,21,22,23]. Indeed, independent studies have reported the feasibility of using circulating miRNAs as potential disease biomarkers of tumor clinical-pathological variables and patient clinical outcomes, including for BC [24,25,26]. In the context of NAC for early BC, the availability of non-invasive biological samples before, during, and after treatment is strongly convenient and provides the opportunity to investigate circulating miRNAs as prognostic and predictive biomarkers for daily clinical practice. This review aims to summarize the major findings regarding circulating miRNAs as diagnostic, predictive, and prognostic biomarkers in BC patients undergoing NAC, by highlighting their potential applicability in clinical practice and their possible limitations. In recent decades, several studies have thoroughly investigated circulating miRNAs as candidates for discriminating between BC patients and healthy controls, and possibly contributing to BC diagnosis in the case of incertitude. As reported in Table 1, the results of these case-control studies have been very heterogeneous and somewhat conflicting. The miRNAs most frequently cited in the literature for their potential diagnostic value are miR-21-5p and miR-155-5p (oncogene-like), and miR-let-7a-5p and miR-34a-5p (tumor suppressor-like). Strong and consistent data from multiple studies have shown that circulating miR-21-5p levels were significantly higher in the serum and plasma of BC patients compared to healthy women, regardless of subtypes [27,28,29,30,31,32]. In addition, Rodriguez-Martinez et al. reported significantly higher miR-21-5p levels in advanced BC patients compared to early BC, suggesting that circulating miR-21-5p may be a promising candidate not only for early tumor detection but also as a marker of tumor burden [32]. Similarly, the analyses of circulating miR-155-5p levels showed highly consistent results across studies, indicating a marked increase in serum miRNA levels in BC patients compared to healthy controls [30,33,34]. In contrast, for the tumor suppressor-like miRNAs miR-let-7a-5p and miR-34a-5p inconsistent results have been reported among different studies. In fact, Marques et al. reported markedly lower levels of miR-let-7a-5p in plasma and serum samples from BC patients compared to healthy controls [35]. However, in other studies significantly higher levels of miR-let-7a-5p were found in blood or serum of BC patients compared to healthy individuals [27,36]. Similarly, circulating miR-34a-5p levels were significantly higher in BC women compared to healthy volunteers in several studies [34,37,38] but, in the case-control series reported by Freres et al., the authors found opposing results [39]. As part of diagnostic evaluation, many studies then analyzed the possible correlation among circulating miRNA levels and some of the key BC clinical-pathological features (Figure 1). The strongest evidence concerns tumor grade, tumor size (T), lymph node involvement, clinical stage, molecular subtypes, HR, and HER2 expression. Several miRNAs have been associated with tumor grade in different studies. In particular, serum miR-125b-5p levels were significantly increased in high pathologic grade compared to low-grade tumors in a series of Luminal B patients [40]. Similarly, serum miR-155-5p, plasma miR-21-5p, and blood miR-195-5p levels were higher in G3 than in G2 tumors [33,41]. Serum levels of miR-21-3p, miR-10b-3p, miR-145-3p, and miR-let-7a-3p were shown to correlate directly with T stage: higher levels were associated with higher tumor size [42]. In addition, serum miR-21-5p [32] and miR-34a-5p [34] levels were significantly higher in T3-T4 stages compared to T1-T2 in BC patients. Similar results were observed by Heneghan et al., who reported a significant increase in the circulating levels of miR-195-5p in blood samples of BC patients with T3-T4 tumors compared to T1-T2 ones [36]. Moreover, Stevic et al. reported that the plasma levels of six miRNAs (-185-5p, -376a-3p, -382-5p, -410-3p, -433-3p, and -628-5p) were significantly associated with a higher tumor stage (i.e., T3-T4 versus T1-T2) in a cohort of HER2-positive BC patients [43]. Across different studies, several circulating miRNAs have been investigated in early BC patients in association with nodal involvement. Increased levels of circulating miR-210-3p [29] in plasma and miR-125b-5p [30] and miR-21-5p [31] in serum directly correlated with the positivity of locoregional nodes. However, several other miRNAs in plasma (-24-3p, -92a-3p, -143-3p, -146-5p, -185-5p, -193b-3p, and -484) [44] and serum (-155-5p [30,45], -182-5p [45], and -3200-3p [46]) have been found to be inversely related to nodal status, with lower levels found in patients with nodal involvement. Multiple circulating miRNAs were also assessed for their possible relationship with clinical stage. Among them, miR-21-5p [31,45,47], miR-155-5p [30,33,45,47], miR-182-5p [45], miR-373-3p [28,48], miR-221-3p [47], miR-125b-5p [30,33,48], and miR-10b-5p [30] had significantly higher levels in both the serum and plasma of BC patients in advanced clinical stages compared to earlier stages (i.e., II versus III, or I-II versus III-IV). Several circulating miRNAs were found to have different levels depending on the BC molecular subtype. Serum circulating miR-200c-3p levels were lower in triple-negative breast cancer (TNBC) patients compared to estrogen receptor (ER)- and progesterone receptor (PgR)-positive BC patients [49]. Conversely, Luminal-like tumors showed lower plasma miR-185-5p levels compared to TNBC [44]. Finally, Rodríguez-Martínez et al. reported definite level profiles of miRNA-222-3p in serum samples according to molecular subtype: it was decreased in Luminal A compared to basal-like and Luminal B tumors [32]. A large number of circulating miRNAs were investigated in relation to ER status. Indeed, high levels of circulating miR-221-3p in serum [50] and miR-185-5p [44] and miR-34a-5p [37] in plasma were associated with the negative expression of both ER and PgR. Correlation among circulating miRNAs and ER was also observed for miR-195-5p (in blood samples) [41], miR-let-7a-5p [47], and miR-145-5p [48] (in plasma samples), with higher levels directly related to higher ER expression. In addition to the aforementioned results, circulating miR-221-3p, miR-185-5p, and miR-34a-5p, and serum miR-21-5p were also found to be inversely correlated with ER positivity (i.e., low circulating levels were related to higher ER expression) in a study conducted by Al-Khanbashi M and colleagues [46]. Concerning PgR expression, serum miR-222-3p [32] and miR-10b-5p [48] showed an inverse association with PgR positivity, with higher levels mostly found in PgR-negative BC. Finally, regarding HER2 status, Zhang et al. identified three circulating miRNAs that were significantly associated with HER2 expression: serum levels of miR-375-3p, miR-718, and miR-4516 were lower in patients with HER2-negative tumors than in those with HER2-positive BC [40]. Moreover, higher plasmatic levels of miR-24-3p and miR-185-5p were associated with HER2-positive tumors [44]. Currently, there are no data on the correlation between circulating miRNA levels and low HER2 status (i.e., tumors expressing HER2 protein at the 1+ or 2+ immunohistochemistry level without HER2 gene amplification). For the prediction of clinical and pathological outcomes in an NAC setting, a plethora of circulating miRNAs have been investigated (Table 2). Here, we report the most extensively researched ones, underlining their potential clinical relevance. Among them, miR-21-5p has emerged as an independent predictor of response in several studies. In fact, low levels of circulating miR-21-5p before NAC (based on paclitaxel and doxorubicin) have been associated with a higher likelihood of favorable response to NAC in HR-positive BC patients [44]. Similarly, in a multicentric prospective study by McGuire and colleagues, which assessed a predefined panel of circulating miRNAs (selected on their reported relevance in BC) as a measure of NAC response in all BC subtypes, the whole-blood miR-21-5p levels of responders (defined as patients who had a complete response or >90% reduction in primary T) were considerably lower than those of non-responders (defined as patients with <90% reduction in primary T) in the HR-positive subtype (p = 0.048). On the contrary, in TNBC and HER2-positive BC, the response to NAC was not associated with circulating levels of miR-21-5p [41]. Likewise, similar results were reported in a Ukrainian retrospective study, which included 182 patients with stage II–III HR-positive/HER2-negative BC undergoing NAC with polychemotherapy with fluorouracil, doxorubicin, and cyclophosphamide or doxorubicin/cyclophosphamide. Patients with chemo-sensitive tumors (evaluation of NAC response performed every two cycles by mammography) showed changing in serum baseline levels of miR-21-5p lower than two-fold, while those with resistant tumors had a change above three-fold [45]. In the same study, serum miR-205-5p levels increased more than four-fold in chemotherapy-sensitive patients with the HR-positive/HER2-negative subtype and decreased lower than 2.5-fold in patients with a poorer response [45]. In contrast, another observational study with a similar cohort of patients (68 luminal A stage II-III BC patients for the discovery group, and 56 patients for the validation one) revealed that serum miR-205-5p levels in patients undergoing epirubicin- and paclitaxel-based NAC were higher in the resistant group (defined as no or minor reduction (≤30%)) compared to the sensitive group (defined as a decrease of >30% or no residual invasive cancer; p < 0.05) [53]. Besides miR-21-5p and miR-205-5p, miR-375-3p has also been shown to be a promising predictive biomarker whose basal levels are associated with response. Zhang et al. prospectively investigated the role of several miRNAs in 37 luminal B BC patients undergoing NAC with taxane- and/or anthracycline-based regimens, plus trastuzumab for HER2-positive cases. In luminal B HER2-negative patients, relatively low baseline serum levels of miR-375-3p were found to be associated with pathological complete response (pCR) (p = 0.043) and “comprehensive response”, defined as partial or complete response in clinical evaluation and loss > 30% or no residual invasive cancer in pathological evaluation (p = 0.023) [40]. Similar results were observed in the luminal A subtype in the abovementioned Ukrainian study, where decreased baseline levels predicted sensitivity to NAC with fluorouracil, doxorubicin, and cyclophosphamide, or doxorubicin and cyclophosphamide [45]. On the contrary, in the study by Wu and colleagues, when applying de novo sequencing to identify circulating miRNAs associated with BC clinical outcome, lower levels of miR-375-3p significantly correlated with not achieving pCR in HER2-positive BC patients receiving doxorubicin/cyclophosphamide treatment followed by carboplatin and nab-paclitaxel plus trastuzumab [54]. Multiple studies are concordant on the possible association between circulating miR-125b-5p and NAC response. In fact, circulating baseline levels of miR-125b-5p were found to be higher in non-responders (defined as stable or progressive disease) compared to responders (defined as partial or complete response; p = 0.008) in serum samples of 56 BC patients with invasive ductal carcinoma undergoing four to six cycles of NAC with 5-fluorouracil, epirubucin, and cyclophosphamide [33]. Similarly, a Chinese study involving 118 patients diagnosed with stage II/III BC and undergoing four to six cycles of NAC with docetaxel, epirubicine, and cyclophosphamide showed that significantly higher levels of serum miR125b-5p were present in patients who had stable or progressive disease compared to those that had reached partial or complete response [30]. The identification of biomarkers that can be repeatedly tested through non-invasive approaches could provide the possibility of analyzing real-time information on disease evolution during treatment [55] (Table 3). In this context, many studies have aimed to identify the predictive biomarkers of NAC response by monitoring miRNA dynamic changes on multiple blood samples collected during therapy. Again, one of the most investigated circulating miRNAs is miR-21-5p. A prospective clinical trial by Davey MG et al. evaluated the possible role of circulating miRNAs in decision making for NAC [56]. Blood miRNA levels were measured at diagnosis (Timepoint 1, or T1), and after two cycles of NAC (T2) in a total of 120 patients (59 luminal A, 21 luminal B, 15 HER2-positive, 25 TNBC). In the overall cohort, no circulating miRNAs were associated with response to NAC, but decreased or increased miR-21-5p levels trended to significance as associated with treatment response in specific BC subtypes. A second analysis evaluated levels of serum miRNAs during NAC in 83 HER2-positive early BC patients treated with four to six cycles of taxane-carboplatin plus trastuzumab [48]. Serum samples were collected before treatment, at the end of the second cycle, and at the end of therapy. The results showed that serum miR-21-5p levels in clinical responders were significantly lower at the end of the second cycle and at the end of therapy compared to baseline level (p < 0.001 for both), while there was no significant difference in non-responders. Dynamic circulating miR-21-5p levels were also investigated by Liu B. et al. in 118 patients affected by early HER2-negative BC [30] receiving four to six cycles of NAC with an association of docetaxel, epirubicin, and cyclophosphamide (TEC regimen). Blood samples were collected before treatment, at the end of the second cycle, and at the end of NAC. In line with the abovementioned results, the mean miR-21-5p level was lower during and after NAC than at baseline in responders (p = 0.016 for both), but not in non-responders. These results suggested that a decreased level of serum miR-21-5p detected after the second NAC cycle (compared to baseline) could be able to predict responder patients. Besides miR-21-5p, other circulating miRNAs have been investigated for their dynamic changes during NAC. The abovementioned prospective trial performed by Zhang Z. et al. evaluated circulating miRNAs in 37 luminal B early BC patients [40]. Blood samples were collected at baseline, and after two/four cycles of NAC. The circulating miR-210-3p levels during NAC were increased in non-responders, while the authors did not find a significant change in responders. In addition, significantly higher plasma miR-210-3p levels were observed in the non-pCR group than in the pCR group. Circulating levels of miR-210-3p associated with sensitivity to trastuzumab were evaluated in another trial involving 29 patients with HER2-positive early BC [29]. Patients received four cycles of taxanes followed by four cycles of anthracycline-based chemotherapy plus trastuzumab. Plasma samples were collected preoperatively and in the second postoperative week. The mean baseline level of plasma miR-210-3p was higher in samples from patients with residual disease than in the pCR group (p = 0.0359). In the above-mentioned study by Liu B. et al. analyzing the dynamic predictive role of circulating miR-125b-5p [30], a significant association was found between miRNA level and NAC response; miR-125b-5p level was higher at all timepoints in non-responders than in responders; however, treatment did not induce statistically significant changes in miRNA levels in either group. Moreover, the analysis by Zhang Z. et al. reported that, in the luminal B/HER2-positive cohort, the levels of circulating miR-125b-5p remained relatively stable from the baseline through the first two/four cycles of NAC, in patients with both complete and partial response [40]. Recently, Todorova K. et al. performed RNA sequencing on plasma samples collected from 20 BC patients before and after the first cycle of NAC (combination of doxorubicin with cyclophosphamide). The authors showed an increased level of circulating miR-34a-5p after the first NAC dose in patients that did not achieve pCR [57]. Similarly, another analysis reported that the miR-34a-5p levels were significantly increased after two/four cycles of NAC (compared to baseline level) in luminal B responder patients, regardless of HER2 status [40]. A further study investigated serum miR-34a-5p levels during NAC in 86 HER2-negative BC patients [38]. All patients received six cycles of chemotherapy (anthracycline plus taxane-based regimen). Serum samples were collected at baseline, at the end of the second cycle, and at the end of NAC. The authors found that changes in miR-34a-5p levels during NAC were significantly associated with the chemotherapeutic responses. At the end of the second cycle and at the end of NAC, almost all responders had decreased serum miR-34a-5p levels compared to baseline (p < 0.001 for both). Finally, Zhu W. et al. evaluated the dynamics of circulating miR-34a-5p during NAC (i.e., epirubicin-paclitaxel regimen) [26]. In the HER2-positive and TNBC cohorts, plasma miR-34a-5p levels were significantly decreased in chemo-insensitive patients after the first two cycles of NAC (p = 0.027 and p = 0.006, respectively), while they remained stable throughout the course of treatment in chemo-sensitive patients (no statistically significant changes). In an analysis by Davey G.M. et al. [56], the authors found that increased miR-let-7a-5p levels (from baseline to after the second cycle of NAC) identified patients who achieved partial or complete response in the luminal HER2-positive cohort (p = 0.049), whereas in the luminal cohort reduced miR-let-7a-5p levels predicted achieving pCR (p = 0.037). Circulating miR-let-7a-5p was also analyzed in the NEOCENT trial, a phase III translational study in which 63 patients (ER-rich) were randomized 1:1 to receive chemotherapy (anthracycline-based regimen, followed by docetaxel in the case of poor response after the first three cycles) or endocrine therapy (letrozole) [58]. Blood samples were collected at baseline, after 8 weeks, shortly before surgery, and 6-monthly for 2 years; miRNA markers were assessed from baseline to the end of treatment for both arms. An increase in circulating miR-let-7a-5p level was associated with objective radiological response in both arms, but it was statistically significant only in the chemotherapy arm (p = 0.008). MiRNAs have been extensively investigated for their promising role as prognostic biomarkers. However, despite several analyses suggesting them as potential prognostic tools in BC, the clinical application of these findings has yet to be verified [60]. A recent meta-analysis by Zhang et al. [61] analyzed 39 miRNAs with a prognostic value from 23 studies and found that 26 miRNAs were associated with survival outcomes. Although the study did not distinguish between tissue and circulating miRNAs, they identified miR-125b-5p, miR-21-5p, and miR-7-5p as the most frequently investigated ones with significant results. After reviewing the most recent literature, we identified seven circulating miRNAs significantly associated with prognosis in BC patients treated with NAC (Table 4). As for diagnosis and prediction, among prognostic miRNAs, circulating miR-21-5p is one of the most extensively studied with the most robust data [28,30,31,48,62]. There is consistent evidence that low circulating miR-21-5p levels are associated with better outcomes, while higher levels are associated with worse outcomes. A study by Muller et al. was the first to investigate the effects of NAC with trastuzumab and lapatinib on serum levels of circulating miR-21-5p, miR-210-3p, and miR-373-3p in 129 HER2-positive BC patients compared with a cohort of 19 healthy controls. One of the aims of the study was to evaluate whether specific miRNA levels were associated with prognosis. Of the miRNAs investigated, only increased levels of circulating miR-21-5p before (p = 0.0091) and after (p = 0.037) NAC were associated with a statistically significantly worse overall survival (OS) [28]. In a similar cohort of HER2-positive BC patients treated with NAC combined with trastuzumab, Liu et al. evaluated the association of circulating miR-21 levels with survival. The authors analyzed blood and serum samples from 83 HER2-positive BC patients during different phases of NAC (at baseline, after two cycles, and at the end of treatment). They demonstrated that changes in serum miR-21-5p levels were significantly associated with survival outcomes. In particular, patients in whom circulating miR-21-5p levels decreased from baseline to the end of the second cycle and to the end of NAC showed better OS and disease-free survival (DFS) than patients with increased levels of this miRNA [48]. Similarly, an earlier study on HER2-negative BC confirmed that among the miRNAs investigated, a decrease in serum miR-21-5p and miR-125b-5p levels during NAC were associated with better DFS [30]. MiR-21-5p has also been proven to be associated with survival outcomes in two other studies conducted on BC patients (any subtype) [31,62]. In a study by Wang et al., conducted on more than 300 patients, high serum miR-21-5p levels were found to be an independent poor prognostic factor for both recurrence (HR 2.9; 95% CI 1.420–8.325; p = 0.008) and DFS (HR 2.7; CI 1.038–7.273; p = 0.003). In addition, patients with high miRNA levels had shorter recurrence-free survival (RFS) and disease relapse-free survival (DRFS) than patients with lower levels [62]. Accordingly, in another study, elevated miR-21-5p levels in blood samples collected before and after NAC from 75 BC patients was found to be significantly associated with poor survival (p = 0.002) [31]. It has been shown that circulating miR-34a-5p levels were associated with better survival outcomes in two studies conducted in HER2-negative BC. Liu et al. analyzed the serum miRNA levels of 86 patients during different phases of NAC and showed that changes in miR-34a-5p expression during treatment were significantly associated with treatment response and DFS. Furthermore, decreased miR-34a-5p levels between the end of the second cycle and the end of NAC compared to baseline levels were associated with improved DFS (p < 0.001) [38]. In a more recent study, blood samples from 20 patients collected before and after the first cycle of NAC were evaluated to investigate whether circulating exosomal miRNAs could predict pCR. The authors showed that decreased levels of circulating exosomal miR-34a-5p were associated with better OS [57]. Checkhun et al. analyzed the expression levels of circulating miRNAs in serum samples from 182 patients with luminal A and B BC undergoing NAC. The authors found that low serum miR-375-3p levels were associated with a lower rate of 3-year DRFS in luminal B patients. In contrast, higher levels of circulating miR-182-5p correlated with a lower 3-year DRFS rate in luminal A BC [45]. Regarding the circulating miR-200 family in BC, a study evaluating serum miR-222-3p levels in a cohort of 65 HER2-positive patients receiving anti-HER2 NAC showed that low serum miRNA levels were associated with better DFS (p = 0.029) and OS (p = 0.0037). Moreover, the study aimed to assess the association between circulating miRNA levels and trastuzumab-related adverse events. For the first time, serum miR-222-3p levels were found to be an independent protective factor for cardiotoxicity (p < 0.05) and anemia (p = 0.013), although the mechanism of action remains to be elucidated [59]. Finally, in a study conducted by Al-Khanbashi and colleagues, tissue and serum samples were collected from 27 BC patients undergoing NAC at four different timepoints (baseline, after the first and fourth cycle of doxorubicin/cyclophosphamide treatment, after the fourth cycle of docetaxel treatment) to assess the correlation between miRNA expression and different endpoints, including survival outcomes. The authors demonstrated that patients with high serum miR-451-5p levels at diagnosis were associated with better DFS (p = 0.046) [46]. The presented results reflect the heterogeneity of available data on this subject. Indeed, these findings mainly come from single studies, involving different BC subtypes and/or patient characteristics (ethnicity, age, tumor stage, grade), different therapeutic regimens, and different definitions of responders and non-responders, thus limiting result comparison. In addition, accordingly to the literature, the majority of the discrepancies could be due to pre-analytical and analytical variables, as well as to patient-related factors that can generate artifacts, thus prejudicing the quantification of circulating miRNAs [20,63,64,65,66,67]. Specifically, one of the major critical issues is represented by the sample itself, including the type (plasma, serum, whole blood), collection (heparin, citrate, ethylenediaminetetraacetic acid, Paxgene tubes, sample handling), storage, processing (miRNA extraction method, timing of extraction), and blood cell contamination in sample preparation. In fact, it has been previously demonstrated that miRNAs contained in blood cells may have an influence on circulating miRNA analysis [68,69,70,71,72]; thus, different biological fluids show different circulating miRNA levels and whole blood may be strongly contaminated by blood cell miRNAs. In particular, the effect of haemolysis and the consequent release in the circulation of red cell miRNAs in impairing circulating miRNA analysis have been deeply investigated and proven [69,70,71,73]. In addition, blood cell miRNA contamination may also occur during sample collection, due to different centrifugation protocols used to separate plasma and serum from whole blood [74] and processing. For example, the correct choice of the anticoagulant for blood collection may, at least in part, prevent the lysis of red blood cells (e.g., citrate may trigger haemolysis) [75]. Another critical issue in circulating miRNA analysis that may, at least in part, explain the inconsistency among studies is the detection method used and the lack of a robust and standardized method for data normalization [64,65,66,67]. Since miRNA discovery, different quantification methods have been developed for miRNA analysis (e.g., quantitative real-time polymerase chain reaction (qRT-PCR), next-generation sequence (NGS), miRNA microarrays). Nowadays, qRT-PCR is the “gold standard” method since it allows a medium/high throughput and sensitive quantification of miRNAs. However, it can detect only annotated miRNAs, and the amplification step may affect miRNA quantification. NGS is a high-throughput method allowing the identification of both known and novel miRNAs with high sensitivity. However, it needs a larger quantity of each sample to be processed, it is the most expensive method, and it requires deep bioinformatics analyses. MiRNA microarrays are able to conduct high-throughput analysis on thousands of known and annotated miRNAs, but need to be constantly updated, thus impairing the comparison of results obtained with different platform versions. In addition, compared to NGS and qRT-PCR, miRNA microarrays show lower dynamic range and specificity [64,65,66,67,76]. Normalization strategy is a key issue for all platforms used for circulating miRNA quantification. Different endogenous miRNAs have been proposed as possible intrinsic controls for circulating miRNA analysis [19,72], and, among them, miR-16-5p is the most commonly used. However, it has to be considered that erythrocytes have high levels of miR-16-5p; thus, haemolysis may impair its use as an internal control [69,70,71,73]. Other normalization methods have been proposed (e.g., spiked-in synthetic RNAs, global normalization, quantile normalization); however, to date, a validated standardized normalization strategy has not yet been globally adopted. In addition, we have to consider that the normalization method may depend on the detection method used, thus further reducing the ability to compare results from different studies. Finally, we have to highlight that in some of the analyzed studies it is not specified which strand of miRNA (-3p or -5p) is under investigation and, therefore, the authors refer to the miRBase alias used for the guide strand. However, considering that it has been demonstrated that both strands can be incorporated into the miRISC complex depending on tissue or cell type and act in gene regulatory networks [8], specific information about the strand is fundamental for adequate result comparison across different studies. On the other hand, considering patient-related factors, as stated above, we should note that many results derived from single studies or from clinical studies enrolling a low number of patients, with no well-defined inclusion criteria [20,24,72], and with heterogeneous treatments. Moreover, we have to consider that circulating miRNA levels could be affected by various physiological conditions and/or comorbidities, such as obesity and diabetes, and these have to be taken into account during analysis [77,78]. In addition, different studies have shown that circulating miRNA levels may be affected by individual factors, such as age, race, drug assumption, smoking habits, diet, and physical activity [63,79,80,81,82,83,84,85]. Finally, we have to point out that BC is a heterogeneous disease, which may, at least in part, further explain the inconsistency of the results [86]. To date, besides few clinical genomic classifiers [87,88], there is no validated predictor associated with NAC benefit in terms of pCR and, ultimately, DRFS. Our review pointed out that circulating miR-21-5p and miR-34a-5p are the most promising non-invasive biomarkers for BC patients in the NAC setting and deserve further investigations. The collection of miRNAs from biological fluids at the beginning of NAC is simple and may aid in the identification of patients who will receive the greatest benefit. On the other hand, assessing miRNA levels during NAC is straightforward and faster than radiological assessment and may serve as a red warning in cases of poor response, particularly when it is clinically challenging to assess. However, due to the disagreement between studies, further clinical trials with more precise patient inclusion criteria and more standardized methodological approaches are definitely needed to better define the predictive/prognostic ability of these promising non-invasive biomarkers in anticipating the treatment response and outcome of BC patients undergoing NAC.

PMC10000238

Chengbo Zhang,Junmin Chen,Qian Wu,Bo Xu,Zunxi Huang

The Gut Microbiota of Young Asian Elephants with Different Milk-Containing Diets

02-03-2023

Asian elephant,intestinal microbiota,diet,elephant milk,goat milk

Simple Summary Insufficient maternal milk is one of the important reasons for the low survival rate of young Asian elephants. Finding the optimal break milk supplementation for young Asian elephants is a matter of urgency. In our study, we investigated the microbiomes of young Asian elephants on different milk-containing diets (elephant milk only, elephant milk–plant mixed feed, and goat milk–plant mixed feed). Our results suggested that goat milk is not suitable for young elephants, and yak milk may be an ideal source of supplemental milk for Asian elephants. Abstract Evaluating the association between milk-containing diets and the microbiomes of young Asian elephants could assist establishing optimal breast milk supplementation to improve offspring survival rates. The microbiomes of young Asian elephants on different milk-containing diets (elephant milk only, elephant milk–plant mixed feed, and goat milk–plant mixed feed) were investigated using high-throughput sequencing of 16S rRNA genes and phylogenetic analysis. Microbial diversity was lower in the elephant milk-only diet group, with a high abundance of Proteobacteria compared to the mixed-feed diet groups. Firmicutes and Bacteroidetes were dominant in all groups. Spirochaetae, Lachnospiraceae, and Rikenellaceae were abundant in the elephant milk–plant mixed-feed diet group, and Prevotellaceae was abundant in the goat milk–plant mixed-feed diet group. Membrane transport and cell motility metabolic pathways were significantly enriched in the elephant milk–plant mixed-feed diet group, whereas amino acid metabolism and signal transduction pathways were significantly enriched in the goat milk–plant mixed-feed diet group. The intestinal microbial community composition and associated functions varied significantly between diets. The results suggest that goat milk is not suitable for young elephants. Furthermore, we provide new research methods and directions regarding milk source evaluation to improve elephant survival, wellbeing, and conservation.

The Gut Microbiota of Young Asian Elephants with Different Milk-Containing Diets Insufficient maternal milk is one of the important reasons for the low survival rate of young Asian elephants. Finding the optimal break milk supplementation for young Asian elephants is a matter of urgency. In our study, we investigated the microbiomes of young Asian elephants on different milk-containing diets (elephant milk only, elephant milk–plant mixed feed, and goat milk–plant mixed feed). Our results suggested that goat milk is not suitable for young elephants, and yak milk may be an ideal source of supplemental milk for Asian elephants. Evaluating the association between milk-containing diets and the microbiomes of young Asian elephants could assist establishing optimal breast milk supplementation to improve offspring survival rates. The microbiomes of young Asian elephants on different milk-containing diets (elephant milk only, elephant milk–plant mixed feed, and goat milk–plant mixed feed) were investigated using high-throughput sequencing of 16S rRNA genes and phylogenetic analysis. Microbial diversity was lower in the elephant milk-only diet group, with a high abundance of Proteobacteria compared to the mixed-feed diet groups. Firmicutes and Bacteroidetes were dominant in all groups. Spirochaetae, Lachnospiraceae, and Rikenellaceae were abundant in the elephant milk–plant mixed-feed diet group, and Prevotellaceae was abundant in the goat milk–plant mixed-feed diet group. Membrane transport and cell motility metabolic pathways were significantly enriched in the elephant milk–plant mixed-feed diet group, whereas amino acid metabolism and signal transduction pathways were significantly enriched in the goat milk–plant mixed-feed diet group. The intestinal microbial community composition and associated functions varied significantly between diets. The results suggest that goat milk is not suitable for young elephants. Furthermore, we provide new research methods and directions regarding milk source evaluation to improve elephant survival, wellbeing, and conservation. The Asian elephant (Elephas maximus) is a large phytophagous mammal that is mainly found in the Xishuangbanna region of Yunnan Province, China, south of 24.6° north latitude, and in parts of south and southeast Asia [1]. The Asian elephant is a Class I protected wildlife species in China and is listed as endangered by the International Union for Conservation of Nature Red List of Threatened Species™ [2,3]. Furthermore, these elephants are in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [4]. There are only approximately 300 Asian elephants left in China [5]. Although the Asian elephant population has rebounded after years of effort, its survival rate still requires improvement. Approximately 25.6% of elephant calves in Myanmar reportedly die before they reach 5 years of age, with a quarter of these deaths attributed to insufficient maternal milk or the inability of the calves to receive the milk properly [6]. Similarly, in wild African elephant populations, an average of 19% of young elephants die before 5 years of age, with a proportion of these deaths attributed to maternal difficulties regarding meeting nursing needs [7]. During droughts, maternal elephants struggle to maintain milk production, when the metabolic demands of young male elephants are greater, making it difficult for maternal elephants to meet their needs. Thus, young male elephants are more likely to die [8]. A major reason for the high mortality rate of elephant calves in zoos, especially in Asia, is the refusal of mothers to nurse their young, resulting in the need for manual intervention to feed the young [9,10]. Inadequate maternal milk in Asian elephants results in the poor survival rate of young elephants, and currently, staff at the Xishuangbanna Asian Elephant Sanctuary are using goat milk to supplement the feeding of rescued infants and young elephants. The large number of microbial communities present in the gastrointestinal tract of animals constitute the microbiota, which contribute to host nutrient acquisition and immune regulation [11,12] and assist in maintaining host homeostasis in response to environmental changes [13,14,15]. Diet, especially early nutrition, influences the composition and metabolic activity of the gut microbial community and is a key factor in the growth and healthy development of newborn elephants [16,17]. Breastfeeding is considered an influential driver of the gut microbiota composition during infancy, potentially affecting the function thereof [18]. The gut microbiota early in life is associated with physiological development, and early gut microbiota is involved in a range of host biological processes, particularly immunity, cognitive neurodevelopment, metabolism, and infant health [19,20]. Early foods can promote the survival rate of infant and young elephants; therefore, it is vital to study the effects of different foods, especially different kinds of milk on the gut microbiota of infant and young elephants. In this study, the gut microbiota composition and function of young elephants fed an elephant milk-only diet, elephant milk–plant mixed-feed diet, and goat milk–plant mixed-feed diet were analyzed using 16S rRNA gene high-throughput sequencing technology. Although there have been studies regarding the use of non-breast milk dairy products for feeding endangered wildlife (e.g., Siberian tigers [16]), only few studies on the gut microbiota of Asian elephants on diets containing goat milk exist. To the best of our knowledge, this study is the first to describe the composition and function of the gut microbiota of young elephants fed a goat milk diet. In March 2019, we collected fresh feces from eight young Asian elephants with different milk-containing diets at Wild Elephant Valley in Xishuangbanna: three in the elephant milk diet-only group (BF1, BF2, and BF2; they are healthy, aged about 6 months, and can freely shuttle below the abdomen of adult female elephants); three in the elephant milk-plant mixed feeding group (BPM1, BPM2, and BPM3; they are healthy, more than one year old, and tall to the base of the forelegs of adult female elephants); and two in the goat milk–plant mixed feeding group (GPM1 and GPM2; they are healthy, more than three year old, and height slightly higher than the previous group). The detailed sampling method was as follows [21]: young elephants were accompanied by the breeder until defecation, samples were collected immediately from the center of fresh feces with sterile tweezers, placed in sterile centrifuge tubes, and stored in liquid nitrogen. Samples were transported in liquid nitrogen, and then stored at −80 °C until DNA extraction. Microbial genetic DNA was extracted from eight fecal samples using the EZNA® Soil DNA Kit (Omega, GA, USA) following the steps in the kit instructions. DNA quality and quantity were assessed using a 1% agarose gel and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with the primer pair 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) using an ABI GeneAmp 9700 PCR thermal cycler (Appliedbiosystems, Foster City, CA, USA). The PCR mix consisted of 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTP, 0.8 μL each of 5 μM forward and reverse primers, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA and ddH2O up to 20 μL. PCR amplification was performed in triplicate under the following conditions: 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 10 min. Purified amplicons were pooled in equimolar aliquots and then sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) to obtain paired-end reads [22]. Raw 16S rRNA gene sequencing reads were demultiplexed and quality-filtered using fastp version 0.20.0 [23] and then merged using FLASH version 1.2.7 [24]. Stringent criteria were established for quality. Three hundred-base pair reads were truncated at any site that received an average quality score <20 over a 50 bp sliding window. Truncated reads shorter than 50 bp and reads with ambiguous characters were discarded. Sequences required an overlap larger than 10 bp for assembly, and the maximum mismatch ratio of the overlap region was 0.2. Reads that could not be assembled were discarded. Samples were distinguished by barcodes and primers, and the sequence direction was adjusted accordingly. Exact barcode matching was required, and a mismatch of two nucleotides in primer matching was allowed. Operational taxonomic units (OTUs) with a 97% similarity cutoff [25,26] were clustered using UPARSE version 7.1 [25]; chimeric sequences were identified and removed. Taxon assignments for each representative OTU sequence were determined using RDP Classifier version 2.2 [27] with the 16S rRNA gene database (Silva v138) with a confidence threshold of 0.7. To investigate the similarity and difference relationship of microbial community structure among different milk-containing diet groups, sample-level clustering analysis was performed using UPGMA method based on the average Bray_curtis distance matrix among groups. Alpha diversity indices including Chao1 index, Shannon index, and Pielou index were calculated using software mothur (version 1.30.2, http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity, accessed on 23 April 2019), and difference tests between multiple groups were performed using Welch’s t-test. The Kruskal–Wallis H test was applied to detect species that exhibited differences in abundance in the microbial communities between groups. In addition, functional prediction results were obtained using PICRUSt2, and the difference significance was detected using the Kruskal–Wallis H test. At the family and genus levels, the samples were analyzed using hierarchical clustering based on the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis method (Figure 1), which indicated that the samples were clearly clustered into two groups: the elephant milk diet group (BF1, BF2, and BF2) and the milk–plant mixed-feed diet group (remaining samples). The milk–plant mixed-feed diet group was clearly further divided into two groups according to the type of supplemented milk: the elephant milk–plant mixed-feed diet group (BPM1, BPM2, and BPM3) and the goat milk–plant mixed-feed diet group (GPM1 and GPM2). These results exhibited that the gut microbial community composition of young elephants in the elephant milk-only diet group and that of young elephants in the milk–plant mixed-feed diet groups differed clearly. Moreover, the gut microbial community composition of young elephants in the elephant milk-only diet group and that of young elephants in the goat milk–plant mixed-feed diet group also differed significantly. An α-diversity test was performed to evaluate the differences in the gut microbial community between the three groups at the family level (Figure 2). Consequently, the richness index (Chao1) and diversity index (Shannon) were significantly different between the three groups (p < 0.05, Figure 2A,B). The richness and diversity indices of the milk–plant mixed-feed diet groups were significantly higher than those of the elephant milk-only diet group (p < 0.05), which was consistent with the richness of dietary diversity in the milk–plant mixed-feed diet groups. In addition, the Shannon and Pielou indices were significantly higher in the elephant milk–plant mixed-feed diet group than in the goat milk–plant mixed-feed diet group (p < 0.05, Figure 2B,C). These findings suggested that supplementation with elephant milk in young elephants resulted in a more diverse and homogeneous gut bacterial community than supplementation with goat milk, and supplementation with goat milk may lead to a highly dominant bacterial taxon in the gut environment of young elephants. Firmicutes and Bacteroidetes represented the dominant phyla in young elephant guts, which was consistent with the dominant phyla in the gut microbiota of adult Asian elephants (Figure 3) [28]. The young elephant intestinal microbiota in the elephant milk-only diet group (BF1, BF2, and BF3) contained a high abundance of Proteobacteria, averaging around approximately 17.3% (Figure 3). The elephant milk–plant mixed-feed diet group (BPM1, BPM2, and BPM3) had a higher abundance of Spirochaetae (approximately 8.8%), Fibrobacteria (approximately 3.8%), and Verrucomicrobia (approximately 3.6%) compared to the elephant milk-only diet group (Figure 3). The BPM1 group had a relatively higher intake of elephant milk and, correspondingly, higher abundance of Proteobacteria, while BPM2 and BPM3, which had lower intakes of elephant milk, had an extremely low abundance of Proteobacteria, indicating that elephant milk is closely related to Proteobacteria levels. The goat milk–plant mixed-feed diet group (GPM1 and GPM2) contained nearly no Proteobacteria, Spirochaetae, and Fibrobacteria (Figure 3), and the considerably low abundance of Proteobacteria indicated that elephant milk is closely related to the abundance of this bacterium. In addition, Synergistetes were abundant in the intestinal microbiota of young elephants in the goat milk–plant mixed-feed diet group compared to the other groups (Figure 3). At the family level, the intestinal bacteria of young elephants in the elephant milk-only diet group consisted mainly of Bacteroidaceae, Enterobacteriaceae, Ruminococcaceae, and Lachnospiraceae, accounting for >75% of intestinal bacteria (Figure 1A). The intestinal bacteria of young elephants in the elephant milk–plant mixed-feed diet group consisted mainly of Lachnospiraceae, Ruminococcaceae, Rikenellaceae, Spirochaetaceae, and Prevotellaceae, accounting for >70% of intestinal bacteria (Figure 1A). BPM1, who consumed a large amount of elephant milk, had an abundance of Enterobacteriaceae, suggesting that Enterobacteriaceae levels are closely related to the elephant milk consumed by young elephants. The intestinal bacteria of young elephants in the goat milk–plant mixed-feed diet group consisted mainly of Ruminococcaceae, Lachnospiraceae, Prevotellaceae, and Synergistaceae, accounting for approximately 60% of intestinal bacteria (Figure 1A). At the family level, differential microbiota analysis of young elephants (Figure 4) revealed that Rikenellaceae, Spirochaetaceae, Fibrobacteraceae, and Bacteroidales_UCG-001 were significantly enriched in the elephant milk–plant mixed-feed diet group (p < 0.05). These bacterial taxa belong to the lignocellulose-degrading bacterial phyla commonly encountered in the gastrointestinal tracts of animals, such as Bacteroidetes, Spirochaetes, and Fibrobacteres, suggesting that elephant milk enriches lignocellulose-digesting bacterial groups in the intestinal tract of young elephants, facilitating the transition from an elephant milk diet to a plant-based diet. Prevotellaceae, Synergistaceae, and Christensenellaceae were significantly enriched in the goat milk–plant mixed-feed diet group (p < 0.05). This indicated that there was a significant difference in the effect of elephant and goat milk supplementation in the diet on the intestinal microbiota of young elephants. Predictive analysis of the intestinal microbiota function in young elephants revealed differences in microbial community functions between different milk-containing diet groups (Figure 5). Carbohydrate and cofactor metabolism, vitamins, and glycan biosynthesis and metabolism were significantly more enriched in the elephant milk-only diet group compared to the mixed-feed diet group (p = 0.044). These function enrichments were beneficial to infant elephant growth and development. The enrichment of nucleotide metabolism (p = 0.044) and biosynthesis of other secondary metabolites (p = 0.044) were significantly higher in the goat milk–plant mixed-feed diet group compared to that of the elephant milk-only diet group, indicating that secondary metabolic pathways occurred during food digestion in the goat milk–plant mixed-feed diet group. The other amino acid metabolic (p = 0.030), transformation (p = 0.046), transcriptional (p = 0.020), replication and repair (p = 0.030), endocrine system (p = 0.044), and cell growth and death metabolic (p = 0.030) pathways were also significantly more enriched in the elephant milk–plant mixed-feed diet group than in the elephant milk-only diet group. The significant enrichment of these functions reflected strong metabolism and good growth and development of the young elephants in this group, indicating that the elephant milk–plant mixed-feed diet promoted the transition of young elephants from an elephant milk-based diet to a plant-based diet. In the elephant milk–plant mixed-feed diet group, enrichment of the membrane transport pathway (p = 0.044) and cell motility pathway (p = 0.044) was significantly higher in the elephant milk–plant mixed-feed diet group than in the goat milk–plant mixed-feed diet group. Meanwhile, the energy metabolic (p = 0.044), amino acid metabolic (p = 0.044), and signal transduction (p = 0.025) pathways were significantly more enriched in the goat milk–plant mixed-feed diet group than in the elephant milk–plant mixed-feed diet group. These results suggested that supplementation of the host’s diet with milk from different sources led to changes in the functional structure of the gut microbiota in Asian elephants. There were significant differences in the composition and function of the gut microbiota between the elephant milk diet groups and the goat milk diet group of young elephants (Figure 4 and Figure 5). Moreover, there was a close correlation between the host’s diet and their gut microbiota [29,30], where diet may have represented the main reason for these differences. Previous studies have shown significant differences in the nutrient composition of Asian elephant milk [6,10,31,32] compared to goat milk [33,34]. In the Asian elephant milk, the total solids (17.56–19.60%), protein (3.30–5.23%), and milk fat (7.70–8.30%) content were significantly higher than those in the goat milk (11.53–13.00%, 3.17–3.75%, and 3.95–4.25% for total solids, protein, and fat contents, respectively), while the water content (81.90–82.44%) was significantly lower than that of the goat milk (88.00%) (Table 1). The differences in the gut microbiota composition and function between the mixed-feed diet groups in this study may be mainly due to the differences in the nutrient composition and content between elephant milk and goat milk. Comparisons of the nutrient composition and content of different kinds of milk and Asian elephant milk have been conducted in previous studies [35,36,37,38]. The nutritional composition and content of yak milk [35,36] was similar to that of Asian elephant milk (Table 1). Water, total solids, protein, milk fat, ash, and lactose accounted for 83.74%, 16.60–18.52%, 4.68–5.41%, 6.72–8.18%, 0.72–1.19%, and 4.40–5.10% of yak milk, respectively (Table 1). Although there has been no study on the intestinal microbiota of Asian elephants supplemented with yak milk, the similarity between the composition and content of yak milk and Asian elephant milk suggests that yak milk may represent a viable choice of milk compared to goat milk for the supplementation of rescued young Asian elephants. Asian elephants are endangered wild animals, and there are few milk-drinking young elephants. Although the number of samples in each group in this study is insufficient, this is all the samples that could be collected in Xishuangbanna region at that time. Here, the diversity of gut microbial communities of young elephants differed significantly between different milk-based diet groups, reflecting the various effects that these diets may have on the growth and development of young elephants. The richness (Chao 1 index) and diversity (Shannon index) of human intestinal microbiota are crucial indicators of health [39]. Claesson et al. [40] reported that preterm infants with necrotizing colitis have a significantly lower diversity of fecal microbiota compared to those without the disease, and young children with lower gut microbiota diversity are at higher risk of developing allergic diseases later in life. Thus, the greater the gut microbiota richness and diversity, the more likely it is that the nutritional status and health of the host will be good. In this study, the elephant milk–plant mixed-feed diet group had higher intestinal microbiota diversity compared to the goat milk–plant mixed-feed diet group; therefore, although it is feasible to feed goat milk to young elephants, these results suggest that more suitable milk sources should be identified to serve as appropriate elephant milk supplementation for Asian elephants. Firmicutes and Bacteroidetes were the dominant phyla in all three groups, which is consistent with the results of Ilmberger et al. [41], and are also the dominant phyla in the adult Asian elephant gut microbiota [21]. Intestinal Firmicutes have many genes encoding fermentable dietary fiber proteins, which can also interact with the intestinal mucosa, contributing to the stability of the host’s internal environment [42]. Bacteroidetes are the main drivers of plant biomass degradation in Asian elephants [21,28,41]. These two bacterial taxa are indispensable for Asian elephants, as they assist plant digestion for energy acquisition. In the goat milk–plant mixed-feed diet group, the dominant phyla in the gut remained Firmicutes and Bacteroidetes, indicating that the use of goat milk to feed young Asian elephants could maintain the stability of the dominant phyla in the intestinal microbiota, allowing digestion and energy acquisition from food. The abundance of Spirochaetae in the intestinal microbiota of young Asian elephants was higher in the elephant milk–plant mixed-feed diet group compared with the goat milk–plant mixed-feed diet group. Spirochaetae are associated with the cell motility pathway, which is required by intestinal microbiota to actively contact their substrates and facilitate the biochemical reactions of the substrates [43,44]. This suggests that goat milk is not the most suitable supplement for elephant milk. In addition, Lachnospiraceae were more abundant in young Asian elephants in the elephant milk–plant mixed-feed diet group compared to in the goat milk–plant mixed-feed diet group, and are closely associated with host mucosal integrity, bile acid metabolism, and polysaccharide catabolism [45]. The low Lachnospiraceae abundance in the goat milk–plant mixed–feed diet group further suggested that goat milk may not be the best choice for feeding young Asian elephants. The abundance of Prevotellaceae and Rikenellaceae was higher in the mixed-feed diet groups than in the elephant milk-only diet group. A low abundance of Rikenellaceae and a high abundance of Prevotellaceae have been associated with obesity [46,47]. Therefore, the lower abundance of Rikenellaceae and higher abundance of Prevotellaceae in the goat milk–plant mixed-feed diet group compared to the elephant milk–plant mixed-feed diet group suggest that goat milk–plant mixed feeding may cause obesity in Asian elephants. In turn, this could lead to a potential risk of obesity-related diseases in Asian elephants. Synergistaceae encode multiple pathways that may be associated with the metabolism of diet-generated compounds [48], and these are predicted to be key factors in dietary detoxification in herbivores. In this study, Synergistaceae were significantly enriched in the goat milk–plant mixed-feed diet group, which was consistent with the significant enrichment of biosynthesis of other secondary metabolites in this group. This was likely due to the excess of secondary metabolism occurring during food digestion in this group. Meanwhile, the reason behind excess secondary metabolism, caused by the supplementation of goat milk or the presence of specific components in the foraged plants, requires further elucidation. Recent studies on the relationship between breast milk and the gut microbiota have revealed a correlation between milk composition and gut microbiota in infants [31], and that milk composition varies by mammalian species [49,50]. The composition and content of Asian elephant [5,10,31,32] and goat milk [33,34] differ significantly. Asian elephant milk is richer in nutrients than goat milk, which may have been the main reason for the difference in the composition and function of the gut microbiota between the elephant milk–plant mixed-feed diet group and the goat milk–plant mixed-feed diet group. Nutrient composition analysis and the content of yak milk [35,36] indicates that it is similar to Asian elephant milk. Furthermore, through the study of yak milk on retinoic acid-induced osteoporosis in mice, it was found that yak milk could improve bone quality and microstructure to promote bone health [51]. The study of Zhang Wei et al. showed that yak milk could improve endurance capacity and relieve fatigue [52]. It is reported that yak dairy products seem to be particularly rich in functional and bioactive ingredients, which may play a role in maintaining the health of nomadic peoples [53]. Nutritional composition analysis of yak milk and its advantages in other animals suggested that yak milk may be an ideal source of supplemental milk for Asian elephants, compared to goat milk. By studying the gut microbiome of Asian elephants on different milk-containing diets, it revealed the fact that the diet supplemented with goat milk diet seems not to be the most indicated to young elephants, and the composition and function of the gut microbiota of young elephants on a supplemented goat milk diet were also revealed for the first time, which were compared with those on an elephant milk diet only and an elephant milk–plant mixed-feed diet. This study presents a breakthrough in a new research area, the gut microbiome, regarding the serious problem of a low survival rate of infant and young elephants due to insufficient breast milk. Furthermore, we demonstrate the importance of finding a more suitable supplemental or alternative source of breast milk for Asian elephants. We believe that, in the future, with the help of wildlife gut microbiome analysis, the best supplemental or alternative sources of milk can be identified for other endangered wildlife infants and young to enhance the wellbeing of wildlife and relieve the threat to survival caused by insufficient breast milk.

PMC10000248

Xiaodong Gao,Xibao Wang,Xiaoyang Wu,Yongquan Shang,Xuesong Mei,Shengyang Zhou,Qinguo Wei,Guolei Sun,Yuehuan Dong,Weijia Cui,Honghai Zhang

Comparative Analyses of the Fecal Microbiome of Five Wild Black-Billed Capercaillie (Tetrao parvirostris) Flocks

03-03-2023

black-billed capercaillie (Tetrao parvirostris),fecal microbiome,function prediction,high-throughput sequencing

Simple Summary Black-billed capercaillie (Tetrao parvirostris) were listed as least concern (LC) class by the International Union for Conservation of Nature (IUCN). However, black-billed capercaillie was an endangered species in China and classified as first-class state protection animal (category I). Only a few studies have focused on the feeding habits of black-billed capercaillie and there is less research content at the molecular level, such as genomics, microbiome, and molecular markers. To improve our understanding of black-billed capercaillie, the fecal microbiome was characterized. This study provides reasonable scientific data for the understanding of black-billed capercaillie and may provide important insights for protecting this endangered species in China. Abstract Black-billed capercaillie (Tetrao parvirostris) was listed as a first-class state-protected animal because it was endangered in China (Category I). This study is the first to examine the diversity and composition of T. parvirostris gut microbiome in the wild. We collected fecal samples from five black-billed capercaillie flock roosting sites (each 20 km apart) in one day. Thirty fecal samples were sequenced with 16S rRNA gene amplicons on the Illumina HiSeq platform. This study is the first to analyze the fecal microbiome composition and diversity of black-billed capercaillie in the wild. At the phylum level, Camplyobacterota, Bacillota, Cyanobacteria, Actinomycetota, and Bacteroidota were the most abundant in the fecal microbiome of black-billed capercaillie. At the genus level, unidentified Chloroplast, Escherichia−Shigella, Faecalitalea, Bifidobacterium, and Halomonas were the dominant genera. Based on alpha and beta diversity analyses, we found no significant differences in the fecal microbiome between five flocks of black-billed capercaillie. Protein families: genetic information processing; protein families: signaling and cellular processes, carbohydrate metabolism; protein families: metabolism and energy metabolism are the main predicted functions of the black-billed capercaillie gut microbiome through the PICRUSt2 method. This study reveals the composition and structure of the fecal microbiome of the black-billed capercaillie under wild survival conditions, and this study provides scientific data for the comprehensive conservation of the black-billed capercaillie.

Comparative Analyses of the Fecal Microbiome of Five Wild Black-Billed Capercaillie (Tetrao parvirostris) Flocks Black-billed capercaillie (Tetrao parvirostris) were listed as least concern (LC) class by the International Union for Conservation of Nature (IUCN). However, black-billed capercaillie was an endangered species in China and classified as first-class state protection animal (category I). Only a few studies have focused on the feeding habits of black-billed capercaillie and there is less research content at the molecular level, such as genomics, microbiome, and molecular markers. To improve our understanding of black-billed capercaillie, the fecal microbiome was characterized. This study provides reasonable scientific data for the understanding of black-billed capercaillie and may provide important insights for protecting this endangered species in China. Black-billed capercaillie (Tetrao parvirostris) was listed as a first-class state-protected animal because it was endangered in China (Category I). This study is the first to examine the diversity and composition of T. parvirostris gut microbiome in the wild. We collected fecal samples from five black-billed capercaillie flock roosting sites (each 20 km apart) in one day. Thirty fecal samples were sequenced with 16S rRNA gene amplicons on the Illumina HiSeq platform. This study is the first to analyze the fecal microbiome composition and diversity of black-billed capercaillie in the wild. At the phylum level, Camplyobacterota, Bacillota, Cyanobacteria, Actinomycetota, and Bacteroidota were the most abundant in the fecal microbiome of black-billed capercaillie. At the genus level, unidentified Chloroplast, Escherichia−Shigella, Faecalitalea, Bifidobacterium, and Halomonas were the dominant genera. Based on alpha and beta diversity analyses, we found no significant differences in the fecal microbiome between five flocks of black-billed capercaillie. Protein families: genetic information processing; protein families: signaling and cellular processes, carbohydrate metabolism; protein families: metabolism and energy metabolism are the main predicted functions of the black-billed capercaillie gut microbiome through the PICRUSt2 method. This study reveals the composition and structure of the fecal microbiome of the black-billed capercaillie under wild survival conditions, and this study provides scientific data for the comprehensive conservation of the black-billed capercaillie. Black-billed capercaillie (Tetrao parvirostris) belong to the family Tetraonidae (distributed in the coniferous forest zone of eastern Asian Russia). However, black-billed capercaillie were listed as in the class of least concern by the International Union for Conservation of Nature (IUCN), and the species was endangered in China and classified as a first-class state-protected animal (category I). According to historical data, the quantity of black-billed capercaillie in northeast China declined by 35.25% between 1970 and 2018 [1]. Black-billed capercaillie live in flocks through the harsh winter, and their night habitat is dominated by arbor forest (larch, birch, poplar) [2]. Black-billed capercaillie can cope with food shortages using predictable browsing and low energy content foods [3]. During the overwintering period (from October to April of the following year), the bud of Scots pine (Pinus sylvestris), Channamu (Pinus koraiensis), Larch (Larix gmelinii), and Asian white birch (Betula platyphylla) are the main constituents of the diets of the black-billed capercaillie [4]. However, there have been few studies on the fecal microbiome of black-billed capercaillie. The gut microbiome can help the host achieve several important biological functions such as digestion [5], absorption [6], and the maintenance of the gut steady-state [7,8]. Bacteroidetes (phylum), Cerea bacillus, and Pseudomonas aeruginosa are significantly enriched in the fecal microbiome of geese, which can digest fiber to provide the energy for the host [6,9,10]. The gut microbiome of animals is affected by many factors, including the environment [11,12,13,14], phylogeny [15,16], flocks [17,18], and diet [19,20]. Many studies have focused on the impact of the environment and diet on the gastrointestinal microbiome of different species or the same species. For example, environmental factors affect the alpha and beta diversity of the gut microbial communities of white-crowned sparrows [21]. Noise pollution from urbanization has also significantly changed the gut microbiome and hormone secretion of birds [11]. Based on the cluster analysis and host phylogeny tree, Laviad-Shitrit found that the eco-evolutionary process termed phylosymbiosis may occur between wild waterbird species and their gut microbial community composition [16]. In a broader phylogenetic context, the diet and host phylogeny affect the gut microbial composition of non-passerine birds, with diet being the main influencing factor [15]. In addition, the flight ability can also affect the gut microbiome composition of birds [22]. There have been few studies of the flock factor, and most of them focused on captive environments [17,18]. Therefore, we raise two scientific questions. First, what is the fecal bacteria composition of black-billed capercaillie? Second, is there any difference in fecal bacteria composition between different wild black-billed capercaillie flocks? In this study, we performed 16S rRNA gene V3-V4 region high-throughput sequencing of the fecal microbiome of black-billed capercaillie. We aim ed to explore the fecal bacteria composition of black-billed capercaillie and the composition differences between five wild flocks. We want to provide a reasonable basis for understanding and protecting black-billed capercaillie. In this article, we first describe the materials and methods used to answer these questions (feces sample collection, bacterial DNA extraction and sequencing, sequence processing, and statistical analyses), then the results of the study (bacterial 16S rRNA gene data, composition of the fecal microbiome, comparison of microbiome for the studied flocks, and the prediction of the gut microbiota function), followed by their discussion and conclusions. The fecal samples of black-billed capercaillie were collected on 1 January 2017, in the Jinhe Forestry Bureau of Genhe City, Inner Mongolia. During the sampling period, the temperature was approximately −30 °C, which ensures the freshness of feces. Before sampling, we found the night-roosting habitat of five black-billed capercaillie flocks using transect method. The distance between night-roosting habitats was of 20 km, ensuring that there is no flock duplication. Six fresh feces samples were collected from the several available habitats. We used alcohol (99%) to wipe the scalpel, which was used to cut across the feces placed on the sterile gloves [5]. We only collected the core of the fecal matter to avoid disturbance from other birds and animals [23]. We used sterile gloves to pick up the feces and placed the fecal samples in a sterile tube. This sampling method is from our published article [24]. All samples were stored at −20 °C during the transit to Qufu Normal University, China. We stored all samples at −80 °C until sequencing. The cetyltrimethylammonium bromide (CTAB) method was used to extract the total DNA and the purity and concentration of DNA were then determined using agarose gel electrophoresis (1%) [25]. We used the polymerase chain reaction (PCR) primers (forward primer: CTACGGGNGGCWGCAG; reverse primer: GACTACHVGGGTATCTAATCC) [14,23,26] to amplify the V3–V4 hypervariable regions of bacterial 16S rRNA genes. The PCR amplification volume was 50 μL:5 μL of microbial DNA (5 ng/μL), 25 μL of 2 × Taq PCR Master Mix (0.1 U/μL); 18 μL of double-distilled water (ddH2O); 1 μL of forward primer and reverse primer (10 μM). The PCR amplification conditions were as follows: 1 min at 98 °C for pre-denaturation; 25 cycles of 30 s at 95 °C for denaturation; 30 s at 55 °C for annealing and 30 s at 72 °C for elongation, followed by 5 min at 72 °C for the final extension. The PCR products were detected by electrophoresis with 2% agarose gel. We used Qiagen Gel Extraction Kit (Qiagen, Dusseldorf, Germany) to purify the DNA target strip (400–450 bp). We used a TruSeq PCR-Free DNA Sample PreparationKit (Illumina, San Diego, CA, USA) to generate the sequencing libraries and following the manufacturer’s recommendations. We used Qubit 2.0 Fluorometer (ThermoScientific, Waltham, MA, USA) and an Agilent Bioanalyzer 2100 system to evaluate the sequencing library quality. Finally, we used Illumina HiSeq2500 PE250 (San Diego, CA, USA) to sequence the sequencing library. We used Fast Length Adjustment of Short reads (FLASH software; V 1.2.11) to cut the barcode and primer sequences and combined the pair-ended reads. We referred to the tags quality control process of Quantitative Insights into Microbial Ecology (QIIME) pipeline (Qiime; V1.7.0) pipeline and performed the following operations: tags interception and tags length filtering. Based on UCHIME algorithm [20], the Parallel-Meta Suite (PMS; V 3.7) [27] was used to eliminate chimeric sequences. Based on the sequence similarity at 97%, we performed PMS to cluster the sequences into operational taxonomic units (OTUs). We used vsearch [28] and SILVA ribosomal RNA databases (v 138.1) built into PMS (V 3.7) to annotate each sample, and we then obtained the set of operational taxonomic units (OTUs) relative abundance table and each taxon relative abundance tables for all data. Based on the OTU level, the rarefaction curve, species accumulation boxplot, non-metric multidimensional scaling analysis (NMDS), principal coordinate analysis (PCoA), and Tukey test of beta diversity were plotted using the ggplot2 package in R software (v 4.2.1). The Kruskal–Wallis test of alpha diversities (richness, chao1, and Good’s coverage indices) among five wild flocks was calculated using the vegan package of R software (v 4.2.1). Rarefaction curve and Good’s coverage index can be used to evaluate whether the sequencing results and depth content meet the requirements of subsequent experiments. The species accumulation boxplot can be used to evaluate whether the sequencing samples are sufficient for analysis. We used PCoA, NMDS, and the Tukey test of beta diversity to verify whether the bacterial compositions of five flocks were different. We used the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software [29] to predict the gut microbiome functions of black-billed capercaillie. In all figures, we represent the samples of black-billed capercaillie by “HZ” and the five flocks by “F” (F1, F2, F3, F4, and F5). We obtained 2,190,160 effective tags from 30 black-billed capercaillie samples, with an average of 73,005 (standard deviation, 9509) sequences per sample. The 30 rarefaction curves (Figure 1) and species accumulation boxplot (Figure 2) became gradually flattened, indicating that the sequencing depth and the number of samples were reasonable for the subsequent experimental analyses. The goods coverage index (Figure 3) of all data was higher than 99.6%, suggesting that all sequencing data effectively represent the fecal microbiome of black-billed capercaillie. At the phylum level, we used bacteria with >1% abundance in each sample to generate a relative abundance column cumulative plot. Camplyobacterota (33.44%) were the most abundant in the gut microbiome of black-billed capercaillie, and the other fecal dominant bacteria of gut microbiome were Bacillota (25.51%), Cyanobacteria (24.12%), Actinomycetota (14.92%), Bacteroidota (1.46%), and Acidobacteriota (0.26%) (Figure 4A). We listed OTUs with an average abundance greater than 1% in Table 1. Among them, six OTUs (OTU_3504_102, OTU_3327_4, OTU_3616_27, OTU_26443_5, OTU_3400_101, and OTU_3616_340) belong to Camplyobacterota, three (OTU_11392_1, OTU_2185_14, and OTU_2184_5) belong to Bacillota, two (OTU_25228_508 and OTU_25228_2257) to Cyanobacteria, and two OTUs (OTU_436_66 and OTU_13901) belong to Actinomycetota. At the genus level, we used the abundance of the top 10 genera to generate a relative abundance column cumulative plot. The Faecalitalea (12.60%, belong to Bacillota) were the most abundant in the gut microbiome of black-billed capercaillie. Escherichia−Shigella (10.67%, belong to Camplyobacterota), Halomonas (5.78%, belong to Camplyobacterota), Bifidobacterium (5.65%, belong to Actinomycetota), and Anaerobiospirillum (3.38%, belong to Camplyobacterota) were the other major genera in the gut microbiome of black-billed capercaillie (Figure 4B). The relative abundance of the top 10 bacteria genera occupied more than 38% in the fecal microbiome relative abundance of the black-billed capercaillie. The richness and chao1 indices can show the alpha diversity of the fecal microbiome, and the Kruskal–Wallis test examined the difference in the alpha diversity of the fecal microbiome of five black-billed capercaillie flocks. The boxplot showed that the indexes of richness (p = 0.42) and chao1 (p = 0.6) were not significantly different between the five wild flocks (Figure 3). Based on the unweighted and weighted unifrac distance, the beta diversity boxplot showed that the fecal microbiome composition of five wild flocks were not significantly different (Figure 5; Tukey test; p > 0.05). The NMDS (Figure 6A) and PCoA (Figure 6B) also indicated that five wild flocks of black-billed capercaillie were grouped together. The results suggest that the fecal microbiome diversity was not significantly different between the five wild flocks. Based on the functional prediction analysis, we predicted the 53 KEGG Level 2 categories. All samples displayed high abundances in KEGG Level2 categories of the following protein families: genetic information processing (15.97%); protein families: signaling and cellular processes (5.58%), carbohydrate metabolism (8.75%), amino acid metabolism (6.64%); protein families: metabolism (6.57%), energy metabolism (4.55%), metabolism of cofactors and vitamins (4.11%), membrane transport (1.35%), and translation (2.87%) (Figure S1). In this study, we first characterized the gut microbiome and potential functions of black-billed capercaillie using the 16S rRNA gene. The relative abundance of Camplyobacterota, Bacillota, Cyanobacteria, Actinomycetota, and Bacteroidota were the major phyla in gut microbiome of black-billed capercaillie. These results were basically consistent with the previous characterizations of birds’ gut microbiome, such as that of Oriental stork (Ciconia boyciana) [12], Zonotrichia leucophrys nuttalli [11], and crested ibis (Nipponia nippon) [30]. There was no significant difference in the alpha and beta diversity of the gut microbiome between the five wild flocks. Gut bacterial diversity was generally associated with the food diversity. There may not have been any appreciable variations in the gut microbiota makeup of the five wild flocks due to the same forest environment, analogous vegetation (similar diet), and severely cold temperatures. Camplyobacterota contains the most OTUs with an average abundance greater than 1% and was the most abundant phylum enriched in the gut microbiome of black-billed capercaillie. Camplyobacterota was a highly functionally diverse group of bacteria and related to the functional variability of the gut [31], and the most abundant phylum in fecal microbiome of many birds [15,22,31]. Camplyobacterota can maximize the function of the microbiome while reducing the diversity and mass [12]. This may be beneficial for the black-billed capercaillie to adapt to the buds of various trees (Scots pine, Channamu, Larch, and Asian white birch). The OTU_436_66 and OTU_24538_7 belong to Actinomycetota, which is associated with carbohydrate metabolism [32]. Bacillota contains three highly abundant OTUs: OTU_11392_1, OTU_2185_14, and OTU_2184_5. The food composition of black-billed capercaillie contains fiber and cellulose, and Bacillota contains a lot of fiber-decomposing bacteria and can decompose the fiber and cellulose to provide energy for the host [33,34]. The highly abundant OTU_25228_508 and OTU_25228_2257 belong to Cyanobacte-ria, which are enriched in the gut microbiomes of passerines [35]. Based on research on the gut microbiome of the American white ibis (Eudocimus albus), it was found that the relative abundance of Cyanobacteria is negatively correlated with urban land cover [36]. n our study, Cyanobacteria was the third-most dominant phylum in the fecal microbiome of the Black-billed capercaillie. We speculate that China’s policy of returning farmland to forests has effectively increased its habitat. This policy may prove to be beneficial to in-crease the wild Black-billed capercaillie population. OTU_11392_1 was annotated as Faecalitalea, which is isolated from the feces of chickens, and can produce butyric acid, lactic acid, and formic acid as the main metabolic end products [37]. These acids are important energy substances for the host and maintain host intestinal homeostasis [38,39]. In our study, the OTU_436_66 in the gut microbiome of black-billed capercaillie was annotated as Bifidobacterium. Complex carbohydrates are the substrate for Bifidobacterium, which are the beneficial bacteria in the host’s gut [40,41]. These two highly abundant bacteria contribute to the energy acquisition and maintenance of gut homeostasis in black-billed capercaillie. Two OTUs were annotated as Halomonas, with a sum of abundances of 3.76%. Halo-monas can balance the cell’s osmotic pressure and protect the structures of enzymes, DNA, and the cytomembrane that may improve cell motility, amino acid and carbohydrate metabolism, and the environmental adaptation of the black-billed capercaillie [42]. In our study, OTU_3504_102 was annotated as Escherichia−Shigella, which comprises the dominant bacteria in the gastrointestinal tract microbiomes of newly hatched chicks [43], Grey catbird (Dumetella carolinensis), and Swainson’s thrush (Catharus ustulatus) [44]. In general, Escherichia−Shigella is considered pathogenic bacteria in the animal gut microbiome. For example, Escherichia−Shigella is negatively correlated with the gut microbi-ome alpha diversity and weights of female steppe buzzards [45]. Ralstonia was present as a highly abundant OTU (OTU_26443_5), which uses volatile fatty acids as substrates [46]. Ralstonia acts as a pathogen or opportunistic pathogen in the host [47]. However, we did not conduct metagenomic sequencing analysis and Black-billed capercaillie health assessments. Therefore, we could not determine whether Escherichia−Shigella and Ralstonia are pathogenic bacteria in the gut microbiomes of the Black-billed capercaillie. The predictions of fecal microbiome functions for the Black-billed capercaillie were performed using the PICRUSt2 method [29]. Carbohydrate metabolism, protein families: metabolism, and energy metabolism were the functions found to be highly abundant based on functional prediction analysis. In an environment with a lack of food resources, the predicted fecal microbiome functions were found to be almost consistent among the five wild black-billed grouse flocks. These predicted functions could allow the black-billed grouse to obtain more energy for life activities in the buds of Scots pines (P. sylvestris), Channamu (P. koraiensis), larch (L. gmelinii), and Asian white birch (B. platyphylla). These findings will help us to better understand how the fecal microbiome and metabolism interact. However, as this was not a real metagenomic sequencing analysis, metagenomics and transcriptomics should be analyzed in the future. Through 16S rRNA gene sequencing analysis, we characterized the gut microbiome composition of the Black-billed capercaillie and analyzed the differences between flocks. Camplyobacterota, Bacillota, Cyanobacteria, Actinomycetota, and Bacteroidota were the major phyla in the fecal microbiome of the Black-billed capercaillie, and we found no no-table differences in the fecal microbiome compositions, diversity, and predictive functions among different wild flocks. Genetic information processing, signaling and cellular pro-cesses, carbohydrate metabolism, amino acid metabolism, metabolism, energy metabo-lism, and metabolism of cofactors and vitamins were the main functions in the gut micro-biomes of different wild flocks. We hypothesize that the reason for this is the extreme cold conditions and similar tree compositions (same diet) between habitats. These results will help researchers further understand the wild Black-billed capercaillie and contribute to the comprehensive conservation of this endangered species.

PMC10000249

Zirong Chen,Yan Fang,Weihong Jiang

Important Cells and Factors from Tumor Microenvironment Participated in Perineural Invasion

21-02-2023

perineural invasion (PNI),tumor microenvironment (TME),single-cell spatial transcriptomics (sc-ST)

Simple Summary Perineural invasion (PNI) as the fourth way for solid tumor metastasis and invasion has attracted a lot of attention, and axon growth and possible nerve “invasion” to tumors has also become an important component of PNI. We aim to summarize the current theories on the molecular mediators and pathogenesis of PNI, add the latest scientific research progress, and especially explore the use of single-cell spatial transcriptomics in this invasion way, to obtain a better understanding of PNI. Abstract Perineural invasion (PNI) as the fourth way for solid tumors metastasis and invasion has attracted a lot of attention, recent research reported a new point that PNI starts to include axon growth and possible nerve “invasion” to tumors as the component. More and more tumor–nerve crosstalk has been explored to explain the internal mechanism for tumor microenvironment (TME) of some types of tumors tends to observe nerve infiltration. As is well known, the interaction of tumor cells, peripheral blood vessels, extracellular matrix, other non-malignant cells, and signal molecules in TME plays a key role in the occurrence, development, and metastasis of cancer, as to the occurrence and development of PNI. We aim to summarize the current theories on the molecular mediators and pathogenesis of PNI, add the latest scientific research progress, and explore the use of single-cell spatial transcriptomics in this invasion way. A better understanding of PNI may help to understand tumor metastasis and recurrence and will be beneficial for improving staging strategies, new treatment methods, and even paradigm shifts in our treatment of patients.

Important Cells and Factors from Tumor Microenvironment Participated in Perineural Invasion Perineural invasion (PNI) as the fourth way for solid tumor metastasis and invasion has attracted a lot of attention, and axon growth and possible nerve “invasion” to tumors has also become an important component of PNI. We aim to summarize the current theories on the molecular mediators and pathogenesis of PNI, add the latest scientific research progress, and especially explore the use of single-cell spatial transcriptomics in this invasion way, to obtain a better understanding of PNI. Perineural invasion (PNI) as the fourth way for solid tumors metastasis and invasion has attracted a lot of attention, recent research reported a new point that PNI starts to include axon growth and possible nerve “invasion” to tumors as the component. More and more tumor–nerve crosstalk has been explored to explain the internal mechanism for tumor microenvironment (TME) of some types of tumors tends to observe nerve infiltration. As is well known, the interaction of tumor cells, peripheral blood vessels, extracellular matrix, other non-malignant cells, and signal molecules in TME plays a key role in the occurrence, development, and metastasis of cancer, as to the occurrence and development of PNI. We aim to summarize the current theories on the molecular mediators and pathogenesis of PNI, add the latest scientific research progress, and explore the use of single-cell spatial transcriptomics in this invasion way. A better understanding of PNI may help to understand tumor metastasis and recurrence and will be beneficial for improving staging strategies, new treatment methods, and even paradigm shifts in our treatment of patients. There are three well-known traditional ways for solid tumor metastasis and invasion: direct invasion of surrounding tissue, lymphatic spread, and haematogenic spread [1]. With the deepening of tumor research, the fourth invasion and metastasis mode has gradually attracted the attention of researchers: perineural invasion (PNI). In the process of tumorigenesis, nerves will be induced to sprout into the tumor during tumorigenesis, which in turn leads to cancer–nerve crosstalk, and assists tumor dissemination [1,2,3,4,5]. PNI was first reported by European scientists who described head and neck cancers that show a tendency to invade along nerves when moving to the intracranial fossa [6,7], which is usually associated with poor prognosis and may lead to severe pain, typically pain such as pancreatic adenocarcinoma (PAAD) [1,4,5,8]. Since then, PNI has become the key pathological feature of many malignant tumors, such as PAAD [8], prostate cancer (PRAD) [9,10], colorectal cancer [11], squamous cell carcinoma (SCC) of head and neck mucosa [4,5,12,13,14,15], adenoid cystic carcinoma (ACC) [2,16], etc. As a unique pathological entity, PNI can be observed without lymphatic or vascular invasion. Nerve sheath was composed of three connective tissue layers, which are the epineurium, the perineurium, and the endoneurium [1]. The endoneurium is the innermost layer formed mainly by axons and Schwann cells, and also contains mast cells, resident macrophages, fibroblasts, and blood vessels [17]. A laminated cylindrical layer derived from fibroblasts formed the perineurium to surround the endocardium, and they connected with each other through tight connections and gap connections to form a layer separated by collagen fibers (type IV) [18]. The epineurium is the outermost layer and includes a collagen tissue sheath, a plexus of blood vessels, lymphatic vessels, resident macrophages, fibroblasts, mast cells, and sometimes adipose tissue [19,20]. The definition of PNI was based on the nerve sheath: the tumor is close to the nerve and involves at least 33% of the circumference of adjacent nerve sheaths, or cancer cells exist in any of the three layers of adjacent nerve sheaths [1]. The incidence of PNI is different among different cancers, such as 70–100% in PAAD [8], 85–100% in PRAD [21], 20–57% in colorectal cancer [11], 25% to 80% in SCC of head and neck mucosa [4], and up to 80% in salivary adenoid cystic carcinoma (SACC) [22]. Cases reported of PNI were commonly associated with poor prognosis and decreased survival [1,11,23,24]. It has been reported that when PNI occurs in nerves with a diameter of >1 mm, there is an independent correlation between a higher local recurrence rate and PNI [25]. It has also been proved that the difference in the position of the PNI site relative to the tumor will also lead to the difference in patient prognosis [26]. Therefore, some researchers use the term “nerve invasion (NI)” instead of “perineural invasion (PNI)” to describe this tumor behavior. Given the importance of the invasion mode and the extremely serious clinical consequences, researches on this point are essential to control tumor dissemination and monitor disease progression. Although most research focused on the invasion of tumor cells to nerves, recent research reported that PNI starts to include axon growth and possible nerve “invasion” to tumors [27]. More studies have recognized PNI as the result of the interaction of tumor cells, nerve cells, and nerve microenvironment, and all parties work together to promote the invasion of tumors into the space around nerves [16]. The tumor microenvironment (TME) plays an important role in PNI. The interaction of tumor cells, peripheral blood vessels, extracellular matrix (ECM), other non-malignant cells [28,29], and signal molecules in TME play key roles in the occurrence, development, and metastasis of cancer [2,22,30,31,32,33]. The nerve barrier constitutes a defense line against tumor invasion. When tumor cells invade, peripheral nerve cells release neurotrophic factors, growth factors, chemokines, and cell-adhesion molecules to promote nerve repair and regeneration and maintain the stability of barrier function. At the same time, nerve cells such as Schwann cells and tumor cells interact with each other through autocrine and paracrine signals, enhancing the adhesion between cancer cells and nerves, and promoting tumor diffusion along nerves [34]. All kinds of cells in the microenvironment, including tumor-associated macrophages and fibroblasts, are activated by chemokines to produce peripheral inflammation, release neurotrophic factors, growth factors, and MMPs, and promote the growth, proliferation, and invasion of tumors, resulting in PNI [34,35,36]. As our understanding of the pathogenesis of PNI continues to progress, so will the definition of PNI [37,38]. The study of TME promotes the understanding of the existence and function of nerves in the TME and leads to the emergence of innovative anticancer therapy [39,40,41]. Based on the cellular components of TME, we aim to summarize the current theories on the molecular mediators and pathogenesis of PNI and add the latest scientific research progress. The application of some new research methods and technologies may not only find new molecular targets for PNI but also provide valuable insights for the development of PNI in the tumor microenvironment and neural microenvironment; to obtain a better understanding of PNI may help to understand tumor metastasis and recurrence and will be beneficial for improving staging strategies, new treatment methods, and even paradigm shifts in our treatment of patients. Infiltrating cells constituting the tumor matrix include fibroblasts, bone marrow-derived cells (BMDCs), tumor-associated monocytes and macrophages, endothelial cells, endothelial progenitor cells (EPC), myeloid-derived suppressor cells (MDSCs), neurons, T regulatory cells (Treg), and pericytes, and different components in neuroimmune axis and many other unrelated pedigrees, and extra-cellular components (cytokines, growth factors, hormones, extracellular matrix, etc. (see Figure 1 and Table 1) [41,42]. Several populations of BMDCs are recruited into TME and niche, where they can differentiate into tumor-promoting populations, such as EPC, MDSCs, and macrophage-like cells [3]. Factors derived by cells in this microenvironment promote the migration and proliferation of adjacent vascular cells, tumor cells, and neurons, and grow together with tumors [43,44]. Monocytes/macrophages are usually the most abundant component in the immune cell population of the TME, and affect tumor progression and metastasis through interaction with tumor cells (see Figure 2) [45]. The main source of tumor-related macrophages (TAMs) is circulating mononuclear cells, which were derived from myeloid progenitors in the bone marrow and differentiated into mononuclear cells under the mobilization of the chemokine (C-C motif) ligand 2 (CCL2)/chemokine (C-X-C motif) receptor 2 (CCR2) axis and then enter the peripheral inflammation site through blood or further differentiate into macrophages in primary tumors [29,41,46], and they actively migrate to the tumorigenesis site and rapidly differentiate into TAMs during tumorigenesis, inhibiting various T-cell reactions, maintaining normal tissue homeostasis in response to various systemic infections and injuries, and helping anti-tumor escape [41]. They showed a high degree of functional plasticity, and can rapidly adapt to the disturbance of the environment around them. TAMs were described into M1 and M2 subtypes according to their polarization state [47]. The M1 cell was activated by Th1 cytokine interferon-γ (IFN-γ), interleukin-12 (IL12), tumor necrosis factor (TNF), and microbial products to exert a tumor-killing effect, while the M2 cell was activated and differentiated by Th2 cytokines (such as IL4, IL5, IL10, IL13, colony-stimulating factor-1 (CSF1), transforming growth factor-1 (TFGβ1) and prostaglandin E2 (PGE2) to exert angiogenesis and immunosuppressive effect [41,43,45,48,49]. Anti-tumor M1 type was recruited at an early stage of tumor development, and the overexpression of the p50 subunit of nuclear factor kappaB (NFκB) in macrophages promoted the repolarization of M1 to M2 [42,50,51,52], gradually differentiated M1 into a tumor-promoting M2 type [53]. TAMs are generally considered to be M2 macrophages in the TME which, when exposed to hypoxia or lactate, secretes a variety of cytokines with metabolic functions, including IL6, TNF, CCL5, and CCL18 to enhance PNI [47]. TAMs express PD-L1 and can directly reduce the activation of T cells, then suppress the anticancer immune responses [42]. In addition, TAMs can promote tumor progression and invasion by up-regulating matrix metalloproteinases (MMPs) [54,55]. Carcinoma-produced MMP1 activates neuronal protease-activated receptor 1 (PAR1) and induces substance P (SP) release, activating carcinoma neurokinin 1 receptor (NK1R), which plays a pivotal role in tumor progression and PNI [42,56]. Macrophage-derived IL-1β induces non-neuronal cells to synthesize nerve growth factor (NGF), and activated macrophages can also release glial cell-derived neurotrophic factor (GDNF)-activated RET receptors to trigger tumor cell migration. Evidence from clinical and experimental studies shows that there is a close relationship between TAM density and cancer cell metastasis in various cancers [28]. In PAAD, inflammatory monocytes (IM) expressing CCR2 can be driven by CCL2 released by Schwann cells at the PNI site to recruit preferentially to the nerve site, then differentiated into macrophages and enhance NI by expressing cathepsin B-mediated process to degrade the protein of nerve bundle membrane [29,39]. Protein species degraded by cathepsin B contained laminin, fibronectin, and collagen IV [29], which are important parts of the protective nerve bundle membrane, and its function as a peripheral nerve protective barrier is weakened when it is destroyed [57]. In addition, it has been reported that endoneurial macrophages (EMφ) can transform into microglia/macrophage subsets involved in cell defense and peripheral nerve regeneration [30]; EMφ around the nerve invaded by cancer is recruited to the tumor front in response to colony-stimulating factor -1(CSF-1) secreted by the tumor, then activated by tumor, secretes a higher level of GDNF as a chemical attractant of cancer cells, which induces phosphorylation of the transmembrane helper receptor RET in PAAD cells and activation of MAPK and PI3K pathways in downstream cancer cells of the extracellular signal-regulated kinase (ERK), promotes the migration to nerves of cancer cells, and enhances the PNI [30]. In addition, macrophages also regenerate injured nerves by secreting VEGF, which was also used by Schwann cells to guide the growth and migration of nerves for tumor axonogenesis [58]. Cancer-associated fibroblasts (CAFs) are an important subpopulation of fibroblasts to support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and mediating tumor-enhancing inflammation and invasion in tumors (see Figure 3) [13,59]. The origin of CAFs in TME has not been clarified, while in PDAC and CCA, they are considered to be differentiated from stellate cells [34,60], and bone marrow-derived circulating progenitor cells of hematopoietic or mesenchymal lineage were also reported to derive from CAFs [61,62]. Currently, it is believed that local tissue fibroblasts are activated into CAFs, which account for a large part of the stromal cells in TME and up to 80% of the tumor volume in advanced squamous cell carcinoma of the head and neck (HNSCC) [13,63], due to the influence of tumor-derived paracrine factors and cytokines [64]. They are responsible for the synthesis, deposition, and remodeling of most of the ECM in the tumor matrix and are thought to be sources of growth factors that affect paracrine, which affect the growth of cancer cells and have been shown to provide key signals supporting tumor progression and allow a small population of cancer cells to escape treatment [65,66,67,68]. Such as transforming growth factor β (TGF-β) secreted by CAFs induced a fibroblastic response in the surrounding environment to synthesize collagen, stiffen the tissue, and destroy microvascular structure by extensive collagen cross-linking, then the mechanical stress of the connective tissue collapses the nearby blood vessels to promote hypoxia [69]. Under hypoxic conditions, interleukin-6(IL-6) produced by cancer cells induces the activation of Schwann cells and triggers the development of PNI [70], which is also accompanied by the activation of a series of hypoxia-associated signaling pathways. In addition, a dense structure formed by fibrosis around a cluster of cancer cells in an adenocarcinoma caused internal high and lead to the rupture of glandular structure, simultaneously, the high-density fibers formed a track that contributed to the movement of the cancer cells to overflow and diffuse quickly [64]. By the anisotropic E-cadherin/N-cadher in junction, the frontier CAFs exert the physical force to enable the synergistic invasion of CAFs and cancer cells through a dual mechanism: CAFs pull cancer cells away from the tumor facilitating the invasion of cancer cells, while cancer cells polarize its migration away from the tumor to enhance the diffusion of CAFs [71,72]. In addition, fibroblasts contribute to Schwann-cell-induced axonal growth and, after nerve injury, ephrin-B on fibroblasts activates EphB2 receptors on Schwann cells, and fibroblasts through the ephrin-B/EphB2 signaling pathway induce Schwann cells to migrate through bridges as dense groups or cords to guide axons through injury site [73]. At the same time, CAFs can regulate the N-cadherin/β-catenin pathway through the production/secretion of the axonal guidance molecule Slit2, affecting the neural remodeling related to glandular catheter adenocarcinoma [74]. On the other hand, CAFs promote cancer invasion by secreting factors that cause cancer cell activation and matrix remodeling. Studies have shown that CAFs exist at the PNI site and the produced inactive matrix metalloproteinase-2(MMP-2) is activated by membrane-type 1 matrix metalloproteinase (MT1-MMP) produced by tumor cells, which degrades extracellular collagen in the perineural niche and promotes the spread of cancer cells in the perineural space [68,75]. Schwann cells are the main cells of peripheral nerves, which wrap the single or multiple axons of peripheral nerves and promote signal transduction (see Figure 4). When it is damaged or invaded by tumor cells, the axonal injury may trigger the dedifferentiation and activation of SCs through various pathways [76]. The myelinated SCs are dedifferentiated into “repair SCs” (RSCs) [77] with a demyelinating phenotype. By producing a variety of neurotrophic factors and cell surface proteins, including GDNF, artemin and BDNF, p75 neurotrophic factor receptor (p75NTR), and N-cadherin, remodeling the matrix and releasing pro-inflammatory mediators to change the local signaling environment, the injured SCs can induce macrophages to synergistically remove myelin debris [77], and guide axonogenesis, participate in axon maintenance and post-injury repair, playing an important role in maintaining axon health and neuron survival, opening the way for subsequent nerve regeneration. In the TME, SCs are thought to drive PNI—cancer cells using normal SCs nerve repair procedures to promote PNI [78]. SCs can directly interact with cancer cells through nerve cell adhesion molecule 1 (NCAM1) to break the connection between cells in the tumor cell cluster, thereby dispersing them into single cells [79]. Contact between SCs and cancer cells induces cancer cell protrusion and guides cancer cells to migrate from the cluster to SCs, thereby promoting the invasion and migration of cancer cells along the nerve [79]. L1 cell adhesion molecule (L1CAM) is overexpressed in SCs adjacent to cancer cells and invaded nerves, which strongly induces cancer cells as a strong chemoattractant by activating MAP kinase signaling, and L1CAM also up-regulates the expression of metalloproteinase -2 (MMP2) and MMP9 in PDAC cells by activating STAT3 and facilitates matrix remodeling along the axon [80,81]. MMPs secreted by SCs, especially MMP2 and MMP9, enhance the degradation of ECM and provide loose channels for the movement of cancer cells [82]. Schwann cells have also been identified as an important source of TGFβ, which can activate SMAD signals that induce migration, invasiveness, and PNI in PAAD cells, increasing the invasiveness of cancer cells [83]. Studies have suggested that before cancer cells migrate to peripheral neurons, SCs migrate to pancreatic or colon cancer cells through the NGF-TrkA-p75NTR signaling pathway, but do not migrate to benign cells, so activated oncogenic SCs are likely to construct a pathway to cancer cells [84]. It has also been proposed in the literature that tumor cells activated SCs by c-Jun, while non-myelinating activated SCs form tumor-activated Schwann cell tracks (TASTs) as active scaffolds, and exert forces on cancer cells to enhance cancer mobility, promote cancer cell migration and invasion, a process similar to their reprogramming during nerve repair, leading to “neurogenesis” of precancerous cells and the periphery of tumor cells [85]. At the same time, SCs were demonstrated to be abundant in the surrounding stroma of the precancerous lesions of PAAD, possibly capable of recruiting immune cells at the PNI site, and macrophages recruited by SCs provided additional and persistent cytokine sources to further enhance neural invasion of tumor cells [20,77,84,86]. In a recent study, SCs activated by tumor produced prostaglandin E, polarized T cells to failure phenotype, leading to tumor-related immunosuppression, and play an important role in tumor–nerve crosstalk [87]. In addition, the differentiation of myoepithelial into SCs may be one of the mechanisms of PNI that occur in SACC2. The interaction between nerve and cancer cells leads to the relationship of mutual growth promotion via the action of neurotrophic factors (NTFs) from nerve and cancer cells, and the pro-invasive and proliferative characteristics of autonomic neurotransmitters from nerve fibers [86,88]. Pathological neurogenesis is not only the physiological basis of chronic pain produced by the tumors but also contributes to the PNI [86,89,90,91]. Sensory nerve fibers undergoing pathological sprouting in cancers has been reported, which was driven by NTFs, especially the nerve growth factor (NGF), released from tumors and their associated stromal cells [92]. The expression of NTFs by cancer cells and nerves at the same time implies that cancer cells can make use of the same repertoire of trophic signals as nerves do to develop themselves [88]. In addition, after partial peripheral nerves were injured, the sustained demyelination state of the peripheral nerve relieves the axon of growth inhibition and encouraged nerve sprouting [93]. In a cancer such as PAAD, increased neural density (neural sprouting) and size (neural hypertrophy) occur, compared to normal pancreas innervation [86,94,95,96]. The mutual trophic interaction changes neural distribution in a solid tumor, which makes it more conducive to the exchange of signals between cancer cells and nerves [88]. In another way, it could be explained as the pathological neural plasticity induced by tumor-derived factors [86,91,97]. These signals were usually transferred via chemical substances with chemoattractive attributes such as NTFs, neuropeptides, neurotransmitters, and so on, leading to the mutual attraction between tumor cells and nerves, and enhancing cancer cells’ chemoattraction and motility. In addition, neurogenesis can also be induced by the recruitment of neural progenitor cells from the central nervous system (CNS), especially from the subventricular zone (SVZ), traveling through the bloodstream attracted by tumor-derived factors [42]. They will colonize in a tumor, and differentiate into functional autonomic neurons to produce adrenergic neurons and release neurotransmitters, and stimulate the growth of the tumor, mainly producing adrenergic neurons and releasing neurotransmitters to enrich the TME [98]. It was also proposed that cancer stem cells can differentiate and acquire an autonomic neuron-like phenotype, which may be able to perform neuron-related functions to influence the progression of tumors [42]. For example, cancer stem cells of gastric cancer and colorectal cancer can differentiate into sympathetic neurons producing tyrosine hydroxylase (TH) and parasympathetic neurons producing vesicular Ach transporter [99]. Studies have shown that in the process of PNI, when PAAD cells are close to neurons and SCs of peripheral nerves, they are attracted by neural components of peripheral nerves and migrate to neurons [84]. Neurotransmitters derived from fibers such as glutamic acid, aminobutyric acid (γ-aminobutyric acid), NE, or Ach stimulate the survival, proliferation, and migration of tumor cells [35]. Neurotransmitters regulate the immune-promoting and anti-immune responses and affect the TME through this indirect mechanism [34]. Sympathetic and parasympathetic nerve fibers release norepinephrine (NE) and acetylcholine (ACh) in tumors and lymphatic organs, as well as other neuromodulators, to reduce the anti-tumor immune response [100,101]. The sympathetic nerve fibers in the tumor are related to the early stage of cancer, and the angiogenesis switch is triggered by adrenergic signals [102]. In the late stage of tumor development, parasympathetic nerve fibers help to stimulate the invasion and metastasis of cancer cells [34,103,104]. NE derived from sympathetic nerve fibers activates β2- adrenergic receptors and activates through PKA/STAT3, which leads to the expression of NGF, MMP2, and MMP9 in PAAD cells, enabling them to migrate and invade and induce PNI [42]. NE can also stimulate the production of IL-6 and activate macrophages in TME, which can promote cancer migration and nerve invasion by releasing GDNF [30]. Parasympathetic nerve fibers release Ach, which inhibits immune response through nicotine receptors, while sensory nerve fibers release substances P and CGRP to activate mast cells and blood vessels [42]. Catecholamine activates the immunosuppressive switch in the TME of lung cancer, causing M1 to M2 macrophages to re-polarize and aggregate M2 polarized macrophages and MDSCs. At the same time, it reduces anti-tumor dendritic cells (DC), which leads to the synthesis and release of IL-10, inhibits immune response, leads to the synthesis and release of VEGF, and promotes angiogenesis [105]. In addition, another β2 Adrenergic-Neurotrophin (NT) loop driven by catecholamine can up-regulate NTs to increase sympathetic innervation and local NE accumulation [106]. Peripheral nerve microenvironment includes neurons, Schwann cells, and microglia/macrophages, which secrete various factors to participate in nerve homeostasis, dendritic growth, and axon germination [30]. Neurotrophic factors (NTFs) are a kind of protein, which play an important role in the development, survival, and apoptosis of neurons. Its members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NRTN), neurotrophins-3(NT-3), NT-4, NT-5, Artemin, etc, which promotes the growth of neurons and PNI together with receptors [100,107]. There are two different membrane protein receptors of NTFs, namely the tyrosine kinase receptor Trk(TrkA, TrkB and TrkC), which bind neurotrophins with higher affinity and specificity, and p75 neurotrophic receptor (p75NTR), which binds all neurotrophins with lower affinity and specificity [108]. NTFs interact with the extracellular domains of these two receptors, and transmit the signals related to the survival and apoptosis of nerve cells to the inside of cells, thus regulating the development and apoptosis of cells. When the nerve is subjected to transverse or focal crush injury, the expression of NGF and p75NTR far away from the injured site is rapidly induced [84,107]. In oral squamous cell carcinoma (OSCC), Trk receptors have been confirmed to be overexpressed: Tropomyosin receptor kinase A (NGF), TrkB (BDNF, NT-4/5), and TrkC (neurotrophins 3, NT3) receptors, TrkC and its ligand NTF3 can promote the proliferation of SCs by inhibiting the formation of myeloid cells in the peripheral nervous system, and TrkC may also participate in PNI by regulating the interaction between SCs and tumor cells [68]. Ovarian cancer cells overexpress TrkB, and BDNF/TrkB can promote the migration and invasion of ovarian cancer cells by affecting myelination during nerve regeneration [67]. Macrophage-derived IL-1β induces non-neuronal cells to synthesize NGF, and tumor cells can also secrete NGF and BDNF to active their Trk receptors to stimulate nerve growth [109,110]. ACC, a neurotrophic tumor, and BDNF receptor, which promotes the survival and differentiation of axons and nerves, are abundant in the nerves invaded around the tumor. The normal prostate is one of the most abundant sources of NGF outside the nervous system [108]. Malignant prostate epithelial cells were reported to overexpress NGF and BDNF, but also the corresponding TrkB and TrkC receptors, which may be related to the migration of malignant cells, frequently occurs along nerves within the prostate, because it may provide abundant neurotrophins to act as chemoattractive guidance clues for tumor migration [111,112]. It was also reported that PAAD cells migrate preferentially toward human glial along a GDNF concentration gradient [113,114]. GDNF promotes the expression of integrin, activates MMP-9, and increases nuclear factor-κB(NF-κB), which affects nerve adhesion and invasion and promotes PNI [115]. By binding to the RET tyrosine kinase receptor, GDNF activates two downstream signaling pathways: the PI3-K-AKT pathway, which is involved in pro MMP-9 expression, and the RAS/RAF-MEK-1-ERK1/2 pathway, which is critical for the activation of MMP-9 [113]. In addition, neurons and their related SCs can also release soluble GFRα1 and GDNF(secreted by nerve macrophages), strongly activate RET in cancer cells and initiate the downstream activation of RAS/ERK, MAPK, JNK, and PI3-K-AKT signaling pathways, cancer cell migration, and PNI effect, and induced migration along the nerve [33,42,114,116,117]. Artemin sends signals through the Ret/GFRα3 receptor complex, as a member of the GDNF family of ligands [118]. In PAAD, over-expressed neurotrophin (NRTN) and Artemin activate RET tyrosine kinase (TK) by binding their homologous GDNF family receptor -α (GFRα) receptors, promoting cancer cell invasion and neuronal plasticity, and promoting the proliferation of nerve fibers around the tumor [119,120]. When the tumor volume increases, the pressure on nerve fibers increases and axon neurotrophic factors increase [89]. When an initial damage to the nerves by cancer cell, neurons and/or SCs produce Artemin/GFRα3 to repair the nerves, but the abundance of Artemin attracts further cancer cells to the site of injury, which produces a vicious cycle [121]. Axon injury may trigger the dedifferentiation and activation of SCs by releasing neuregulin (NRG) from the remaining neurons [20]. Neuregulin 1 (NRG1), which is the best studied of several neuregulin genes, can active ErbB receptor tyrosine kinases situated on glial membranes and signal to adjacent glial cells [122,123]. NRG1 forms is an EGF-like domain, binding to and activating receptors belonging to the EGF family of receptor tyrosine kinases erbB2, erbB3, and erbB4, after which it can produce erbB3/erbB2 or erbB4/erbB2 heterodimers or erbB4 homo-dimers and generate active signaling complexes and active an intracellular kinase domain present on erbB2 and erbB4, which phosphorylates specific tyrosine residues within the cytoplasmic tail of the receptor [124,125]. Then it leads to the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3K) pathways [126,127,128]. A research work about PAAD has reported that nuclear FGFR1 regulates the transcription of NRG1, which generates an autocrine loop through ERBB2/4 to further drive invasion [129]. The expression and rapid release of NRG1 in neurons and their axons can be stimulated by NGF, BDNF, NT-3, and GDNF, which are extremely abundant in tumor microenvironment. NRG1 regulates the proliferation, migration and survival of developing SCs [130], which was one of the important members in PNI. Tumor-derived factors and inflammatory mediators activate peripheral sensory fibers, resulting in the release of the substance P(SP), a neuropeptide that promotes tumor growth. SP enters the tumor, activates NK1R in cancer cells, and activates growth factor receptor through Src (EGFR, HER2) [3], and activates the MAPK pathway including extracellular signal-regulated kinases 1 and 2 (ERK1/2), to stimulate mitogenesis, induce cell proliferation, and avoid apoptosis by increasing the mRNA expression of MMP-2, MMP-9, VEGF, and VEGFR [131], which can also be produced by transactive EGFR and activation of NK-1R/Akt/NF-κB signaling pathway [132,133]. By these pathways, SP mediate the interaction between cancer cells and nerves, and promote the proliferation, invasion, and neurotropism of cancer cells, and PNI [132]. It was shown that SP promotes PAAD cell clusters gradually migrating to the dorsal root ganglions (DRGs) and SP-induced neurite regeneration extended to the clusters from the DRGs which provides an invasive pathway for the clusters [132,134]. In addition, neurogenic galanin (GAL) activates the G protein-coupled receptor galanin receptor 2 (GALR2) in tumors to induce NFATC2-mediated transcription of cyclooxygenase-2 and GAL, which causes the crosstalk between nerves and cancer cells [14]. Prostaglandin E2 promotes cancer invasion by promoting the secretion of pro-inflammatory mediators and neuropeptides by tumor cells, GAL released by cancer induces neuritogenesis, facilitating PNI [14,22]. In addition, GALR2-RAP1-p38MAPK-mediated inactivation of Tristetraprolin(TTP), an RNA-binding protein that promotes decay of transcripts of proangiogenic factors (including IL-6, VEGF, IL-8) [135,136,137], and then induces angiogenesis, which facilitates tumor progression by supplying oxygen and nutrients [138]. Chemokines play an important role in cell–cell interaction, which may mediate the chemical attraction of neurons and/or SCs to cancer cells. Infiltration of tumor cells into nerves can lead to nerve injury and release CCL, which induces inflammatory reaction of nerve repair, and then induces the migration of cancer cells expressing CCR to injured nerves, and finally promotes the PNI effect [139]. In prostate cancer (PRAD) cells, the expression of CCR2 (the receptor of CCL2) promotes NI, and the expression of CCR2 is closely related to the activity of MAPK and Akt pathways and the migration of cancer cells to chemokine (C-C motif) ligand 2 (CCL2) and DRG [140]. In PAAD, sensory-neuron-derived mediators CXCL10 and CCL21 pass through complementary receptors CXCR3 and CCR7 on tumor cells, activating AKT, MEK, and RAC signaling pathways in tumor cells to mediate migration [141]. CXCL12 derived from PAAD cells can induce SCs to infiltrate the tumor in the early canceration process and promote cancer cells to attract and migrate to the nerves [141,142]. In addition, the main chemokines include chemokine CCL2 and matrix-derived factor 1 (SDF-1/CXCL12), which induce cancer cell migration under the action of CCR2 and CXCR4 receptors respectively, and can also recruit bone marrow-derived cells (BMDC) and M2 macrophages, and the recruited macrophages secrete GDNF, which can activate RET-GDNF receptor α1 (GFRα1) in cancer cells to promote PNI and the invasion of cancer cells [30,143]. Nerve-related macrophages accumulate in the nerves invaded by tumor cells along the gradient of CCL2 and CSF-1 recognized by CCR2 and CSF-1R receptors, respectively [29,144]. Nerve-derived C-X3-C motif chemokine ligand 1 (CX3CL1) and NT-3 further support the interaction between nerve tumors [145], and the former enhances the adhesion between cancer cells and nerves, while the latter regulates the interaction between SCs and cancer cells [3]. As a family of membrane-related or secreted glycoproteins, it was reported semaphorin can participate in axon guidance and regulate cell migration such as WBCs, neurons, and endothelial cells, to attend cancer progression [146]. Axonal guidance was the most important function, Semaphorin family can mediate axonal guidance by combining plexin family members, such as Sema3D and plexin D1 (PlxnD1) [147], and semaphorin-3A (Sema3A) binding to plexin A1 [148], and the tripartite complexes formed by semaphorins-3 (Sema3s), plexin receptor and neuropilin coreceptor can also mediate axon guidance and invasion [148,149,150]. Besides, there still are some other pathological processes mediated by this combination of the Semaphorin family and plexin family, such as that semaphorin-4D (Sema4D) induce tumor angiogenesis and vascular maturation by binding to the plexin B1 receptor on endothelial cells [42]. They can also have an effect on regulating tumor cell survival, such as Sema3E-PlxnD1 signaling suppressing apoptosis in breast cancer [151]. Over-expression of Semaphorin-4F (Sema4F) in PRAD cells contributes to the communication between nerve fibers and cancer cells and induces the proliferation and migration of PRAD cells [152]. In solid tumors, the rapid growth of tumor tissue, high expansion, and incomplete vascular system in tumor tissue, will lead to insufficient oxygen supply in tumor tissue, and the TME presents overall hypoxia [153,154]. Due to hypoxia, tumor cells can only metabolize energy through anaerobic glycolysis, which will lead to the accumulation of lactic acid [155,156]. At the same time, ion-exchange proteins on tumor cell membranes are constantly transporting H+ inside cells to outside cells to avoid self-acidosis [157]. These cellular reactions also caused the PH of the tumor microenvironment to decrease to different degrees, and the overall environment was acidic [158]. In the microenvironment of tumor occurrence and development, hypoxia, and acidity, a lot of apoptosis will occur in tumor tissues and peripheral tissues, releasing cell fragments and chemokines, leading to infiltration of inflammatory cells and secretion of inflammatory factors [158]. At the same time, the occurrence and development of the tumor itself will also trigger the immune response of the immune system, causing inflammatory cells to gather in this area, and triggering a severe inflammatory response [159]. This local microenvironment tumor promotes the growth and infiltration of tumor cells into nerve tissue [3,29]. It interacts with the perineural environment (nerve cells, glial cells, and their products) to further change the microenvironment and promote PNI [3]. Mitochondrial dysfunction and altered glucose metabolism are considered the typical changes in the tumor microenvironment, and this metabolic change is potentially related to the etiology of nervous system degeneration and nerve injury [160,161]. Oxidative stress was reported to induce chronic neuroinflammation, which contributes to the functionality switch of astrocytes from neurotrophic to neurotoxic, which can release more lactate to reinforce their energetic support to neurons, although upregulating the detrimental pathway [162]. An important feature of energy metabolism in tumor cells is called the “Warburg effect”, which is characterized by heavy dependence on glycolysis and the production of a large amount of lactate [163]. Lactate is easily absorbed and decomposed by tumor cells to produce energy [164]. Lactate can promote tumor invasion and metastasis, play an immunosuppressive role, and promote tumor development by inducing and recruiting immunosuppressive-related cells and molecules [165,166]. In particular, lactate is abundant in the nervous system, which was provided by myelinating Schwann cells (mSCs) using aerobic glycolysis to support action potentials propagation along axons [167,168]. In addition, glycogen is indicated to be present in the peripheral nerve, primarily in mSCs [168]. We speculate that this is helpful for the tumor to invade the nerve and obtain a faster spread speed after the tumor invades the nerve. Recently, single-cell RNA sequencing (scRNA-seq) has provided unprecedented resolution for revealing complex cellular events and deepening our understanding of biological systems [169,170]. However, most scRNA-seq protocols require the complete recovery of cells from tissues and the guarantee of cell survival, which excludes many cell types from the scope of research, and largely destroys the spatial background that could have provided information for cell identity and function analysis. However, the functions of many biological systems, such as embryos, liver lobules, intestinal villi, and tumors, depend on the spatial organization of their cells. In the past decade, high-throughput technology has been developed to quantify gene expression in space, and computational methods can be used to identify genes with spatial patterns and describe the neighborhood in tissues, which is called “spatial transcriptomics (ST)”. The emergence of this new technology has improved the spatial resolution and high-dimensional evaluation of gene transcription [171]. Some researchers have used single-cell spatial transcript information (sc-ST) obtained by combining single-cell sequence information with spatial transcriptome technology to decipher cell components in the tumor invasion niche, and its transcription reprogramming and potential crosstalk, so that the resolution of research becomes higher and accurate spatial positioning can be obtained. It is well known that hypoxia, EMT, and inflammation signatures contributed to intra-tumor spatial variations, which led to functional differences in different niches [172]. In a study of skin cancer, the author confirmed that tumor cells showed a collective migration phenotype and strongly expressed cytokine A, which contributed to the spatial organization of the invasive niche of basal cell carcinoma [5]. ST showed that tumor cell subgroups in the hypoxia group changed, and different subgroups showed their location characteristics and different gene markers. Subgroups at the front of invasion showed higher proliferation ability, invasiveness, and response to stress under hypoxia [155]. This tech has been also used to explore the spatial landscape of multiple cell subpopulations in esophageal squamous cell carcinoma (ESCC). A study reported the intra- and inter-tumoral heterogeneity of ESCC, which, exploring inflammatory fibroblasts (iCAFs), were mostly clustered in the stromal regions, whereas no difference was found in the distribution of myofibroblast (myCAFs) between cancer and stromal regions [173]. Some specific pathways enriched in iCAF subpopulations may be candidates for future research in the progression of ESCC. Another scRNA-seq analysis study also reported the difference in cells between the primary and metastatic sites of HNSCC. Previous studies have also confirmed that the position of macrophages relative to tumor cells is different in various characteristics [45]. In an experiment in diffuse gastric cancer, fibroblasts, endothelial cells, and bone marrow cells were found to be enriched in the deep layer, and it was found that cell-type-specific clustering further revealed that the transition from the shallow layer to the deep layer was related to the up-regulated enrichment of CCL2 transcript in inflammatory endothelial cells and fibroblasts [40]. ST analysis confirmed that stromal cell clusters located at the front of the tumor invasion were identified, which expressed genes related to hypoxia signal transduction, angiogenesis, and cell migration, which proved that hypoxia signal was involved in the metastasis process of invasive gastric cancer [174]. Another study also testified to this view by sc-ST technique, which showed that tumor cells at the outermost edge responded strongest to their local microenvironment, behaved most invasively, and activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to low-confluence areas, and induced similar phenotypic plasticity in neighboring regions [175]. In a study of oral squamous cell carcinoma (OSCC), the spatial localization of nerves in TME was evaluated and successfully summarized into four types of PNI to provide more detailed and accurate pathological information [38]. For the exploration of the interaction between nerve and tumor, the conventional laboratory methods are limited; we could only observe an averaged biological signal over a large number of cells or conventional description based on morphological layer, so the sc-ST should be used to explain the communication between nerve and tumor cells and provide more exact information of tumor cells and nerve [176]. In future research, we expect to make full use of sc-ST to further explain the phenomenon of a tumor’s perineural infiltration, observe its interaction at different spatial points, and explore its mechanism. A lot of early studies have been carried out to explore the inner pathology process of the perineural invasion of some cancers. Important cells, such as SCs, TAMs, CAFs, and some other related cells have been thoroughly studied or are on the way. However, all of these studies were limited and it appears we cannot go any further at present. In this review, we try to introduce a new technology into this pathology process, single-cell spatial transcriptomics. This technology has been used in some studies of common tumors; we can observe the cell components in tumors and their distribution, and then explore the information that we had no chance to capture before through conventional research techniques. According to some studies that have been reported, we hope we can find some transforming cells with an important effect on the PNI process, such as some special CAFs explored in gastrointestinal cancer and the cell-type-specific clustering characteristics. We are trying to understand PNI with this new technology. Maybe soon, we can find a way to stop PNI and the distant metastases that follow.

PMC10000250

Mit Joshi,Bhoomika M. Patel

Unveiling the Role of the Proton Gateway, Uncoupling Proteins (UCPs), in Cancer Cachexia

23-02-2023

cancer cachexia,uncoupling protein,mitochondria,muscle atrophy,metabolism,wasting syndrome,adipose tissue wasting

Simple Summary Cancer cachexia is a wasting syndrome mainly driven by chronic inflammation and high energy expenditure. The hyperactivation of the catabolic pathway leads to higher energy utilization by the body. Uncoupling proteins are involved in uncoupling the electron transport chain and thereby halting the ATP production and releasing energy in the form of heat, which increases the body’s overall energy utilization. UCPs may play an important role during cancer cachexia. This review aims to highlight the role of UCPs in cancer and cancer cachexia and provide new knowledge to tackle this wasting syndrome. Abstract Uncoupling proteins (UCPs) are identified as carriers of proton ions between the mitochondrial inner membrane and the mitochondrial matrix. ATP is mainly generated through oxidative phosphorylation in mitochondria. The proton gradient is generated across the inner mitochondrial membrane and the mitochondrial matrix, which facilitates a smooth transfer of electrons across ETC complexes. Until now, it was thought that the role of UCPs was to break the electron transport chain and thereby inhibit the synthesis of ATP. UCPs allow protons to pass from the inner mitochondrial membrane to the mitochondrial matrix and decrease the proton gradient across the membrane, which results in decreased ATP synthesis and increased production of heat by mitochondria. In recent years, the role of UCPs in other physiological processes has been deciphered. In this review, we first highlighted the different types of UCPs and their precise location across the body. Second, we summarized the role of UCPs in different diseases, mainly metabolic disorders such as obesity and diabetes, cardiovascular complications, cancer, wasting syndrome, neurodegenerative diseases, and kidney complications. Based on our findings, we conclude that UCPs play a major role in maintaining energy homeostasis, mitochondrial functions, ROS production, and apoptosis. Finally, our findings reveal that mitochondrial uncoupling by UCPs may treat many diseases, and extensive clinical studies are required to meet the unmet need of certain diseases.

Unveiling the Role of the Proton Gateway, Uncoupling Proteins (UCPs), in Cancer Cachexia Cancer cachexia is a wasting syndrome mainly driven by chronic inflammation and high energy expenditure. The hyperactivation of the catabolic pathway leads to higher energy utilization by the body. Uncoupling proteins are involved in uncoupling the electron transport chain and thereby halting the ATP production and releasing energy in the form of heat, which increases the body’s overall energy utilization. UCPs may play an important role during cancer cachexia. This review aims to highlight the role of UCPs in cancer and cancer cachexia and provide new knowledge to tackle this wasting syndrome. Uncoupling proteins (UCPs) are identified as carriers of proton ions between the mitochondrial inner membrane and the mitochondrial matrix. ATP is mainly generated through oxidative phosphorylation in mitochondria. The proton gradient is generated across the inner mitochondrial membrane and the mitochondrial matrix, which facilitates a smooth transfer of electrons across ETC complexes. Until now, it was thought that the role of UCPs was to break the electron transport chain and thereby inhibit the synthesis of ATP. UCPs allow protons to pass from the inner mitochondrial membrane to the mitochondrial matrix and decrease the proton gradient across the membrane, which results in decreased ATP synthesis and increased production of heat by mitochondria. In recent years, the role of UCPs in other physiological processes has been deciphered. In this review, we first highlighted the different types of UCPs and their precise location across the body. Second, we summarized the role of UCPs in different diseases, mainly metabolic disorders such as obesity and diabetes, cardiovascular complications, cancer, wasting syndrome, neurodegenerative diseases, and kidney complications. Based on our findings, we conclude that UCPs play a major role in maintaining energy homeostasis, mitochondrial functions, ROS production, and apoptosis. Finally, our findings reveal that mitochondrial uncoupling by UCPs may treat many diseases, and extensive clinical studies are required to meet the unmet need of certain diseases. “Cachexia” is a Latin term meaning wasting or weakness of the body due to a prolonged disease condition. Cachexia is diagnosed in many chronic illnesses such as cancer, AIDS, chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF), stroke, and chronic kidney conditions [1]. Cancer cachexia can be defined as irreversible weight loss, loss of appetite, and loss of skeletal muscle along with a reduction in fat depots [2]. In the last decade, cachexia has been considered a clinical condition in cancer patients that results in early mortality, resistance to chemotherapeutic treatment, and a substantial decrease in quality of life [3]. In 2011, Fearon et al. published an article focused on the classification and diagnosis of cachexia in different populations [4]. In recent years, much preclinical and clinical research has been conducted to identify the underlying mechanism of cachexia in cancer patients. The main culprits of cancer cachexia are systemic inflammation [5], host immune response [6], and host–tumor interaction [7]. Due to the activation of these pathways, there is an increase in catabolic cellular and molecular pathways increasing the energy expenditure of the whole body. The imbalance between catabolic and anabolic pathways leads to the wasting of skeletal muscle and adipose tissues [8]. To date, many interventions have been conducted to target the pathophysiology of cachexia, but they have all failed. Drugs such as cytokine inhibitors, thalidomide, NSAIDs, melatonin, anamorelin, corticosteroids, omega-3 fatty acids, and progesterone analogs showed much less evidence in the amelioration of cachectic conditions in cancer patients [9]. Nutritional supplements and exercise are the only possible choice to treat cachexia, although the results are controversial. To develop a new intervention, there is an urgent need to identify new targets in cancer cachexia pathophysiology. As indicated in [10], UCPs are present in the inner mitochondrial membrane [11]. Mitochondria have a diverse role in biochemical processes such as the generation of adenosine triphosphate (ATP) through oxidative phosphorylation, involved in several steps of the citric acid cycle, urea cycle, and gluconeogenesis also take place in mitochondria. The outer membrane is permeable toward small molecules, whereas the inner membrane has selective permeability to generate an electronic gradient between the outer and inner membrane for the synthesis of ATP [12]. The main task of the mitochondrion is to regenerate the energy required for the normal functions of the cell and body. The cellular mechanism through which it facilitates the regeneration of energy in a form of ATP is known as the “electron transport chain” (ETC) or “cellular respiration” [13]. We will use the term “ETC” for the rest of the article. As the name suggests, the electron transport chain involves the transfer of electrons through the chain of different complexes ranging from complex I to complex IV. Reduced cofactors, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), obtained from oxidized molecules via glycolysis and citric acid cycle, undergo a series of electron transfers. NADH yields three molecules of ATP, while FADH2, which enters into ETC at complex II, yields two molecules of ATP. The transfer of electrons through ETC controls the pumping of H+ ions from the mitochondrial matrix to the intermembrane space, thereby creating a proton gradient. The proton gradient generates energy, which is utilized by ATP synthase to phosphorylate ADP to generate ATP [13]. ETC is not accurate, and the energy liberated from the oxidation of molecules does not convert into ATP; instead, some energy is lost in a form of heat. H+ ions re-enter the matrix independent of ATP synthesis, and this is known as a proton leak [14]. There are two types of proton leak: inducible and non-inducible (basal). Non-inducible or basal proton leak is not regulated by a particular transporter, and the transfer of H+ ions across the membrane is not mediated by UCP. The transfer of H+ ions depends on the composition of fatty acyl present in the inner membrane and the presence of adenine nucleotide translocase. The inducible proton leak is highly controlled, with UCPs playing an important role in this process. The proton gradient created from proton transfer across the inner mitochondrial membrane acts in a supply-and-demand manner. The transfer of electrons through different complexes pumps the proton from the matrix to the inner membrane, while ATP synthase drives the proton from the inner membrane to the matrix to generate ATP [15]. The exact mechanism behind basal proton leak is not yet deciphered. Few studies have enlightened the role composition of inner membrane lipids, mainly fatty acyls. Although only one-third of protons leak through the lipid bilayer [16], and the majority of proton leak is facilitated by the abundance of adenine nucleotide translocase in the inner membrane, it should be noted that proton leaks are dependent upon the abundance of adenine nucleotide translocase (ANT) and not on their activity. Compared with inducible proton leak, the conductance of proton through ANT is considered negligible, as an abundance of ANT is considered very low in the inner membrane of mitochondria [17,18]. The inducible proton leak is mainly facilitated by uncoupling proteins [15]. Researchers are now curious to identify the exact role and pathophysiology of UCPs and inducible proton leaks in different diseases. Few studies have been carried out to identify the role of the different types of UCPs in the pathophysiology of different diseases. The role of UCPs in cancer and cachexia is discussed in the current article. Brown adipose tissue (BAT) has been found to be activated in hibernating animals, in small rodents during cold exposure, and in infants at birth. The only role of BAT found in different physiological conditions is to induce thermogenesis. Brown adipose tissues are abundant with mitochondria and small lipid droplets [19]. UCP-1 is the first uncoupling protein found in brown adipose tissue, where it represents 10% of the mitochondrial content and is involved in the thermogenesis process via proton leak. There are two types of adipose tissue in which the expression of UCP-1 was predominantly found: brown adipose tissue and beige adipose tissue. The expression of UCP-1 in brown adipose tissue remains constant even at the basal level, while the expression of UCP-1 in beige adipose tissue depends on the phenotype change from white adipose tissue to beige adipose tissue, the activation of the sympathetic nervous system, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists. However, both types of tissue show an equal level of activation and distribution when stimulated and result in the same level of thermogenic activity [20]. The activation of the sympathetic nervous system in response to overfeeding and cold conditions stimulates BAT, which further activates UCP-1 and initiates non-shivering thermogenesis. The sympathetic nervous system stimulates thermogenesis by activating the classic cAMP pathway, which increases the mitochondrial number and size, activates the transcription and translation of UCP-1 protein, and increases the flow of free fatty acids to the mitochondria for heat generation. Prolonged exposure to a cold environment results in the phenotype change from white adipose tissue to beige adipose tissue, which increases the thermogenic capacity of the body [21]. In the beginning, it was thought that sympathetic stimuli are the only way to stimulate non-shivering thermogenesis, although many other factors, molecules, and hormones are involved in the activation of UCP-1 and further thermogenesis. The activation of UCP-1 is highly dependent upon the environmental temperature. The UCP-1 present in brown adipose tissue is considered a major mediator of thermogenesis under two major stimuli: prolonged cold exposure and overfeeding. Prolonged exposure to cold temperatures results in the activation of the sympathetic nervous system, which facilitates lipolysis in WAT. Free fatty acids are released through the lipolysis process utilized by BAT and beige adipocytes to generate heat by increasing resting energy expenditure. These phenomena raised interest among researchers in the treatment of obesity. Early research work was focused on identifying the role of UCP-1 in the regulation of thermogenesis. However, the preclinical data retrieved from different studies highlight the significant influence of ambient temperature. UCP knockout mice with C57BL/6J background demonstrated resistance to diet-induced obesity at subthermoneutral temperature (20 °C). The possible mechanism behind the resistance was the alternate mechanism that maintains the body temperature. Thus, resistance was quickly reversed at a thermoneutral temperature (27 °C) [22]. A similar type of study also showed that at a thermoneutrality temperature (30 °C), the ablation of UCP-1 in mice resulted in obesity under the influence of a high-fat diet. The reason behind UCP-1 ablation resulting in obesity is that under thermoneutrality conditions, increased metabolism is not required to maintain the temperature, but under subthermoneutrality conditions, thermal stress is present, and the body utilizes either brown fat in the presence of UCP-1 or other mechanisms such as shivering thermogenesis in the absence of UCP-1, so the exact role of UCP-1 in BAT may be masked by other complementary mechanisms [23]. Having a close homology to UCP-1, the exact role of UCP-2 is still debatable. The distribution of UCP-2 had been found in adipose tissue, the central nervous system, the immune system, the kidney, and the brain. Based on its high distribution throughout the body, UCP-2 may have a role in several diseases such as obesity, diabetes, cardiovascular disease, neurodegenerative, and psychological disease [24]. Many research groups have published contrasting results regarding the role of UCP-2 in different diseases. The polymorphism of UCP-2 gene such as −866G>A (rs659366), Ala55Val (rs660339), −5331G>A, exon 8 deletion/deletion, and 45 bp insertion/deletion in 3′UTR results in obesity, type-2 diabetes, and neural tube defects [25,26]. Discovered in 1997, UCP-3 has been found in the skeletal muscle, the heart muscle, and adipose tissue. Initially, UCP-3 was thought to have a thermogenic effect, as it has a similar homology to UCP-1 [27]. However, unlike UCP-1, which has a life cycle of 30 h [28], UCP-3 has a very short half-life of 30 min, which makes it difficult for any molecular analysis [29,30]. A study using UCP-3 knockout (KO) mice revealed that UCP−/− 3 mice did not exhibit thermogenesis problems and did not show obesity. Increased levels of reactive oxygen species (ROS) were identified in UCP-3 KO mice, as well as in UCP-3 Tg mice. Although the role of UCP-3 as an antioxidant has remained controversial due to the lack of data regarding the correlation between UCP-3 and ROS production, UCP-3 is also thought to be involved in β-oxidation [27]. Same as other UCP proteins, UCP-4 is thought to be involved in the thermogenesis process in brown adipose tissue, as UCP-4 has a similar homology to other UCPs [31]. Earlier studies have revealed that UCP-4 and UCP-5 are mainly present in the brain [32]. UCP-4 is mainly expressed in neurons, the hippocampus, the cortex, the substantia nigra, the striatum, and the cerebellum [32,33,34], while UCP-5 is expressed in the amygdala, the hippocampus, the mediodorsal and paraventricular thalamic nucleus, and the dorsomedial hypothalamic nucleus [35,36]. Both UCP-4 and UCP-5 are involved in the uncoupling of oxidative phosphorylation by providing a gateway to H+ ions and reducing oxidative stress and thereby protecting the mitochondria from an overload of oxidative stress [37]. UCP-4 is able to protect mitochondrial depolarization and decrease oxidative stress against MPP+-induced toxicity in SH-SY5Y cells, while another study showed the involvement of UCP-4 in maintaining calcium homeostasis and apoptosis in PC12 cells. UCP-5 is also involved in protecting SH-SY5Y cells from MPP+ and dopamine toxicity by preserving mitochondrial membrane potential and decreasing oxidative stress and ATP levels. These studies highlight the function of UCP-4 and UCP-5 in brain homeostasis and their possible role in neurodegenerative diseases. A study evaluating the role of UCP-2 and UCP-3 in obesity found that the overexpression of UCP-2 and/or UCP-3 resulted in a decrease in fat mass with an increase in LDL cholesterol in mice, thus highlighting the role of UCPs in alleviating obesity [38]. Another study evaluating the role of UCP-1 and BAT in an obesity-resistant 129S mice strain found that the ablation of UCP-1 resulted in obesity even in obesity-resistant mice. That study also highlighted that the expression of UCP-1 increased, having a positive correlation with weight, and UCP-1 may counteract weight gain in mice fed with a high-fat diet and cafeteria diet [39]. Another study demonstrated that the gut microbiota was able to enhance the effect of curcumin by activating UCP-1-dependent thermogenesis, which prevents weight gain in diet-induced obese mice [40]. These studies revealed that the activation of UCPs in WAT and BAT leads to high energy expenditure followed by weight loss. The activation of UCPs in obese individuals plays an important role to halt weight gain but has a detrimental effect during cancer cachexia. Several studies involved in identifying the role of UCPs in cardiovascular complications [41,42,43,44,45,46,47,48], neurodegenerative disorders [49,50,51], and kidney diseases [52,53,54,55] found that the activation of UCPs decreases oxidative stress, suppresses inflammation, and induces protective effects. However, in cancer and cancer cachexia, UCPs play a negative role and accelerate cachexia in cancer patients. Warburg et al. found that cancer cells use the glycolysis pathway for energy production even in an aerobic environment. The uniqueness of cancer cells is now known as the “Warburg effect” [56]. According to the phenomena, cancer cells utilize the glycolysis pathway due to defects in mitochondrial respiration. The Warburg effect is one of the important hallmarks of cancer and plays a central role in providing energy to fast-growing and differentiating cancer cells [57]. The study conducted by Negre-Salvayre et al. was one of the first studies to identify the role of UCPs in ROS generation. This study found that UCP-2 was able to decrease ROS production in the mitochondria [58]. Another study found that the formation of tumors in a colon carcinoma mice model lacking UCP-2 resulted in higher ROS production, proliferation, and NF-кB production, and decreased apoptosis. The study concluded that higher ROS levels may contribute to tumor progression in UCP-2-deficient mice. Nevertheless, no tumor invasion was detected, which questions the role of UCP-2 in the tumor microenvironment [59]. Subsequent studies revealed that the low expression or deletion of UCP-2 resulted in increased ROS production in the mitochondria. The activation of UCP-2 may play an important role in decreasing ROS production but has a detrimental effect on cancer because many studies have established that the overexpression of UCP-2 in cancer cells decreases ROS production in cancer cells and helps them to thrive [60]. An in vitro study on human colon cancer, using the HCT116 cell line, showed that the overexpression of UCP-2 protects the cells from apoptosis as well as oxidative stress, while the in vivo data showed resistance to anticancer drugs against HC-16-induced cancer in NCr nu/nu mice [61]. Another study also demonstrated that low UCP-2 levels in lung cancer cells can facilitate STAT3 activation and subsequent ROS generation by a chemotherapeutic agent. This study also showed the role of UCP-2 in cancer cell survival and drug resistance [62]. Another research group demonstrated that gemcitabine-induced UCP-2 mRNA expression, which in turn resists gemcitabine-induced damage to cancer cells, elucidates UCP-2-induced resistance [63]. The upregulation of UCP-3 changes the mitochondrial-induced oxidative stress during hypoxia/reoxygenation in vitro in partial H/R-resistant proximal convoluted tubule (PT) cells. As tumor hypoxic cells accelerate malignancy and drug resistance, targeting UCP-3 may help develop anticancer therapies [64]. Another study showed that UCP-2 expression in cancer cells determined the immunomodulatory function of the tumor microenvironment (TME) and had a direct effect on the survival of cancer cells. The activation of UCP-2 altered the cytokine signaling in an interferon-regulatory-factor-5-dependent manner. UCP-2 activated type-1 dendritic cells and CD8+ T cells and normalized TME. That study concluded that the induction of UCP-2, either through genetic modification or a pharmacological approach, resulted in UCP-2 making melanomas cells prone to programmed cell death via protein-1 blockade treatment and thus led to antitumor effects [65]. These studies found that the expression of UCP-2 is initially suppressed, which allows ROS production by cancer cells, and its overexpression in later stages leads to the inhibition of apoptosis and drug resistance. A clinical trial study revealed that HER2-positive breast cancer patients had overexpressed UCP-2 in tumor samples receiving the trastuzumab drug. Treatment with genipin, a UCP-2 inhibitor, significantly increased the antitumor effect of trastuzumab and increased the apoptosis of cancer cells. This study highlighted the role of UCP-2 as a potential target to overcome drug resistance in HER2-positive breast cancer [66]. Another study showed that mitochondrial uncoupling through the activation of UCPs protects CD133(+) colon cancer cells from ROS generation. This study suggested that the use of genipin with a ROS-inducing agent could be useful to eliminate stem-like colon cancer cells [67]. The pathophysiology of cancer cachexia mainly involves higher catabolic signaling compared with anabolic signaling, which leads to a higher energy expenditure of the body. The hypermetabolic state of the body results in the wasting of the skeletal muscle and adipose tissues. Proinflammatory cytokines and other mediators released by the tumor itself into the bloodstream are involved in skeletal muscle and adipose tissue wasting [5,68]. Although the host immune system is predominantly involved in releasing catabolic mediators in response to the tumor, another important factor is radiotherapy or chemotherapeutic drugs, which cause the activation of danger-associated molecular patterns, thus leading to the activation of cytokines and systemic inflammation [69]. Skeletal muscle wasting is mainly mediated through the autophagy–lysosomal pathway and the ubiquitin–proteasomal pathway [70]. Shreds of evidence have suggested that muscle-specific E3 ubiquitin ligases specifically present in muscle such as MuRF-1 and atrogin-1 are the main culprits of skeletal muscle wasting through activating the proteolysis pathway [71]. The activation of molecular pathways such as NF-кB, p38 MAPK, and STAT3 pathways leads to the overexpression of catabolic proteins such as MuRF-1, MAFBX, and atrogins, which further activates autophagy and the proteasomal pathway for muscle atrophy. The activation of autophagy and the proteasomal pathway mainly depends upon the type of cancer cells and the response of an immune system to it. Mitochondrial alterations are another important factor in the promotion of skeletal muscle wasting. Increased mitochondrial oxidative stress results in the degeneration of the mitochondrial network, and the overexpressed Fis1 gene leads to the autophagy and apoptosis of the skeletal muscle [72]. The role of the mitochondria in skeletal muscle wasting in cachectic conditions has caused the mitochondria to be a potential novel target in treating skeletal muscle wasting [73]. Systemic inflammation is another important mediator of skeletal muscle wasting [5]. Preclinical lines of evidence have found elevated levels of TNF-α and that the blockade of TNF-α with antibodies leads to the alleviation of muscle wasting [74]. TNF-α also contributes to the activation of other cytokines such as IL-1 and IL-6, which accelerate skeletal muscle wasting [75,76]. TNF-α and IL-1 further activate the classic NF-кB signaling pathway. The activation of the NF-κB pathway leads to the activation of the ubiquitin–proteasomal pathway through the overexpression of E3 ligase genes atrogin-1 and MuRF1 [77,78]. NF-κB also inhibits the Akt pathway, which leads to increased FOXO activity, further activating UPS- and ALP-related genes (LC3 and Bnip3) [79]. NF-κB also inhibits myogenesis-related genes such as MyoD, Myf5, and MRF4 and decreases myoblast differentiation [80]. The leukemia inhibitory factor (LIF) secreted by tumors belongs to the IL-6 family [81] and has been reported to induce muscle atrophy in animal models. The immunological inhibition of LIF resulted in reduced skeletal muscle loss, thus confirming LIF’s role in cancer cachexia [82]. LIF is also involved in lipolysis and lipid catabolism by acting on adipocytes as well as the hypothalamus [83]. Parathyroid-related protein (PTHrP)m, along with other tumor-derived factors, causes severe skeletal muscle loss by increasing the activity of atrophy-related genes [84]. Another important complication in cancer cachexia is the loss of adipose tissues [85]. Adipose tissues are mainly known to be involved in the storage of excessive fat. However, many other functions are reported in the last few decades. One of the important functions is the browning of white adipose tissues. Many studies have reported the browning of adipose tissue during prolonged cold exposure. The study conducted by Wagner et al. reported a phenotype change from white adipose tissue (WAT) to brown adipose tissue in cachectic patients [86], while another study conducted by Patsouris et al. observed a WAT-to-BAT phenotypic change in burn patients [87]. This study concluded that the WAT-to-BAT change occurs during a hypermetabolic state irrespective of pathological conditions. During cancer cachexia, the tumor and host-derived factors lead to a phenotypic change from WAT to BAT. The activation of BAT leads to more heat production by the mitochondria through upregulating UCPs and thereby increasing the total energy expenditure of the body [88]. During cachectic conditions, the tumor and host-derived factors, such as inflammatory cytokines, catecholamine, the leukotriene inhibitory factor (LIF) [89], and parathyroid-hormone-related protein (PTHrP) [84], lead to an increase in lipolysis in adipocytes, which results in increased liberation of free fatty acids in the circulation. These FFAs are taken up by beige or brown adipocytes and utilized to generate excessive heat [90,91] (Figure 1). UCPs play a central role in inducing thermogenesis in humans, thereby increasing the energy expenditure of the body. One of the key features of cachexia is higher resting energy expenditure and thereby causing the atrophy of skeletal muscle and draining the adipose tissue. The involvement of UCPs in cancer cachexia was identified in different studies. As discussed above, the primary role of UCPs is to induce non-shivering thermogenesis. The upregulation of UCPs in the skeletal muscle during cancer progression is poorly understood. In one of the early studies, Sanchis et al. demonstrated that the rats bearing Yoshida Ah-130 ascites hematoma showed overexpression of UCP-2 and UCP-3 in the skeletal muscle after tumor induction. The study concluded that the overexpression of UCP-2 and UCP-3 in the skeletal muscle was due to the anorexia induced by the burden of the tumor. This was the first study highlighting the role of UCPs in cachectic conditions during cancer [92]. However, the same researchers further demonstrated contradictory results and showed that anorexia and high circulating FFAs were not directly linked with the activation of UCPs in an LLC tumor model [93]. Another study found decreased TCA cycle flux and ATP synthesis rate in Lewis lung carcinoma (LLC)-bearing mice compared with the normal control. The same study also found the overexpression of UCP-3, atrogin-1, FOXO3α, and PDK4 genes. The study reported that the overexpression of UCPs and mitochondrial uncoupling might be involved in the wasting of the skeletal muscle [94]. Mitochondrial alterations and impairment were observed in tumor-bearing cachectic mice. A previous study demonstrated that mitochondrial alterations led to muscle atrophy in LLC-bearing mice. In C26 tumor-bearing mice, the overexpression of the genes related to proteolysis, autophagy, and mitophagy was associated with decreased mitochondrial fusion proteins. Decreased levels of respiratory complexes I and II was observed in the muscle of tumor-bearing mice, which may be induced by increased levels of BNIP3-mitochondria (SDHa) colocalization. Previous studies also confirmed the downregulation of MFN2 and OPA1 in the atrophied muscle, which leads to altered mitochondrial function and muscle atrophy [95]. Another study showed a lower mitochondrial respiration rate and reduced mitochondrial coupling and found lower mitochondrial protein abundance in the soleus muscle of C26 tumor-bearing mice [96]. These studies showed that in cancer-bearing mice, the induction of the wasting of the skeletal muscle was due to a decrease in mitochondrial function, and the overexpression of UCPs was not the sole contributor to skeletal muscle loss. The same conclusion was drawn by another study conducted on Berlin–Druckrey IX rats with peritoneal carcinosis (PC), which showed that cancer cachexia was able to induce alteration in mitochondrial biogenesis and bioenergetics in skeletal muscles. The study found that alterations in mitochondrial activity were not due to the wasting or browning of AT but due to a decrease in the activity of complex IV, which resulted in decreased oxidative phosphorylation. This resulted in decreased ATP production in the mitochondria of the skeletal muscle. The overexpression of UCP-2 in the skeletal muscle was not associated with the wasting of the skeletal muscle. The overexpression of UCP-2 in quadriceps muscles did not change ROS production in PC rats compared with healthy pair-fed rats [97]. These studies showed that the overexpression of UCPs was found in the skeletal muscle during cancer progression. However, their direct involvement in the wasting process is still debatable, and other factors, such as mitochondrial dysfunction due to autophagy, mitophagy, decreased protein synthesis, and decreased activity of mitochondrial complexes, may be involved in skeletal muscle wasting during cancer. Wagner et al. revealed that the browning of WAT starts at the early stage of cachexia and contributes to more energy expenditure and lipid mobilization. Increased energy expenditure is associated with increased mitochondrial activity and uncoupling mechanism in adipocytes. The thermogenic capabilities of interscapular BAT and subcutaneous WAT were found to be increased. The overexpression of UCPs and the browning of WAT contribute to increasing wasting in cancer conditions. Increased UCP-1 staining in the adipose tissue of cancer cachectic cancer patients showed that increased UCP activity was associated with more thermogenic activity in cancer cachexia and the inhibition of UCPs or the browning of adipose tissue may be beneficial to treat cancer cachexia [86]. A small clinical pilot study comparing cancer patients without weight loss vs. cancer patients with weight loss showed that patients with cancer-associated weight loss had decreased abdominal adipocytes, higher circulating IL-6, increased lipolysis through the overexpression of the ATGL gene, and the browning of adipose tissue through the UCP-activating gene PGC-1α. That study also found that Cidea may be involved in promoting WAT browning by inhibiting UCP-1 repression activity, indicating that the UCP-1 protein was detected in peritumoral white adipocytes in seven out of the eight patients having different types of the tumor but was not detected in ten weight-stable patients [98]. MAC16 tumor-bearing mice showed the overexpression of UCP-1 in BAT, which may be to counter the effect of the hypothermia generated by tumor-derived factors. BAT thermogenesis results in increased energy expenditure, leading to wasting syndrome. The overexpression of UCP-2 and UCP-3 in the skeletal muscle may result in decreased food intake and increased lipid mobilization through increased lipolysis from WAT [99]. A lipid-mobilizing factor was extracted and purified from the urine of cancer cachexia patients who had a weight loss of more than 10%. When repeated i.v. injections of LMF were given to NMRI mice, they showed significant weight loss in gonadal fat mass as well as interscapular BAT. The secretion of leptin from adipocytes was significantly reduced, and mRNA levels of UCP-1, UCP-2, and UCP-3 were significantly increased. The upregulation of UCP-1, UCP-2, and UCP-3 in BAT and UCP-2 in the skeletal muscle and liver suggested that UCPs were involved in utilizing an excess of fat to induce heat production and thereby increased catabolism and energy expenditure in cancer cachexia [100]. Many different interventions have been assessed to inhibit the browning of adipose tissue by inhibiting the overexpression of UCP-1 as well as the factors involved in the phenotype change from WAT to BAT. Luan Y et al. first reported that cachectic mice showed increased expression of p38 MAPK signaling, which activates thermogenesis in adipocytes by activating UCP-1 expression [101]. Another study reported that the p38 MAPK inhibitor SB203580 was able to block the activation of the MAPK pathway by inhibiting pancreatic tumor release exosomes in humans and 3T3-L1 adipocytes. P38α, a member of the p38 MAPK signaling pathway, is known to be involved in activating several proteins in lipid metabolism [102]. p38 MAPK inhibitors VCP979 and SB203580 were able to inhibit p38α and the β subunits of the p38 MAPK pathway, thereby decreasing the overexpression of UCPs and the browning of adipose tissue. These studies have shown the role of UCPs in activating the browning of WAT and increased energy expenditure due to it. The inhibition of UCPs or their upstream pathway may be beneficial to ameliorating the progression of cachexia in cancer [103]. A study conducted by Rohm et al. showed that decreased AMPK activity in the wasting of adipose tissue during cancer cachexia and targeting the WAT AMPK–Cidea interaction may prevent WAT browning in cachectic conditions. That study suggested that AMPK decrease occurs during the late stage of cachexia. It also questioned the stand-alone role of the overexpression of UCP-1 as a driving force of energy expenditure in cachexia. The study concluded that the inactivation of AMPK results in the browning and wasting of adipose tissue, and the stabilization of the AMPK activity in WAT prevents the browning and wasting of WAT [104]. Another study also supported the claims of the previous study and demonstrated that the extracts of Arctium lappa L. were able to lower the expression of UCP-1 by activating AMPK in WAT and BAT [105]. A study evaluating the role of free fatty acid (FFA) receptors FFA1 and FFA4 in cancer cachexia found that UCP-1 was not expressed in the interscapular WAT. Epididymis WAT showed UCP-1 expression in tumor-free mice, while it was downregulated in Lewis lung carcinoma-bearing mice regardless of treatment with GW9508 [106]. The same kind of result was obtained by another study on pancreatic cancer cachexia, which showed a reduction in UCP-1 expression in BAT and WAT with the progression of cachexia. The author concluded that the expression of UCP-1 increases in the early stage of cachexia, which may be due to sympathetic modulation [107] (Figure 2). The current review sheds light on the role of UCPs and mitochondrial uncoupling. Very limited studies have been carried out to evaluate the exact role of UCPs in cancer-induced cachexia. UCPs are highly abundant in WAT and BAT and play important roles in non-shivering thermogenesis in the expanse of FFAs. Many studies have shown the upregulation of UCPs in adipose tissue. However, several studies highlighted the fact that even though UCPs were upregulated in WAT and BAT in cancer cachectic conditions, they were not exclusively involved in inducing cachexia. The upregulation of UCPs may be due to other factors involved during cachexia, and UCP activation just exaggerates the cachectic condition. In the context of the skeletal muscle, detailed studies are required to identify the role of UCPs in the context of cancer-associated cachexia. As discussed earlier, the upregulation of UCP-2 and UCP-3 was found to contribute to skeletal muscle loss during cancer cachexia, although some studies indicated that the upregulation of UCPs does not have any direct effect on skeletal muscle loss. More robust preclinical and clinical trials are required to unlock the mystery of UCPs in cancer cachexia. Based on the available studies, we can conclude that UCPs have two important roles. For diseases such as obesity, cardiovascular complications, and neurological complications, UCPs act as protagonists, while in cachexia and cancer, UCPs act as antagonists. The main role of UCPs is to maintain the proton flux across the mitochondrial membrane; nevertheless, UCPs impart many physiological changes in a particular disease condition. Different studies have revealed different responses from different UCPs, and most of the signaling remains complex and controversial and depends upon a vast number of other parameters. Thus far, UCP-1, UCP-2, and UCP-3 have been studied in a very limited way in cancer cachexia, and only their expression has been measured and identified, but no drug or molecule has been developed. The development of drugs acting on UCPs, along with targeting other molecules, could be a potential therapeutic option to halt or cure cachexia in cancer patients.

PMC10000275

Ping Xiao,Zhangyue Shi,Chenang Liu,Darren E. Hagen

Characteristics of circulating small noncoding RNAs in plasma and serum during human aging

22-02-2023

aging clock,circulating sncRNAs,human aging,machine learning

Abstract Objective Aging is a complicated process that triggers age‐related disease susceptibility through intercellular communication in the microenvironment. While the classic secretome of senescence‐associated secretory phenotype (SASP) including soluble factors, growth factors, and extracellular matrix remodeling enzymes are known to impact tissue homeostasis during the aging process, the effects of novel SASP components, extracellular small noncoding RNAs (sncRNAs), on human aging are not well established. Methods Here, by utilizing 446 small RNA‐seq samples from plasma and serum of healthy donors found in the Extracellular RNA (exRNA) Atlas data repository, we correlated linear and nonlinear features between circulating sncRNAs expression and age by the maximal information coefficient (MIC) relationship determination. Age predictors were generated by ensemble machine learning methods (Adaptive Boosting, Gradient Boosting, and Random Forest) and core age‐related sncRNAs were determined through weighted coefficients in machine learning models. Functional investigation was performed via target prediction of age‐related miRNAs. Results We observed the number of highly expressed transfer RNAs (tRNAs) and microRNAs (miRNAs) showed positive and negative associations with age respectively. Two‐variable (sncRNA expression and individual age) relationships were detected by MIC and sncRNAs‐based age predictors were established, resulting in a forecast performance where all R 2 values were greater than 0.96 and root‐mean‐square errors (RMSE) were less than 3.7 years in three ensemble machine learning methods. Furthermore, important age‐related sncRNAs were identified based on modeling and the biological pathways of age‐related miRNAs were characterized by their predicted targets, including multiple pathways in intercellular communication, cancer and immune regulation. Conclusion In summary, this study provides valuable insights into circulating sncRNAs expression dynamics during human aging and may lead to advanced understanding of age‐related sncRNAs functions with further elucidation.

Characteristics of circulating small noncoding RNAs in plasma and serum during human aging Aging is a complicated process that triggers age‐related disease susceptibility through intercellular communication in the microenvironment. While the classic secretome of senescence‐associated secretory phenotype (SASP) including soluble factors, growth factors, and extracellular matrix remodeling enzymes are known to impact tissue homeostasis during the aging process, the effects of novel SASP components, extracellular small noncoding RNAs (sncRNAs), on human aging are not well established. Here, by utilizing 446 small RNA‐seq samples from plasma and serum of healthy donors found in the Extracellular RNA (exRNA) Atlas data repository, we correlated linear and nonlinear features between circulating sncRNAs expression and age by the maximal information coefficient (MIC) relationship determination. Age predictors were generated by ensemble machine learning methods (Adaptive Boosting, Gradient Boosting, and Random Forest) and core age‐related sncRNAs were determined through weighted coefficients in machine learning models. Functional investigation was performed via target prediction of age‐related miRNAs. We observed the number of highly expressed transfer RNAs (tRNAs) and microRNAs (miRNAs) showed positive and negative associations with age respectively. Two‐variable (sncRNA expression and individual age) relationships were detected by MIC and sncRNAs‐based age predictors were established, resulting in a forecast performance where all R 2 values were greater than 0.96 and root‐mean‐square errors (RMSE) were less than 3.7 years in three ensemble machine learning methods. Furthermore, important age‐related sncRNAs were identified based on modeling and the biological pathways of age‐related miRNAs were characterized by their predicted targets, including multiple pathways in intercellular communication, cancer and immune regulation. In summary, this study provides valuable insights into circulating sncRNAs expression dynamics during human aging and may lead to advanced understanding of age‐related sncRNAs functions with further elucidation. Heterogeneity of human lifespan and health outcomes occurs due to differential aging process. , , Organismal aging is often accompanied by dysregulation of numerous cellular and molecular processes that triggers age‐related pathologies such as tissue degradation, tissue fibrosis, arthritis, renal dysfunction, diabetes, and cancer. The highly proactive secretome from senescent cells, termed the senescence‐associated secretory phenotype (SASP), is one of main drivers that cause age‐related pathogenesis through intercellular communication. The classical SASP includes secretome of soluble factors, growth factors, and extracellular matrix remodeling enzymes, and it can transmit age‐related information to the healthy cells via cell‐to‐cell contact. As one of the emerging SASP components protected by extracellular vesicles (EVs), ribonucleoprotein (RNP) complexes, and lipoproteins, extracellular RNAs (exRNAs) are found in many biological fluids and can bridge the communication between “donor” and “recipient” cells through endocytosis, inducing paracrine senescence and pro‐tumorigenic processes. , Deep sequencing of human plasma exRNA revealed more than 80% of sequencing reads mapped to small noncoding RNAs (sncRNAs) in human genome, including microRNAs (miRNAs), PIWI‐interacting RNAs (piRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs). Extracellular miRNA expression in plasma of mice changes with age and cellular senescence can affect age‐related homeostasis throughout the body by circulating miRNA. Other studies uncovered the roles of circulating miRNAs in age‐related dysfunction such as osteogenesis imperfecta, decreased myelination, tumorigenesis, and cardiovascular disease. However, the molecular function of other circulating sncRNAs in aging and age‐related diseases has been overlooked, and their expression profiles during human aging process must be further characterized. In this study, we determined the extracellular sncRNAs landscape during healthy human aging. Furthermore we generated an aging clock based on dynamic changes in extracellular sncRNAs and identified putative core sncRNAs with larger contribution weights in machine learning models for age‐related risks prediction. To achieve this, we used 446 pre‐selected small RNA‐seq data from plasma and serum samples (age: 20–99 years) and employed differential expression analysis and linear or nonlinear association measurements to determine age‐related sncRNAs as primary inputs for comprehensive machine learning modeling. Based on supervised machine learning models, aging estimators were created in high accuracy and sncRNAs candidates with top importance values in built models were considered as final age‐related biomarkers. Additionally, pathway enrichment of targets of core miRNAs strengthens our viewpoint that extracellular sncRNAs change with age‐related processes. To profile sncRNAs features during human healthy aging, we obtained small RNA‐seq datasets from the Extracellular RNA (exRNA) Atlas data repository (https://exrna‐atlas.org). This work includes the studies for which information on age, health status, and gender, but only individuals having healthy aging process were retained for analysis. For datasets meeting the quality control standards established by the Extracellular RNA Communication Consortium (ERCC) (see experimental procedures), we created a bioinformatics procedure for reads mapping, processing, normalizing, categorizing, and modeling (Figure 1A). As a result of these criteria, 302 plasma and 144 serum samples (Figure 1B) were used in this study, with a similar number of samples representing each gender ranging from 20–99 years old (Figure 1C, Table S1). As these datasets originate from distinct studies with multiple sampling and library preparations, there are clear batch effects after Counts Per Million (CPM) normalization (Figure S1A,B). The ComBat function from the R package sva (v3.40.0) in Bioconductor was employed to reduce or eliminate batch effect that may deviate from actual cross‐study results (Figure S1C,D). These corrected data were used for correlation measurements and machine learning training described below. To determine sncRNAs expressed during aging, we considered sncRNAs with ≥1 CPM in at least 30% of individuals within an age group (young (20–30), adult (31–60), and aged (61+) groups) as expressed sncRNAs. As a result, there were 7953 and 6476 sncRNAs observed in plasma and serum samples respectively (Figure 1A). Further, we identified highly expressed sncRNAs by increasing minimal CPM to 10, resulting in 1243 and 1139 sncRNAs retained in plasma and serum samples respectively (Figure 1A, Table S2). In terms of distribution of sncRNAs subtypes in three age groups, miRNAs account for a high proportion (26.5%–63.4%) of all sncRNAs in both plasma and serum, and their abundance consistently decreased with age (Figure 2A,B). tRNAs increased and became the dominant sncRNA in aged group while expression of miRNAs were reduced in older individuals (Figure 2A,B). The corresponding mapped reads are proportional to the number of each highly expressed subtype, even though miRNA showed relatively more sequencing reads than others in both plasma and serum (Figure 2C,D). We calculated the maximum information coefficient (MIC) (D. N. ) to investigate both linear and nonlinear associations between sncRNAs expression and corresponding individual age. By employing batch‐corrected data of expressed sncRNAs, we identified 364 and 1941 age‐related sncRNAs from plasma and serum respectively (Figure 3A,B, Table S3). Intriguingly, piRNAs became the most abundant sncRNAs in MIC measurement, with the number of snRNAs representing the second largest (Figure S2A,B). Similarly, the over‐represented biological processes of miRNA targets were identified, and cellular response and epigenetic modification were enriched in plasma (Figure 3C), while biosynthetic processes were significantly observed in serum samples (Figure 3D). As the expression of sncRNAs changes with age, further data‐driven analysis was conducted to construct a human aging clock. MIC‐based age‐correlated sncRNAs were used as inputs to train regression models in plasma and serum samples. Compared to the linear models, such as Linear Regression (without feature selection) and Elastic Net (feature selection through regularization), the tree‐based ensemble machine learning methods (including Adaptive Boosting, Gradient Boosting, and Random Forest regressors) showed stronger power of prediction with better performance in accuracy (Figure 4) since its great capability of learning the underlying nonlinear patterns. With stably ideal performance in test subsets (Table S4), all models inputting age‐correlated sncRNAs (MIC_plasma and MIC_serum) accurately predicted the ages of corresponding individuals in test sets, with average R 2 values greater than 0.96, root mean squared error (RMSE) values less than 3.7 years and mean absolute error (MAE) values less than 1.9 years (Figure 4A–C). Due to the strong generalization ability in all ensemble learning methods, core sncRNAs associated with aging processes were determined by combined statistics and sum of importance ranks in the three methods was used as the criteria for core sncRNAs identification. As a result, there were 222 and 321 core sncRNAs overlapped in all three methods with MIC_plasma and MIC_serum as the inputs respectively (Table S5). Particularly, four snRNAs, three piRNAs, two small cytoplasmic RNAs, and one miRNA were identified as top core sncRNAs in plasma (Table 1). In serum samples, seven snRNAs, two tRNAs, and one small cytoplasmic RNA identified as top core sncRNAs in serum samples (Table 2). Notably, we also observed a gender‐specific model performance. When male‐only samples were used as training set for predicting female‐only test sets or vice versa, there were core sncRNAs unique to one gender (Figure S3A,B and Table S6), with slightly lower performance in R 2 and RMSE values compared to the models trained in gender‐mixed data (Figure S3C,D). To gain further insight into extracellular sncRNAs potential functions in a microenvironment, we focused on miRNAs, which are well characterized in post‐transcriptional gene regulation. The most ranked miRNA with the largest importance score in plasma and serum, hsa‐miR‐11,181‐3p and has‐miR‐7845‐5p (Table S5), were selected and their targets were separately predicted via the integration of eight miRNAs databases. The expressional profile of these two miRNAs in three age groups is in Figure S4 and corresponding targets are included in Table S7. As expected, these miRNA targets are enriched in canonical cell–cell communication pathways such as Sulfur relay system and Endocytosis pathways, as well as Immune development, Asthma and Ras signaling pathways that closely related to immune dysfunction and tumorigenesis during aging process (Figure 5A). We also investigated the association between miRNA targets and protein coding genes previously validated in the human aging process from Human Aging Genomic Resources (HAGR), and we found targets, including DDIT3, HLA‐DQA1, PTK2B, TTR, and YWHAG, were experimentally identified to be associated with cancer progression, senescence, aging, and longevity (Table S8). Based on protein–protein interaction enrichment analysis, these targets were demonstrated to have regulatory relationship with hallmark proteins, such as PIK3R1, STAT3, IL7R, and JAK2 (Figure 5B and Table S9), which have function in cancer, immune response, and intercellular transduction, bolstering the probability that other non‐miRNA sncRNAs also have functions in aging and aging‐related diseases. Our study comprehensively profiled the relationship of extracellular sncRNAs with age in blood and built an aging clock of healthy individuals using sncRNAs linear and nonlinear correlated with age. Previously, age predictors were developed through DNA methylation sites, transcriptome expression, , repeat elements, microRNAs, and protein abundance. This study provides the first detailed analysis of relationship between circulating sncRNAs and age based on regression models and core sncRNAs whose expression changes with age, allowing reliable age prediction. From previous human biofluids studies, differential composition of small RNA has been reported in multiple biofluids. Godoy et al. used 12 normal human biofluids including plasma and serum in their study and for mapping reads of corresponding RNA sequencing (RNA‐seq), miRNA showed relative high fraction (63.8906%, median) in adult plasma compared to serum (36.0154%, median). However, the percentage of tRNA mapped reads in serum increased (42.2067%, median) and became the most abundant RNA biotype, while median value was 0.7759% in adult plasma. One study determined the diversity of small RNA in different biofluids, and tRNA showed the largest percentage of mapped reads (39.7%) in serum compared to plasma (5.8%) and whole blood (2.1%). Also, in the Max et al. study, they characterized extracellular RNAs (exRNAs) from both plasma and serum samples of the same healthy volunteers, and interestingly they showed substantial differences of small RNA composition, with higher proportion of miRNA in plasma and more tRNA reads in serum. We have some serum and plasma samples from the same individuals (Table S1) and consistent results were observed (Figure 2). Max et al. also concluded that different biofluid types, even though they come from the same origin, plasma and serum show significant variable that impact exRNA profile. One of the reasons is that additional absorption and continuous degradation of exRNAs by retained blood clot will reduce exRNA abundance. So proper exRNA isolation is essential and immediate platelet and cell debris depletion for plasma collection may avoid losses of exRNA characteristics as much as possible. It is of interest to identify a detectable increase of highly expressed tRNAs in aged individuals, and it has been reported that spleen and brain had the highest tRNA expression, which may indicate unique and differential biological process happen as individuals age. A previous report similarly finds tRNAs were the second most abundant sncRNAs in healthy adults (20–40 years) when small cytoplasmic RNA was not mentioned. Unlike tRNAs driving protein synthesis, tRNA‐derived small RNAs (tsRNAs), including tRNA‐derived fragment (tRF) and stress‐induced tRNA halves (tiRNA), have been uncovered as aging process related sncRNAs. Similar as human studies, the expression of tsRNAs increased during aging in Drosophila, C. elegans, and mouse brain cells. Compared with healthy controls, differential expression of tsRNAs in age‐related diseases has been employed in disease prediction such as Alzheimer's disease and Parkinson's disease, ischaemic stroke, and osteoporosis. tsRNAs have roles not only in potential biomarkers, but also in expressional regulation of age‐related mRNAs. For example, 5′‐tRFTyr from tyrosine pre‐tRNA can silence PKM2, which is the inhibitor of p53, to cause p53‐dependent neuronal death. The number of highly expressed miRNA in our study displayed a decreased tendency in older group, and it has been observed in both plasma and serum. Both core miRNAs identified by machine learning models were found to have reduced expression as age increased, similar to decreased expression of a majority of age‐associated miRNAs in whole‐blood, serum, and peripheral blood mononuclear cells. It has been previously demonstrated that circulating sncRNAs from serum samples show strong association with human aging, while the human aging modeling based on regression relationship was not yet built. In our study, potential function of core sncRNAs was predicted via miRNA target prediction, and these targets showed enrichment in cancer, cell cycle, and longevity regulating pathways. There are overlapping genes included in both cancer and longevity regulation pathways, and this result was consistent with early study that profiled miRNAs expression between young and old individuals. For example, increased PIK3R1 expression has been identified to impair anti‐tumor effect through PI3K‐Akt activation in breast and ovarian cancer chemotherapy. , Previous research determined that protein level of p85α, which is the subunit of PIK3R1, was elevated with age, and age‐associated miRNAs that potentially target PIK3R1 were downregulated. Studies in human aging also show that sequence variations within PIK3R1 gene are significantly correlated with longevity, and individuals with different genotypes of PIK3R1 were associated with longevity through reduced mortality risk in cardiovascular disease. Interestingly, both core miRNAs (hsa‐miR‐11,181‐3p and has‐miR‐7845‐5p) that are potentially involved in PIK3R1 regulation (Figure 5B) showed lower expression in aged individuals (Figure S4). The hsa‐miR‐11,181‐3p has been used as biomarker for identification of glioma brain tumors from other brain tumor types. By suppressing Wnt signaling inhibitor APC2, overexpression of hsa‐miR‐11,181‐3p can promote Wnt signaling pathway and increase cell viability in colon malignant tumor cell line. For has‐miR‐7845‐5p, its expression in serum has been applied in constructing diagnostic classifier of ovarian cancer, and higher expression was also observed in serum of patients with persistent atrial fibrillation. Some direct targets of core miRNAs have been determined as drivers of age‐related process. For example, protein tyrosine kinase 2β (PTK2B) is a tyrosine kinase activated by angiotensin II through Ca2+‐dependent pathways to mediate ion channels as well as map kinase signaling pathway. PTK2B is involved in cell growth, inflammatory response, and osmotic pressure regulation after activation and mutated PTK2B is statistically associated with hypertension in Japanese population. PTK2B has also been reported in memory formation and corresponding protein variants can trigger cognitive dysfunction and higher prevalence of Alzheimer's disease. As a nuclear protein that activated by DNA damage, DNA‐damage inducible transcript 3 (DDIT3) shows increased expression and prevents gene transcription by dimerizing with transcription factors. Specifically, DDIT3 plays role in endoplasmic reticulum (ER) protein processing and resulted ER stress promotes cardiomyocyte senescence in mouse hearts. The function of most of age‐associated sncRNAs identified in this study is unknown and further investigation into their function may provide meaningful results. We also observed the mild sex‐dependent differences in the aging clock modeling. Similarly, a previous study indicated that sncRNAs differences between genders were minor and sex‐specific training sets have relatively low performance score in prediction compared to the gender‐mixed training sets. During this process, some gender‐dependent core sncRNAs were identified, including male‐specific sncRNAs piR‐31,143 and piR‐48,977 in plasma, male‐specific sncRNAs piR‐33,527 and piR‐57,256 in serum, female‐specific sncRNAs hsa‐miR‐3789 and U5‐L214 in plasma and female‐specific sncRNAs U6‐L989 and piR‐30,597 in serum (Table S6). Further mechanistic study is needed to uncover their prospective role in aging and aging‐related disease. A major limitation of our current study is the corresponding datasets utilized were developed by researchers for different, unique projects and with multiple RNA extraction protocols, which may bias extracellular RNA abundance. Furthermore, trait information such as ethnicity, body mass, and smoking habits were not considered in our study due to the lack of information, and a more sophisticated and systematic sample processing and recording would help future research on big data‐based human aging modeling. In conclusion, we provide a novel insight into the circulating sncRNAs profile of human aging. We developed predictive models in uncovering core sncRNAs and estimated age by utilizing meta‐analysis based correlation measurement and machine learning modeling. The sncRNA dynamics with age provide valuable references for extracellular RNA study in aging, and the potential mechanisms of age‐related intercellular communication by sncRNAs need further investigation. Human small RNA‐Seq datasets in the extracellular RNA (exRNA) Atlas data repository (https://exrna‐atlas.org) were queried with studies filtered using the following requirements: (1) data were sequenced from plasma/serum samples; (2) samples have definitive age and gender information within each study; and (3) the donor of corresponding samples should have a healthy status and was sampled as a control individual for the study. As a result, two studies (Accession ID: EXR‐MTEWA1ZR3Xg6‐AN and EXR‐TTUSC1gCrGDH‐AN) were included in both plasma and serum studies, and two studies (Accession ID: EXR‐TPATE1OqELFf‐AN and EXR‐KJENS1sPlvS2‐AN) were obtained with only plasma and serum samples respectively and 366 plasma and 188 serum samples passed preliminary filtration. To avoid genes' expressional bias due to the low sequencing reads and host genome contamination, we only retained samples that met the quality control (QC) standards developed by Extracellular RNA Communication Consortium (ERCC). Briefly, individual dataset should have a minimum of 100,000 reads that aligned to annotated RNA transcript (including miRNAs, piRNAs, tRNAs, snoRNAs, circular RNAs, protein coding genes, and long noncoding RNAs), and ratio of transcriptome reads over total sequencing reads should be more than 0.5. Consequently, 302 plasma and 144 serum samples (Table S1) were retained for further analysis. To generate expression matrices of sncRNAs, read adaptors and low quality bases were removed using the Trim Galore (v0.6.5) wrapper. Clean reads were aligned and quantified with bowtie2 (v2.4.4) and samtools (v1.1.4) through miRNAs and other sncRNAs annotation file from miRBase (Release 22.1) and the DASHR (v2.0) database, respectively. The raw sncRNAs expression results were integrated and processed in R (v4.1.1) computational environment for identifying age‐related sncRNAs after preprocessing. To correct for actual expression characteristics masked by sequencing depth variability, gene read counts were transformed into CPM values after measuring normalized library sizes by edgeR (v3.14) package. Since there were still obvious batch effects observed via principal component analysis (Figure S1), we conducted batch removal using the ComBat function in sva package (v3.40.0) and processed CPM‐based data showed improved sample clustering by age (Figure S1). Batch‐effect corrected data were used for identifying maximum information coefficient and constructing machine learning models described below. To select the sncRNAs representative of the age prediction model, the maximal information coefficient (MIC), which permits the identification of important, difficult‐to‐detect associations, was used to identify and screen the linear or nonlinear correlations between each sncRNA expression (X) and the individual's chronological age (Y). Reshef et al. reported that MIC − ρ2 to be near zero for linear relationships and MIC − ρ2 > 0.2 for nonlinear relationships, where ρ2 is the coefficient of determination (R 2). We also employed total information coefficient (TIC) to evaluate the power of independence testing between X and Y. The sncRNAs having both MIC and TIC values greater than 0.7 with actual age were retained for building models. The corrected expression data of sncRNAs selected from differential expression analysis and MIC‐based correlation measurement were used for machine learning modeling. Since sncRNAs expression inputs could be seen as the explanatory variable X, which is a high dimensional vector, the modeling process was performed as a regression analysis problem and was formularized as:where X denotes the sncRNA inputs, y denotes individual's age, and f^ denotes the fitted mapping function. Ensemble learning including Adaptive Boosting, Gradient Boosting, and Random Forest were leveraged in this study, taking advantage of their strong generalization ability achieved by multiple weak learners combination. Based on manual parameter tuning, the parameter “number of estimators,” which is the number of weak learners (i.e., the regression tree in this study) to be integrated in model fitting, was determined in each specific model based on the overall performance (RMSE, R 2, and MAE, showed in Table S10). The performance of ensemble learning is compared with linear regression and elastic net. The corresponding importance of each sncRNA was calculated as impurity‐based feature score (sum to 1), which can be used to determine the fraction of sncRNA that it makes contribution to distinguish. Potentially core sncRNAs were determined by sorting the corresponding sum of ranks of their importance values in each ensemble learning model. Since the number of samples is different in each age group (young, adult, and aged), simple k‐fold cross‐validation may cause uneven sampling and then trigger bad model performance due to over‐fitting. Therefore, stratified k‐fold cross‐validation is a better option to avoid this issue by selecting approximately the same proportions of samples in each pre‐set age group to the training set (Figure S5). In this study, we stratified fivefold cross‐validation based on the overall sample size. The regression modeling was conducted under Python 3.8.8 and scikit‐learn 0.24.1. To better understand the potential function of circulating sncRNAs changing with age, we primarily predicted the targets of miRNA candidates by using multiMiR R package (V3.14), which integrates eight microRNA‐target databases (DIANA‐microT, ElMMo, MicroCosm, miRanda, miRDB, PicTar, PITA, and TargetScan). Functional enrichment analyses of genes targeted by age‐related miRNAs performed through Enrichr gene list‐based enrichment analysis tool. We used the combined score, which is a combination of the P value and z‐score, to offset the false positive rate caused by the different length of each term and input sets. For direct miRNAs functional enrichment, an over‐representation analysis was performed via miRNA Enrichment Analysis and Annotation Tool (miEAA 2.0), with expressed miRNA sets as the background set and P values were adjusted using Benjamini‐Hochberg (BH) procedure. PX performed the experiments and contributed to project design, data collection, execution of machine learning modeling and analysis, and manuscript writing. ZS and CL contributed to experimental design and execution of machine learning modeling and analysis. DEH contributed to data collection, analysis, and manuscript writing. Not applicable. This research did not receive external funding. The authors have no conflicts of interest to declare. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

PMC10000287

Sakshi Mathur,Chaitali Gawas,Iffat Zareen Ahmad,Minal Wani,Heena Tabassum

Neurodegenerative disorders: Assessing the impact of natural vs drug‐induced treatment options

22-02-2023

herbal,mechanistic approach,neurodegenerative disorders,synthetic,treatment

Abstract Neurodegenerative illnesses refer to the gradual, cumulative loss of neural activity. Neurological conditions are considered to be the second leading cause of mortality in the modern world and the two most prevalent ones are Parkinson's disease and Alzheimer's disease. The negative side effects of pharmaceutical use are a major global concern, despite the availability of many different treatments for therapy. We concentrated on different types of neurological problems and their influence on targets, in vitro, in vivo, and in silico methods toward neurological disorders, as well as the molecular approaches influencing the same, in the first half of the review. The bulk of the second half of the review focuses on the many categories of treatment possibilities, including natural and artificial. Nevertheless, herbal treatment solutions are piquing scholarly attention due to their anti‐oxidative properties and accessibility. However, more quality investigations and innovations are undoubtedly needed to back up these conclusions.

Neurodegenerative disorders: Assessing the impact of natural vs drug‐induced treatment options Neurodegenerative illnesses refer to the gradual, cumulative loss of neural activity. Neurological conditions are considered to be the second leading cause of mortality in the modern world and the two most prevalent ones are Parkinson's disease and Alzheimer's disease. The negative side effects of pharmaceutical use are a major global concern, despite the availability of many different treatments for therapy. We concentrated on different types of neurological problems and their influence on targets, in vitro, in vivo, and in silico methods toward neurological disorders, as well as the molecular approaches influencing the same, in the first half of the review. The bulk of the second half of the review focuses on the many categories of treatment possibilities, including natural and artificial. Nevertheless, herbal treatment solutions are piquing scholarly attention due to their anti‐oxidative properties and accessibility. However, more quality investigations and innovations are undoubtedly needed to back up these conclusions. In the modern world, neurological disorders are regarded as the second greatest cause of death. “Neuro‐,” which refers to nerve cells and “degeneration,” in the context to tissues or organs, denotes losing structure or function. Thus, neurodegeneration can be explained as a gradual loss in the functionality as well as the structure of neurons, ultimately resulting in death. There are significant medical and health care costs associated with degenerative disorders of the neurological system for people all over the world. Three of the main neurodegenerative illnesses include Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Given that the frequency and incidence of these diseases grow sharply with aging, reported cases are anticipated to climb soon as life expectancies continue to rise in many nations. For the therapeutic treatment of neurodegenerative disorders, there are many medications in the form of drugs like apomorphine, baclofen, donepezil, entacapone, etc. However, these medications offer very little benefit from the illness and are frequently accompanied by a wide range of side/harmful effects to the patient. Due to their accessibility, minimum of side effects compared to pharmaceuticals, and antioxidant qualities, natural products may present chances in the prevention and control of neurological illness symptoms. For instance, P. ginseng has been shown to have positive benefits on neuronal degeneration illnesses because of its antioxidative and antiproliferative properties. Mechanism of action investigations showed they also regulate mitochondrial activity, cell surveillance or gene death, and intrinsic neuronal signaling as well as metabolism. The objective of this review paper is to compare natural (herbal) and drug‐based treatments for neurological illnesses as well as to highlight the differences the benefits that the former holds. People across the globe are affected by neurodegenerative disorders. The two most prevalent neurological illnesses are AD and PD. According to an estimate from the Alzheimer's Disease Association, 6.2 million people in the United States might suffer from AD in 2022. According to the Parkinson's Foundation, about a million Americans suffer from PD. The number of people dying from neurological illnesses rose by 36.7% between 1990 and 2015; the majority of these increases were caused by stroke and infectious neurological diseases. The most common neurological conditions were: Headache due to stress, migraine, and headache due to excessive use of medication for AD and dementia. The medium and maximum levels of SDI had the greatest and lowest incidence of stroke, respectively. Years of life lost (YLL) changed more than Disability‐Adjusted Life‐Years (DALY) rates of neurological illnesses with development. To the total illness burden in India, non‐communicable and concussion‐related neurological diseases contributed more than twice as much over the course of 29 years, from 4%in 1990 to 8% in 2019. In India, the neurological disorders that contributed the most DALYs overall in 2019 were: Stroke, headache, epilepsy, cerebral palsy, and encephalitis. Poor birth weight, shortened pregnancy, and poor air quality were the recognized risk factors for communicable illnesses with moderate impacts to DALYs(Table 1). The business for neurodegenerative diseases is predicted to grow at a compound annual growth rate (CAGR) of 6.93% from 2022 to 2027, from an average of US $45,441.30 million in 2021 to US $67,525.41 million in 2027. In terms of the market for neurodegenerative diseases, North America dominates. Increased awareness, an increase in the incidence of neurological disorders, and a robust treatment option for neurodegenerative diseases are the main drivers of market expansion. According to data from the National Institute of Environmental Health Sciences updated in June 2022, an estimated 6.2 million Americans may have AD. The number of individuals above and/or 65 years of age with AD dementia is assumed to increase to 12.7 million by 2050, according to the Alzheimer's Association's 2021 Alzheimer's Disease Facts and Figures. The growing prevalence of AD increases the need for cutting‐edge treatments to combat the status of the illness. The influence of the pandemic on the industry was considerable because it affected clinical trial procedures, research and development (R&D) operations, and mainline products. As a result, development even during this time was rather moderate despite a strong late‐stage product pipeline. A 140‐amino acid protein called α‐synuclein is hypothesized to control release of neurotransmitter dopamine, alterations that take place at synapses, the connectors between neurons that enable communication, and differentiation of cell. Although its biological form (non‐toxic) does not stimulate reactive oxygen species (ROS) generation in neurons and glia, it can cross plasma and intracellular membranes and elicit the release of ROS. In fact, the monomeric form agglomerates to a framework that develops insolvable harmful oligomeric intermediates. Overproduction of ROS within cells damages lipids, nucleic acids, amino acids, and membranes, which may trigger programmed cell death. ROS encourages poly (ADP‐ribose) polymerase (PARP) activity, which alters the energy homeostasis by lowering the NAD+ content, in PD‐related Fbxo7‐deprived cells. Oligomerized and transformed α‐synuclein induces PARP‐1 to create PAR, which in turn mediates cell death by continuing the synthesis of pathologic α‐synuclein. Both neurons and non‐neuronal cells like oligodendrocytes and astrocytes produce tau protein sparingly. It is a protein that binds to microtubules and maintains their structure, which is necessary for neural growth. Post‐translational modification (PTM), conformational alterations, and tau's misfolded conformation are involved in tau alteration, which causes tau to abnormally accumulate into neurofibrillary tangles (NFTs) and cause neurodegeneration. The complexity of AD and PD, relates to the amount of NFTs as well as tau alterations. Tau changes are actively promoted by ROS. Insoluble tau aggregation integrates into the membrane, thereby altering electrical currents and cell membrane voltage, and triggering voltage‐gated calcium (Ca2+) channels (VGCC). This results in an intracellular calcium influx that stimulates NADPH oxidase to produce ROS. Acute toxicity is likely to result from the interaction of each of these effects. Slight suppression of mitochondrial complex I cause the F1Fo ATPase to convert to backward mode in order to maintain the potential gradient of membrane of mitochondria. In cells with complex I inactivation, this form of mitochondrial potential maintenance could result in mitochondrial hyperpolarization, which increases the development of ROS (Figure 1). The amyloid precursor protein is broken down into 39–43 amino acid peptides called Aβ peptides by the enzymes β‐ and γ‐secretases (APPs). In grown neuronal cells, β‐amyloid most efficiently binds to astrocyte membranes and can trigger ASK1 primarily through the release of ROS but not by inducing endoplasmic reticulum (ER) stress. The processes that astrocytes and neurons use to regulate internal levels of cholesterol and its transportation within the brain are very distinct, despite the fact that both types of cells generate cholesterol from start, albeit in varying quantities. Similar to the peripheral circulation, lipoproteins, which are made by astrocytes rather than neurons, interact with cholesterol in the brain. In contrast to neurons, which is solely triggered by lipoproteins, astrocytes are driven to release cholesterol through both lipid‐free apolipoproteins and lipoproteins. Therefore, astrocytes have elevated levels of cholesterol than neurons, which then creates porous structure in their membranes that can induce a calcium signal. Such pores also induce GSH to drop significantly, the DNA‐repairing enzyme poly(ADP‐ribose) polymerase to be activated, mitochondria to depolarize, and the death of neurons in astrocytes. In parallel to Aβ‐peptides producing ROS in the presence of metallic ions, mitochondrial dysfunction is also involved in the development of AD through the formation of ROS by the mitochondria. Accordingly, ER stress, nitric oxide (NO), and ROS have been linked to Aβ‐induced neurotoxicity (Figure 1). Oxidation of phospholipid bilayer, also known as lipid peroxidation, is a quasi‐mechanism that can be aided via COX, CYP, and LOX as well as variables of ROS. Oxidative deterioration of DNA, peptides, or phospholipid bilayer may result from excessive ROS generation. The tissue's makeup and the intrinsic antioxidant system's capacity to return ROS generation to baseline levels determine the degree of tissue oxidation degradation. As it contains a significant amount of oxidation‐prone polyunsaturated fatty acids (PUFAs). With greater contents of palmitate, omega‐6 PUFA arachidonic acid, and DHA but small amounts of EPA, the brain has a distinctive fatty acid profile that suggests the brain regulates concentrations through nutritional absorption. Because DHA serves as the most significant omega‐3 PUFA in terms of quantity in the brain, deficiencies in DHA and even EPA levels have been observed in neurological conditions. A growing body of research backs up the positive effects of increasing PUFA intake in a number of neurodegenerative disorders due to their preventative capabilities. Numerous neurogenic illnesses, such as AD, PD, Huntington's disease, ALS, and Spinocerebellar ataxia, and the preponderance of neurodegenerative disorders have also been demonstrated to include abnormalities in mitochondrial redox equilibrium and excessive generation of ROS. During enzymatic processes, GSH efficiently neutralizes free radicals as well as ROS and RNS. Its function as a co‐substrate for GPX has been acknowledged as the most significant pathway for the reductions of hydrogen peroxide and lipid hydroperoxides. It also serves as an antioxidant in the purification of byproducts resulting from ROS‐induced oxidation of lipids. Lack of GSH leads to oxidative stress, which is linked to the pathophysiology of illnesses, notably malignancy and dementia. By using GSH as a coenzyme to decrease lipid peroxides to the appropriate R‐OH or R‐CHO, glutathione peroxidase (GPx) uses the results of lipid oxidation to produce hydroxyl acids catalytically (Figure 2). Researchers have discovered that antioxidant nutraceuticals neutralize ROS or are necessary as coenzymes for antioxidative enzymes when consumed daily. Nutraceuticals are essential for proper aging and play important roles in the treatment of a range of age‐related illnesses such as neurodegenerative illnesses. Nutraceuticals are those foods that help with illness treatment as well as prevention. These are distinct from nutritional supplements, unlike the latter, which should just serve to enhance the diet, the former is involved in the treatment of the disorder. Examples of such substances include vitamin E, soy, vitamin D, green tea, etc. The endocrine mechanisms mediated by curcumin may directly decrease the synthesis of Aβ peptides. Tannic acid, a polyphenolic substance originating from plants, has been shown to have neuroprotective, anti‐inflammatory and antioxidant properties because it reduces toxic Aβ fibril accumulation. In addition to these, melatonin, berberine, and genistein displayed a notable mitigative impact. When specific DNA lesions, such as base modification, mutation double or single stranded break occur, transcription and replication are disrupted, which causes cell death, senescence, and aging. DNA damage causes chromosomal abnormalities that can result in either cell malfunction or development of cancer. Cerebellar neurons especially are affected by the predominance of neurodegenerative illnesses brought on by abnormalities in DNA repair genes, as opposed to motor neurons. The exact causes of defects in DNA repair in neurological system as a whole and the cerebellum in specifically are yet unclear. As a result of these flaws, reactive DNA deterioration gradually increases, which is a primary lead to cellular malfunction. This results in the loss of appropriate neurotransmission, which may then lead to the activation of defense pathways responsible for preserving genomic integrity thereby stimulating apoptosis and the death of neurons. In base excision repair and single stranded break repair, DNA production related to repair is mediated by the polymerase Polβ. Directed deletion of polβ results in new‐born mortality and extensive neuronal death in the growing central nervous system (CNS) and peripheral nervous system (PNS). Etiologies as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), each of which has neurologic elements, are caused by mutations in nucleotide excision repair components. Mice with downregulation of Ercc1 exhibit age‐dependent motor neuron deterioration and increase in the number of astrocytes as a consequence of neuron destruction from CNS all of which is comparable to ALS, this suggests that motor neurons might also be impacted by deficits in DNA repair. It is yet unknown if damage to DNA results from other pathogenic mechanisms or whether it directly causes motor neuron atrophy in ALS (Figure 3). The pathogenic hallmark of the majority of neurodegenerative disorders is a build‐up of protein aggregates, which may be a factor in synaptic atrophy and neuronal injury. Evidence suggests that overly produced ROS and reactive nitrogen species (RNS)‐dependent PTM, such as S‐nitrosylation and tyrosine nitration, cause this improper protein folding to aggregate in the brain. As they are important enablers of protein folding, molecular chaperones and the co‐chaperones are crucial in both health and sickness. Chains of HSP70 molecular chaperones have been linked to neurological disease brought on by misfolded protein. Congenital cardiac and neurological illnesses have been linked to abnormalities in multiple HSP70 co‐chaperones, directly connecting the HSP70 chaperone pathway to disease pathogenesis. The core of the cell connection of signaling molecules is made up of Hsp70 proteins. Through the interaction of their substrate binding domain with small non‐polar peptide sequences inside the substrate proteins, they support a wide range of protein folding activities in the cell. Because ATP drives the cellular cycle of binding and liberation of substrate, ATP binding and breakdown are crucial for their activity. The translational apparatus is connected directly to chaperones linked to protein synthesis (CLIPs). The proteome is guarded against environmental stressors like temperature, oxidative and hypoxic stresses by the second group of chaperones, known as heat shock proteins (HSPs). It has been discovered that a subgroup of chaperones, particularly CLIPs, are suppressed as the human brain ages and are triggered by simulating stress; these variations are especially prominent in the brains of patients with AD, Huntington's disease, or PD. These signs of physiological stress are thought to be a factor of neurodegeneration because they frequently appear in the progression of the disease (Tables 2, 3, 4, Figure 4). Curcumin (CUR) was created as a nanodrug form coupled with cholesteryl‐hyaluronic acid (CHA), which improved cell penetration and antitumor efficacy. Enhanced circulatory characteristics of CHA‐CUR at oral, i.p., and intravenous delivery ways were found by pharmacokinetic/pharmacodynamic (PK/PD) tests of the drug. CHA‐CUR has demonstrated tumor sites deposition and efficient tumor growth suppression in animal studies of severe orthotropic murine mammary carcinoma 4T1 and human pancreatic adenocarcinoma MiaPaCa‐2. CHA‐CUR therapy was very well accepted; hence, it could be used to cure and reduce the risk of cancer. To increase blood brain barrier (BBB) permeability, TSI nanoemulsion (TSI‐NE) was created and enhanced with a brain targeted agonist, lactoferrin (Lf). To make the approach more effective, pseudo‐ternary phase diagrams were used. Findings indicate that the Lf‐modified sustained optimum formation of the O/W nanoemulsion had been created and enhanced satisfactorily. Fluorescence intensity rose throughout the subsequent intervals in the sequence. The findings revealed that Coumarin‐6‐Lf‐BBB NE's permeability was superior to that of Coumarin‐6‐NE and Coumarin‐6 solution. Future research may improve TSI brain delivery using Lf tailored nanoemulsion. In order to create products that could heal injuries, aloe vera extract was sonochemically microemulsified with tragacanth gum. The notable benefit of the newly created injury healing product includes its relatively high antimicrobial properties, which result in microbial reductions of 84, 91, and 80% against E. coli, S. aureus, and C. albicans, a cell viability of 98% against human fibroblast cells, and a decent wound healing action with a considerable pace of fibroblast cell migration. To increase brain absorption, curcumin was enclosed in solid lipid nanoparticles (SLNs) and nanostructured lipid carrier (NLC). Curcumin has indeed been spread as an unstructured substance in the nanocarriers, according to the findings of various scanning calorimetry and X‐ray diffraction experiments. A study using the free radical scavenger 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) showed that the synthesis of curcumin had no discernible impact on its antioxidant properties. The utilization of curcumin‐loaded NLCs in the management of CNS illnesses both showed encouraging results. Appropriate chitosan coated nanostructured lipid carrier (CS‐NLC) formulation that can enter the brain following intranasal administration. When the nanoparticles were treated with erythrocytes, neither hemagglutination nor hemolysis events were seen, nor did any toxic signs manifested in the mouse nasal mucosa after CS‐NLC delivery. Last but not least, the bioavailability analysis of CS‐NLC‐DiR showed that the particles were effectively delivered to the brain following intranasal administration. In conclusion, CS‐NLC can be regarded as a secure and reliable nanocarrier for N to B drug delivery. In order to enable nanoparticle receptor mediated transcytosis (RMT) across the BBB, 80‐nm gold nanoparticles with transferrin (Tf) and an acid‐cleavable connection between the Tf and the nanoparticle core were used. These findings are in line with prior findings of BBB endothelium. The acid‐cleavable linkage prevents substantial endothelium retention by losing surface Tf during their transcytosis. The heterobifunctional polyethylenglycol (PEG) linker NHS‐PEG‐MAL was used to connect lactoferrin (Lf) to β‐cyclodextrin (β‐CD), resulting in Lf conjugated ‐β cyclodextrin (Lf‐CD). According to the findings of tissue dispersion experiments, Lf‐CD/IR therapy significantly increased BBB efficiency levels. Additionally, a greater area under the curve (AUC) in brain tissue was a beneficial outcome of the inclusion of the Lf‐CD drug‐delivery system. These findings demonstrate the possibility of Lf‐CD nanoparticles as a hydrophobic drug delivery method that might reach the brain. Role of glial cells and cAMP as progenitors of neural tissue in EIN was investigated. Under conditions of maximum vital activity, it was discovered that the cAMP/PKA signaling induced the NSC to proliferate and hindered the NCP's mitotic activity. EIN causes the cAMP/PKA pathway, which controls NSC activities, to be inverted. The cAMP‐pathway has a variety of effects on how glial cells secrete NTF. Astrocytes and microglial cells in the EIN are stimulated to produce neurotrophins and to realize the growth prospects of NSC and NCP. These results demonstrate the potential of AC antagonists as novel, very successful medications for the treatment of alcoholic encephalopathy. Synthetic chaperone made of mixed‐shell polymeric micelles (MSPMs) with adjustable structural characteristics was developed to prevent AD. These MSPM‐based chaperones might retain Aβ homeostasis by a conjunction of Aβ fibrillation inhibition and Aβ aggregate removal as well as simultaneous reduction of Aβ ‐promoted neurotoxicity. Researchers capitalized on biocompatibility, specificity against anomalous proteins, and blood circulation. To enhance the clinical efficacy of MSPMs, the hydrophilic/hydrophobic moieties on their interface must be balanced. O‐GlcNAc was shown to trigger specific small heat shock proteins' (sHSPs') anti‐amyloid action using synthetic protein chemistry. Whereas O‐GlcNAc levels are generally decreased in the brains of patients with AD, the alteration of pertinent sHSPs is either retained or enhanced, suggesting a pathway to retain possible protective O‐GlcNAc alteration. O‐GlcNAc boosts the capacity of sHSPs to suppress the amyloid creation of both α‐synuclein and Aβ(1–42). The findings had significant outcome for neurodegenerative conditions linked to amyloid production. One study concentrated on creating new lysosome/ER‐targeted compounds of 4‐phenylbutyric acid (4‐PBA). They prevented the neurodegenerative activity of the fly retina to varying degrees of effectiveness. Compound 9, an ER‐targeted peptide analogue, was the most effective chemical chaperons. The lysosome was the focus of yet another active compound 4. Novel chemical chaperons may provide a new category of therapeutic options for the treatment of ALS and other misfolded protein associated conditions as they were found to behave more efficiently than 4‐PBA. To prevent the production of Aβ‐(1–42) fibrils, biocompatible nanogels called cholesterol‐bearing pullulan (CHP) were created. These nanogels have a polysaccharide pullulan framework and hydrophobic cholesterol moiety. A shift from a random coil to a configuration rich in helixes or sheets as a result of the CHP‐nanogels. Additionally, the introduction of methyl‐cyclodextrin led the nanogels to dissociate, releasing monomeric Aβ molecules. During normal settings, amino‐group‐modified CHP (CHPNH2) nanogels had stronger suppressive activities than CHP‐nanogels, indicating the significance of electrostatic interactions between CHPNH2 and Aβ for preventing fibril development. A neutral counterpart of MKT‐077 called YM‐08 was created and manufactured. It bound to Hsp70 in vitro and decreased the levels of phosphorylated tau in cultivated brain slices. YM‐08 passed the BBB, according to pharmacokinetic analysis in CD1 mice. All these results pointed to YM‐08 as an exciting framework for the creation of Hsp70 inhibitors safe for usage in the CNS. However, drug‐based treatment does also lead to variety of side effects ranging from insomnia, dizziness, and hormonal imbalance. Some such as promazine, haloperidol, chlorpromazine, and pimozide may result in drug‐induced parkinsonian disorder. The prevalence of neurodegenerative diseases has increased, which has caused widespread worry. It is now of the utmost importance to find the right treatment for these illnesses. The strength of this review is to provide a comprehensive distinction between the herbal and drug‐based treatment options. Although wide variety of drug‐based options are available for the treatment of neurodegenerative disorders but the negative effects that these prescriptions hold cannot be neglected. These medications only provide temporary relief from the symptoms but cannot stop their progression. Therefore, finding an alternative treatment option that might not only keep the symptoms from becoming worse but also guarantee fewer side effects becomes crucial. Many herbal compounds such as curcumin, aloe vera, epigallocatechin gallate, etc., due to their antioxidative qualities and accessibility, are now piquing the interest of academics. Despite wide range of benefits provided by herbal compounds, it is their low rate of absorption which imposes a restriction to their use in medicinal applications. To support these findings, however, additional high‐quality research and innovation are unquestionably required in order to understand the efficacy of these natural compounds. Equal involvement and contribution from authors and have given acceptance to final manuscript. This review received no specfic grant from any funding agency. The authors declare no conflict of interest.

PMC10000298

Tian Tian,You Cai,Xin Qin,Jiangang Wang,Yali Wang,Xin Yang

Forebrain E-I balance controlled in cognition through coordinated inhibition and inhibitory transcriptome mechanism

24-02-2023

homeostasis,GABA,forebrain,cognition,E-I balance significance statement

Introduction Forebrain neural networks are vital for cognitive functioning, and their excitatory-inhibitory (E-I) balance is governed by neural homeostasis. However, the homeostatic control strategies and transcriptomic mechanisms that maintain forebrain E-I balance and optimal cognition remain unclear. Methods We used patch-clamp and RNA sequencing to investigate the patterns of neural network homeostasis with suppressing forebrain excitatory neural activity and spatial training. Results We found that inhibitory transmission and receptor transcription were reduced in tamoxifen-inducible Kir2.1 conditional knock-in mice. In contrast, spatial training increased inhibitory synaptic connections and the transcription of inhibitory receptors. Discussion Our study provides significant evidence that inhibitory systems play a critical role in the homeostatic control of the E-I balance in the forebrain during cognitive training and E-I rebalance, and we have provided insights into multiple gene candidates for cognition-related homeostasis in the forebrain.

Forebrain E-I balance controlled in cognition through coordinated inhibition and inhibitory transcriptome mechanism Forebrain neural networks are vital for cognitive functioning, and their excitatory-inhibitory (E-I) balance is governed by neural homeostasis. However, the homeostatic control strategies and transcriptomic mechanisms that maintain forebrain E-I balance and optimal cognition remain unclear. We used patch-clamp and RNA sequencing to investigate the patterns of neural network homeostasis with suppressing forebrain excitatory neural activity and spatial training. We found that inhibitory transmission and receptor transcription were reduced in tamoxifen-inducible Kir2.1 conditional knock-in mice. In contrast, spatial training increased inhibitory synaptic connections and the transcription of inhibitory receptors. Our study provides significant evidence that inhibitory systems play a critical role in the homeostatic control of the E-I balance in the forebrain during cognitive training and E-I rebalance, and we have provided insights into multiple gene candidates for cognition-related homeostasis in the forebrain. Cognitive health is essential for human and animal survival. Cognitive dysfunction is a core symptom in many neurological disorders, including Alzheimer’s disease (AD) (Yang et al., 2018) and schizophrenia (SZ) (Bhat et al., 2021). Excitation-inhibition (E-I) balance in the hippocampus and cerebral cortex is essential for normal cognition (Vogels et al., 2011; Yu et al., 2014; Barron et al., 2016). A functional balance between excitatory and inhibitory synapses (E-I balance) is established and maintained throughout life (Turrigiano and Nelson, 2004). Among them, excitatory synaptic transmission is driven mainly by glutamatergic synapses, whereas inhibitory synaptic transmission involves GABAergic and glycinergic signaling. A major function of homeostasis is to regulate neuronal activity in a negative feedback manner, thus playing an important role in maintaining forebrain E-I balance (Gilbert et al., 2016) and neuronal activity at the appropriate cognitive level in the ever-changing world (Huber, 2018). At the individual synaptic level, the E-I ratio of neural inputs can be locally regulated by plasticity (Eichler and Meier, 2008). At the neuronal level, E-I balance is globally controlled via an excitable threshold and is homeostatically regulated by glutamatergic and GABAergic transmission (Cagetti et al., 2004; Eichler and Meier, 2008). Furthermore, experience or the environment shapes the forebrain E-I balance through homeostatic regulation (Bateup et al., 2013; Herstel and Wierenga, 2021). For example, brief (2–3 days) deprivation of the vision of one eye in rodents (monocular deprivation) reduces network activity (Miska et al., 2018) and improves the acuity of the non-deprived pathway (Fischer et al., 2007). However, the responses of forebrain E-I balance and transcriptome to cognitive experience are not well understood. Inwardly rectified potassium channel 2.1 (Kir2.1) was wide used to study synaptic and neuronal homeostatic compensative mechanism. Overexpression of Kir2.1 causes hyperpolarization to inhibit neural excitation (Okada and Matsuda, 2008) and network balance control (Paradis et al., 2001). Adult-onset expression of Kir2.1 could induce homeostatic plasticity and presynaptic transcriptional changes in the fruit fly brain (Harrell et al., 2021). Silencing pyramidal neurons with Kir2.1, after synapse formation, causes a homeostatic increase in synaptic inputs to stabilizes network activity (Burrone et al., 2002; Turrigiano, 2011). However, homeostatic regulation of E-I balance in the hippocampus when Kir2.1 is specifically overexpressed in forebrain excitatory neurons remains unknown. Furthermore, the pattern of this rebalance of neural activity in response to subsequent spatial training or environmental changes remains unclear. Here, we assessed the homeostatic regulation of E-I balance in the forebrain at the electrophysiological and transcriptomic levels by spatial training and overexpressing of Kir2.1 in forebrain excitatory neurons. We also indicated that inhibitory transmission and inhibitory transcriptome mechanisms are important for the homeostatic control of forebrain E-I balance and cognitive experience. This study may provide a reference for understanding the homeostatic control of specific types of neuronal activity abnormalities in diseases including AD and SZ and also suggests a number of potential candidate genes related to neural homeostasis control and cognition. All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Adult (90 ± 2 days old) male C57BL/6 mice were used in this study. The Rosa-CAG-Flag-Kir2.1-2A-tdTomato-WPRE targeting vector (Figure 1A) was designed with a CMV-IE enhancer/chicken-actin/rabbit β-globin hybrid promoter (CAG), FRT site, loxp-flanked STOP cassette (with stop codons in all three reading frames and a triple poly(A) signal), Flag-Kir2.1-2A-tdTomato sequence, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE, to enhance mRNA transcript stability), a poly(A) signal, and attB/attP-flanked PGK-FRT-Neo-poly-A cassette. The entire construct was inserted between exons 1 and 2 of the Gt (ROSA)26Sor locus via electroporation into C57BL/6-derived embryonic stem (ES) cells. Targeted ES cells were selected and injected into C57BL/6 blastocysts, and chimeric animals were bred into C57BL/6 mice to generate Kir2.1loxp/loxp mutant mice. The conditional mutant strain of theKir2.1 mice was developed by crossing Kir2.1loxp/loxp mice with Tg (CaMK2α-Cre/ERT2) mice (Stock number: 012362, Jackson Laboratory, Bar Harbor, Maine, USA). Male adult (90 ± 2 days old of age) Kir2.1 (+) mice and their Kir2.1 (–) littermates were treated with tamoxifen (Tam) dissolved in core oil (100 mg/kg, i.p., once per day for 5 consecutive days). After recovery for 2 days, the animals were used for the following experiments. The water maze task was performed with a circular tank (120 cm diameter) filled with opaque water (21–23°C) and a hidden platform (6 cm diameter) submerged 1 cm below the surface of the water. The devices (tank and platform) and software [WMT-100] were purchased from Tai Meng Technology Co., Ltd. (Chengdu, China). Before the start of the training trials, the mice were allowed to acclimate to the testing room for 30 min. Mice were trained to find the invisible platform within 70 s on six consecutive days, with four trials per day. If a mouse failed to find the platform within 70 s, it was guided to find the platform and allowed to remain there for 15 s. Escape latency to find the hidden platform, path length, and swim velocity were recorded. After 2 days of rest, the platform was removed, and the mouse was allowed to search for the pool for 70 s (probe tests). The time spent in each quadrant was analyzed. No training means no learning and probe process in MWM, but the animals still swim in the water, while training means an intact MWM experiment. And mice sacrificed at day 6 after probe test. Locomotion activity was measured in clear boxes measuring 42 cm × 42 cm, outfitted with photo-beam detectors for monitoring horizontal and vertical activity. Data were collected via a PC. Mice were placed in a corner of the open-field apparatus and left to move freely. Mice were not exposed to the chamber before testing. Data were individually recorded for each animal during 30 min. As described previously (Gilbert et al., 2016; Yang et al., 2018), slices (350 μm) of the hippocampus were obtained from male mice at 90 days of age and were placed in a holding chamber for at least 1 h, and saturated with 95 and 5% CO2. Artificial cerebrospinal fluid (ACSF, in mM): 124 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose. The internal solution contained (in mM): 140 potassium gluconate, 10 HEPES, 0.2 EGTA, 2 NaCl, 2 MgATP, and 0.3 NaGTP, 5 QX-314was used for sEPSCs or mEPSCs, and 140 CsCl, 10 HEPES, 0.2 EGTA, 2 NaCl, 2 MgATP, and 0.3 NaGTP, 5 QX-314 was used for sIPSCs or mIPSCs. The temperature for the patch clamp recording is maintained at about 30 degrees, the electrode enters the liquid surface and corrects the liquid junction potential, and the cell with a resistance less than 20 MΩ after breaking through the cell membrane is considered to be usable. The current-voltage relations for transfected and control neurons were calculated by recording whole-cell currents under a voltage clamp. Voltage steps in 10 mV increments were applied every 250 ms. To measure the amount of current required to reach the action potential threshold, a series of current steps (2 ms or 500 ms duration, 0–1,000 pA range with 100 pA step increments) were injected into the cell until an action potential was generated. Under voltage clamp, the patched cell was held at −70 mV. A bipolar stimulating electrode (FHC Inc., Bowdoin, ME, USA) was placed in the Schaffer collaterals to deliver the stimuli, and the evoked excitatory postsynaptic currents (EPSCs) or inhibitory postsynaptic currents (IPSCs) were recorded under 30 μM bicuculline (Tocris Bioscience, Bristol, UK) or 10 μM CNQX (Tocris Bioscience, Bristol, UK), respectively. Without TTX (Tocris Bioscience, Bristol, UK), spontaneous EPSCs or spontaneous IPSCs were recorded with an external solution containing 30 μM bicuculline or 10 μM CNQX (Tocris Bioscience, Bristol, UK). Miniature EPSCs or miniature IPSCs were recorded with an external solution containing 1 μM TTX, 30 μM bicuculline (Tocris Bioscience, Bristol, UK), or 10 μM CNQX (Tocris Bioscience, Bristol, UK). The synaptic currents were monitored using an Axon 200 B amplifier (Molecular Devices). Spontaneous events were handpicked and analyzed using the Clampfit 10.2 software (Molecular Devices, Sunnyvale, CA, USA), using template matching and a threshold of 5 pA. All data were acquired at 10 kHz and filtered using a low-pass filter at 2 kHz. Uncaging of MNI-glutamate, calcium imaging, and whole-cell recordings were performed under a DIC/fluorescence Olympus microscope (FV1000-BX61WI). Slices with a thickness of 300 μm were prepared as described above. The slices were kept at room temperature for at least 1.5 h before being transferred to the recording chamber. Twenty ml of oxygenated magnesium-free ACSF containing 0.2 mM caged glutamate (Tocris Bioscience, Bristol, UK), 1 μM TTX, 30 μM bicuculline, and 10 μM CNQX at 30°C was perfused into the slice recording chamber through a custom-designed flow system driven by pressurized 95% O2–5% CO2 at roughly 2 ml/min. Slices were examined under a 20 × objective for proper targeting of tdTomato-expressed CA1 pyramidal neurons. To target whole-cell recordings, cells were visualized at a high magnification (60 × objective, 1.0 NA; LUMPLFLN60XW, Olympus). Kir2.1-positive neurons, which were selected on the basis of their pyramidal somata detected under DIC, were RFP-positive on the monitor. Patch pipettes (3–5 MΩ resistance) made of borosilicate glass were filled with an internal solution containing (in mM): 140 potassium gluconate, 10 HEPES, 2 NaCl, 2 MgATP, and 0.3 aGTP, with 20 μM Alexa594 and 50 μM Fluo-5F. For calcium imaging, the neurons were filled via the patch electrode for 10–20 min before imaging. The dendrites of the recorded neurons were 30–60 μm below the surface of the slice. Laser scanning and photo stimulation were performed using a 60 × objective lens. One laser was tuned to 405 nm (FV5-LD405-2) to activate MNI-glutamate. The second was tuned to 800 nm (Ti: sapphire laser; 100 fs pulses; Mai Tai HP DeepSee, Spectra-Physics) for the excitation of Alexa Fluor-594 and Fluo-5F. Calcium signals were used to locate dendritic spines 30–80 μm from the soma. The selected dendrite was scanned repeatedly with a series of 200 scans (scan dimensions: 30 μm × 30 μm, 5 μm × 5 μm; 100 ms per scan). The uncaging laser spot was located ∼1 μm away from the spine in Figure 1E. In the first scan, acquisition of NMDAR currents (Axon200B and FV10-ANALOG in our BX61WI microscope) and calcium signals began, and in the 10th frame, uncaging occurred with a constant area 0.25 μm2. Glutamate was uncaged using 5–30 ms pulses to generate the I-O curve of the laser strength calcium-NMDAR current under voltage clamp (Vh = −70 mV). To ensure that uncaging was performed in a constant location, image drift was corrected before each line scan acquisition by collecting a frame scan and calculating the cross-correlation to a reference image region of interest (ROI) analysis from these scans, as explained below. Fluorescence changes were analyzed off-line with laboratory-written software using the IGOR-Pro programming environment (Wavemetrics). To study the time course and amplitude of Ca2+ rise, the average fluorescence was measured in small “ROIs” (2.25-4 μm2) and converted to the percentage change in fluorescence: , where F is the measured fluorescence signal at any given time, Fr is the average fluorescence from the scans preceding the stimuli, and B is the average value of the background fluorescence in the scanned field that does not contain any part of the dye-filled cell. Total RNA from the hippocampus and cortex was purified using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). RNA quality (RIN 8–9) and quantity were analyzed on a 2,100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using RNA 6,000 Pico chips; ds-cDNA was produced using the Ovation RNA-seq system V2 (NuGEN, San Carlos, CA, USA) and fragmented using a Covaris S-Series System (Covaris, Woburn, MA, USA). DNA fragments in the 150–300 bp size range were recovered to construct a sequencing library using the Encore NGS Library System I (NuGEN, San Carlos, CA, USA) for 100 bp paired-end RNA-seq using the Illumina HiSeq-2500 sequencer. Raw data were processed using the cutadapt (Martin, 2011) software to remove joints and filter low-quality reads, and 77–121 M clean reads were obtained for each sample with an average Q30 > 95%. RNA-sequencing data alignment and differential gene expression analysis were performed using Tophat2 (Kim et al., 2013) and edgeR (Robinson et al., 2010). Specifically, RNA-sequencing data were aligned to the reference genome (mm10 NCBI buid 38.1) using Tophat2, using default parameters. Uniquely mapped and properly paired alignments were used for further analyses. FeatureCounts (Liao et al., 2013) were used to count the number of reads mapped to each gene. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted using the edgeR program package through one scaling normalized factor. Differential expression analysis under the two conditions was performed using the edgeR R package with bcv = 0.1. P-values were adjusted using the Benjamini and Hochberg method. A corrected P-value of 0.05 and absolute fold change of 2 were set as the thresholds for significantly differential expression. Sample cluster analysis of all detected genes was performed using the pheatmap R package. Gene Ontology (GO) enrichment analysis of differentially expressed genes was performed using the clusterProfiler R package (Yu et al., 2012; Wu et al., 2021), in which the gene length bias was corrected. As described previously (Shi et al., 2021; Tian et al., 2021), brain tissue was perfused and fixed with 4% paraformaldehyde (PFA) solution, dehydrated with 30% sucrose, and frozen to obtain 30 μm sagittal sections with a cryostat microtome (Leica CM1950). Fluorescence images were obtained using a FV1000-BX61WI microscope. Statistical analysis of the transcriptomics can be found in the RNA-sequencing and data analysis sections. Other statistical analyses were performed using Graphpad Prism version 8. The amount of data collected is described in the corresponding figure legend. The statistical variables in Figures 1–3 are all continuous variables, described using mean and standard deviation, represented as mean ± SEM. In Figures 1, 2, one way ANOVA were used to compare the differences between the Tam (+) group and the other three groups. In Figures 3A, B, an independent samples t-test was used to compare the differences between the Kir2.1 (+) and Kir2.1 (-) groups. One-way analysis of variance (ANOVA) was used in Figures 3C, D, and the Bonferroni correction was used for multiple comparisons between different groups. The description of the significance levels of p-values is also given in the corresponding figure legends. To study the homeostatic regulation of E-I balance in the forebrain, we genetically suppressed forebrain excitatory neural activity by conditional expression of the inward rectifying potassium channel. Targeting strategies and corresponding genotypic assays are described in detail in the (Section “Materials and Methods). Adult mice received 5 days of tamoxifen (Tam) or vehicle (Veh) treatment, showing successful expression of Kir2.1 channels by morphological and functional confirmation (Figure 1, A–F). First, we showed the presence of a tdTomato-tagged Kir2.1 protein in the forebrain excitatory neurons (Figure 1A). Second, The Kir2.1 currents were plotted against the holding potentials in CA1 pyramidal neurons from Kir2.1 (+) and Kir2.1 (–) mice. The (+)/Tam group mice exhibited large inward rectifying potassium currents (green) recorded from a 250 ms voltage clamp from −160 to −10 mV with Kir2.1 overexpression compared to other groups (Figure 1B). Third, the (+)/Tam group mice had an increased threshold for action potential firing with a 2 ms current step injection compared to the other groups (Figure 1C). The CA1 pyramidal neurons in the Kir2.1 (+) mice required more currents to elicit firing with a 2 ms current step and less firing probabilities in vitro with 500 ms current step injection in the (+)/Tam group mice than in others (Figure 1D). Finally, the (+)/Tam group mice showed less calcium influx in Kir2.1 (+) neurons (Figures 1E, F) compared to others with MNI-glutamate uncaging (>10 ms) under a two-photon microscope. Together, these data demonstrate that excitatory neural activity in the forebrain is selectively inhibited by Kir2.1 overexpression. To study the changes in E-I homeostatic balance after inhibition of forebrain excitatory neural activity, we evaluated the changes in spontaneous and miniature neural transmission in the hippocampus, which is one of the most important areas of the brain for spatial cognition. We observed a reduction in spontaneous EPSCs, but not spontaneous IPSCs in the hippocampal principal neurons (Figures 2A, B). However, the frequency of both miniature mEPSCs and mIPSCs decreased after Kir2.1 overexpression in forebrain pyramidal neurons, and the amplitude of mIPSCs decreased after Kir2.1 overexpression in the forebrain (Figures 2C, D). Furthermore, both evoked IPSCs and evoked EPSCs were reduced after Kir2.1 overexpression in the forebrain of Kir2.1 (+)/Tam group mice compared to those of the other groups, but the E-I currents remained balanced (Figure 2E). These data indicate that reduction in GABA transmission plays an important role in compensating for the E-I imbalance in CA1 elicited by pyramidal neural inhibition. In an ever-changing world, experiences regulate E-I homeostatic balance, and rebalanced E-I networks are often thought to optimize learning and memory (Mizumori and Jo, 2013; Huber, 2018; Girardeau and Lopes-dos-Santos, 2021). To study the association between E-I homeostatic balance and cognition, we evaluated the cognitive level in Kir2.1 (+) mice and tested the changes in miniature postsynaptic currents in the hippocampus with or without spatial training. As many of the previous tests showed no differences in neuronal activity-related parameters among the negative control groups, we only examined the parameter changes between Kir2.1 mice with (Kir2.1 (+)) or without tamoxifen (Kir2.1 (–)) induction treatment in the follow-up experiments. First, we showed that compared with Kir2.1 (–) mice, Kir2.1 (+) mice spent more time reaching the escape platform during the 5-day training session (Figure 3A) and less time in the target quadrant during probe testing (Figure 3B), indicating learning and memory impairments in Kir2.1 (+) mice, while no obvious difference in swimming speed in MWM and travel distance in the open field was found between Kir2.1 (–) and Kir2.1 (+) mice (Supplementary Figure 1). Next, we scarified them and compared the miniature postsynaptic currents in trained mice or untrained Kir2.1 (+) and Kir2.1 (–) animals. We found that both excitatory and inhibitory transmission increased after 5-day spatial training in the control group Kir2.1 (–) mice (Figure 3C). However, only GABA transmission, that is, the amplitude of hippocampal mIPSCs, was enhanced after the 5-day spatial training in Kir2.1 (+) mice (Figure 3D). These data indicate that homeostatic modulation of forebrain E-I balance via GABA transmission may be related to cognitive level and experience. The changes of mIPSC amplitude could be correlated to reduction in inhibitory/GABA receptors in response to cognitive experiences and E-I balance, we determined the underlying genome-wide transcriptome difference in the cortex and hippocampus of trained or untrained Kir2.1 (+)/Kir2.1 (-) mice via RNA sequencing. Details of sample grouping and naming, as well as sequencing data, can be found in Figure 4A and Supplementary Table 1; 77–121 M clean reads were obtained for each sample, with an average Q30 > 95%. The count expression matrix for all samples is shown in Supplementary Table 2. Unsupervised clustering of all gene expression profiles between samples showed that spatial training induced more severe transcriptome perturbation in the hippocampus than the overexpression of Kir2.1 in the cortex (Figure 4B). This indicates that spatial training has a more direct effect on the hippocampus, while Kir2.1 overexpression has a more direct effect on the cortex. A detailed list of differentially expressed genes (DEGs) in each group is shown in Supplementary Table 3. From the results of the GO analysis of DEGs caused by Kir2.1 overexpression, we found that the Kcnj2 gene (Kcnj2 is the gene symbol name of Kir2.1) was significantly upregulated, this was expected given the fact that they are overexpressing Kir. Hence it served as a control. However, GABA and glycine receptors were significantly downregulated in both the cortex and hippocampus (Figure 4C). However, in the spatial training group, GABA and glycine receptors were significantly upregulated in both the cortex and hippocampus (Figure 4D), particularly in the Kir2.1 (+) group (Figure 4E). This indicates that the transcription mechanism for inhibitory transmission or chloride homeostasis is an important strategy for homeostatic control of the E-I balance in the forebrain network and also suggests many gene candidates for cognition-related homeostasis in the forebrain. In the present study, we identified that inhibitory systems, including inhibitory transmission and transcription of inhibitory receptors, are important for forebrain E-I balance and cognitive experience, and propose a number of potential genes involved in cognition and homeostasis. This study may provide a reference for understanding the homeostatic control of specific types of neuronal activity abnormalities in diseases, including AD and SZ, and also suggest a number of potential candidate genes related to neural homeostasis control and cognition. Ion channels play important roles for homeostatic E-I balance. Previously, overexpression of Kir2.1 in forebrain excitatory neurons in mature neurons, inhibited the excitability of excitatory neurons (Burrone et al., 2002) induce homeostatic plasticity (Harrell et al., 2021) and network re-stable (Burrone et al., 2002; Okada and Matsuda, 2008). In this study, we showed a reduction in neural activity in forebrain excitatory neurons lead to the reduction of inhibitory transmission in Kir2.1 (+) mice. Second, in the hippocampus (Figures 4D, E), spatial training increased the transcription of anion and cation channel-related genes, including cholinergic receptors (Chrna9), voltage-dependent sodium and potassium receptor (Kcne2), calcium receptor (Cacng), chloride ions receptor channel (Clcnka), and GABA receptor (Gabrr). The increasing trend of these receptors is not only consistent with the enhancement of synaptic transmission by spatial training, but may also be related to remodeling of cellular ion homeostasis (including cationic sodium and calcium and anionic chloride ions) and the formation of a new E-I balance after spatial training. Furthermore, artificial inhibition of forebrain excitatory neurons in Kir2.1 (+) mice weakened the transcription levels of anion and cation channel-related proteins, including voltage-dependent calcium, GABA, and sodium receptors, which also supported the weakening of inhibitory synaptic transmission and the change in ion homeostasis in Kir2.1 (+) mice. The reduction of inhibitory transmission is consistent with suppressing pyramidal cell activity reduces inhibitory system (such as parvalbumin cells) in the visual cortex from Kir2.1 overexpression mice (Xue et al., 2014). In addition, these ion channels are often important risk factors for diseases with E-I imbalance, suggesting that the cognitive and homeostasis candidate genes identified in this study may also be targets for diseases characterized by E-I imbalance, such as scn-related channels, which have been associated with Dravet syndrome or other epilepsy syndromes (Meisler et al., 2010). It is suggested that influxed/outfluxed anion and cation or related ion channels may serve as a reference for E-I global homeostatic controlled gene compensation in forebrain, which may also be a mechanism in Dravet syndrome or other epilepsy syndromes. In our study, GABA transmission (mIPSC) homeostatic changes in cognitive experience and E-I balance via disinhibition or inhibition enhancement way in direct interneurons-pyramidal neurons or indirect interneurons-interneurons-pyramidal neurons network. Supporting this conclusion, previous studies have suggested that synaptic transmission of GABA can quickly feedback to abnormal neuronal activity homeostasis in the cortex and plays an important role in maintaining E-I balance (Le Roux et al., 2006; Wilhelm and Wenner, 2008). In trained Kir2.1 (+) mice, the amplitude of mIPSC increased but the amplitude of mEPSC is unchanged and frequency still stay lower level in pyramidal neuron in hippocampus, this unchanged mEPSC may result from neutralizing effect of activity inhibition of pyramidal neuron induced by overexpression of Kir2.1 (+) in forebrain excitatory neurons. Furthermore, in the sequencing results, we mainly compared hippocampal data for a single factor, and also tested and analyzed gene transcription (such as GABA receptors) in the cortex, obtaining ion channel changes similar to those in the hippocampus, which improved the problem of low repetition of sequencing data. Interestingly, the inhibitory system is more likely to be disordered in many diseases, and the resulting E-I imbalance is often an important mechanism in these diseases, such as lots of evidence for primary inhibitory dysfunction in autism spectrum disorders (Gilby and O’Brien, 2013; Nelson and Valakh, 2015), less inhibition linked to SZ (Bhat et al., 2021), parvalbumin cells or parvalbumin cells innervated pyramidal neurons pathway selective degenerated lead to E-I imbalance in AD (Yang et al., 2018). Thus, this study suggests the particularity of the inhibitory system in the process of E-I homeostatic balance and cognitive experience, which is of great significance for subsequent studies of the inhibitory system in this type of disease model. Homeostatic control is essential for adaptation to experience and the accurate regulation of forebrain E-I balance (Turrigiano, 1999; Fong et al., 2015). The forebrain neural networks (Kir2.1 (–) trained) and rebalanced forebrain networks (Kir2.1 (+) trained) may recruit homeostasis to improve the efficiency of cognitive information processing. Although the E-I currents remained in a balanced status in Kir2.1 (+) mice, it recruited evoked IPSCs and mIPSC reduction after Kir2.1 overexpression in the forebrain. And, we suggest that the narrowed neural transmission in Kir2.1 (+) mice may service less bandwidth to processing cognitive information, which supports the impairment of learning and memory in Kir2.1 overexpression mice. Furthermore, spatial training increased neural transmission, such as the amplitude enhancement of mIPSC may be related in part to the increase in transcription of GABAergic, glycinergic and calcium channels related genes after training, which also may increase the bandwidth of cognitive information processing recruited for homeostasis in CA1 from trained Kir2.1 (–) mice compared with that from untrained Kir2.1 (–) mice. Therefore, the level of inhibitory systems (including inhibitory transmission and transcription of inhibitory receptors) that are controlled by homeostasis may be linked to cognitive function. We did not screen out any significant changes in the transcriptome of excitatory receptor, suggesting that different level of mechanisms homeostatic regulating E-I balance. A transcriptome-level mechanism, which may be more sensitive to global regulation of E-I balance via inhibitory system. Another protein-level receptor trafficking mechanism may sensitive to local (such as synapse level) regulation of E-I balance that recruits both excitatory and inhibitory receptors in protein level (Wilhelm and Wenner, 2008; Hou et al., 2011). In this study did not test the total and membrane amounts of glutamate and GABA receptors at the protein level in each group to verify these possible different mechanism. However, we suggested that inhibitory transmission compensation is important for forebrain E-I rebalance and cognitive experience and suggested multiple candidate genes associated with homeostasis and cognitive levels. The forebrain excitatory-inhibitory (E-I) homeostatic balance is an important regulator of normal cognition; however, the underlying changes and transcriptomic mechanisms that maintain E-I homeostatic balance and cope with cognitive experiences are largely unknown. This work elucidates that the inhibitory systems, including inhibitory transmission and transcription of inhibitory receptors, are important regulators to forebrain E-I homeostatic balance and cognitive experience, and proposes a number of potential genes involved in cognition and homeostasis. The original contributions presented in the study are publicly available. This data can be found here: https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA013698. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences (SIAT-IACUC-210303-NS-YXA1694). TT was the main investigator in this study. YC analyzed the RNA sequencing data. XQ, JW, and YW revised the manuscript. XY designed the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.

PMC10000302

Jianwei Zhao,Weihua Wang,Ke Yan,Haifeng Zhao,Zhen Zhang,Yu Wang,Wenyu Zhu,Shiwen Chen

RNA-seq reveals Nup62 as a potential regulator for cell division after traumatic brain injury in mice hippocampus

07-03-2023

Traumatic brain injury,RNA-seq,Nup62,Bioinformatics,Hippocampus

Background Hippocampus impairment is a common condition encountered in the clinical diagnosis and treatment of traumatic brain injury (TBI). Several studies have investigated this phenomenon. However, its molecular mechanism remains unclear. Methods In this study, Illumina RNA-seq technology was used to determine the gene expression profile in mice hippocampus after TBI. We then conducted bioinformatics analysis to identify the altered gene expression signatures and mechanisms related to TBI-induced pathology in the hippocampus. Real-time quantitative polymerase chain reaction and western blot were adopted to verify the sequencing results. Results The controlled cortical impact was adopted as the TBI model. Hippocampal specimens were removed for sequencing. Bioinformatics analysis identified 27 upregulated and 17 downregulated differentially expressed genes (DEGs) in post-TBI mouse models. Potential biological functions of the genes were determined via Gene Set Enrichment Analysis (GSEA)-based Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, which suggested a series of functional changes in the nervous system. Specifically, the nucleoporin 62 (Nup62) DEG was discussed and verified. Gene ontology biological process enriched analysis suggests that the cell division was upregulated significantly. The present study may be helpful for the treatment of impaired hippocampus after TBI in the future.

RNA-seq reveals Nup62 as a potential regulator for cell division after traumatic brain injury in mice hippocampus Hippocampus impairment is a common condition encountered in the clinical diagnosis and treatment of traumatic brain injury (TBI). Several studies have investigated this phenomenon. However, its molecular mechanism remains unclear. In this study, Illumina RNA-seq technology was used to determine the gene expression profile in mice hippocampus after TBI. We then conducted bioinformatics analysis to identify the altered gene expression signatures and mechanisms related to TBI-induced pathology in the hippocampus. Real-time quantitative polymerase chain reaction and western blot were adopted to verify the sequencing results. The controlled cortical impact was adopted as the TBI model. Hippocampal specimens were removed for sequencing. Bioinformatics analysis identified 27 upregulated and 17 downregulated differentially expressed genes (DEGs) in post-TBI mouse models. Potential biological functions of the genes were determined via Gene Set Enrichment Analysis (GSEA)-based Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, which suggested a series of functional changes in the nervous system. Specifically, the nucleoporin 62 (Nup62) DEG was discussed and verified. Gene ontology biological process enriched analysis suggests that the cell division was upregulated significantly. The present study may be helpful for the treatment of impaired hippocampus after TBI in the future. Temporary or permanent cognitive impairment is one of the most common complications of traumatic brain injury, mainly manifested as memory loss, poor concentration, and reduced executive ability (Maas, Stocchetti & Bullock, 2008; Paterno, Folweiler & Cohen, 2017; Sinke et al., 2021). The hippocampus is crucial to memory (Kim et al., 2015). It is a vital brain region involved in the physiological circuitry of memory and is often damaged after TBI (Graham et al., 1995). The destruction of the hippocampus is a pathological feature of the human and animal models of brain injury (Carbonell & Grady, 1999; Graham et al., 1995). TBI includes primary and secondary injuries, and the hippocampus is highly susceptible to TBI (Ansari, Roberts & Scheff, 2008). The primary brain injury is caused at the moment of the impact, which involves direct tissue damage, impaired regulation of cerebral blood flow, and diffuse axonal injury due to shearing, tearing, or stretching (Prasetyo, 2020). The secondary injury was complex and involved a cascade of ischemia, anoxia, and cytotoxic and inflammatory processes (Prasetyo, 2020). After TBI, hippocampal atrophy is a common problem in various experimental models of TBI (Bramlett & Dietrich, 2002; Saber et al., 2017) that contribute to hippocampal-dependent memory impairments. Changes were also observed in the hippocampal structure at the cellular level. Fourier transform infrared imaging (FTRI) identified TBI-caused significant structural changes with respect to total protein content, lipid content, lipid/protein ratio, and membrane lipid order (Cakmak et al., 2016; Ustaoglu et al., 2021). The dysregulation of neurotransmitters, including gamma-aminobutyric acid (GABA) and glutamate (Almeida-Suhett et al., 2015; Harris et al., 2012), led to hippocampal deficits. Also, biochemical compounds and changes in electrical neural activity affected different subsections of the rodent hippocampus following TBI (Girgis et al., 2016). As mentioned above, pathological changes occur in the hippocampus after TBI at all levels (Almeida-Suhett et al., 2015; Bramlett & Dietrich, 2002; Cakmak et al., 2016; Girgis et al., 2016; Harris et al., 2012; Saber et al., 2017; Ustaoglu et al., 2021). However, the mechanism of injury and modulation in the hippocampus post-TBI is yet controversial. Our results identified the putative genetic changes and potential therapeutic targets of hippocampal injury in mice after moderate TBI using Illumina RNA-seq technology. Strikingly, RNA sequencing technology has become a powerful research tool for exploring disease pathogenesis. In previous studies, transcriptome analyses were used to identify gene changes in the hippocampus post-TBI at later time points, such as 3 or 7 days (Attilio et al., 2021; Todd et al., 2021). As the effect of TBI on hippocampal brain tissue was time-dependent (Ustaoglu et al., 2021), in this study, 24 h post-TBI was chosen as the time point. As affected by primary and secondary injuries, the whole hippocampus tissue was examined in this research. We used a systematic approach to identify novel molecular biomarkers. In addition, the differently expressed gene (DEG) profiles were explored based on sequencing data. Functional enrichment analyses determined the hub gene-related functions. The top hub gene Nup62 was chosen, and the upregulation of Nup62 was determined by both real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) assay. These findings could provide a novel insight into TBI-induced hippocampus impairment. Male C57B6/J 8–10-weeks-old mice (20–25 g; SLAC Laboratory Animal Corporation, Shanghai, China) used in this work were bred and housed (five per cage) in standard cages under a 12-hour light-dark cycle with the controlled temperature of 23 ± 2 °C and full access to food and water. A total of 24 mice were randomly divided into two groups: TBI (n = 12) and sham-operated (n = 12). In each group, three mice as biological replicates were used for RNA sequencing, three used for (RT-qPCR), four for WB, and two for hematoxylin-eosin (HE) staining. All procedures in these studies involving animals followed the protocols approved by Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China (Approval No: 2021-B28). Samples were collected 24 h after the models were established. All mice were euthanized humanely via cervical spondylectomy. Controlled cortical impact (CCI) was adopted as the TBI model. After the mice were anesthetized with 1% ketamine (75 mg/kg) and xylazine (10 mg/kg), their heads were fixed in a stereotaxic frame (Stoelting, Wood Dale, IL, USA), and their body temperature was kept at 37 °C placing a warming pad under the body. Then, a 10-mm-long incision was made from the midline of the skull under aseptic conditions. After separating the skin and fascia using a vascular clamp, we performed a craniotomy over the center of the right parietal bone, one mm lateral to the sagittal suture using a 4-mm trephine. Mice were excluded from the study if the dura mater was damaged during surgery. The TBI mouse model was established using a CCI device (PinPoint Precision Cortical Impactor PCI3000; Hatteras Instruments Inc., Cary, NC, USA) to simulate a moderate TBI based on our previous studies (Gong et al., 2022; Jing et al., 2020; Yuan et al., 2016). The brain injury of moderate severity was induced using CCI with the following parameters: impact velocity 1.5 m/s, dwell time 100 ms, and striking depth 1.5 mm. The bleeding was staunched with a sterile cotton compress. Bone wax was covered over the surface of the cortex, and the incision was closed carefully with interrupted 6–0 silk sutures under aseptic conditions. The sham group underwent the same procedure except for the impact injury. During the experiment, the body temperature of the mice was stabilized using a heating pad until their consciousness recovered fully; then, all mice were tagged and returned to their cages. The mice were anesthetized and sacrificed 24 h after TBI, and their brains were harvested immediately. The hippocampal tissues were separated from the brains according to Paxinos and Franklin’s Mouse Brain Atlas (Paxinos & Franklin, 2013). The scalp of the mice was cut to expose the head after they were killed by cervical spondylectomy humanely. The mouse skull was pulled apart with tweezers. Then, the superficial cerebral cortex was carefully removed with straight forceps to expose the underlying hippocampal tissue. Similarly, the contralateral hippocampal tissue was also exposed. Finally, the hippocampus was isolated from the surrounding tissue. The brain was sectioned and stained with HE (see the photograph and HE stained section in the Supplemental Files). Total RNA was extracted using MolPure® Cell/Tissue Total RNA Kit (Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China). After generating cDNA libraries, RNA-seq was performed on the Illumina NovaSeq platform according to the manufacturer’s protocol. The RNA-seq datasets have been deposited in the Gene Expression Omnibus (GEO) database under GEO: Message Body GSE214701. In the current analysis, FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) method was adopted for quality control. HISAT2 (http://ccb.jhu.edu/software/hisat2) was used to align the raw RNA-seq reads to the mouse reference genome (Mus musculus, GRCm38) and StringTie (http://ccb.jhu.edu/software/stringtie) to assemble and quantify the transcripts. The DEGs between the TBI and sham groups were analyzed using the “DESeq2” R package (version 1.38) in R (version 4.2.2) statistical software (R Core Team, 2022) (settings: P < 0.05; fold-change (FC)>1.5 or <0.667; false discovery rate (FDR)<0.1). The principal component analysis (PCA) plot was used to determine the principal components using the DEGs list and visualized using the “ggbiplot” R package (version 0.55). The clustering analysis of DEGs was carried out using the “pheatmap” package (version 1.0.12) of R. The GSEA method with all genes was used for gene enrichment analysis. Functional terms were retrieved from the GO database that describes the gene attributes, including the biological process (BP), molecular function (MF), and cellular component (CC). Then, the analysis was performed to identify significantly different regulatory pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG), a major public pathway-related database. GSEA-based GO and KEGG analyses were performed using the “clusterProfiler” package (version 4.4.4; https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) of R statistical software. For RT-qPCR, total RNA was isolated using the MolPure® Cell/Tissue Total RNA Kit (Yeasen Biotechnology) and reverse transcribed into cDNA. qPCR was carried out using gene-specific primers and α-tubulin as the reference gene. Gene-specific primers used for amplification are listed in the Supplemental Files. Then, the mRNA relative expression levels were analyzed via RT-qPCR on a LightCycler 480 (Roche). The isolated hippocampal tissue was homogenized in lysis buffer by ultrasonication, diluted in loading buffer (Cat# P0015; Beyotime) to estimate the protein concentration, and separated by SDS-PAGE. Subsequently, the protein was transferred to the PVDF membrane. The membranes were blocked for 1.5 h with 5% non-fat milk and probed with primary antibodies (Nup62; Proteintech, Rosemont, IL, USA, 1:10000) and α-tubulin (Proteintech, 1:15000)) overnight at 4 °C. The membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody for one hour at room temperature, and the immunoreactive bands were visualized using Amersham Imager 680. The relative optical density was calculated by ImageJ software (NIH, USA). Continuous variables were presented as means ± SD, and categorical variables were displayed as a percentage. The statistical difference between TBI and sham-operated groups was analyzed using the two-tailed unpaired t-test. GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) was used for analyses and graphic representation of data. P-values <0.05 were considered statistically significant. At 24 h after surgery, both the sham and TBI animals were sacrificed, and hippocampus samples were removed rapidly. The size of all hippocampus samples was >0.5 mg, and the 28s/18s value was >0.7 on the Illumina NovaSeq 6000 sequencer used for sequencing. A total of 53.06 million, 49.11 million, 48.80 million, 53.32 million, 40.73 million, and 44.63 million reads were obtained from Sham1, Sham2, Sham3, TBI1, TBI2, and TBI3, respectively, of which 97.63% (Sham1), 97.67% (Sham2), 97.64% (Sham3), 97.61% (TBI1), 97.40% (TBI2), and 97.55% (TBI3) were aligned to the mouse reference genome using HISAT (Table 1). After the reads were compared to the mouse genome, StringTie was used to annotate and quantify the expression (Pertea et al., 2016). The statistical power of our RNA-seq data calculated by “RNASeqpower” package was 0.8601569. A total of 44 genes were identified to be differentially expressed between TBI and sham groups using the “DESeq2” package of R; of these, 27 were upregulated and 17 were downregulated (settings: P < 0.05; fold-change (FC) < 1.5 or < 0.667; FDR < 0.1). Nup62 was the most differentially upregulated gene. Pcdhgb8 was the most differentially downregulated gene. The normalized list of all gene data is available in the Supplemental Files. A PCA plot (Fig. 1) was drawn to assess the variability of the data. The PC1 and PC2 explained 36.3% and 32.6% of the variance in the data, respectively, and the TBI group was isolated from the sham group. The top five most significantly up/downregulated DEGs between TBI and sham groups are listed in Table 2. The volcano and heat maps of all the DEGs are depicted in Fig. 2. Top five significant DEGs identified were labeled in the volcano map. In our study, the GSEA method was used for gene enrichment analysis. The list of genes used in GSEA was ranked according to log2FC. Then GSEA was conducted with two gene set collections: GO and KEGG. The enrichment dot plot of GO was used to describe the BP, CC, and MF entries of all the genes between the TBI and sham groups. Figure 3 illustrates the top 10 upregulated GO terms, including “intermediate filament-based process”, “keratin filament” and “keratin filament binding”, the most significantly enriched GO terms belonging to BP, CC, and MF, respectively. BP refers to a series of events accomplished by one or more ordered assemblies of MFs. In this study, we focused on the BP of Nup62. Table 3 lists the top eight significantly enriched functions that Nup62 is involved in the GO term of BP. “Positive regulation of cytokinesis”, “regulation of cytokinesis” and “positive regulation of centriole replication” were most significantly enriched in the BP group. After the KEGG pathway enrichment analysis, Fig. 4 shows the top 10 most enriched KEGG pathways of activated and suppressed enrichment hallmarks terms, respectively. These included “Glyoxylate and dicarboxylate metabolism”, “Renin-angiotensin system”, “Cytosolic DNA-sensing pathway” and “Olfactory transduction.” Biochemical experiments verified the results (Fig. 5). The relative mRNA expression level measured by RT-qPCR showed significantly upregulated Nup62 in the TBI groups compared to the sham groups (Fig. 5A, P < 0.05). In WB assays, Nup62 also increased considerably in the TBI groups (Fig. 5B, P < 0.01). In this study, we performed a bioinformatic analysis of high-throughput sequencing to determine the gene expression profiles of mice in the hippocampus 24 h after TBI. The results showed critical roles for several genes and pathways in the hippocampus pathology after TBI. A total of 27 up- and 17 downregulated DEGs were detected in the hippocampus of TBI mice. Several pathways enriched for TBI were identified. The GO analysis demonstrated that the “intermediate filament-based process”, “keratin filament” and “keratin filament binding” were the most significantly enriched GO terms containing the ranked genes belonging to BP, CC, and MF, respectively. After TBI, a series of functional changes occur in the nervous system, including “central nervous system neuron differentiation” and “nerve growth factor receptor binding”. The KEGG pathway analysis revealed that “Glyoxylate and dicarboxylate metabolism” and “Cytosolic DNA-sensing pathway” were the most enriched pathways. These bioinformatics analyses may improve the understanding of hippocampal pathological processes in TBI, especially in the acute phase. According to the DEGs analysis, Nup62 levels differed significantly between the TBI and sham-operated groups. These findings were validated by RT-qPCR and WB analyses. Therefore, we were concerned about the role of Nup62 in hippocampal pathology after TBI. Nup62, located on human chromosome 19, is a component of the nuclear pore complex (NPC) and is expressed in various human tissues (Fagerberg et al., 2014). NPC is the only channel responsible for material transport in the nuclear membrane that is conserved across all eukaryotes (Huang et al., 2020). It also plays a critical role in regulating gene expression, and its abnormal function gives rise to a variety of diseases (Huang et al., 2022; Huang et al., 2020). Recent studies have investigated the fine structure of NPCs using a comprehensive technique (Allegretti et al., 2020; Beck & Hurt, 2017; Fontana et al., 2022; Tai et al., 2022). The molecular mass of vertebrate NPCs ranges from 110–125 MDa and the diameter is about 120 nm (Fontana et al., 2022). The NPCs are divided into four main rings: the cytoplasmic ring (CR) on the cytoplasmic side, the inner ring (IR) and luminal ring (LR) on the nuclear membrane plane, and the nuclear ring (NR) facing the nucleus (Fontana et al., 2022). The CR provides docking sites for cytoplasmic filaments. It consists of the Nup214 complex formed with Nup62 together with Nup214 and Nup88 (Tai et al., 2022; Wang et al., 2016; Wu et al., 2016). Nup62 plays a critical role in nuclear transportation, cell migration, and cell cycle regulation (Wu et al., 2016). A previous study showed that the neuropathology of chronic traumatic encephalopathy (CTE) patients is associated with the upregulation of the Nup62 gene (Anderson et al., 2021). Increased Nup62 in vivo and in vitro may trigger TDP-43 cytoplasmic and nuclear mislocalization, abnormalities, perinuclear accumulation, and reduced motor ability and lifespan of animals. Thus, modulating Nups, including Nup62 post-trauma, exerted a protective effect following head trauma (Anderson et al., 2021). In the chronic stress model, the altered Nup62 levels may affect the architecture and plasticity of apical dendrites in the hippocampus (Kinoshita et al., 2014). Another study reported that depletion and cytoplasmic mislocalization of Nup62 contributes to TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration (FTLD) (Gleixner et al., 2022). Nup62 is also associated with other neurodegenerative diseases, such as Alzheimer’s and Huntington’s disease (Nag & Tripathi, 2022). In conclusion, Nup62 pathology may be a common event in various nervous system disorders. Thus, we speculated that changes in Nup62 are associated with a poor prognosis of TBI. Furthermore, our data in GO analysis suggested that altered Nup62 may cause changes in the cell cycle that lead to TBI pathology. In the GO_BP group, “positive regulation of cytokinesis”, “regulation of cytokinesis”, “positive regulation of centriole replication” and other cell division-related pathways were enriched post-TBI compared to the sham group. Cell division depends on the activation of the cell cycle. After TBI, cell cycle activation (CCA) occurs in the hippocampus cells, including neurons, glial cells, and progenitor cells, and contributes to secondary brain injury (Redell et al., 2020; Stoica, Byrnes & Faden, 2009). In the animal models of TBI, CCA in the brain has been well-demonstrated experimentally (Kabadi et al., 2012a; Kabadi et al., 2014; Skovira et al., 2016). Reportedly, cell cycle proteins are upregulated in post-mitotic cells, including neurons and mature oligodendrocytes, and proliferating cells, including microglia and astrocytes (Skovira et al., 2016). In the proliferating cells, CCA induced the formation of glial scar and produced the neuroinflammatory factor that ultimately led to neuronal degeneration (Kabadi et al., 2012b; Loane & Byrnes, 2010; Stoica, Byrnes & Faden, 2009). For post-mitotic cells, re-entry into the cell cycle is associated with apoptotic cell death (Skovira et al., 2016). Specifically, enhanced neurogenesis and increased proliferation of progenitor cells are observed in the hippocampus after TBI (Liu & Song, 2016). The magnitude of injury is correlated with the degree of post-TBI neurogenesis in the hippocampus (Girgis et al., 2016; Wang et al., 2016). Abnormal neurogenesis in the hippocampus may result in detrimental effects, including aberrant sprouting and migration, reduced dendritic outgrowth, and loss of newborn neurons (Gibb et al., 2015; Ibrahim et al., 2016; Robinson, Apgar & Shapiro, 2016). Taken together, CCA has an adverse impact on hippocampal function. Moreover, the cell cycle inhibitors improve the functional outcomes following TBI in several models (Kabadi et al., 2012b; Kabadi et al., 2012c; Kabadi et al., 2014). Previous studies proved that NPCs, including Nup62, regulate the gene expression at the NPC and within the nucleoplasm (Kalverda et al., 2010). Nucleoporin-chromatin interactions stimulate the cell-cycle gene expression directly inside the nucleoplasm (Casolari et al., 2004; Kalverda et al., 2010; Taddei et al., 2006). Therefore, we speculated that Nup62 affects the hippocampal function after TBI by activating the cell cycle. Nonetheless, additional in vivo and in vitro studies are required to verify this finding further. Since the present study only involves the changes in the acute phase after TBI, we observed the alteration 24 h after TBI. One limitation of the present study was the lack of experimental ethology. The experiments, such as Morris water maze and T-maze, are required to evaluate hippocampus impairment, which leads to cognitive deficits, memory difficulties, and behavioral disorders. In addition, further study is needed to address whether Nup62 dysfunction is a cause or a consequence of the hippocampus pathology in TBI to understand the exact mechanism. The correlation between the specific CCA mechanism and central nervous system damage also needs to be explored at the molecular level. The bioinformatics analysis of DEGs showed that Nup62 mRNA was significantly upregulated in the acute stage. The biochemical experiments confirmed this conclusion at the RNA and protein levels. Post-trauma, the Nup62 protein may be upregulated at the transcriptional level. The GO_BP enrichment analysis showed that the cell division of mice after TBI treatment was significantly elevated. The data from our experiments suggest that Nup62 enhances cell division in TBI mice. Further experimental investigations on cell division after TBI should be considered. Also, the long-term effect of Nup62 after TBI needs further investigation. 10.7717/peerj.14913/supp-1 Click here for additional data file. 10.7717/peerj.14913/supp-2 Click here for additional data file. 10.7717/peerj.14913/supp-3 Click here for additional data file. 10.7717/peerj.14913/supp-4 Click here for additional data file. 10.7717/peerj.14913/supp-5 Click here for additional data file. 10.7717/peerj.14913/supp-6 Click here for additional data file. 10.7717/peerj.14913/supp-7 Click here for additional data file. 10.7717/peerj.14913/supp-8 Click here for additional data file.

PMC10000306

Tian Xiang,Longjiang Wu,Murtala Bindawa Isah,Chen Chen,Xiaoying Zhang

Apocynum venetum, a medicinal, economical and ecological plant: a review update

07-03-2023

Apocynum venetum L,Medicinal plant,Fibrous plant,CiteSpace

Apocynum venetum L. is an important medicinal perennial rhizome plant with good ecological and economic value. Its leaves have many pharmacological effects such as anti-inflammatory, anti-depression, anti-anxiolytic, etc., while its fibers have the title of “king of wild fibers”. Furthermore, it was suitable for the restoration of degraded saline soil in arid areas. An increasing studies have been published in the past years. A scientometric analysis was used to analyze the publications of Apocynum venetum L. to clearly review the pharmacology, fiber application of Apocynum venetum L. and the potential value with its similar species (Apocynum pictum Schrenk) to the environment.

Apocynum venetum, a medicinal, economical and ecological plant: a review update Apocynum venetum L. is an important medicinal perennial rhizome plant with good ecological and economic value. Its leaves have many pharmacological effects such as anti-inflammatory, anti-depression, anti-anxiolytic, etc., while its fibers have the title of “king of wild fibers”. Furthermore, it was suitable for the restoration of degraded saline soil in arid areas. An increasing studies have been published in the past years. A scientometric analysis was used to analyze the publications of Apocynum venetum L. to clearly review the pharmacology, fiber application of Apocynum venetum L. and the potential value with its similar species (Apocynum pictum Schrenk) to the environment. Apocynum venetum L. (A. venetum), commonly known as “Luobuma” in Chinese and “Rafuma” in Japanese is a perennial herbaceous shrub (Fig. 1) widely distributed in the temperate regions of Asia, Europe and North America, especially in saline-alkali land, river-banks, fluvial plains and sandy soils (Grundmann et al., 2007; Jiang et al., 2021b; Xie et al., 2012). The species Apocynum venetum L. (Apocynaceae) currently includes 9 subspecies documented on World Flora Plant List (Table S1) (World Flora Online, 2022) A. venetum can adapt to extreme conditions where the surface salinity is up to 20% and the annual average precipitation is more than 250 mm, making the plant of high ecological value for the transformation of coastal saline and barren lands (Thevs et al., 2012; Yuan, Li & Jia, 2020a). A. venetum leaves has been used to produce herbal drugs and tea (Chinese Pharmacopoeia, 2020). Furthermore, since 2002 luobuma tea has been included in the list of health-care food in China (National Health Commission of the People’s Republic China, 2002). Lau et al. (2012) confirmed that A.venetum leaf extract could stimulate vascular receptor (alpha-adrenergic and angiotensin II receptors) and inhibit vasoconstriction, suggesting antihypertensive properties of the plant. Modern pharmacological investigations confirmed that A. venetum has, among other effects, anti-inflammatory, anti-depression, anti-anxiolytic, anti-ageing, antioxidants, cardiotonic and hepatoprotective effects (Du et al., 2020; Grundmann et al., 2007; Xie et al., 2015; Xie et al., 2012). A. venetum fiber, known as the “king of wild fibers”, is receiving increasing attentions in the apparel industry owing to its additional advantage of possessing antibacterial properties (Han et al., 2008; Wang, Han & Zhang, 2007; Xu et al., 2020a). Alongside the rapid increase in A. venetum-related studies, systematic and comprehensive analyses on A. venetum publications is essential. We have previously reviewed the traditional uses, phytochemistry and pharmacology of A. venetum (Xie et al., 2012). As a timely update, this article aims to respond to the rapidly increasing literature on A. venetum studies by: (i) conducting scientometric analysis of the publications on A. venetum and (ii) reviewing the progress recorded on the exploration of the medicinal, economical and ecological benefits of the plant from 2012 to date. For the scientometric analysis, we used Citespace, which is a specifically designed to facilitate the detection of emerging trends and mutations in the scientific literature (Chen et al., 2012). Web of Science Core Collection (WoSCC; Clarivate Analytics, London, UYK) is the premier resource on the Web of Science platform. It is considered as the most trusted citation index on many research topics (Wu, Yakhkeshi & Zhang, 2022). This work can provide researchers and readers with a comprehensive information on A. venetum, covering the areas of phytopharmacy and pharmacology, functional food, ecology, and applications in textile and fiber industry. Data were collected from WoSCC with the following search strategy: Topical Subject = (“Apocynum venetum” OR “Luobuma”) OR Title = (“Apocynum venetum” OR “Luobuma”) OR Abstract = (“Apocynum venetum” OR “Luobuma”). The searched time spans 1987–2022, the type of literature was article and review, and the language was English. Our search strategy did not limit the impact factor of journals and the affiliation of authors. A total of 200 publications were obtained, including 190 articles and 10 reviews, and their full record with the cited references was exported in plain text format. CiteSpace 6.1.3 was used to analyze keywords of the literatures, with the time partition set to 1987–2022, the time slice set to 1, the node types set to keyword, G-index set to 25, and the pathfinder, pruning sliced networks and pruning the merged network were used to trim the atlas. Based on the result of keywords analysis of CiteSpace, the chapter topics were divided into the pharmacological effects and related components of A. venetum, A. venetum fiber, other Apocynum species similar to A. venetum: Apocynum pictum Schrenk, and the ecological value of A. venetum and A. pictum, and the topics were discussed. The discussion on the bioactive components cover the period 2012–2022. Keywords represent the core content of an article and provide information on the topic or the important category to which an article belongs. The keywords with high frequency and highly mediated centrality were analyzed and presented in the form of a visual mapping through the Citespace software (Fig. 2). The most frequent keywords from 1987–2022 were Apocynum venetum L. (111), Apocynum venetum leaves (52), Apocynum venetum leaf extract (31). The keywords with the highest centrality before 2018 include Apocynum venetum L. (0.49), component (0.28), hepatoprotective activity (0.27), identification (0.25) and antioxidant (0.23) (Fig. 2A, Table 1). However, after 2018, among the top ten keywords showing the highest centrality, two words that are poorly correlated with Apocynum venetum leaves appeared: Apocynum venetum fiber (0.28) and Apocynum pictum schrenk (0.27). These results implied that the studies before 2018 mainly focused on the components and the pharmacological effects of Apocynum venetum leaves while Apocynum venetum fiber and Apocynum pictum Schrenk have also attracted the attention of researchers in recent years. Based on the keyword co-linear graph (Fig. 2A), the parameter of “burstiness” was set to γ = 0.5, minimum duration = 1. Sixteen burst entries were generated. Among them, the words that have kept the outbreak status were oxidative stress (3.93), Apocynum venetum fiber (3.26), identification (2.68) and tolerance (2.0) (Fig. 2B). These data confirmed that apart from the further in-depth pharmacological investigations, the fiber of this plant has recieved attention in recent years. In addition, the ecological value of Apocynum venetum L and Apocynum pictum Schrenk L has attracted increasing attentions. With the deepening of research and the technological improvement in high performance liquid chromatography, mass spectrometry etc., many phytochemicals of A.venetum have been identified and isolated. Some of these phytochemicals were flavonoids such as hyperoside and isoquercetin, which bioactivities have been comprehensively reviewed previously (Xie et al., 2016a; Xie et al., 2016b; Xie et al., 2012). Since then, more studies have reported on the isolation and bioactivities of known and novel flavonoids from A.venetum. The flavonoids isolated from A. venetum since 2012 are listed in Table 2 and their structures shown in Fig. 3. Kaempferol, quercetin, isoquercitrin (quercetin-3-O-β-D-glucose) and astragalin (kaempferol-3-O-β-D-glucose) isolated from A. venetum leaves have significant anti-depressant activities in mice (Yan et al., 2016). Hyperoside isolated from the leaves of A. venetum showed antidepressant-like effect in P12 cell lines which could improve neuronal viability by protecting neurons from corticosterone damage (Zheng et al., 2012). Hyperoside had protective effect on H2O2-induced apoptosis of human umbilical vein endothelial cells (Hao et al., 2016). For acetaminophen-induced liver injury, both hyperoside and isoquercetin exerted hepatoprotective effect by upregulating the expression and activity of detoxifying enzymes such as sulfotransferases (hyperoside could also increase activities of UDP-glucuronosyltransferase) in liver microsomes and inhibited the activity of cytochrome P450 2E1, accelerating the harmless metabolism of acetaminophen. Additionally, isoquercetin could significantly inhibit acetaminophen induced oxidative stress and nitrosative stress (Xie et al., 2016a; Xie et al., 2016b). Isoquercitrin, isolated from the A. venetum leaf aqueous extract exerted anti-obesity effect in high fat diet induced obese mice by inhibiting adenosine 5′-monophosphate-activated protein kinase (AMPK)/sterol regulatory-element binding protein (SREBP-1c) signaling pathway, glucose uptake, and glycolysis flux. C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P are potential target proteins of isoquercitrin (Manzoor et al., 2022). 8-O-methylretusin (Fig. 3) isolated from A venetum leaves showed antifouling activity (Kong et al., 2014). On the other hand, 4′,7-dihydroxy-8-formyl-6-methoxyflavone isolated from A venetum leaves showed high anti-inflammatory activity via significant inhibitory effect on the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) (IC50 values were 9.0 ± 0.7 and 42.1 ± 0.8 µM, respectively) in lipopolysaccharide-induced mouse peritoneal macrophages (RAW 264.7) (Fu et al., 2022). Wang et al. (2020) investigated the absorption and metabolism of quercetin-3-O-sophoroside, isolated from the leaves of A. venetum, in rats. The results indicated that quercetin-3-O-sophoroside was completely absorbed in the small intestine and metabolized in the jejunum to sulfated quercetin-3-O-sophoroside, methylated quercetin-3-O-sophoroside, and methylated quercetin-3-O-sophoroside sulfate. Quercetin-3-O-sophoroside was deglycosylated to aglycones by the cecal microbiota to form derivatives of benzoic, phenylacetic and phenylpropionic acids (Wang et al., 2020). To obtain larger amounts of flavonoids, A. venetum hairy roots were induced with Agrobacterium rhizogenes strain Ar.1193, and 117 kinds of flavonoids were detected in the roots. The flavonoid content and antioxidant activity of the roots were significantly increased as compared to field-planted roots, therefore, this technique could be used for large-scale production of flavonoids from A. venetum (Zhang et al., 2021). Natural polysaccharides have been proved to possess, among other effects, immune regulatory, anti-oxidative and anti-inflammatory activities, as well as having the advantages of being safe and non-cytotoxic (Liu et al., 2022). Zhou et al. (2019) used different concentrations and kinds of solvents (HCl, H2O, NaOH) to extract polysaccharides from A. venetum leaves. The results showed that the polysaccharide yield was the highest with 21.32% (w/w), 0.5 M NaOH at 90 °C, and the bioactivity of the alkaline extracted polysaccharides was the strongest, which was reflected in the antioxidant capacity (DPPH and ABTS radical scavenging activities) and α-glucosidase and lipase inhibitory activities. The 0.5 M NaOH extracted polysaccharides showed a strong inhibitory activity on α-glucosidase (IC50 value of 16.75 µg/mL), which was better than the positive control, acarbose (IC50 value of 1,400 µg/mL). In addition, the alkaline polysaccharide-rich extracts were proved to possess hypoglycemic and hypolipidemic effects on mice with high fat diet induced and streptozotocin-induced type 2 diabetes. Moreover, the extract reversed intestinal dysbiosis by increasing the abundance of Odoribacter, Anaeroplasma, Muribaculum, Parasutterella and decreasing the abundance of Enterococcus, Klebsiella, Aerococcus in diabetic mice (Yuan et al., 2020b). Some polysaccharides were also isolated from various parts of A. venetum and validated for bioactivity. These are summarized in Table 3. ALRPN-1 and ALRPN-2 exerted a significant anti-inflammatory activity in lipopolysaccharide-induced macrophages by regulating the levels of pro-inflammatory mediators (NO) and cytokines (TNF-α, interleukin-6, interleukin-1 β) and the mechanism may involve, in part, extracellular signal-related kinase (ERK)/mitogen-activated protein kinases (MAPKs) signaling pathway (Liu et al., 2022). Vp2a-II and Vp3 obtained from the flowers of A. venetum showed anticoagulant activity and immunoregulation. The anticoagulant activities of Vp2a-II and Vp3 were assayed in vitro by plasma coagulation parameters (activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), fibrinogen). The results showed that Vp3 significantly prolonged TT and PT, while Vp2a-II significantly prolonged APTT and TT, indicating that the two polysaccharides could inhibit blood coagulation (Wang et al., 2019b). In addition, the polysaccharides could exert immunomodulatory effects by promoting phagocytic activity, enhancing NO secretion and mRNA expression of inducible nitric oxide (iNO) synthase, interleukin-6 and TNF-α which activate RAW264.7 cells. Vp2a-II might activate the MAPK signaling pathway, which then induce the nuclear translocation of NF-κB p65 (Wang et al., 2022). In addition to the pharmacological effects of A. venetum polysaccharides, researchers have also began exploring their other properties. The polysaccharide conjugates (ATPC-A) extracted from A. venetum tea residues with an alkaline solution (0.10 M NaOH) had emulsifying properties and stabilized the emulsion which comprised of amphipathic polysaccharides covalently bound to proteins. The stability of the neat ATPC-A emulsions with a concentration equal to or greater than 1.00 weight % was higher than 5.00 weight % gum arabic during storage at different temperatures and pH values (Chen et al., 2022b). Many studies have reported other phytochemicals from A. venetum leaf extracts and their pharmacological effects. The ethanol extract of A. venetum leaf possesses anti-cancer activity. A fraction separated from the extract could inhibit the proliferation of Human PCa cells tumor cells. Lupeol accounted for approximately one-fifth (19.3% w/w) of the components of the fraction and was implicated for the induced cytotoxicity against PCa cells. The fraction and lupeol elicited similar anti-proliferative mechanisms, involving: regulating apoptosis signal molecules (P53, cytochrome c, Bcl-2, and caspase 3 and 8), promoting G2/M arrest through impairing the DNA repair system via downregulating the expression of uracil-DNA glycosylase, as well as downregulating the expression of β-catenin (Huang et al., 2017). In preventing D-galactose-induced oxidative damage in mice, the polyphenol extract of A. venetum was superior to the antioxidant vitamin C (Guo et al., 2020). Within its safe concentration range (0–100 µg/ml), the polyphenol extract of A. venetum inhibited U87 glioma cell proliferation and caused cell apoptosis by affecting NF- κB and genes of other relevant pathways (Zeng et al., 2019). Additionally, A. venetum leaf extract inhibited doxorubicin induced cardiotoxicity through (protein kinase B) Akt/(B-cell lymphoma-2) Bcl-2 signaling pathway (Zhang et al., 2022). The efficacy and mechanism of action of individual chemical components, as well as their possible synergistic effects, of A. venetum leaf extract need to be further investigated. In addition to flavonoids, polysaccharides and polyphenols, sterols (β-sitosterol, sitgmasterol), triterpenoids (lupeol, uvaol), glycolipids (apocynoside I), natural lignan glycoside (alloside of benzyl alcohol) and amino acids have been isolated from A. venetum (Huang et al., 2017; Sun et al., 2022). A. venetum flowers are rich in free amino acids, accounting for about 3% of the total dried weight, including leucine (13.71 µg/mg), isoleucine (7.86 µg/mg), lysine (2.22 µg/mg), tryptophan (1.67 µg/mg) and valine (1.20 µg/mg) (Jin et al., 2019). Uvaol from A. venetum leaves had potent anti-inflammatory effects on dextran sulfate sodium-induced experimental colitis and lipopolysaccharide-stimulated RAW264 cells (Du et al., 2020). Validation of the activities of other components in A. venetum should be the focus of future studies. The fiber of A. venetum has been used in textile and paper industries with superior properties compared to other commonly used fibers. Fiber from Apocynum species has a higher average length to diameter ratio (up to 1219) compared to kenaf (209), another natural plant fiber (Liu et al., 2020; Wang, Han & Zhang, 2007; Xie et al., 2012). Another reason for the popularity of A. venetum fabric is the antibacterial effect that A. venetum fiber naturally possesses (Li et al., 2012; Song et al., 2019). Such antibacterial activity might be because: (i) A. venetum fiber has small openings between microstructures, which improve the breathability of the A. venetum fabric, which subsequently destroy the environment for bacterial growth (Han et al., 2008); (ii) the A. venetum stem cells contain tanning agents, which is resistant to microbial decomposition (Thevs et al., 2012); (iii) the presence of water-insoluble polyphenol derivatives confers antimicrobial properties to the fabric (Xu et al., 2020a). A. venetum is rich in cellulose, but impurities such as pectin, lignin, and waxes must be removed to produce clean fibers (Lou et al., 2019). In the direction of environmental safety and high efficiency, various degumming methods have been proposed, including chemical degumming, biological degumming and microwave-assisted ultrasonic degumming. A study revealed that microwave-assisted ultrasonic degumming showed the advantages of requiring less chemical reagents during degumming (1 kg raw A. venetum bast needed 0.6 kg of reagents while the chemical degumming treatment required 1.34 kg) and shorter time, as well as higher quality (low residual gum content of 5.15%; lignin content less than 3%; whiteness more than 80% in the refined A. venetum fibers) (Li et al., 2020). Degumming methods and the fiber quality of A. venetum reported from 2012 to 2022 are listed in Table 4. In addition to the textile industry, A. venetum fiber also has many potential applications in medicine as well as in the construction industry. Microcrystalline cellulose (MCC-N) from A. venetum fibers was shown to have a rougher structure and less macrostructure than commercially available microcrystalline cellulose (MCC-C). MCC-N had a crystallinity of up to 78.63% and a thermal stability comparable to that of MCC-C, which made it suitable as a load-bearing material for composite structures, and could be used in polymer composites with high temperature resistance (Halim, 2021). Furthermore, cellulose nanofibers (CNFs) from A. venetum straw were added into poly lactic acid (PLA), and the prepared PLA/CNFs film did not only improve the wettability and permeability of PLA, but also had superior antibacterial properties (the antibacterial growth inhibition rate on Escherichia coli and Staphylococcus aureus were 96.31% and 92.83% at PLA/6% (w/w) CNFs film, respectively). Then, polyvinyl pyrrolidone was added to this film to form a sustained-release nanofiber membrane (PLA/drug-loaded PVP nanofiber membranes), and a purified sea buckthorn was embedded in the drug-loaded film to evaluate its performance. The nanofiber membrane extended and sustained the release of purified sea buckthorn, and the cumulative release reached a maximum of 75.41%. It showed the advantage of a profile with a high initial release followed by a slow diffusion phase (Wang et al., 2021b; Wang et al., 2019a). In addition, when the hydrogel was prepared with chitosan as the matrix, the addition of CNFs improved the mechanical properties and swelling rate of the chitosan-based hydrogel. As the CNFs was 1.5%, the compressive strength of the hydrogel increased by nearly 20%, the swelling capacity reached 140%. In this form, the antibacterial efficacy against Escherichia coli and Staphylococcus aureus were 98.54% and 96.15%, respectively (Wang et al., 2021a). See Abubakar, Gao & Zhu (2021) for further details on the composition, properties and degumming methods of A. venetum fiber. Due to excessive exploitation, wild A. venetum has declined in recent years. A similar species, Apocynum pictum Schrenk (Apocynum hendersonii Hook) is often used in the market as a substitute for A. venetum due to their similarity in morphological characteristics and geographical distribution. The incorporation of A. pictum may affect the safety and effectiveness of A. venetum (An et al., 2013; Chan et al., 2015; Zheng et al., 2022). Although A. pictum has not been included in the Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2020), some studies have reported that it is an important medicinal plant (Gao et al., 2021; Jiang et al., 2021a). For the quality control of A. venetum and to explore the potential application of A. pictum, some studies compared the similarities and differences between the two species in terms of genome size, flavonoid content, chemical composition and biological activity. The whole genomes of the two species were both small and similar, with 232.80 megabase (A. venetum) and 233.74 megabase (A.pictum). The contents of quercetin, hyperoside and total anthocyanin in A. venetum were much higher than those of A. pictum, which was considered to be the reason for the difference in color between the two species (Gao et al., 2019). Hyperoside could be a suitable chemical marker to distinguish between the two species (Gao et al., 2019). In addition, A. venetum has a better antioxidant activity than A. pictum (Chan et al., 2015). However, recent studies have shown that the flavonoids from A. pictum (quercetin-3-sophoroside, isoquercetin, quercetin-3-O-(6-O-malonyl)-galactoside) and A. venetum (hyperoside, isoquercetin, quercetin-3-O-(6-O-malonyl)-galactoside, quercetin-3-O-(6-O-malonyl)-glucoside, and quercetin-3-O-(6-O-acetyl)-galactoside) both exhibited significant antimicrobial activity against methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa and the fungus, Aspergillus flavus, but A. pictum was superior to A. venetum in terms of antimicrobial capacity (Gao et al., 2021). Apart from the pharmacological value, in recent years, A. pictum is often studied together with A. venetum because of its high ecological value. Phytoremediation is one of the appropriate ways to deal with land problems such as drought, salinity and metal pollution (Pilon-Smits, 2005). Apocynum spp. were selected to stabilize sands and restore the degraded saline lands due to their advantages of easy propagation, resistance to harsh environment, and high economic value (Jiang et al., 2021a; Jiang et al., 2021b). The matured seeds of A. venetum appeared to possess higher drought tolerance than seeds of A. pictum. The simulation of the critical values of Apocynum spp. seeds under PEG-6000 simulated drought conditions are summarized in Table 5. Different PEG-6000 concentrations (0%–35%) was used to simulate natural drought conditions to study the effect of drought stress on the germination of Apocynum spp. seeds. The results showed that low concentrations PEG (0–20%) had no significant impact on the germination rate of Apocynum spp. seeds. However, when the concentration was more than 20%, the germination rates of the seeds were reduced, and the negative impact on A. pictum seeds was higher than that on A.venetum. In addition, after the drought stress was alleviated, the seeds were able to germinate under appropriate conditions (Han et al., 2021; Jiang et al., 2021a). Moreover, the membership function (A mathematical tool for representing fuzzy sets) was used to comprehensively evaluate the drought resistance of A. venetum and another desert economic plant, Lycium ruthenicum, by analyzing the physiological and biochemical indices (the content of chlorophyll a, chlorophyll b, proline and soluble sugar, antioxidant enzyme activity, etc.). The results showed that when the soil moisture content was 9.70%, 6.89% and 5.54%, the drought resistance of A. venetum was stronger than that of Lycium ruthenicum (Wang, 2017). Low concentration of salt solution (0–200 mM NaCl) had no significant effect on the germination rate of current season mature seeds the two species (Jiang et al., 2021a; Shi et al., 2014). However, another study showed that under 200 mM NaCl stress, the growth and development of A. venetum seedlings were inhibited, the phenotypic characteristics (plant height, root length, leaf length, leaf width) were damaged, and the total flavonoid content decreased. However, salt stress increased the content of quercetin and kaempferol in seedlings (Xu et al., 2020b). In addition, the seeds of Apocynum spp. both exhibited high tolerance to lithium salts during germination, particularly LiCl (Table 5) (Gao et al., 2020; Jiang, Wang & Tian, 2018a; Jiang et al., 2018b). The simulated critical value of A. venetum was as high as 196 mM (Jiang, Wang & Tian, 2018a). To put the salt tolerance of A. venetum into perspective, Brassica carinata, another heavy metal tolerant plant with phytoremediation potential, has a germination rate of less than 50% at LiCl concentration above 120 mM (Li et al., 2009). Notably, the addition of lithium in soil did not reduce the concentrations and antioxidant capacity of total flavonoids, rutin and hyperoside in A. venetum leaves (Jiang et al., 2019). Therefore, Apocynum spp. are suitable for the restoration of degraded saline soil in arid areas, and are promising species in the remediation of lithium pollution in the environment (Jiang et al., 2021a; Jiang et al., 2021b; Rouzi et al., 2018). Looking back on the research history of A. venetum, the research focuses mainly on the components and pharmacological effects of A. venetum leaves. At present, many of the pharmacological effects are attributable to flavonoids, however these active components and their synergistic mechanism need to be further studied. In addition to flavonoids, some polysaccharides (Vp2a-II, Vp3) and triterpenoid (uvaol) from A. venetum have also shown pharmacological effects. However, the current research in this area is still lacking. In recent trends, the fiber of A. venetum have attracted attention. Apart from its textile value, the potential application of the fiber in other industries needs further exploration in future studies. The ecological value of Apocynum spp. is gradually being revealed by multiple research. This study provided rich and rigorous CiteSpace analysis on A. venetum. However, as a limitation, we analyzed only the papers written in English, and within the WoS database, therefore it may not be comprehensive enough to reflect the entire research status. For example, we searched a major Chinese scientific literature database, the China National Knowledge Infrastructure (CNKI), and more than 2,000 Apocynum related publications were retrieved, although these were not within the analysis scope of the current study. This further attests to the interest Apocynum species have received from the scientific community over the past decades. 10.7717/peerj.14966/supp-1 Click here for additional data file. 10.7717/peerj.14966/supp-2 Click here for additional data file.

PMC10000335

Abstracts from the World Congress of Cardiology/Brazilian Congress of Cardiology 2022

07-03-2023

Cardiology,Global Health,Heart Health,Cardiovascular Disease,Conference

These are the abstracts from the combined 77th Brazilian Congress of Cardiology, together with the World Congress of Cardiology, held in October 2022. From 1950 to today, the World Heart Federation’s World Congress of Cardiology (WCC) has been a key event on the cardiovascular calendar, offering a global perspective on cardiovascular health and bringing together thousands of cardiology professionals from all over the world with one common goal: to reduce the global burden of cardiovascular disease and help people live longer, healthier lives.

Abstracts from the World Congress of Cardiology/Brazilian Congress of Cardiology 2022 These are the abstracts from the combined 77th Brazilian Congress of Cardiology, together with the World Congress of Cardiology, held in October 2022. From 1950 to today, the World Heart Federation’s World Congress of Cardiology (WCC) has been a key event on the cardiovascular calendar, offering a global perspective on cardiovascular health and bringing together thousands of cardiology professionals from all over the world with one common goal: to reduce the global burden of cardiovascular disease and help people live longer, healthier lives. 107880 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANTONIO DE PADUA MANSUR1, Carlos Henrique Del Carlo1, José Antonio Ramos Neto1, André Barbosa de Abreu1, Airton Roberto Scipioni1, Antonio Carlos Pereira Barreto1 (1) Insituto do Coração – HC FMUSP Background: Chronic Chagas cardiomyopathy (CCC) is one of the leading causes of congestive heart failure (CHF) in Latin America and carries a high morbidity and mortality burden. Previously, it was believed that there was no epidemiological and clinical evidence of a gender-associated risk of death in patients with CCC. Purpose: To analyze the mortality of congestive heart failure due to CCC in women and men. Methods: From February 2017 to September 2020, we followed a cohort of patients with CHF (Framingham criteria) due to CCC in a single-center outpatient clinic. Appropriate serologic tests defined Chagas disease. Baseline data included clinical characteristics and echocardiographic findings. Statistical analyses were performed with the Kaplan-Meier (K-M) method to analyze time-to-event data and the Cox proportional hazards methods to search for predictors of death. Results: We studied 733 patients, mean of 61.4 ± 12.3 years, 381 (52%) males. Females were older (63.0 ± 11.9 vs. 60 ± 12.4 years; p = 0.01), had a higher baseline mean left ventricular ejection fraction (LVEF) (44.5 ± 14.6% vs. 37.3 ± 14.8%; p < 0.001), and a lower left ventricular diastolic diameter (LVDD) (56.7 ± 8.9 vs. 62.4 ± 9.4 mm; p < 0.001). Over a 3-years follow-up period, 168 (44%) men and 126 (36%) women died (K-M: log-rank p = 0.002; Figure). Women had more implantable pacemakers (PM) (26.1% vs. 16.5%; p = 0.002) and men more implantable cardioverter-defibrillators (ICDs)(20.7% vs. 12.5%; p = 0.003). Heart transplant occurred in 10.8% of men and 7.4% in women (p = NS). Cox regression for death adjusted for age, previous myocardial infarction, diabetes, previous stroke, chronic kidney disease (CKD), atrial fibrillation, PM, ICD, heart transplant and LVEF, showed, in descending order, previous stroke (HR = 2.4; 95%CL:1.5–3.6), diabetes (HR = 2.0; 95%CL: 1.3–3.1), and CKD (HR = 1.8; 95%CL:1.3–2.6) as the main predictors of death in men, and in women diabetes (HR = 2.2; 95%CL:1.4–3.4), previous stroke (HR = 1.8; 95%CL:1.1–2.9), and CKD (HR = 1.7; 95%CL:1.1–2.7). Conclusions: Women had a better prognosis than men but similar predictors of death. Control of diabetes and prevention of stroke and CKD could significantly reduce the death rate in CHF due to CCC. 108498 Modality: WHF Abstracts – Young Researcher Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 14/10/2022 H: 16:50/17:50 L: Auditório 14 JOSÉ LUCAS PERES BICHARA1, José Lucas Peres Bichara1, Luiz Antônio Viegas de Miranda Bastos1, Paolo Blanco Villela1, Glaucia Moraes de Oliveira1 (1) Universidade Federal do Rio de Janeiro – UFRJ Introduction: Cardiovascular diseases (CVD) are the main causes of death in the world and in Brazil, and ischemic heart diseases (IHD) are one of the main responsible for these statistics. Previous studies have already suggested a relationship between the evolution of IHD mortality rates and socioeconomic indicators. Objective: To relate the evolution of IHD mortality rates and the sociodemographic index (SDI) from 2000 to 2019 in Brazil and in its federative units (FUs). Methods: Ecological time series study of deaths from IHD in Brazil. Crude and standardized mortality rate for IHD were analyzed by sex, age group and FU between 2000 and 2019. Data were correlated with the SDI. Deaths and population were taken from DATASUS to estimate crude and standardized mortality rates per 100,000 inhabitants (direct method with Brazilian population in 2000). The SDI for each UF was extracted from the Global Health Data Exchange website. Results: In the period, there were 1,968,160 deaths from IHD in Brazil, 58.19% of which were male. The national SDI ranged from 0.538 in 2000 to 0.64 in 2019, with constant growth in the period. Concomitantly, the age-standardized mortality rate for IHD decreased from 46.12/100,000 inhabitants to 36.42/100,000 inhabitants. Thus, IHD has become the leading cause of mortality in the country. In the FUs, all states in the North and Northeast regions showed improvements in the SDI, however, the best indicators continued to be concentrated in the other regions. In 2000, the highest mortality rates from IHD were found in the South, Southeast and Midwest regions of the country, but they showed a significant reduction. When evaluating the variation of the standardized mortality rate in the period, it was noted that the FUs with the best SDI were responsible for the greatest drops (graph 1). Conclusion: During the period, the country showed a significant improvement in socioeconomic indicators accompanied by a reduction in IHD mortality rates. When evaluating the FUs, it was noted that those with better socioeconomic indicators were able to obtain a greater reduction in these mortality rates. 108502 Modality: WHF Abstracts – Researcher Category: NEGLECTED CARDIOVASCULAR DISEASES D: 14/10/2022 H: 13:50/14:50 L: Auditório 14 ADRIANA SOARES XAVIER DE BRITO1, Renata Junqueira Moll-Bernardes1, Martha Valeria Tavares Pinheiro1, Paulo Henrique Rosado de Castro1, Gabriel Cordeiro Camargo1, Adriana Pereira Glavam1, Sergio Altino de Almeida1, Fabio Paiva Siqueira1, Marcelo Teixeira de Holanda2, Luiz Henrique Conde Sangenis2, Fernanda de Souza Nogueira Sardinha Mendes2, Andrea Silvestre de Sousa2 (1) D’Or Institute for Research and Education (IDOR), Rio de Janeiro; (2) Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation Introduction: Chagas disease continues to be an important cause of morbidity and mortality in Latin America. Sudden cardiac death (SCD) represents the most dramatic course of Chagas cardiomyopathy (CC), and it is closely related to the presence of ventricular arrhythmias and myocardial dysfunction. However, there is also a high incidence of SCD when left ventricular ejection fraction (LVEF) is normal or mildly depressed. Unfortunately, despite its significant mortality, there is no clear recommendation for early cardio-defibrillator implantation in patients with CC. Ideally, the risk of SCD should be evaluated in earlier stages of the disease, but important questions remain unresolved regarding its pathophysiological mechanism and the diagnostic tools. New imaging parameters to identify the genesis of arrhythmia such as fibrosis, inflammation and dysautonomia can be a promising strategy. Purpose: We tested the correlation between the extent of myocardial sympathetic denervation, myocardial perfusion, fibrosis and the severity of the ventricular arrhythmia in patients in the early phase of CC. Methods: Twenty-nine patients with CC and LVEF >45% prospectively underwent magnetic resonance (MRI), SPECT imaging of myocardial sympathetic innervation using 123Iodine-MIBG (MIBG) and rest myocardial perfusion with 99mTc-sestamibi (MIBI), and were divided into two groups: arrhythmic group (n = 15): >120 ventricular ectopic beats and/or Non-Sustained Ventricular Tachycardia (NSVT) and non-arrhythmic group (n = 14): <120 ventricular ectopic beats without NSVT on 24-h Holter monitoring. Results: Compared to non-arrhythmic, the arrhythmic group had significantly higher denervation score (mean ± SD 23.2 ± 18.7 versus 5.6 ± 4.9; p < 0.01), higher summed rest perfusion score (mean ± SD 4.7 ± 6.8 versus 0.3 ± 0.6; p < 0.05) as well as the higher mismatch in the innervation/perfusion score between MIBG and MIBI images, which evaluates the extent of denervated but viable myocardium (mean ± SD 18.4 ± 17.5 versus 5.3 ± 4.7; p < 0.01). There was a correlation between myocardium denervation (r 0.555; p < 0.01), hypoperfusion (r 0.562; p < 0.01), and interstitial fibrosis evaluated by extracellular volume at MRI. Conclusion: The combination of different imaging parameters to assess autonomic innervation, myocardial perfusion and fibrosis may allow better understanding of the pathophysiology and risk stratification for SCD in the early stages of CC. 108593 Modality: WHF Abstracts – Researcher Category: NEGLECTED CARDIOVASCULAR DISEASES D: 14/10/2022 H: 13:50/14:50 L: Auditório 14 BRUNO OLIVEIRA DE FIGUEIREDO BRITO1, Bruno Oliveira de Figueiredo Brito1, Emilly Malveira de Lima2, Elsayed Z. Soliman3, Maria Fernanda Lima-Costa4, Antonio Luiz Pinho Ribeiro1 (1) Hospital das Clínicas and Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; (2) Department of Statistics, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; (3) Department of Epidemiology and Prevention, Wake Forest School of Medicine, Winston-Salem, NC, USA; (4) Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Brazil The electrocardiogram (ECG) plays a key role in the evaluation of Chagas disease (ChD). The ECG shows progressive abnormalities that indicate worsening myocardial damage. There is no information about the evolution of the ECG of elderly individuals with ChD. Objective: To compare the evolution of electrocardiographic abnormalities in Trypanosoma cruzi chronically infected individuals to that of the non-infected (NChD) elderly in a follow-up of 14 years of the Bambui Cohort Study of Aging in Brazil. Methods: A digitally recorded 12-lead ECG of each individual was obtained at the baseline examination in 1997, in 2002 and in 2008, and was classified by the Minnesota Code criteria. The influence of ChD on the ECG evolution was assessed by the semi-competing risks methods considering a new ECG abnormality as the primary event and death the terminal event. The group with ChD was compared with the NChD group. A Cox regression model was conducted separately in ChD and NChD groups, in a landmark point at 5.5 years until the end of follow-up. The individuals of both groups were compared according to the following categories: No major: individuals without major abnormalities in the first and second visits; Maintained major: individuals who had the number of major abnormalities in second visit equal to the first visit, More major: individuals who had major abnormalities in the first visit and gained more abnormalities in the second visit and New major: individuals who had no abnormalities in the first visit and gained any major abnormality in the second visit. Results: Among the 1,462 participants, 557 had ChD. The median age was 68 years for patients with ChD and 67 for patients without ChD. Chagas disease was independently associated to the occurrence of new major ECG abnormalities in the multivariate analysis HR: 2.89 (95% CI 2.28–3.67). Compared with the group of ChD No major, the risk of death was HR: 2.48 (95% CI 1.43–4.28) for the Maintained, HR: 1.95 (95% CI 1.04–3.69) for the New major, and HR: 2.77 (95% CI 1.57–4.88) for the More Major. Compared with the group of ChD Maintained major, the risk of death was HR: 0.79 (95% CI 0.44–1.42) for the group New Major and HR: 1.11 (95% CI 0.66–1.89) for the group More Major. Conclusion: Even in advanced ages, the patients with ChD have higher risk than NChD of developing new abnormalities in the ECG. It is enough for individuals with ChD to have ECG abnormalities for their risk of death to increase. 108671 Modality: WHF Abstracts – Young Researcher Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 14/10/2022 H: 16:50/17:50 L: Auditório 14 GAUTAM SATHEESH1, Mohammad Abdul Salam2 (1) The George Institute for Global Health, Hyderabad, India; (2) The George Institute for Global Health, University of New South Wales, Sydney, New South Wales, Australia Introduction and/or rationale: Heart failure (HF) is a leading global health burden that disproportionately affects patients in low- and middle-income countries with weaker health systems, such as India. Underutilization of guideline-recommended medical therapy (GRMT)—causing large gaps in evidence and clinical practice—can be mitigated through the adoption of GRMT in the EMLs. Objectives: To compare the adoption of GRMT across the global (WHO) EML and the national and state EMLs of India. Methods: We collated a list of medicines recommended by American (American College of Cardiology Foundation, the American Heart Association, and the Heart Failure Society of America; 2017), European (European Society of Cardiology; 2021), and relevant Indian guidelines (Cardiological Society of India; 2018). We assessed the adoption of these medicines in WHO EML (2021), India’s latest national EML (2015), Ministry of Health and Family Welfare’s free medicines list (2020), and 23 Indian state EMLs. We only included ‘evidence-based medicines’, i.e., those supported by relevant randomized controlled trial data for HF. Therefore, we did not include medicines listed as ‘commonly used’ without relevant evidence. We also excluded medicines listed for use in selected patients only (e.g., milrinone, levosimendan, nesiritide etc.). Results: The adoption of GRMT by WHO EML 2021 was 48% (n = 17), ranging from 75% (beta-blockers) to 0% (SGLT-2 inhibitors). GRMT adoption by Indian EML was 35% (n = 11), ranging from 50% (mineralocorticoid-receptor antagonists) to 0% (SGLT-2 inhibitors). On average, Indian state EMLs contained 36% [18% (Punjab)–53% (Rajasthan)] of GRMT. The lowest adoption (16%; n = 6) was observed in the Ministry of Health’s free medicines list, which omitted several major first-line medicine classes. Newly included GRMT, including angiotensin-receptor neprilysin inhibitors and SGLT-2 inhibitors, are yet to be listed in global and national EMLs. Further, at least one emergency medicine was included in all EMLs. Conclusion: Inclusion of GRMT for HF remains suboptimal in the global as well as the national and state EMLs of India. EMLs guide medicine selection and procurement in limited-resource settings. Considering India’s increasing cardiovascular disease burden and the potential for EMLs to improve availability and affordability of GRMT, optimizing India’s national and state EMLs and particularly its public sector (free medicines) list, is vital. 109112 Modality: WHF Abstracts – Young Researcher Category: NEGLECTED CARDIOVASCULAR DISEASES D: 14/10/2022 H: 16:50/17:50 L: Auditório 14 DENISE MAYUMI TANAKA1, Camila Godoy Fabricio1, José A. Marin-Neto1, Antônio Carlos Leite de Barros Filho1, Luciano Fonseca Lemos de Oliveira3, Jorge Mejia4, Rafael Ribeiro Almeida2, Maria de Lourdes Higuchi2, Edecio Cunha Neto2, Minna Moreira Dias Romano1, Marcus Vinícius Simões1 (1) Medical School of Ribeirao Preto – University of São Paulo, Sao Paulo, Brazil; (2) Heart Institute (InCor), Faculty of Medicine – University of Sao Paulo, Sao Paulo, Brazil.; (3) Physiotherapy Department – Federal University of Minas Gerais, Brazil; (4) Hospital Israelita Albert Einstein, Sao Paulo, Brazil. Background: Microvascular myocardial perfusion defect (MPD) is frequent in chronic Chagas cardiomyopathy (CCC) and may be involved with the progression of left ventricular systolic dysfunction (LVSD). However, the histopathological meaning of MPD in CCC and its correlation with endothelial activation is scarce. Purpose: To investigate the correlations between MPD detected in vivo with functional and histopathological changes in the model of CCC in hamsters. Methods: 24 female hamsters were studied 8-months after intraperitoneal infection with 35,000 trypomastigote forms of T. cruzi. All animals were submitted to rest high-resolution 99mTc-Sestamibi-SPECT myocardial perfusion scintigraphy and echocardiography in vivo. The area of MPD was assessed by calculating polar maps using dedicated software (MunichHeart®). After euthanasia, we performed a histopathological study of cardiac inflammation and fibrosis and mRNA expression for TNF-alfa and ICAM to assess inflammation and endothelial activation, respectively. Results: 17 animals presented MPD (71%) – extension ranging from 1.4 to 30.3% of LV surface. Animals with MPD present lower values of LVEF (38.5 ± 11.2%) when compared with animals without MPD 48.4 ± 9.1%, p = 0.04), and a trend to higher intensity of myocardial inflammation in animals with MPD (540.4 ± 153.6 cell/mm2) vs. without MPD (409.6 ± 130.3 cell/mm2), p = 0.09. In addition, animals with MPD presented a higher ICAM (0.02 ± 0.01) expression when compared with animals without MPD (0.01 ± 0.01, p = 0.02). There was no difference between groups regarding the extent of fibrosis. There was a negative correlation between individual values of MPD with LVEF (R = –0.6, p = 0.001), wall motion score index (WMSi, R = 0.5, p = 0.007), and the number of mononuclear cells (R = 0.5, p = 0.01). Moreover, an analysis based on myocardial segments (n = 312) showed that segments with MPD (n = 54) in comparison to those without MPD (n = 258) presented a higher number of mononuclear cells (608 ± 299.9 cell/mm2 and 478.3 ± 201.1 cell/mm, respectively, p < 0.0001) and higher WMSi (1.8 ± 0.9 and 1.2 ± 0.4, respectively, p < 0.0001. Conclusions: MPD is a common finding in the experimental model of CCC in hamsters and is correlated with inflammation, endothelial inflammatory activation, and systolic ventricular dysfunction. These results suggest that MPD may be an in vivo surrogate marker for inflammation with potential translational implications for monitoring disease activity. 109166 Modality: WHF Abstracts – Researcher Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 14/10/2022 H: 13:50/14:50 L: Auditório 14 MELANIE B. TURNER, MPH, FAHA1, Laura L. Hayman, PhD, MSN, FAHA2, Dhruv Kazi, MD, MSc, MS, FAHA3, Laxmi S. Mehta, MD, FAHA4 (1) American Heart Association; (2) University of Massachusetts Boston; (3) Beth Israel Deaconess Medical Center; (4) The Ohio State University Medical Center Introduction: Cardiovascular disease (CVD) is the leading cause of death (LCOD) in women globally, causing ≈9 M deaths annually. Global community awareness data of CVD and its risk factors are limited. Objective: Determine global awareness of CVD incidence and risk factors among women and examine regional variation in awareness. Methods: An online native language survey was administered among nationally representative samples of women ≥18 years of age in 50 countries across 6 global regions. The survey was administered June-July 2021 using the YouGov real time omnibus service. The survey included an open-ended question regarding LCOD for women in the country (coded as CVD, cancer, or other/don’t know), and a multiple choice question on CVD risk factors. Results were weighted by country, then combined by region. Results: Of 24,100 women, 15.5% reported CVD as LCOD (regional range 6.9–35.0%), 49.4% cancer (23.9–62.9%) and 18.0% other/don’t know (9.1–34.4%). Cancer was perceived as LCOD in all regions except South Asia and Middle East and North Africa where other/don’t know was the most common response. Regarding CVD risk, 69.5% identified family history as a risk (regional range 51.0–77.1%). Most recognized hypertension, overweight, high cholesterol, and stress. Awareness of diabetes as a CVD risk was low (32.0%, 26.5–44.1%). Conclusions: Awareness of CVD as LCOD among women is low in all global regions. Most women recognize hypertension, high cholesterol, and overweight as risk factors, but awareness of diabetes as a risk factor is low. Efforts are needed in all regions to create awareness of lifetime CVD risk in women, focusing on modifiable risk factors. As an increasingly prevalent global chronic condition, diabetes merits raised attention in community heart health education. 109180 Modality: WHF Abstracts – Researcher Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 14/10/2022 H: 13:50/14:50 L: Auditório 14 CHARLE ANDRE VILJOEN1, Karen Sliwa1, Peter van der Meer2, Alice M Jackson3, Mark C Petrie3, Cecile Laroche4, Jolien W Roos-Hesselink5, Petar Seferovic6, Alexandra Frogoudaki7, Bassem Ibrahim8, Hasan Al-Farhan9, Johann Bauersachs10 (1) Cape Heart Institute, Department of Medicine and Cardiology, Faculty of Health Sciences, University of Cape Town, South Africa; (2) Department of Cardiology, University Medical Center Groningen, University of Groningen, The Netherlands; (3) Institute of Cardiovascular and Medical Sciences, Glasgow University, Glasgow, United Kingdom; (4) EurObservational Research Programme, European Society of Cardiology, Sophie Antipolis, France; (5) Department Cardiology, Thoraxcenter, Erasmus Medical Center, Rotterdam, the Netherlands; (6) University of Belgrade Faculty of Medicine, Belgrade, Serbia; (7) Attikon University Hospital, Athens, Greece; (8) North Cumbria University Hospitals, Carlisle, UK; (9) Iraqi Board of Medical Specilazations, Baghdad Heart Center, Iraq; (10) Department of Cardiology and Angiology, Medical School Hannover, Hannover, Germany Background: Peripartum cardiomyopathy (PPCM) is a global disease associated with substantial morbidity and mortality. The aim of this study was to analyze to what extent country- and individual-level socioeconomic factors were associated with maternal and neonatal outcomes. Methods: In 2011, >100 national and affiliated member cardiac societies of the European Society of Cardiology (ESC) were contacted to contribute to a global registry on PPCM, under the auspices of the ESC EORP Programme. We investigated the characteristics and outcomes of women with PPCM and their babies according to individual (income and educational attainment) and country-specific (Gini coefficient [GINI], health expenditure [HE] and human developmental index [HDI]) socioeconomic status. Results: 739 women from 49 countries were enrolled (Europe [33%], Africa [29%], Asia-Pacific [15%], Middle East [22%]). Women from countries with low HDI had lower income and educational attainment (p < 0.001). Low HDI was associated with greater LV dilatation at time of diagnosis (p < 0.001), but LV ejection fraction (LVEF) did not differ according to HDI, HE or GINI. Countries with low HE prescribed guideline-directed heart failure therapy less frequently (p < 0.001). Low HE was associated with more frequent mortality (p < 0.002), whereas HDI and GINI were not. Women from countries with low HDI and low HE had significantly less recovery of LV function (p < 0.001). Analysis of maternal outcome as per highest level of educational attainment showed significant differences in LVEF at 6 months (43.7 ± 12.9% [primary], 46.5 ± 13.0% [secondary], 48.9 ± 11.7 [tertiary education] respectively, p = 0.022). Low maternal income, irrespective of region of origin, was independently associated with poor outcome (OR 1.99 [95% CI 1.1–3.6] for composite of maternal death, re-hospitalization, or LV non-recovery). Neonatal death was more prevalent in countries with low HE (p = 0.009) and low HDI (p = 0.023) but was not influenced by maternal sociodemographic parameters. Conclusion: Maternal and neonatal outcomes depended on country-specific socioeconomic characteristics, with a greater prevalence of maternal and neonatal deaths in women from countries with low HE. Globally, women with low income and lower levels of educational attainment had poorer outcomes, irrespective of region. Attempts should be made to improve patient education, and allocation of adequate health resources to improve maternal and neonatal outcomes in PPCM. 111430 Modality: WHF Abstracts – Young Researcher Category: NEGLECTED CARDIOVASCULAR DISEASES D: 14/10/2022 H: 16:50/17:50 L: Auditório 14 WHESLEY TANOR SILVA1, Matheus Ribeiro Ávila1, Pedro Henrique Scheidt Figueiredo1, Lucas Frois Fernandes de Oliveira1, Vanessa Amaral Mendonça1, Ana Cristina Rodrigues Lacerda1, Vanessa Pereira Lima1, Henrique Silveira Costa1 (1) Universidade Federal dos Vales do Jequitinhonha e Mucuri UFVJM Introduction: In the functional assessment of patients with Chagas cardiomyopathy, peak oxygen consumption (VO2peak) and ventilatory equivalent of carbon dioxide (VE/VCO2 slope) have already been shown to be parameters of clinical and prognostic relevance. However, other variables assessed by the Cardiopulmonary Stress Test need to be investigated. The peak end-tidal carbon dioxide pressure (PETCO2 peak) has been prominent in the therapy of patients with heart failure. However, its behavior is still unknown in patients with Chagas cardiomyopathy and systolic dysfunction. Objective: To verify, in patients with Chagas cardiomyopathy and systolic dysfunction, the association between peak PETCO2 and functional and echocardiographic parameters. Methods: Seventy-six patients with Chagas cardiomyopathy and systolic dysfunction (49.9 ± 10.8 years, 60% male, NYHA I to III) were recruited and underwent clinical evaluation, echocardiography and Cardiopulmonary Stress Test. Systolic dysfunction was defined as a left ventricular ejection fraction (LVEF) of less than 52 or 54% for men and women, respectively. The variables of interest were PETCO2 peak, VO2peak and VE/VCO2 slope (on Cardiopulmonary Stress Test) and LVEF and left ventricular diastolic diameter (LVd) (on echocardiogram). Results: In the sample, the mean peak PETCO2 was 33.6 ± 4.9 mmHg. In the correlation analysis, PETCO2 peak was associated with VO2peak (r = 0.355; p = 0.008), VE/VCO2 slope (r = –0.626; p < 0.001) and with LVEF (r = 0.299; p = 0.029). There was no correlation between peak PETCO2 and VEd. Conclusion: These results suggest that peak PETCO2 is associated with important clinical and functional parameters of patients with Chagas cardiomyopathy and should be used in the management of patients with Chagas cardiomyopathy. 112213 Modality: WHF Abstracts – Young Researcher Category: NEGLECTED CARDIOVASCULAR DISEASES D: 14/10/2022 H: 16:50/17:50 L: Auditório 14 JULIANA DA ROCHA FERREIRA1, Juliana da Rocha Ferreira1, Julia Passarelli Perreira1, Marcelo Machado Melo1, Helena Cramer Veiga Rey1, Glauber Monteiro Dias2 (1) Instituto Nacional de Cardiologia – INC; (2) Universidade Estadual do Norte Fluminense Darcy Ribeiro – UENF Aortopathies are a silent disease with a high natural fatality rate. Multiple genes have been linked to hereditary aortic thoracic disease (HATD), and it is believed that 30% of people have a deleterious mutation. This study aimed to uncover genetic variations related to aortopathies using genomic and molecular analysis. Seventy-nine people with aortopathies were studied using clinical data, target-NGS (tNGS), and the Sanger sequencing. The ACMG classification was used to find causal variations. This study defined a severe phenotypic population. Aortic dissection occurred in 49.4% of patients diagnosed at 44.59 years old, mainly after 40, and required surgery at 72.2%. Seven pathogenic variants (PV), 4 likely pathogenic variants (LPV), and 22 variants of uncertain significance (VUS) were discovered. tNGS found 10.37 percent of ATDs in this cohort. Direct sequencing of the fibrillin-1 gene (FBN1) yielded 37.5% diagnostic yield for Marfan syndrome suspect individuals, with two PV and one LPV. PV/LPV variants were identified in 6 genes (ACTA2, FBN1, MYLK, SMAD3, TGFB2, TGFBR2), being FBN1 the most prevalent (6 var). Seven PV/LPV are novel variants. Patients with VP/PPV had a younger mean age (39.3 years vs. 44.4 years) and a larger mean aorta diameter (39.3 years vs. 44.4 years) (6.56 vs 5.66 cm), however no statistic significance was achieved. Both groups required surgery (81.8%), with the VP/PVP group having a worse prognosis and severity. An emphasis is placed on clinical suspicion in selecting the genetic test to use and increasing the yield. 111396 Modality: Best Abstracts Oral – Researcher Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION D: 14/10/2022 H: 10:40/11:40 L: Auditório 14 MARTIN BOBAK1, Steven Hageman2, Hynek Pikhart1, Abdonas Tamosiunas3, Andrzej Pajak4, Ruzena Kubinova5, Wentian Lu1 (1) University College London (UCL), United Kingdom; (2) Utrecht University, Utrecht, The Netherlands; (3) Lithuanian University of Health Sciences, Kaunas, Lithuania; (4) Jagiellonian University Medical College, Krakow, Poland; (5) National Institute of Public Health, Prague, Czech Republic Introduction: The cardiovascular disease (CVD) risk prediction model SCORE2 uses several biomedical and behavioural factors. It is likely that other factors may also contribute to CVD risk prediction. Cognitive impairment and CVD are associated and share some risk factors. While the causality of this relationship remains unclear, adding cognitive measures to risk prediction may be useful for predicting future CVD events. Objective: To test the hypothesis that including simple measures of cognitive functions improve the prediction of SCORE2 for future CVD events. Methods: We used data on 13,391 Polish, Lithuanian and Czech adults without CVD at baseline (45–69 years) from the Health, Alcohol and Psychosocial factors In Eastern Europe cohort. Incident cases of myocardial infarction (MI), stroke, CVD mortality and composite (fatal and non-fatal) CVD events were identified over a 10-year follow-up. Baseline cognitive measures included immediate and delayed word recall (verbal memory and learning), animal naming (verbal fluency) and letter cancellation (attention, mental speed and concentration). Using competing-risks regression, relationships between cognitive functions and CVD outcomes were examined. Improvement in Receiver Operating Characteristic (ROC) was used to compare predictive performance for CVD events of models including SCORE2 variables combined with cognitive tests vs SCORE2 alone. Results: Incidence rates of MI, stroke, CVD mortality and composite CVD events were 5%, 4%, 4% and 11%, respectively. All four cognitive measures were inversely associated with CVD outcomes; e.g. one standard deviation increase in immediate word recall score was associated with a 28% and 17% reduction in risk of CVD mortality and composite CVD events, respectively. Adding cognitive measures to SCORE2 variables improved the ROCs for prediction of MI, CVD mortality and composite CVD events. E.g. for the composite CVD measure, inclusion of all four cognitive measures increased the ROC area from 0.6948 to 0.7030 (p < 0.001) and similar improvement was seen for word recall. Conclusion: Including even a single (and simple) cognitive test in assessment of cardiovascular health can improve prediction of future CVD risk and it may be potentially feasible to do so in general practice. 110777 Modality: Best Abstracts Oral – Researcher Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT D: 14/10/2022 H: 10:40/11:40 L: Auditório 14 MARCUS VINICIUS SIMÕES1, Pedro Gabriel Barros e Silva2, Denilson Campos Albuquerque3, Renato Delascio Lopes4, Luis Eduardo Paim Rohde5, Lidia Zytinsky Moura6, Fabiana Goulart Marcondes-Braga3, Evandro Tinoco Mesquita3, José Albuquerque de Figueiredo Neto7, Ricardo Mourille Rocha8, João David de Souza Neto9, Mucio Tavares Oliveira Junior10 (1) Faculdade de Medicina de Ribeirão Preto – USP, Ribeirão Preto, SP, Brasil; (2) Brazilian Clinical Research Institute (BCRI), Sao Paulo, Brazil; (3) Sociedade Brasileira de Cardiologia, Departamento de Insuficiência Cardíaca – DEIC, Rio De Janeiro, Brazil; (4) Duke Clinical ResearchInstitute, Durham, United States of America; (5) Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; (6) Santa casa, Curitiba, Brazil; (7) Centro de Pesquisa Clínica doHospital Universitário da Universidade Federal do Maranhão (CEPEC-HUUF, Sao Luis, Brazil; (8) PedroErnesto University Hospital, Rio De Janeiro, Brazil; (9) Messejana Hospital, Fortaleza, Brazil; (10) Heart Institute (InCor), University of São Paulo Medical School, Brazil Background: Chagas cardiomyopathy (CC) is a prevalent cause of heart failure in Latin America countries. Studies describing clinical manifestations and outcomes of heart failure associated to CC are scarce. Purpose: Report the results of the I Brazilian Heart Failure Registry (BREATHE) addressing the clinical and laboratorial characteristics, and outcomes of patients with acute heart failures (AHF) due to CC in comparison to other etiologies. Methods: BREATHE was a multicenter nationwide prospective registry, enrolling 3,013 adult patients hospitalized with AHF, median follow-up of 346 days. We proceeded the comparative analysis between 261 (8.7%) patients with CC and 2,752 (91.3%) patients with other etiologies, concerning clinical, demographic, cardiac structure/function on Echocardiogram, death rate or heart transplantation during hospital stay and death rate at 3, 6 and 12 months after discharge. The categorical variables were compared by using Fisher Exact test and the continuous variables were compared by using Mann-Whitney test. A multivariate logistic model was used to estimate the odds ratio of CC in 12-month mortality adjusted for clinically relevant variables. Results: CC patients, in comparison to other etiologies, were younger (60.6 ± 13.9 vs 65.7 ± 15.7 y.o., p < 0.001), presented lower systolic blood pressure (108.3 ± 26.1 vs 128.3 ± 30.3 mmHg, p < 0.001), lower heart rate (77.3 ± 22.1 vs 88.5 ± 23.2 bpm, p < 0.001), higher rate of jugular vein distension (54.8% vs 38.9%, p < 0.001) and hepatomegaly (47.9% vs 25.6%, p < 0.001), higher rate of “cold and wet” clinical hemodynamic profile (27.2 vs 10.6%, p < 0.001); larger diastolic left ventricular (LV) diameters (65 [57–72.8] vs 59 [51–66] mm, p < 0.001), and lower LV ejection fraction (25.4 [19–36]% vs 37 [27–54]%, p < 0.001), with higher rates of dobutamine use (23.8% vs 6.8%, p < 0.001); presented higher rate of death or heart transplantation during hospital stay (11.1% vs 17.4%, p = 0.004), and higher cumulative death rate after discharge at 3-months (16.5% vs 10.8%, p = 0.017, at 6-months (25.7% vs 17.5%, p = 0.006, and at 12-months (40.8% vs 27.8%, p < 0.001). In a multivariate analysis, CC was independently associated with 12-month mortality risk with odds ratio = 2.02 [95% IC: 1.47;2.77]. Conclusions: Patients hospitalized with AHF with CC etiology, in comparison to other etiologies, presented higher-risk profile that was associated with a poorer outcome during hospital stay and after discharge. 111321 Modality: Best Abstracts Oral – Researcher Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS D: 14/10/2022 H: 10:40/11:40 L: Auditório 14 PEDRO GABRIEL MELO DE BARROS E SILVA1, Ana Amaral3, Antonielle Figueiredo Macedo4, Celso Musa Correa5, Eduardo Zincone6, Marcelo Paiva Cury7, Gustavo Augusto Lopes Rosa8, Alexandre de Matos Soeiro9, Carlos Alexandre Lemes de Oliveira10, Augusto Celso De Araujo Lopes Junior11, Adriana Bertolami7, Renato Delascio Lopes12 (1) Hospital Samaritano Paulista; (2) Cardiologia Americas; (3) Hospital Pro-cardíaco; (4) Hospital da Luz; (5) Americas Medical City; (6) Hospital Santa Helena; (7) Metropolitano Lapa; (8) Ipiranga Mogi; (9) Incor HC FMUSP; (10) Hospital Paulistano; (11) Hospital Monte Klinikum; (12) Brazilian Clinical Research Institute Background: Chest pain is a major cause of medical evaluation at emergency department (ED) and demands observation in order to exclude the diagnosis of acute myocardial infarction (AMI). Recent algorithms using high-sensitivity cardiac troponin assays at 0 h and 1 h are accepted as a rule-out/rule-in strategy but there is a lack of validation in specific populations. Methods: IN-HOPE was a multicentre prospective study that included patients admitted to the ED due to suspected symptoms of AMI at 16 sites in Brazil. All patients followed the standard approach of 0–3h but, in addition, blood samples were also collected at 0 and 1 hour and sent to a core laboratory to measure high sensitivity troponin T (hs-cTn T) Elecsys (Roche). Troponin <12 ng/L with a delta <3 was considered rule out while a value ≥52 and/or a delta ≥5 was considered rule in for AMI. The main objective of the study was to assess the accuracy of 0–1 h rule-out/rule-in algorithm in comparison to the standard of care (0–3h). All patients were followed for 30 days. In addition to the prospective cohort, a retrospective analysis was performed assessing all patients with hs-cTn T measured during 2021 but not included in the prospective cohort. Results: A total of 5.497 patients were included (583 in the prospective and 4.914 in the retrospective analysis). The prospective cohort had a mean age of 57.3 (±14.8) and 45.6% of females with a mean HEART score of 4.0 ± 2.2. By the core lab analysis, 71.6% would be eligible for a rule-out approach while 7.3% would fit the rule-in criteria. At 30 days, no death or AMI occurred in the rule-out group while 64.9% of the patients in the rule-in group were considered as AMI. In the retrospective analysis, 1.091 patients had a troponin value <5 ng/L without cardiovascular deaths in this group. Among all 4.914 patients, the 30-day risk of AMI or cardiovascular death increased according to the level of troponin: 0% in the group <5 ng/L, 0.6% between 5 and 14 ng/L, 2.2% between 14 and 42 ng/L, 6.3% between 42 and 90 ng/L and 7.7% in the level ≥90 ng/L. Conclusions: In this large multicentre Brazilian study, a 0–1h algorithm was effective for classifying as rule in or out almost 80% of the patients. The rule-out protocol had high negative predictive value regardless clinical risk scores. Categories of levels of hs-cTn T also showed good accuracy in discriminating risk of the patients with a very favourable prognosis for the group with values <5 ng/L. 112023 Modality: Best Abstracts Oral – Researcher Category: CARDIOVASCULAR SURGERY D: 14/10/2022 H: 10:40/11:40 L: Auditório 14 LUIS EDUARDO PAIM ROHDE1, Marcio R. Martins1, Luis E. Rohde1, Flávia K. Borges2, Andre Lamy2, Richard Whitlock2, P. J. Devereaux2, Carisi A. Polanczyk1 (1) Hospital de Clinicas de Porto Alegre, Post Graduate Program in Cardiovascular Sciences and Cardiology, UFRGS; (2) McMaster University, Hamilton Health Sciences Background: Previous studies addressing the association of chronic kidney disease (CKD) and prognosis after open-heart surgery had limited sample sizes and retrospective designs. Methods: We investigated the association of preoperative renal function and in-hospital mortality, and major cardiac and cerebrovascular events (MACCE) in patients enrolled in the prospective multicentric VISION Cardiac Surgery Study. Patients were divided in 5 CKD stages according to preoperative estimated glomerular filtration rate (eGFR in mL/min/1.73 m2): Stage I(>90; n = 1914), Stage II(60 to 89; n = 8122), Stage III(30 to 59; n = 3406), Stage IV(<30; n = 352) and Stage V(dialysis; n = 227). Results: 15,837 were included in the current analysis (71% male, 66% hypertensive and 20% >75 y.o.). Mortality and MACCE during the first 30 days occurred in 480(3%) and 1727(11%) patients, respectively. Logistic regression models adjusted for EuroSCORE demonstrated increased 30-day mortality in CKD Stage III (odd ratio[OR], 1.82; 95% confidence internal[CI], 1.36–2.41), CKD Stage IV (OR, 2.62; 95% CI, 1.66–4.15) and in patients in dialysis (OR, 3.56; 95% CI, 2.17–5.85) In analysis across the whole spectrum of renal function (Figure), mortality was increased particularly when eGFR was <45 mL/min/1.73 m2, while MACCE risk was observed in less severe stages of CKD. Conclusion: In this contemporary cohort, CKD was significantly associated with morbidity and mortality after open-heart surgery. 112135 Modality: Best Abstracts Oral – Researcher Category: DIGITAL HEALTH/INNOVATION D: 14/10/2022 H: 10:40/11:40 L: Auditório 14 LUIZ SÉRGIO FERNANDES DE CARVALHO1, Gustavo Alexim5, Ana Claudia Cavalcante Nogueira3, Marta Duran Fernandez1, Tito Barbosa Rezende4, Sandra Avila4, Ricardo Torres Bispo Reis6, Alexandre Anderson Munhoz Soares3, Andrei Carvalho Sposito4 (1) Clarity: Inteligência em Saúde; (2) Universidade Católica de Brasília; (3) Instituto Aramari Apo; (4) UNICAMP; (5) Hospital de Base do Distrito Federal; (6) Universidade de Brasília Introduction: Among individuals with premature acute coronary syndromes (prACS, <55 years-old), prevalent risk factors and the magnitude of their impact on recurrent ischemic events critically differ from older subjects. In that sense, it is plausible to develop risk prediction rules specific for young individuals. Our aim was to evaluate potential improvements in risk prediction quality among prACS by (i) developing models specifically in prACS subjects versus in the global cohort; (ii) splitting predictive rules into two models (short- and long-term prediction windows [STWm and LTWm]) versus a global follow-up model (GFm). Methods: Consecutive individuals with ACS who undergone coronarography up to 48h after hospital admission from January/2011 to February/2020. 6341 subjects (2242 with prACS) admitted into public hospitals in Brasília (Brazil). The observation window in STWm and GFm included the first 48h upon hospital admission, and LTWm included all in-hospital information. prACS cohort was divided into train/validation-set (70%, n = 1569) and test-set (30%, n = 673); global cohort was divided in training/validation-set (70%, n = 4439) and test-set including the 673 prACS subjects. Models were repeated over five cross-validation folds and then assessed in the test-set. C-statistics was the evaluation metric for STWm and time-dependent concordance (Ctd-index) for LTWm. STWm evaluated the occurrence of in-hospital cardiovascular deaths and recurrent ACS (MACE) and LTWm estimated events occurring post-discharge from index ACS hospitalization considering time-to-event with competing risks (MACE versus non-cardiovascular deaths). Results: Median follow-up of 6.7 years (95%CI 5.6–7.2). Among prACS and older subjects, respectively, in-hospital MACE occurred in 180 and 493 individuals, post-discharge MACE in 454 and 881; and post-discharge non-cardiovascular death in 47 and 285. The best strategy was to design models specifically in prACS individuals combining STWm and LTWm. Among prACS subjects STWm and LTWm, respectively, TabNet and DeepHit yielded the best C-statistics [0.921 (95%CI 0.889–0.953)] and Ctd-index [0.722 (95%CI 0.678–0.760)], while the best Ctd-index in GFm was 0.681 (95%CI 0.654–0.703). There was very low concordance among top predictors of MACE for prACS versus global cohort, as well as for STWm versus LTWm. Conclusions: Risk prediction in ACS is optimized by using specific rules for prACS and combining short-term and long-term prediction windows. 109042 Modality: Best Abstracts Oral – Young Researcher Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE D: 14/10/2022 H: 09:00/10:00 L: Auditório 14 GUSTAVO AUGUSTO FERREIRA MOTA 1, Sérgio Luiz Borges de Souza1, Danielle Fernandes Vileigas2, Vitor Loureiro da Silva1, Paula Grippa Sant’Ana1, Licia Carla Silva Costa3, Silméia Garcia Zanatti Bazan1, Marília Afonso Rabelo Buzalaf4, Lucilene Delazari dos Santos5, Marina Politi Okoshi1, Mariana Gatto1, Antonio Carlos Cicogna1 (1) São Paulo State University, UNESP, Botucatu Medical School, Brazil; (2) University of São Paulo, USP, Institute of Chemistry, Brazil; (3) University of Campinas, UNICAMP, Institute of Biology, Brazil; (4) University of São Paulo, USP, Bauru Dental School, Brazil; (5) São Paulo State University, UNESP, Biotechnology Institute, Brazil Introduction: The beneficial effect of aerobic exercise training (ET) on cardiac remodeling induced by aortic stenosis (AS) has been observed in experimental studies. However, the mechanisms involved in cardiac function improvement are not fully understood. In this study we investigated the myocardial proteoma in rats with AS subjected to ET. Methods: Wistar rats (n = 60, 21 days) were divided into 2 groups: operated control (C) and supravalvar aortic stenosis (AS). AS was induced by the insertion of a stainless-steel clip, 0.60 mm, around the ascending aorta. Two weeks after surgery, rats were assigned into 4 groups: C, exercised C (C-Ex), AS and exercised AS (AS-Ex). Exercised rats underwent treadmill exercise 5 days a week for 4 months, at 60% of the maximal functional capacity. Two and 18 weeks after surgery, rats were subjected to echocardiogram. Functional capacity was analyzed by treadmill maximum exercise testing and blood lactate concentration. Myocardial proteome was assessed by label-free shotgun approach using mass spectrometry. Protein expression was quantified by Western blotting. Statistical analysis was performed by ANOVA or Kruskal-Wallis; significance level of 5%. Results: Two weeks after AS induction, AS rats had diastolic and systolic dysfunction and concentric hypertrophy. At the end of the protocol, AS maintained the same remodeling pattern; the AS-Ex group presented lower left atrium diameter-to-aortic diameter and lactate concentration; and higher E/E’ ratio, percentage of midwall fractional shortening, and functional capacity than AS. After enrichment analysis by Gene Ontology, myocardial proteome analysis showed higher expression of proteins related to glycolytic pathway, oxidative stress, and inflammation, and lower expression of proteins associated with beta-oxidation in AS than C. The AS-Ex had higher expression of proteins related to mitochondrial biogenesis and lower expression of proteins related to oxidative stress and inflammation than AS. Expression of proteins of physiological and pathological hypertrophic pathways did not differ between groups, except for p-p38, which was higher in the AS than C group. Conclusion: Aerobic exercise training improves cardiac remodeling and mitochondrial biogenesis, and attenuates oxidative stress and inflammation in rats with ascending aortic stenosis. Financial support: CNPq, FAPESP, and UNESP. 108618 Modality: Best Abstracts Oral – Young Researcher Category: PHYSICAL EDUCATION D: 14/10/2022 H: 09:00/10:00 L: Auditório 14 FRANCIS RIBEIRO DE SOUZA1, Carlos Eduardo Rochitte1, Douglas Carli Silva1, André Matheus Rodrigues Gomes1, Marcelo Rodrigues dos Santos1, Guilherme Wesley Peixoto da Fonseca1, Antonio Carlos Battaglia Filho1, Kelly Thayane Souza Correa1, Maurício Yonamine2, Rosa Maria Rodrigues Pereira3, Carlos Eduardo Negrão1, Maria Janieire de Nazaré Nunes Alves1 (1) Instituto do Coração do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (InCor, HCFMUSP); (2) Faculdade de Ciências Farmacêuticas da Universidade de São Paulo (FCF/USP); (3) Laborátorio de Reumatologia e Metabolismo Ósseo da Faculdade de Medicina da Universidade de São Paulo (FMUSP) Background: The illicit use of anabolic androgenic steroids (AAS) has been associated with diminished cholesterol efflux mediated by HDL, remarkable decrease in high-density lipoprotein (HDL) plasma concentration and subclinical coronary artery disease (CAD). Inflammation is the key to the atherogenic process associated with atherosclerotic plaque vulnerability. The pericoronary mean fat attenuation (pFAM) has emerged as a marker of coronary inflammation and can predict future cardiovascular events, which is measurable from standard coronary computed tomography angiography (CCTA). However, whether AAS abuse has a role in pFAM in young male AAS users is unknown. Purpose: The aim of this study was to evaluate whether AAS abuse could leads to higher pFAM and premature coronary inflammation in young male AAS users. Methods: Twenty strength-trained AAS users (AASU) age 29 ± 5 yr, 20 age-matched strength-trained AAS nonusers (AASNU), and 10 sedentary controls (SC) were enrolled in this study. Coronary inflammation was assessed by pFAM-CCTA in the right coronary artery (RCA), left anterior descending artery (LDA) and left circumflex coronary artery (Cx). Results: pFAM in RCA was significantly higher in AASU compared with AASNU and SC (–64.59 ± 9.45 vs. –79.21 ± 6.67 vs. –80.97 ± 7,91 Hounsfield Units (HU), respectively, p < 0.001]. Also, the pFAM in the LAD was higher in AASU compared with AASNU and SC (–72.83 ± 7.21 vs. –79.41 ± 6.72 vs. –80.97 ± 7.72 HU, p = 0.006). However, no difference to pFAM in the Cx between AASU, AASNU and SC (–74.30 ± 5.85 vs. –79.77 ± 7.13 vs. –78.19 ± 5.82 HU, respectively, p = 0.069) was found. Conclusion: This study indicates that AAS abuse may be associated with higher pFAM and premature coronary inflammation in the RCA and LAD. In addition, the higher pFAM may be linked to early development of CAD in young AAS users. 109119 Modality: Best Abstracts Oral – Young Researcher Category: DIGITAL HEALTH/INNOVATION D: 14/10/2022 H: 09:00/10:00 L: Auditório 14 KELSEY BROWN1, Pooneh Roshanitabrizi, PhD3, Alison Reese, MS1, Andrea Beaton, MD2, Emmy Okello, PhD4, Joselyn Rwebembera, MMed4, Peter Lwabi, MMed4, Emma Ndagire, MMed4, Craig Sable, MD1, Marius George Linguraru, DPhil3 (1) Children’s National Hospital; (2) Cincinnati Children’s Hospital Medical Center; (3) Sheikh Zayed Institute for Pediatric Surgical Innovation; (4) Uganda Heart Institute Introduction: Rheumatic heart disease (RHD) is the number one cause globally of morbidity and mortality from heart disease in children and young adults. The mitral regurgitation (MR) jet length on color Doppler echocardiography is an important index for diagnosis, but its measurement and interpretation are variable. Objective: Develop an automatic machine learning approach to identify and measure the MR jet length on color Doppler for RHD detection. Methods/Design: We used 316 echocardiograms in video format from 95 children (mean age 12 ± 2 years; range 5 to 17 years) with DICOM color Doppler images of the mitral valve taken from parasternal long axis (PLAX) and apical 4 chamber (AP4) views. All echocardiograms were independently reviewed by an adjudication panel which consisted of four expert pediatric cardiologists to determine maximum MR jet length and diagnosis (RHD positive or not). Among 95 cases, 29 were normal and 66 had RHD. Our automated method included. (1) Selection of frames during ventricular systole using a convolutional neural network architecture based on the ResNet-50. (2) Localization of left atrium using convolutional neural networks with LinkNet structure. (3) Measurement of MR jet length using image color analysis. (4) Detection of RHD by applying a generalized regression model based on the maximum MR jet length measured on each view and maximizing the balanced accuracy using cross validation. Results: Machine learning selected the correct systolic frame with an average accuracy of 0.95 (sensitivity 97%/specificity 93%) and 0.94 (sensitivity 94%/specificity 94%) for the AP4 and PLAX view, respectively. It localized the atrium with an average Dice coefficient of 0.89 and 0.9 for the AP4 and PLAX view, respectively. We estimated the length of the MR jet with an average absolute error of 0.33 ± 0.4 cm (p-value = 0.15 compared to manual measurements). Our deep learning approach performed similar to or better than previously published manual methods for categorization of RHD positive vs negative. The accuracy of RHD detection was 0.84 (sensitivity 86%/specificity 79%). Conclusion: Our automatic method has the potential to reliably detect RHD as accurately as expert cardiologists. This innovative approach holds promise to scale echocardiography screening for RHD and greatly expand prophylaxis to prevent progression of RHD globally. 109213 Modality: Best Abstracts Oral – Young Researcher Category: CARDIO-ONCOLOGY D: 14/10/2022 H: 09:00/10:00 L: Auditório 14 JAVIER ELIECER PEREIRA RODRIGUEZ1, Fernando Rivera-Theurel2, Jorge Antonio Lara-Vargas3, Devi Geesel Peñaranda-Florez1, Pedro Pereira-Rodriguez4, Karla Dominguez-Gomez1, Kelly Perez-Diaz1, Dafne Palacios-Toledo1, Hiady Rivera-Lopez1, Alondra Mijangos-Dolores1, Isaias Sánchez-García1 (1) Centro de Estudios e Investigación FISICOL; Colombia; (2) University of Toronto; Canada; (3) Cardiofit; México; (4) Clínica Medical Duarte; Colombia Introduction: Cardio-Oncology aims to prevent and treat cardiac dysfunction induced by anti-oncological therapies. One emergent strategy is the physical activity, which has been considered an important strategy to improve quality of life and outcomes in the susceptible population. Objective: Determine the cardiovascular effects of Moderate Intensity Continuous Training (MICT) and High Intensity Interval Training (HIIT) on oncological patients newly diagnosed with cardiovascular disease (CVD). Methodology: Randomized controlled trial with sample of 690 with oncological patients with a new diagnose of CVD (heart failure and ischemic heart disease) distributed in 3 groups (MICT, HIIT and control group). We identified the echocardiogram, cardiac biomarkers, stress test, clinical and hemodynamic parameters, hematological samples were identified. In addition, 6-minute walk, anthropometry, quality of life, fatigue, sarcopenia. The tests were performed pre and post 36 sessions of 70-minute training. Then, descriptive statistics were carried out to estimate, the normality of the data was assessed by the Kolmogorov-Smirnov test and the indication of specificity was evident for all analyzes. Also, the ANOVA analysis of variance (one-way analysis of variance) was used, and subsequently, post hoc tests with Tukey test. In all cases, a significance level was 5% (p = <0.05). Results: After an structured exercise program, there was an increase in left ventricular ejection fraction (LVEF) (EG1: 40 ± 5 vs 44 ± 4%; EG2: 41 ± 4 vs 47 ± 3%; CG: 40 ± 3 vs 40 ± 1%; p < 0.05). In fact, higher values of functional capacity (VO2peak) were noted in the experimental group 2-HIIT (7.0 ± 5.2 vs 9.7 ± 3.7) compared to experimental group 1-MICT (8.0 ± 4 vs 8.5 ± 3) and control group (9.0 ± 5.0 vs 9.2 ± 4.0). Other parameters results incuded systolic blood pressure (135 ± 10 vs 127 ± 2 mmHg), diastolic blood pressure (85 ± 5 vs 80 ± 2 mmHg), maximum heart rate (166 ± 10 vs 179 ± 5bpm) and quality of life (106 ± 8 vs 73.2 ± 11). It was possible to show significant changes in all variables comparing HIIT vs MICT groups (p = <0.05), and also compared to the control group. Conclusions: HIIT and MICT in oncological patients with newly diagnosed CVD improved the LVEF, functional capacity (VO2peak), hemodynamic parameters (systolic and diastolic blood pressure), exercise tolerance, depression, anxiety, strength, Cancer related fatigue improved as well as other specific cancer variables such as sarcopenia, and quality of life. 109929 Modality: Best Abstracts Oral – Young Researcher Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION D: 14/10/2022 H: 09:00/10:00 L: Auditório 14 LETICIA MARA DOS SANTOS BARBETTA1, Letícia Mara dos Santos Barbetta1, Eduardo Thadeu de Oliveira Correia1, Ronaldo Altenburg Odebrecht Curi Gismondi1, Antônio José Lagoeiro Jorge1, Evandro Tinoco Mesquita1 (1) Universidade Federal Fluminense; (2) Instituto Cardiovascular do Complexo Hospitalar de Niterói; (3) Centro de Educação e Treinamento Edson Bueno – UnitedHealth Group; (4) Hospital Niterói D’Or Introduction: Previous randomized trials showed conflicting results regarding the effectiveness of influenza vaccination on cardiovascular outcomes in patients with coronary artery disease (CAD). These studies were not adequately powered to show the effectiveness of influenza vaccine on major adverse cardiovascular events (MACE); all-cause mortality or cardiovascular mortality. Therefore, this up-to-date meta-analysis combines data from randomized trials to assess the effectiveness of influenza vaccination on endpoints in patients with CAD. Methods: We performed a search of the Cochrane Controlled Register of Trials, Embase, MEDLINE, www.ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform from inception to September 2021. Two authors performed the screening, quality analysis and data extraction. A p-value <0.05 was defined as statistically significant. This study protocol is available in PROSPERO under the following registration number CRD42021282917. Results: We identified 355 records through search of databases. After duplicate analysis, 258 articles remained, of which 239 were excluded based on title and/or abstract analysis. Nineteen full-text articles were assessed for eligibility, and 5 articles were selected for inclusion. Five trials were included in this meta-analysis, comprehending 4238 patients, from which 2116 were controls and 2122 received influenza vaccination. Influenza vaccination reduced all-cause mortality compared with placebo (RR 0.50, CI 0.29–0.88, p = 0.02). However, in a prespecified subgroup analysis, influenza vaccination only reduced all-cause mortality in patients with acute coronary syndrome (ACS) (RR 0.44, CI 0.23–0.82, p = 0.01), but not in stable CAD (RR 1.02, CI 0.32–3.31, p = 0.97). Moreover, influenza vaccination reduced cardiovascular mortality compared with placebo (RR 0.54, CI 0.37–0.80, p = 0.002). Regarding MACE, influenza vaccination was effective compared with placebo (RR 0.65, CI 0.48–0.88, p = 0.005), especially in ACS (RR 0.58, CI 0.39–0.87, p = 0.007), although it was not effective in stable CAD (RR 0.91, CI 0.54–1.54, p = 0.72). Conclusions: The results of this meta-analysis shows that influenza vaccination is effective to reduce all-cause mortality, cardiovascular mortality and MACE among patients with CAD, especially those with ACS. Healthcare strategies must be outlined to deliver this cheap and effective intervention in order to reduce hard outcomes for CAD patients. 109253 Modality: Best Abstracts Oral – Scientific Initiation Category: COVID-19 AND CARDIOVASCULAR SYSTEM D: 13/10/2022 H: 15:40/16:40 L: Auditório 05 NICHOLAS VINCENT LEE1, Nicolas Henrique Borges1, Thiago Mateus Godoy1, Sabriany Nunes Mendes1, Anna Flavia R S Miggiolaro1, Rafaela C Zeni1, Lucas Baena Carstens1, Marcos Roberto Curcio Pereira1, David Batista Wiedmer1, Marina Luise Viola de Azevedo1, Lúcia de Noronha1 (1) PONTIFICIA UNIVERSIDADE CATÓLICA DO PARANÁ – PUC/PR Introduction: Vascular endothelial cells are involved in the immune response mediated by SARS-CoV-2, which, when activated, has a significant influence on cell stability, culminating in edema, thrombosis, and endothelial dysfunction. The pathways responsible for this endotheliopathy process are still being discussed in the literature. Therefore, the analysis of the vascular events involved in the disease is necessary to concatenate findings that justify the cardiovascular repercussions. Objective: Evaluate the tissue expression of endothelium dysfunction markers, Intercellular Adhesion Molecule 1 (ICAM-1), angiotensin 2 (ANGIO-2), interleukin-1-β (IL1β), and Von Willebrand Factor (VWF) and correlate with endothelial activation/dysfunction in the vascular endothelium of lung samples from COVID-19 patients and compare with H1N1 and control cases. Method: The study analyzed post mortem lung samples (COVID-19 group = 20 cases; Group H1N1 = 10 cases and control group = 11 cases) through immunohistochemistry, using the primary monoclonal antibody of anti-ICAM-1, anti-ANGIO 2, anti-IL1β, and anti-VWF. The immunostained slides were scanted and selected by blinding. Then, software was responsible for quantifying the tissue expression of ICAM-1, ANGIO-2, IL-1β, and VWF obtained in each case. The findings were compared using the Kruskal-Wallis nonparametric test. Results: The histopathological characteristics of pulmonary vascular damage caused by H1N1 differ from those observed in the COVID-19 group. For ICAM-1, an increase in expression was observed with statistically significant (p < 0,0001) when comparing the COVID group with both the control and H1N1 groups. The same pattern was repeated in the expression of IL-1β. Although, for ANGIO-2 and VWF, there was no statistical significance when comparing the COVID group with H1N1 (p > 0,05), when comparing the COVID group with the control group, the expression of these markers was higher (p < 0,05). In addition, there are no significant fibrinous thrombi or neutrophilic endotheliitis present in patients infected with H1N1. Conclusions: Our results demonstrated endothelium activation and dysfunction secondary to cytokine storm. The endothelial injury and the state of hypercoagulability caused by COVID-19, when added to the blood stasis present in bedridden patients, culminating in the formation of the Virchow triad, capable of elevating the chances of systemic thrombotic events and cardiovascular repercussions. 111707 Modality: Best Abstracts Oral – Scientific Initiation Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT D: 13/10/2022 H: 15:40/16:40 L: Auditório 05 JORGE TADASHI DAIKUBARA NETO1, Jorge Tadashi Daikubara Neto1, Matheus Bissa Duarte1, Gustavo Sarot Pereira da Cunha1, Leonardo Henrique dos Santos de Melo1, Michelle Bozko Collini1, Carolina Ruschel Senger1, Jessica Tamires Reichert1, Raphael Henrique Déa Cirino1, Sabrina Bernardez2, Fábio Papa Taniguchi2, Miguel Morita Fernandes-Silva1 (1) Hospital de Clínicas – UFPR; (2) Hospital do Coração – HCor Introduction: Acute Heart Failure (AHF) has high mortality and identifying those patients with worse prognosis helps guiding their management. However, current available prognostic assessment scores have sub-optimal performance, so they are rarely used in clinical practice. Objective: Developing and validating a machine learning-based prognostic score to predict in-hospital death in patients with AHF and compare its performance with the Acute Decompensated Heart Failure National Registry (ADHERE) and the Get With the Guidelines–Heart Failure (GWTG-HF) scores. Methodology: We included patients admitted with AHF in 17 brazilian hospitals participants of a multicenter study from 2016 to 2019. Clinical, laboratory and echocardiographic data and the WHOQOL-BREF quality of life questionnaire at hospital admission were used as covariates. The outcome was in-hospital death from any cause. Machine learning prediction models – using Random Forest, Gradient Boosting Machines, and Deep Neural Networks – were applied in 70% of the sample (training set) to develop the score (ML-HF score), which was validated on the 30% remaining of the sample (test set). Results: From 2657 patients hospitalized for AHF, we included 887 [59% men, 61.6 ± 14.5 years, ejection fraction 41.8 ± 17.2%, 84 (9%) died] who had complete data. The five most important variables of the ML-HF score were: Physical Health Domain quality (WHOQOL-BREF), serum sodium, serum urea, serum creatinine and systolic blood pressure at hospital admission. In the test set, the ML-HF score showed good model calibration (Hosmer-Lemeshow test p value = 0.124) and good model discrimination area under the ROC curves [(AUC) = 0.739 (95%CI,0.652–0.825)], which was better than the GWTG-HF [AUC = 0.601 (IC95%, 0.459–0.743)], p = 0.05 and the ADHERE [AUC = 0.594(IC95%, 0.445–0.742)], p = 0.05 scores (figure). Conclusion: We developed and validated a score using machine learning to predict in-hospital death in patients with AHF, which outperformed the ADHERE and GWTG-HF scores. 111880 Modality: Best Abstracts Oral – Scientific Initiation Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 13/10/2022 H: 15:40/16:40 L: Auditório 05 BEATRIZ SALES DE FREITAS1, Ana Carolina Sampaio Freire1, Caio Resende da Costa Paiva1, Isabel Gomes da Silva1, Gabriel Haiek Fernandes1, Gabriela Gonçalves Almeida1, Maria Luíza Marinho de Sá de Paula Lima1 (1) Universidade de Brasília (UnB) Introduction: Heart transplant (HT) is one of the main surgeries by which patients with advanced and refractory heart failure can prolong their lives. Nonetheless, its complexity requires a well prepared staff and abundant financial resources, which can be a challenge in developing countries such as Brazil. In this scenario, searching for trends in epidemiology and regional disparities is key to scale the problem and propose interventions. Objectives: To analyze data of HT in Brazil’s different regions between January 2008 and December 2021. Methodology: Epidemiological inquiry based on data related to mortality rates and annual HT absolute numbers from 2008 to 2021 in Brazil. The data was obtained through the public domain national platform TABNET from the Informatic Department of the Brazilian Universal Healthcare System. A comparative analysis of data was made, in addition to complementary research in recent literature. Results: From January 2008 to December 2021, 3.368 HTs were performed in Brazil. The main causes for HT were Ischemic Cardiomyopathy, followed by Congestive Heart Failure and Chagasic Cardiomyopathy. The majority of HTs were in the Southeast (n = 1792). In contrast, in the North, which doesn’t have specialized centers, no surgeries were performed (n = 0). It is possible to recognize an increase in the absolute number of procedures throughout the period analyzed followed by a decrease in the last two years. This decrease may be a consequence of the COVID-19 pandemic. Despite the upward trend described, the number of capacitated teams or centers remained fairly the same from 2014 to 2021. Regarding the mortality rates, 13,62% of the patients who underwent HT died in the procedure or in post-op care at the health service. In general, these mortality rates have been declining, going from 20,99% in 2018 to 9,27% in 2021, a possible effect of the evolution in transplant techniques. HTs represent a challenge for Brazil’s health system. In December 2021, 321 patients were waiting for the surgery. Compared to kidney, cornea and liver transplants, HT boasts one of the higher discrepancies between performed versus demanded transplants. Conclusion: HT is a highly complex procedure that requires proper logistics, training, resources and specific post-op care, representing a challenge for Brazil’s public health system. Considering the demand of HTs in Brazil, it is necessary to create new centers and to train new teams, especially in the North. 112045 Modality: Best Abstracts Oral – Scientific Initiation Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 13/10/2022 H: 15:40/16:40 L: Auditório 05 LUIZ FELIPE FAÇANHA RAMOS1, Karen Tássia Façanha Ramos1, Hildeman Dias da Costa2, Ayrison Melo Sousa3, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia; (3) Centro Universitário UNINORTE Introduction: The increase in the amount of low-density lipoproteins (LDL cholesterol) or hypercholesterolemia is considered one of the factors in the occurrence and mortality of cardiovascular and cerebrovascular diseases, especially ischemic heart disease (IHD). Objective: To relate hypercholesterolemia and IHD mortality in men in the countries of America in the year 2018. Methodology: This is an analytical ecological study of geographic distribution with secondary data from the Health Information Platform for the Americas (PLISA), of the Pan American Health Organization (PAHO), of the year 2018, on the averages of total cholesterol and fractions and the mortality rates due to IHD in men in the countries of the American continent. The rates were standardized per 100,000 inhabitants with a confidence interval (CI) of 95%. Results: Guyana had the highest IHD male mortality rate (228.9; 95% CI 175.8–312.7) in 2018 in America, followed by Haiti with the 2nd highest rate (190.3; 95% CI 124.6–278.8). In contrast, Peru has the lowest rate (46.6; 30.4–66.6) in the same year. In addition, it was shown that Guyana has men with the highest mean LDL cholesterol (x̄ = 3.81; 95% CI 3.56–4.05). It is noteworthy that Venezuela was the country with the lowest mean LDL cholesterol (x̄ = 3.23; 95% CI 3.03–3.41), despite having the third highest mortality rate in South America (130.9; 95% CI 98.9–169.1). Conclusions: There is a positive linear correlation between high LDL cholesterol and mortality in men from IHD in America, as Guyana has the highest mortality rate and the highest mean LDL cholesterol in men in 2018. This suggests that public policies should be directed to combat hypercholesterolemia that can significantly increase mortality from ischemic heart disease. 112186 Modality: Best Abstracts Oral – Scientific Initiation Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT D: 13/10/2022 H: 15:40/16:40 L: Auditório 05 DHAYN CASSI DE ALMEIDA FREITAS1, Jefferson Luiz Vieira2, Sabrina Bernardez Pereira3, Fabiana Goulart Marcondes-Braga4, Wilson Nadruz Junior5, Silvia Marinho Martins Alves6, Gabriela Arcoverde Wanderley7, Jessica Tamires Reichert8, José Albuquerque de figueiredo neto9, Alana de Oliveira Castro9, Miguel Fernandes da Silva Morita8, Odilson Marcos Silvestre1 (1) Universidade Federal do Acre; (2) Hospital de Messejana – Dr. Carlos Alberto Studart Gomes; (3) Hospital do Coração; (4) Instituto do Coração – Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; (5) Universidade Estadual de Campinas,; (6) Universidade de Pernambuco; (7) Pronto-Socorro Cardiológico Universitário de Pernambuco; (8) Universidade Federal do Paraná; (9) Universidade Federal do Maranhão Background: There are still no studies that characterize regional differences in HFrEF in Brazil. Objective: To compare regional characteristics in the socioeconomic, clinical, and treatment aspects of HFrEF in Brazil. Methods: Rosa dos Ventos is a cohort study in all Brazilian states which will include 3,000 patients with outpatient HFrEF (EF < 50%). We carried out a cross-sectional study to compare the characteristics of patients (n = 853), according to the Brazilian region of origin: North (n = 115), Northeast (n = 351), Center-west (n = 140), Southeast (n = 77) and South (n = 170). We investigated socioeconomic characteristics, clinical presentation of HF, and use of at least four medications of guideline-directed medical therapy (GDMT) in HF (beta-blockers, mineralocorticoid antagonists, SGLT2 inhibitors, and ACE inhibitors or ARBs or ARNIs). We used Student’s T and Chi-square statistical tests. Results: Comparing the patients, the youngest belonged to the southeast region (55 ± 14, p < 0.001), and were less frequently white (19%, p < 0.001) and with lower monthly familiar income (R$ 2176 ± 3015, p < 0.001) in the Northeast region. There was a higher percentage of chagasic etiology in the Central-West region (42%, p < 0.001) and of ischemic etiology in the North region (39%, p = 0.021), the latter region had the highest mean ejection fraction (37 ± 8, p < 0.001). The use of at least four GDMT drugs was more frequent in the Northeast (23%, p = 0.033) and South regions (22%, p = 0.033), with a higher frequency of ARNI use in the South (45%, p < 0.001). Conclusions: The Rosa dos Ventos Study aims to determine regional differences in HFrEF, and the results will help in planning its prevention and treatment. These preliminary data suggest regional differences in the etiology and use of therapy. 108365 Modality: Best Poster – Researcher Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION D: 14/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres ANA CLÁUDIA CAVALCANTE NOGUEIRA1, Joaquim Barreto2, Beatriz Luchiari2, Isabella Bonilha2, Luiz Sérgio Fernandes de Carvalho3, Andrei Carvalho Sposito2 (1) Escola Superior de Ciências da Saúde – ESCS; (2) Atherosclerosis and Vascular Biology Laboratory (Aterolab), Cardiology Division, Universidade de Campinas – UniCamp; (3) Clarity Healthcare Intelligence Background: We sought to compare the effectiveness and cost-effectiveness of cardioprotective glucose-lowering therapies in individuals with T2D in a middle-income country. Methods: A systematic search was performed for randomized clinical trials published until April 2021 reporting the incidence of MACE for pioglitazone, GLP1A, or SGLT2i. Using date from two national cohorts of T2D, we developed a Markov model to estimate the outcomes for each treatment based on incremental cost-effectiveness ratio (ICER) and the disease-adjusted life years [DALYs] gain per dollar spent projected over a lifetime horizon using a 5% annual discount rate. Results: 157 RCT including 267,508 patients and 176 active arms were considered. Compared with sulfonylureas, SGLT2i, GLP1A and pioglitazone reduced the relative risk of non-fatal MACE with HR of 0.81 (95% CI 0.69 to 0,96, p = 0.011), 0.79 (95% CI 0.67 to 0,94, p = 0.0039) and 0.73 (95% CI 0.59 to 0.91, p = 0.0057), respectively. Pioglitazone resulted in incremental effectiveness of 0.2339 DALYs per patient, at a mean incremental cost of US$1660 and a US$ 7,082 (95% CI: 4,521; 10,770) incremental cost per DALY gained, when compared to standard care. The addition of SGLT2i or GLP1A led to more evident (0.261 and 0.259, respectively) but with higher ICERs [US$ 12,061 (95% CI: 7,227; 18,121) and US$ 29,119 (95% CI: 23,811; 35,367) per DALY gained, respectively]. Compared to SGLT2i and GLP1A, pioglitazone had the highest probability of being cost-effective based on the estimated maximum willingness-to-pay threshold. Conclusions: The three therapies bear similar effectiveness in reducing cardiovascular events. In a middle-income country, pioglitazone presents a higher probability of being cost-effective followed by SGLT2i and then GLP1A. 109342 Modality: Best Poster – Researcher Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY D: 14/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres MARCOS DANILLO PEIXOTO OLIVEIRA1, Lélio Lemos Pinto Neto1, Ednelson Navarro2, Adriano Caixeta1 (1) Universidade Federal de São Paulo, UNIFESP; (2) Hospital Regional do Vale do Praíba Background: Distal transradial access (dTRA) as a refinement of the conventional transradial approach has several potential advantages in terms of patient and operator comfort, faster hemostasis, and lower risk of proximal radial artery occlusion. We aim to describe our prospective real-world experience with dTRA as default for routine coronary angiography and percutaneous coronary interventions (PCI) in a broad sample of all-comers patients. Material and methods: From February 2019 to April 2022, 3,991 consecutive all-comers patients submitted to coronary angiography and/or PCI via dTRA have been enrolled into the DISTRACTION registry. Results: Mean patient age was 63.36 ± 13.3-year-old, most male (65.4%) and with acute coronary syndromes (48,8%) at admission. Overall, 843 (20.1%) patients had non-ST-elevation myocardial infarction, 799 (20,0%) had ST-elevation myocardial infarction, and 114 (2.6%) presented in cardiogenic shock. There were only 94 (2.3%) access site crossovers, in only 79 (1.9%) patients dTRA sheath insertion could not be obtained. Right dTRA was the most frequent access (80.1%), followed by redo right dTRA (10.8%), left dTRA (8,1%) and simultaneous bilateral dTRA (0.7%). In 2,210 (60.0%) of all patients, PCI was performed and left anterior descending was the most prevalent target coronary territory (29.1%). No major adverse cardiac and cerebrovascular events and no major complications directly related to dTRA were recorded. Conclusions: The adoption of dTRA as default for routine coronary angiography and PCI in a real-world fashion of all-comers patients by experienced transradial operators appears to be feasible and safe. 111318 Modality: Best Poster – Researcher Category: DIGITAL HEALTH/INNOVATION D: 14/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres ELIZABETH ANNE CALLEJA1, Elizabeth A Calleja1, Dr Amanda K Buttery1, Teresa Gadaleta1, Sheree Hughes1, Jarrod Leggett1, Associate Professor Trevor Shilton1 (1) National Heart Foundation of Australia Physical activity is an important modifiable risk factor for the prevention and management of cardiovascular disease and can reduce the risk of morbidity by 35% [1]. However, only 15% of Australian adults meet the physical activity guidelines [2]. A national walking program was rolled out across Australia led by a national voluntary group-based community walking program in 2007. In response to the COVID-19 pandemic, we aimed to develop a digital program option for people to walk independently. Personal Walking Plans (PWP) were launched in Australia as a free six-week web-based digital program for adults to address this need. Methods: We designed personal walking plans on behaviour change principles and theory. This included four graded walking programs with strengthening and flexibility exercises, supported with online instructional videos and motivational messaging via email or text (SMS) messages. A digital self-report survey was conducted on enrollment to the program, and after completion at six weeks. These were completed between 11th of May and 30th June 2021. Survey questions included established items on participant goals, physical activity levels, influence of motivational messaging (text and email) and intention to continue walking following program completion. Results: Of 2338 (mean 65 years; 87% female) participants completing the survey, 74% completed the program. Of these, 86% achieved their goal and 69% met the Australian physical activity guidelines by the end of the six-weeks. On average, participants increased the number of days they engaged in physical activity from 2.7 days to 4.4 days per week by the end of the six weeks. Strength exercise sessions increased from 0.9 days to 2.4 days per week. Participants reporting they paid attention to motivational messages increased their physical activity (30min physical activity, 4.4 days, strength 2.4 days) more than those who ignored/did not receive texts (30min physical activity, 3.9 days, strength 2.0 days). Nearly all (99%) reported that they would continue walking following the intervention. Conclusion: A six-week digital personal walking plan was effective in improving participants’ attainment of the Australian adult physical activity guidelines. Motivational text messaging and emails appeared to enhance engagement with the program and physical activity levels. Long-term follow-up surveys at 6 and 12 months will evaluate if benefits are sustained over time. 111421 Modality: Best Poster – Researcher Category: CARDIOVASCULAR IMAGING D: 14/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres RONALDO DE SOUZA LEAO LIMA1, Andre Luiz Bezerra2, Claudio Domenico2, Andrea Rocha de Lorenzo2 (1) Fonte Imagem; (2) Universidade Federal do Rio de Janeiro Introduction: CZT cameras have higher sensitivity for photon detection, as well as higher temporal and spatial resolution. These have enabled que noninvasive quantification of myocardial flow reserve (MFR), which may increase the accuracy of myocardial perfusion SPECT (MPS) for the detection of obstructive coronary artery disease (CAD). This study aimed to compare the accuracy of CZT MPS and of MFR for the detection of obstructive CAD. Methods: 66 patients with CAD (>50% obstruction) detected at invasive coronary angiography or CT angiography underwent dipyridamole MPS and MFR evaluation within 30 days. A 1-day protocol (rest-stress) was used to quantify MFR. The acquisition of dynamic rest and stress images was initiated simultaneously to 99mTc sestamibi injection (10mCi e 30mCi, respectively), both lasting for 11 minutes, followed by 5-minute imaging. Pharmacologic stress with dipyridamole (0,56 mg/kg for 4 minutes) was performed with the patient positioned in the CZT camera. The images were processed and time-activity curves were generated, calculating global and regional MFR in a semiautomatic software. A global or regional MFR <2.0 was considered abnormal. The MPS perfusion images were classified as normal or abnormal and perfusion scores were calculated. The images were interpreted by experienced physicians blinded to the results of MFR and coronary angiography/CT. Results: Mean age of the population was 63 ± 3 years, 51.5% male. Hypertension, hypercholesterolemia and diabetes were the most frequente risk factors (80.3%, 45.4%, and 42.4%, respectively). Thirty patients (45.5%) had single-vessel CAD, 28 (42.4%) 2-vessel CAD and 8 (12.1%), triple-vessel CAD. Among the 110 vessels with obstruction, 67 had perfusion abnormalities in MPS and 81 had reduced MFR, while among the normal vessels, 76 had normal MPS and 74 had preserved MFR. The sensitivity of MFR (73,6%) was higher than that of MPS (60.2%), without significant changes in specificity (86,4 vs 84.1%). Conclusions: MFR in the CZT camera is an absolute, physiologic, quantifiable measure which is more sensitive for the detection of obstructive CAD than perfusion abnormalities in MPS, especially in patients with multivessel CAD. 111997 Modality: Best Poster – Researcher Category: COVID-19 AND CARDIOVASCULAR SYSTEM D: 14/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres WARLEY CEZAR DA SILVEIRA1, Manuela Furtado Sacioto2, Luiza Margoto Marques2, Mateus Chaves Ferreira1, Beatriz Figueiredo Lima1, Maria Izabel Alcântara Cunha4, Bruno Barbosa Miranda de Paiva1, Marcos André Gonçalves1, Polianna Delfino Pereira1, Ana Beatriz de Castro Feres2, Magda Carvalho Pires1, Milena Soriano Marcolino1 (1) Universidade Federal de Minas Gerais (UFMG); (2) Faculdade Ciências Médicas de Minas Gerais (FCMMG); (3) Institute for Health Technology Assessment (IATS/CNPq); (4) Centro universitário de Belo Horizonte (UNIBH) Introduction: COVID-19 patients present a high incidence of venous thromboembolism (VTE), which requires early recognition and treatment. In those patients, the diagnosis of pulmonary embolism is a challenge, as its symptoms and signs overlap with the ones of the severe acute respiratory syndrome (SARS). Several studies have tried to identify risk factors of VTE in hospitalized COVID-19 patients, as a path to promote prevention, early diagnosis and treatment. However, such studies have shown inconsistent results. Objective: To identify VTE predictors by both logistic regression (LR) and machine learning (ML) approaches and report the incidence of thromboembolic complications in COVID-19 and their prognostic impact. Methods: This substudy of a large Brazilian COVID-19 Registry included consecutive COVID-19 adult patients from 16 hospitals, admitted between March and September, 2020. Symptomatic VTE was confirmed by objective imaging. LR analysis, tree-based boosting and bagging were used to investigate the association of variables upon hospital presentation with VTE. Results: Among 4,120 patients (median age was 61 years [IQR, 48–72] years-old, 55.5% men, 39.3% critical patients), VTE was confirmed in 6.7%. In multivariate LR analysis, obesity (OR 1.50, 95%CI 1.11–2.02); being an ex-smoker (OR 1.44, 95%CI 1.03–2.01); surgery ≤90 days (OR 2.20, 95%CI 1.14–4.23); axillary temperature (OR 1.41, 95%CI 1.22–1.63); D-dimer ≥4 times above the upper limit of reference value (OR 2.16, 95%CI 1.26–3.67), lactate (OR 1.10, 95%CI 1.02–1.19), C-reactive protein levels (CRP, OR 1.09, 95% CI 1.01–1.18); and neutrophil count (OR 1.04, 95%CI 1.005–1.075) were independent predictors of VTE. Temperature at admission, SF ratio, neutrophil count, D-dimer, CRP and lactate levels were also identified as predictors by ML methods. Patients with confirmed VTE had higher mortality (28.4% vs 18.5%, p < 0.001) and had a higher frequency of mechanical ventilation support (58.4% vs 26.4%, p < 0.001) and renal replacement therapy (21.5% vs 9.7%, p < 0.001), when compared to the group without confirmed VTE. Conclusion: By using ML and LR analysis, we showed that D-dimer, axillary temperature, neutrophil count, CPR and lactate levels are risk factors for VTE in COVID-19 patients. Therefore, we suggest that patients presenting these risk factors at admission should be more closely monitored for VTE development, considering the importance of prevention, early diagnosis and treatment of VTE. 108390 Modality: Best Poster – Young Researcher Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE D: 14/10/2022 H: 16:10/16:50 L: Área de exposição de pôsteres MARINA GAIATO MONTE 1, Carolina Rodrigues Tonon1, Anderson Seiji Soares Fujimori1, Ana Paula Dantas Ribeiro1, Katashi Okoshi1, Paula Schmidt Azevedo1, Marcos Ferreira Minicucci1, Leonardo Antonio Mamede Zornoff1, Sergio Alberto Rupp de Paiva1, Bertha Furlan Polegato1 (1) Faculdade de Medicina de Botucatu Introduction: Doxorubicin (DOX) is widely used effective chemotherapy drug; however, it can cause cardiotoxicity which is a very serious side effect. There is no effective therapy for cardiotoxicity. Omega-3 fatty acid (O3) supplementation may act in the sphingomyelin-ceramide pathway. We aimed to evaluate the influence of O3 in attenuating DOX-induced cardiotoxicity. Methods: Male Wistar rats (n = 60) were divided into 4 groups: control (C), administration of O3 only (O3), DOX only (D), and DOX and O3 (DO3). O3 (400 mg/kg/day, gavage) was administered for 6 weeks. DOX (3.5 mg/kg, IP, once a week) was administered for the last 4 weeks of the experiment. At the end of 6 weeks, rats were submitted to echocardiogram and euthanized (thiopental 120 mg/kg, ip). Statistical analysis: 2-way ANOVA (pi: p value for the interaction between DOX and O3; pD: p value for the effect of DOX; pO3: p value for the effect of O3). Results: Group D exhibited increased left atrium diameter/aorta diameter ratio (C 1.31 ± 0.11; D 1.45 ± 0.11; O3 1.36 ± 0.11; DO3 1.27 ± 0.11; pD = 0.467, pO3 = 0.028, pi < 0.001) and decreased left ventricular fractional shortening (C 0.57 ± 0.07; D 0.46 ± 0.07; O3 0.56 ± 0.08; DO3 0.53 ± 0.08; pD = 0.002; pO3 = 0.164; pi = 0.046) compared to Group C, characterizing diastolic and systolic dysfunction, respectively. DOX increased neutral sphingomyelinase activity (nSMase, C 2283 ± 412; D 2879 ± 680; O3 2461 ± 639; DO3 3319 ± 284 UI fluorescence; pD < 0.001, pO3 = 0.087, pi = 0.461) and decreased myocardial nSMase protein quantification (C 0.05 ± 0.03; D 0.04 ± 0.02; O3 0.05 ± 0.02; DO3 0.03 ± 0.02 arbitrary units; pD = 0.009, pO3 = 0.455, pi = 0.275). There were no differences between groups in myocardial ceramide deposition evaluated by immunohistochemistry. Conclusion: O3 supplementation attenuates DOX-induced diastolic and systolic dysfunction with no changes in neutral sphingomyelinase activity or expression in the myocardium. Financial support: FAPESP 2018/25677-7 and CNPq 407201/2021-1. 108392 Modality: Best Poster – Young Researcher Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE D: 14/10/2022 H: 16:10/16:50 L: Área de exposição de pôsteres MARINA GAIATO MONTE 1, Carolina Rodrigues Tonon1, Tatiana Fernanda Bachiega Pinelli1, Anderson Seiji Soares Fujimori1, Ana Paula Dantas Ribeiro1, Nayane Maria Vieira1, Natalia Fernanda Ferreira1, Danilo Malmonge Barbosa Luciano1, Ronny Peterson Cabral1, Katashi Okoshi1, Bertha Furlan Polegato1, Leonardo Antonio Mamede Zornoff1 (1) Faculdade de Medicina de Botucatu Background: Doxorubicin (dox) is used in the treatment of several types of cancer. However, cardiotoxicity is a common side effect of the drug. The pathophysiology of cardiotoxicity is not clearly understood. Neutrophils produce attractant substances such as NETs (neutrophil extracellular traps), that are involved in immune response and inflammation which could mediate myocardial extracellular matrix remodeling. Purposes: To analyze the role of NETs in the pathophysiology of acute dox-induced cardiotoxicity. Methods: 60 male Wistar rats were allocated into 3 groups: Control (C), Dox (D), and Dox + DNAse (DD). D and DD groups received an intraperitoneal injection of dox 10 mg/kg, and 2h later, DD received a subcutaneous injection of DNAse 20 mg/kg (NETs inhibitor). Rats were submitted to cardiac function evaluation and euthanasia 48h after dox injection. Statistical analysis: one-way ANOVA. Results: D showed increased NETs production compared with C and DD (C = 2997 ± 810; D = 5955 ± 1906; DD = 4108 ± 1674 pg/mL; p < 0.001). Transthoracic echocardiogram showed no differences in systolic parameters, but isovolumetric relaxation time corrected by heart rate was higher in D and DD than C (C = 46 ± 4.6; D = 51 ± 7.9; DD = 51 ± 7.7, p = 0.039). Additionally, in isolated heart study, area under curve for diastolic pressure-volume ratio was reduced in D, indicating lower ventricular compliance, compared with C and DD (C = 827 ± 74; D = 670 ± 109; DD = 966 ± 218; p = 0.007). Dox induced increased malondialdehyde myocardial concentration in D and DD compared to C (C = 48 ± 28; D = 73 ± 32; DD = 82 ± 25 nmol/mg of protein; p < 0.05). Regarding extracellular matrix, dox increased and DNAse attenuated collagen in cardiac tissue (C = 2.88 ± 0.97, D = 3.51 ± 0.7, DD = 2.99 ± 0.66%; p < 0.05). Additionally, dox increased matrix metalloproteinase activity (MMP)-2 (C = 1.01 ± 0.28, D = 2.04 ± 0.47, DD = 2.36 ± 0.6; p < 0.001), but DNAse did not interfere with this parameter. Evaluation of protein expression of |Type 2 and 4 MMP tissue inhibitors (TIMP) showed no differences between groups. Conclusions: Dox-induced cardiotoxicity is associated with diastolic dysfunction, cardiac fibrosis, increased MMP-2 activity, and oxidative stress. NETs are involved in the pathophysiology of dox-induced cardiotoxicity. Netosis inhibition improved diastolic function, associated with decreased myocardial collagen content. However, this effect was not mediated by oxidative stress, MMP-2 activation, or TIMP-2 and –4 protein expression. 109335 Modality: Best Poster – Young Researcher Category: CARDIO-ONCOLOGY D: 14/10/2022 H: 16:10/16:50 L: Área de exposição de pôsteres RENATA ALVES1, Cláudia de Morais Sequeira1, Jefferson Fernandes Evangelista1, Ana Lucia Rosa Nascimento1, Cristiane Matsuura1 (1) Universidade do Estado do Rio de Janeiro Introduction/aim: It is well known that doxorubicin (DOX) elicits toxic effects on the heart limiting its use in cancer treatment. Here, we investigated the effects of resistance training on left ventricle (LV) ultrastructure and function in DOX-induced cardiotoxicity in rodents. Methods: Male adults Sprague Dawley rats were divided into three groups (n = 10): control, DOX that remained sedentary (DoxSed), or DOX submitted to resistance training (DoxTr). Resistance training (5 d/wk for 8 wks) consisted of climbing a ladder with weights placed on the tail, with progressive increase in the training load (number of repetitions and weight). DOX was administered for 10 consecutive days (1 mg/kg/d, i.p.) and it initiated concomitantly with training. At the end, cardiac function was measured by echocardiography, and LV fragments were processed for transmission electron microscopy. Results: There was a reduction in mortality in DoxTr compared to DoxSed (20 vs. 38%, P < 0.001, log rank test). The decrease in LV ejection fraction observed in DoxSed was attenuated in DoxTr (control: 76 ± 1; DoxSed: 64 ± 1; DoxTr: 71 ± 1%; P < 0.05, one-way ANOVA). The most striking effects were seen in LV ultrastructure (Fig 1). The control group showed a normal structural arrangement, with myofibrils arranged in parallel, preserved sarcomeres with uniform distance between Z lines. Intact mitochondria were seen in parallel arrangement to the myofibrils. The cardiomyocytes of the DoxSed group showed severe cellular disruption, with fragmentation of the myofibrils, disappearance of some sarcomeres, increased electron-lucid cytoplasmic content and the presence of autophagosomes, and degenerated mitochondria. Resistance training resulted in positive effects on the ultrastructural morphology of cardiomyocytes, with intact mitochondria and large areas of preservation of the structural organization of the sarcomere, although it was still possible to observe a non-linear arrangement and reduction in the density of myofibrils. Conclusion: Resistance training can be a non-pharmacological strategy to prevent the deleterious effects of DOX on the heart. 110132 Modality: Best Poster – Young Researcher Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION D: 14/10/2022 H: 16:10/16:50 L: Área de exposição de pôsteres FILIPE FERRARI1, Henrique C. da Silva2, Luiz G. M. Emed3, Guilherme D. Dilda4, Haroldo C. Aleixo5, Márcio Dornelles6, Fernando Bassan7, Felipe E. F. Guerra8, Frederico P. L. Coimbra9, Mateus F. Teixeira10, Anderson D. da Silveira11, Ricardo Stein1 (1) Hospital de Clínicas de Porto Alegre (HCPA), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS – Brazil; (2) Universidade do Estado do Pará (UEPA), Belém, PA – Brazil; (3) Hospital Cardiológico Costantini, Curitiba, PR – Brazil; (4) Hospital das Clínicas da Universidade de São Paulo (USP), São Paulo, SP – Brazil; (5) Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG – Brazil; (6) Grêmio Foot-Ball Porto-Alegrense (GFPA), Porto Alegre, RS – Brazil; (7) Universidade do Estado do Rio de Janeiro (UERJ), RJ – Brazil; (8) Clínica Biocardio, Natal, RN – Brazil; (9) Hospital de Urgências de Goiânia, Goiânia, GO – Brazil; (10) Clube de Regatas Vasco da Gama, RJ – Brazil; (11) Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS – Brazil Introduction: The 12-lead ECG is a useful tool for screening cardiac abnormalities in athletes. We aimed to describe physiological ECG findings in young Brazilian football players (YBFP) based on the “2017 International Criteria for Electrocardiographic Interpretation in Athletes”. Methods: Cross-sectional/descriptive study. Intra-group differences were estimated by linear models or binomial and multinomial logistic regressions. Results: 3.490 YBFP from 41 clubs, aged 15–35 years (median: 19 y) were evaluated. 1.668 were Caucasians, 1.154 Mixed-race (MR) and 668 Afro-Brazilians (AB). Prevalence: sinus bradycardia (50%), incomplete RBBB (12%), first-degree AV block (3%), Mobitz type I AV block (0.1%), and increase QRS voltage for left or right ventricular hypertrophy (34% and 15%, respectively). ST-elevation followed by T-wave inversion confined to V1–V4 leads were identified in 2% of AB. Early repolarization (ER) was present in 35% of athletes (AB versus Caucasians and MR: P = 0.002 and P = 0.004, respectively), which was similar to the PR interval (P < 0,001 for both comparisons). There was no difference between Caucasians and MR for ER or PR intervals. For all remaining variables, there was no difference among races. Conclusions: This is the first large study to describe the prevalence of physiological electrocardiographic findings in YBFP. Further studies comparing the frequency of these findings with the prevalence observed in other cohorts are welcome. 112183 Modality: Best Poster – Young Researcher Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT D: 14/10/2022 H: 16:10/16:50 L: Área de exposição de pôsteres PEDRO HENRIQUE DE BORBA ENGSTER1, Pedro Henrique de Borba Engster1, André Zimerman1, Anderson Donelli da Silveira1, Marina S. Borges1, Thomas U. Schaan1, Gabriel C. de Souza1, Giovanni Donelli Costa1, Luís Eduardo Rohde1 (1) Hospital de Clínicas de Porto Alegre; (2) Universidade Federal do Rio Grande do Sul Background: For patients with heart failure (HF), the validity of the New York Heart Association (NYHA) functional class to assess prognosis may be limited when compared with the objective cardiopulmonary exercise test (CPET). Purpose: To investigate the prognostic value of NYHA classification and CPET parameters. Methods: We included the first CPET of every adult patient with HF who in a tertiary care center in Brazil. NYHA class was determined on the day of CPET or during the prior ambulatory visit. NYHA and Weber classes were stratified into “favorable” (NYHA I or II; Weber A or B) or “poor” (NYHA III or IV; Weber C or D), and subjects with discordant classes were compared in a survival analysis. Primary endpoint was all-cause mortality at 5 years. We used a Cox proportional hazards model to estimate the probability of death in 5 years according to relative peak VO2 and NYHA class, adjusted for age and sex. Results: We included 855 patients, of which 30% (255) were classified as NYHA I, 43% (368) as NYHA II, 24% (202) as NYHA III, and 4% (30) as NYHA IV. Mean age was 56 years (±13), 42% (359) were female, and mean LVEF was 36% (±15). Mean relative peak VO2 ranged from 19.6 (NYHA I) to 14.0 (NYHA IV) ml/kg/min. Patients with poor NYHA and favorable Weber classes displayed similar rates of all-cause mortality as patients with favorable NYHA and poor Weber classes (hazard ratio 1.54 [95% CI 0.88–2.70]. In the multivariable model, both NYHA and relative peak VO2 significantly predicted mortality in 5 years after mutual adjustments (Figure 1). The distinction between NYHA I and II did not, however, improve prognostic assessment. Conclusions: Physician-assigned NYHA class and objective CPET measures provide complementary prognostic information, and NYHA classification may be particularly limited for mild cases of HF. These findings question the use of NYHA as the main determinant to guide HF therapy. 108597 Modality: Best Poster – Scientific Initiation Category: DIGITAL HEALTH/INNOVATION D: 13/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres MARIA EDUARDA QUIDUTE ARRAIS ROCHA1, Bruna Sobreira Kubrusly2, Fernanda Pimentel Arraes Maia2, Rodrigo Carvalho Paiva3, Alessia de Alencar Araripe Gurgel3, Davi Sales Pereira Gondim1, Juvêncio Santos Nobre2, Luis Gustavo Bastos Pinho2, Ana Gabriela Ponte Farias2, Maria Camila Timbó Rocha3, Eduardo Augusto Quidute Arrais Rocha3, Francisca Tatiana Moreira Pereira2 (1) Universidade de Fortaleza (Unifor); (2) Universidade Federal do Ceará (UFC); (3) Centro Universitário Christus (UNICHRISTUS) Introduction: The remote monitoring (RM) of implantable electronic cardiac devices (IECD) has shown advantages in the reduction of morbidity and mortality. The early detection of alterations and the possibility of treatment before the occurrence of symptoms has been a great attractive of this follow-up method. Meanwhile, not all the groups benefit, there are costs associated to these devices and the large number of events may determine an overload to the professionals. This work aimed to identify the predictive factors of larger benefit during the IECD follow-up through RM. Methods: This is a cohort prospective study. The statistical analysis used logistic regression models, with p < 5% for statistical significance. The explanatory variables were selected by a stepwise selection method based on the Akaike Information criterion as a measure to choose the best explaining model. The analyzed variables were age, sex, functional class (FC) ≥ II, ejection fraction (EF), type of IECD, sustained ventricular tachycardia and red alert transmission, indicating important events detections. Three different models were created, with the response variable given by, respectively, elective therapy change (model 1), urgent therapy change (model 2) and whether event detections prevented hospitalizations (model 3). Results: There were 119 patients, with mean age of 72 ± 14 years, 69.8% males. The most common pathologies were ischemic cardiomyopathy 28.6%, dilated cardiomyopathy 22.7% and Chagas disease 6.7%. The groups using a biventricular pacemaker(PM) (75.0%, p = 0.02), reduced EF (76.5%, p = 0.01) FC ≥ Il (75.0%, p < 0.01) had the larger rates of events. The red alert transmission variable was statistically significant in the three models (p-values of, respectively, 0.048;0.007 and 0.048), while the presence of ventricular tachycardia was statistically significant in two models, with p-vales of p = 0.039 (changes in elective therapy) and p = 0.039 (prevented hospitalizations). The FC ≥ II was associated to the urgent therapy change (p = 0.047). Conclusion: Patients with biventricular PM, reduced EF, and more advanced FC had higher event rates in the RM of IECD. The detection of alerts considered by the system as important (red alerts) was associated with changes in immediate, elective conduct and was able to reduce hospitalizations. 109148 Modality: Best Poster – Scientific Initiation Category: HYPERTENSION/RENAL DENERVATION D: 13/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres JOÃO GABRIELL BEZERRA DA SILVA1, João Gabriell Bezerra da Silva1, Gabriela da Silva Nascimento1, Hugo Farah Affonso Alves1, Lucca Hiroshi de Sá Kimura1, João Gabriel Rega do Nascimento Vallaperde1, Vitor de Melo Nolasco1, Bianca Botelho Viegas1, Carlos Filipe dos Santos Pimenta1, Bernardo Chedier1, Arthur Fernandes Cortez1, Elizabeth Silaid Muxfeldt1 (1) Universidade Federal do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho – ProHArt Background: Resistant hypertension (RHT) defined as an uncontrolled blood pressure (BP) despite the use of 3 or more antihypertensives presents a high cardiovascular (CV) morbidity and mortality and high prevalence of chronic kidney disease (CKD). High BP levels and kidney injury appear to be strongly associated with inflammatory biomarkers. Objective: To evaluate the relationship between inflammatory markers and subclinical and established CKD in a large cohort of patients with RHT. Methods: Cross-sectional study that evaluated 423 resistant hypertensives (30.5% male, mean age 64.0 ± 10.8 years) submitted to renal function assessment (albuminuria dosage and evaluation of glomerular filtration rate calculated from the CKD-EPI formula) and dosage of inflammatory markers: TNF-alpha, MCP-1, E-selectin and PAI-1. Socio-demographic characteristics, anthropometric measurements and CV risk factors were recorded. We considered subclinical CKD those patients with moderately high albuminuria (30–300 mg/g) and/or GFR between 30 and 60 ml/min/1.73 m2 and established CKD those who presented albuminuria >300 mg/g and/or TFG < 30 ml/min/1.73 m2. Variance analysis compared serum levels of the 4 inflammatory markers and bivariate analysis compared patients with and without subclinical and established chronic kidney disease. Results: The prevalence of established CKD was 7.3% (31 patients) and subclinical CKD was 47% (187 patients). Patients with subclinical CKD were older and with higher arterial stiffness (higher pulse wave velocity). TNF-alpha (7.1 [4.4–8.6] vs 51, [3.2–7.5]) and MCP-1(284 [220–379] vs 260 [185–359] were significant higher in this group of patients. When we analyzed patients with established CKD, we observed that they have higher BP levels and that TNF-alpha values (7.8 [5.6–14.0] vs 5.6 [3.5–8.3]) and E-selectin (54.4[41.2–61.3] vs. 47.8 [32.0–65.3]) were significantly higher in this group. Conclusion: Among the inflammatory markers evaluated, which was most strongly correlated with subclinical CKD were TNF-alpha and MCP-1, while those with established CKD have higher TNF-alpha and E-selectin levels, possibly pointing out that the MCP-1 is an earlier marker of kidney injury. 111855 Modality: Best Poster – Scientific Initiation Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH D: 13/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres LUIZ FELIPE FAÇANHA RAMOS1, Hildeman Dias da Costa2, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia Introduction: Mortality from cardiovascular diseases (CVD) is aggravated by arterial hypertension, as a consequence of high consumption of table salt. Worldwide salt intake is above the 5 grams (2 grams of sodium) per day recommended by the World Health Organization (WHO), and its consumption varies from 8.5 to 15 grams per person. Objective: To relate CVD mortality with policies to reduce salt/sodium consumption in South American countries in the year 2019. Methodology: This is an analytical ecological study of geographic distribution with secondary data from the Information Platform in Health for the Americas (PLISA), from the Pan American Health Organization, of the year 2019, with CVD mortality indicators and policies to reduce salt/sodium consumption in South American countries. In addition, mortality rates (MT) were standardized per 100,000 inhabitants, with a confidence interval of 95%, classified by quintile. These data were tabulated in the Microsoft Excel software and the variables were analyzed in the GNU SPPP software version 1.5.3, using the Pearson Correlation Test, with a significance level of 5%. Results: In 2019, in South America, the highest CVD mortality rates occurred in Guyana (TM = 448.3; 95% CI 363.3–548.5; Q5) and Suriname (TM = 270.8; 95% CI 212.1–337.6; Q5), on the other hand, the lowest mortality occurred in Peru (TM = 77.8; 95% CI 56.9–103.9; Q1) and in Chile (TM = 95. 5; 95% CI 74.4–118.9; Q1). It is noteworthy that the countries that had the highest mortality are the nations that did not have any (n = 0) initiative to reduce salt/sodium consumption in 2019, however, the countries with the lowest mortality were those that developed 3 policies to reduce this excessive consumption. It is evident that Brazil developed 2 reduction policies, but had a high number of deaths from CVD (TM = 217.1; 95% CI 189.9–239.9; Q3). Analyzing the bivariate correlation of the two variables, a negative Pearson coefficient was obtained (r = –0.59; p = 0.043). Conclusion: Countries that developed more initiatives to reduce excessive salt consumption had significantly lower mortality rates from CVD, showing the importance of reducing this consumption to avoid hypertensive complications that lead to death. Thus, the inversely proportional relationship between the number of these policies and the number of deaths from CVD is confirmed. 111704 Modality: Best Poster – Scientific Initiation Category: CARDIOVASCULAR PHARMACOLOGY D: 13/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres PIETRA ARISSA COELHO MATSUNAGA1, Evellyn Cristiny Pereira Marinho Bezerra1, Júlia Fonseca da Silva Saad1, Lara da Silva Soledade1, Ana Cláudia Cavalcante Nogueira3, Alexandre Anderson de Souza Munhoz Soares3, Gustavo Alexim2, Luiz Sérgio Fernandes de Carvalho5, Renato Barros4 (1) Universidade Católica de Brasília UCB; (2) Hospital de Base do Distrito Federal HB-DF; (3) Instituto Aramari Apo; (4) Hospital Regional de Sobradinho do Distrito Federal HRS-DF; (5) Clarity Healthcare Intelligence Introduction: Prasugrel is an thienopyridine antiplatelet agent, which can be an important alternative to clopidogrel in patients with Acute Coronary Syndrome (ACS) treated through percutaneous coronary intervention. In spite of the role of clopidogrel in these conditions, there are no long-term real-world studies comparing clopidogrel versus prasugrel. Objective: To evaluate the long-term risk of major adverse cardiovascular events (MACE) in patients with ACS who are making use of prasugrel or clopidogrel. Methods: This study is a retrospective analysis of all the individuals that were admitted with the principal diagnosis of acute coronary syndrome within the period of 2013 to 2015 and were submitted to cardiac catheterization in public hospitals of Brasilia, FD, Brazil. The total group was divided into two: the first group being of patients who took clopidogrel and the second group formed by patients on prasugrel. The primary clinical outcome was comprised by cardiovascular (CV) deaths and recurrent ACS (MACE). Multivariate analyses on 16 covariates were carried and followed by a Propensity Score Matching analysis (PSM). Results: A total of 2,491 patients with a principal diagnosis of ACS were included (1,616 with clopidogrel and 875 with prasugrel) and monitored for a median of 6.5 (IQR 1.9) years with 742 primary events. Clopidogrel was associated with high rates of MACE in 12 months (20.1% vs 11.8%, p < 0.001, 428 MACE) and 24 months (23.8% vs 17.7%, p < 0.001, 540 MACE) compared to prasugrel, but the curves equalized at 36 months (25.9% vs 26.6%, p = 0.740) and remained equalized until median follow-up time. In multivariate analysis, prasugrel showed a hazard ratio (HR) 0.61 (95% CI 0.47–0.78 at 12 months), 0.78 (95% CI 0.63–0.96, p < 0.001 at 24 months) and 0.94 (95% CI 0.78–1.1 at 36 months). The results were maintained in the PSM analysis. The mean time on prasugrel in this cohort was 22.1 (IQR 5.9) months. Conclusion: Stemming from these findings, despite the already known role that prasugrel has in reducing the risk of MACE in ACS, these results do not seem to be sustained in the long term. The data reveal that after the interruption of drug use there was an increase in the amount of major adverse cardiovascular events in the second and third year of monitored patients in the prasugrel group. Therefore, the benefit of using prasugrel is neutralized over time. 111863 Modality: Best Poster – Scientific Initiation Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE D: 13/10/2022 H: 10:00/10:40 L: Área de exposição de pôsteres RAFAEL DIAS DE BRITO OLIVEIRA1, Denise Mayumi Tanaka2, Thayrine Rosa Damasceno1, Camila Godoy Fabrício2, Alessandra Arantes Resende2, Eduardo Elias Vieira de Carvalho2, Dawit Albieiro Pinheiro Gonçalves1, Enrico de Francisco Magnani1, Mariana Duarte de Souza1, Marcus Vinícius Simões2, Luciano Fonseca Lemos de Oliveira1 (1) Universidade Federal de Minas Gerais – UFMG; (2) Faculdade de Medicina de Ribeirão Preto – FMRP/USP Introduction: Denervation of atrial parasympathetic and ventricular sympathetic fibers is common in chronic Chagas cardiomyopathy (CC) and may be associated with increased ventricular arrhythmias and sudden death. However, it is unclear if the heart rate variability (HRV) present potential to detect these alterations. Objective: We aimed to evaluate the applicability of a low-cost, non-invasive, and unrestricted method to identify animals with ventricular sympathetic denervation and severe cardiac dysfunction in an experimental model of CC in hamsters. Methods: In the study’s first phase, 10 control hamsters were used to validate the non-invasive electrocardiogram (ECG) acquisition system using a vest. Signal quality and heart rate variation were evaluated in anesthetized and dobutamine-infused animals. The vest was also compared to the acquisition of invasive electrodes in conscious animals. In the second phase, HRV was analyzed in an experimental model of CC (CC; n = 6) and its respective controls (CT; n = 6). Myocardial function and morphology were evaluated by two-dimensional echocardiography in dedicated equipment (VEVO2100). The intensity of ventricular fibrosis and/or inflammation was assessed by the extent of perfusion defects at rest using high-resolution myocardial perfusion scintigraphy, acquired by locally constructed equipment and validated system of small animal images. Results: The ECG acquired by the vest showed excellent signal quality, detecting both the increase in HR during dobutamine infusion and HRV in awake animals. In CC animals, left ventricular ejection fraction (LVEF) correlated with indicators of parasympathetic activity (LF; r = 0.72, p = 0.01) and sympathetic activity (HF; r = –0.72, p = 0.01), as well as with the LF/HF ratio (r = 0.70, p = 0.01). In addition, a significant correlation was observed between left ventricular perfusion defect (LVPD) with LF (r = 0.72, p < 0.00), HF (r = –0.72, p < 0.00), and LF/HF (r = 0.71, p = 0.01). A significant correlation was also observed between left ventricular diastolic diameter (LVDD) and LF (r = 0.59, p = 0.04) and HF (r = –0.59, p = 0.04). Conclusion: The low-cost, non-invasive and unrestricted vest method effectively detected ECG signals from animals at rest, during dobutamine infusion and in motion. The vest was also able to detect autonomic changes resulting from ventricular dysfunction, and was sensitive in identifying sympathetic denervation resulting from myocardial fibrosis and/or inflammation. 107748 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES IRENA MITEVSKA1, Elena Grueva1, Irina Kotlar1, Emilija Lazarova1, Marijan Bosevski1 (1) University Cardiology Clinic Introduction: In patients with pulmonary embolism (PE), assessment of right ventricular (RV) function on transthoracic echocardiography has been shown to be an independent predictor of 30-day mortality. Objective: We wanted to assess how echocardiographic assessment for RV function compares with other PE prognostic parameters used in ICU during the COVID-19 pandemic. Methods: This is retrospective cohort study of patients with confirmed PE hospitalized in Intensive Care Unit between January and December 2020. All unstable patients underwent 2-dimensional FOCUS echocardiography before CT pulmonary angiography (CTPA). FOCUS included assessment for right ventricular dilation, McConnell’s sign, septal flattening, tricuspid regurgitation, and tricuspid annular plane systolic excursion (TAPSE). Adverse outcomes were defined as shock, respiratory failure requiring intubation, death, or major bleeding. We have evaluated FOCUS markers of PE diagnosis and prognosis, as well as independent ICU mortality predictors. Results: We studied 47 patients, with mean age 58.6 ± 19.4 years. Eight patients (17%) had massive PE (3 patients positive for COVID-19) and 39 (83%) had sub massive PE confirmed by CTPA. Twelve patients (25,5%) were tested for COVID 19 with PCR assay, and 3 come positive (12.5%). Eight patients were high risk with shock (17%), 29 were intermediate high risk (61.7%) and 10 patients were intermediate low risk (21.3%). Abnormal RV function with TAPSE <17 was found in 32 patients (68%). Five high risk patients died within 72 hours, resulting in an overall ICU mortality rate of 10,6% and 62.5% for patients with cardiogenic shock. We found FOCUS echocardiography 93% (95% confidence interval [CI] = 72% to 98%) sensitive and 69% specific (95% CI = 55% to 81%) for PE. Mean TAPSE values in patients who died were 13+–/2, comparing to 18+/–2, p < 0.001 in survivals. Multivariate logistic regression analysis showed thrombolytic therapy OR 2.145 (95% CI: 1.105–4,512), TAPSE <14 OR 2.893 (95% CI: 0.932–3.241), and acute renal failure OR 1.821 (95% CI: 1.105–4.762) as an independent mortality predictors. Conclusions: RV dysfunction is significant predictor of intrahospital adverse PE outcomes. A negative FOCUS examination significantly lowers the likelihood of PE in most unstable patients. Focus echocardiography is very useful tool for fast assessment of RV function, risk stratification and PE management decision in unstable patients during the COVID-19 pandemic. 107749 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ROQUE DANIEL GONZÁLEZ1, Cayetano Bellomio2, Mercedes Ríos2, Carlos Ramiro Castellanos1, Manuel Parajón Viscido2, Roque Daniel González1 (1) Instituto de Cardiología Tucumán; (2) Sociedad Tucumana de Medicina del Deporte It has been reported that ultramarathons can elevate plasmatic levels of troponins and natriuretic peptides. The mechanisms and significance of these findings is debated. The objective of this study was to investigate the behavior of these markers before and after a mountain ultramarathon in young and inexperienced runners, unaccustomed to altitude. 20 runners (36.7 ± 6.3 years) were evaluated (6 females). They reported 4.0 ± 3.4 years competing in ultramarathon with a training load of 43.7 ± 16.8 km/week. The plasmatic values of troponin T (Trop-T HS) and N-terminal pro-brain natriuretic peptide (NT Pro-BNP) were determined before, immediately after the race and 24 hours later. The race was completed in 637.9 ± 197.5 minutes, and 55.1 ± 7.3 kilometers were covered. Both markers raised significantly after the competition. There was a statistically significant difference between the values of both determinations before the competition and at the end of the race (p < 0.0001) and between these and those after 24 hours (p < 0, 0001). At the end of the run, 95% of the sample presented NT Pro-BNP values above the diagnostic cut-off point of heart failure. All athletes presented values above the upper reference limit for Trop-T HS and 80% exceeded the diagnostic cut-off value for acute myocardial infarction. Troponin levels showed an inverse relation with weekly training (p = 0.04). There was a significant association between relative intensity of exercise and NT Pro-BNP levels after the competition (p = 0.001), where the group that completed the race with a heart rate higher than 80% of its maximum, significantly raised NT Pro-BNP. Conclusions: Trop-T HS and NT Pro-BNP significantly raised after the race. The extreme conditions and some characteristics of the athletes (poor habituation to altitude, short experience and relatively low volume of training) could justify these results. The clinical significance and mechanisms of these findings are still unknown, but the uniformity and brevity of the responses, as well the absence of clinical manifestations, suggest that they could represent an adaptive phenomenon. 107765 Modality: E-Poster Researcher – Non-case Report Category: ANTICOAGULATION TAMRAT ASSEFA1, Gobezie Temesgen1, Dejuma Yadeta2, Legese Chelkaba1, Teferi Gedif1 (1) School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia; (2) School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia Background: Oral anticoagulation therapy with warfarin requires frequent monitoring level of anticoagulation by the international normalized ratio (INR). Aims: This systematic review aimed to systematically summarize anticoagulation control, treatment outcomes, and associated factors in long-term patients receiving warfarin in Africa. Methods: The literature search was conducted in PubMed, Cochrane Library, African Journal of Online databases, Google Scholar, and Google. An advanced search strategy was computed to retrieve relevant studies related to anticoagulation control and outcomes. Duplication, title and abstract screening, and full-text assessment were conducted in Covidence software. Study quality was assessed using the Joanna Briggs Institute Critical appraisal quality assessment tool. The systematic review is registered in PROSPERO (CRD42021260772) and performed based on the PRISMA guideline. Results: Out of 298 identified articles, 18 articles were eligible for the final review and analysis. The mean of 39.4 ± 8.4% time in therapeutic range (TTR) (29.4% to 57.3%), 36.7 ± 11.5% TTR (range 25.2–49.7%) and 46% TTR (43.5–48.5%) was computed from studies that determined TTR by Rosendaal, direct and cross-section-of-the-files methods, respectively. The lowest percentage of TTR was 13.7%, while the highest was 57.3% was observed in this review. The highest percentage of patients (32.25%) who had TTR ≥ 65% was reported in Tunisia, but the lowest percentages were in Namibia (10%, TTR ≥ 65%) and Kenya (10.4%, TTR ≥ 70%). Generally, 10.4–32.3% of study participants achieved desired optimal anticoagulation level. Regarding secondary outcomes, 1.6–7.5% and 0.006–59% of patients experienced thromboembolic complications and bleeding events, respectively. The presence of chronic comorbidities, taking more than two drugs, and the presence of medications that potentially interact with warfarin were the frequently reported predictors of poor anticoagulation therapy. Conclusion: Oral anticoagulation control was suboptimal in patients taking warfarin as evidenced by low TTR in Africa. Therefore, there is an urgent need for further improving oral anticoagulation management service. 107767 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM TAMRAT ASSEFA1, Ashenafi Teklu1, Alfoalem Araba1 (1) School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia Background: Reports indicated association of COVID-19 with coagulation dysfunction and venous thromboembolism (VTE) prevalence was in the range of 20–40%. All patients with COVID-19 who are hospitalized should receive pharmacologic prophylaxis unless they have contraindications. Aims: To assess VTE risk, incidence, and its management in patients with COVID-19 admitted to Tikur Anbessa Specialized Hospital (TASH). Methods: A retrospective study was conducted among 146 COVID-19 patients admitted to TASH. A pre-tested data abstraction format was used to collect patients’ clinical information and VTE risk by using the modified Caprini Risk Score in COVID-19. We used Statistical Package for the Social Sciences (SPSS) version 26 for data analysis. Descriptive statistics were used to summarize the findings and binary logistic regression analysis to assess the association between the variables of interest. Results: Out of 146 patients, 57.53% were males and the mean age was 45.56 ± 18.17 years. All patients were at risk of developing VTE. The most often observed VTE risk factors were being COVID-19 symptomatic (88.40%), serious lung diseases (56.2%), and age >40 years (52.10%). The incidence of VTE was 23 (15.75%) and majorly (91.3%) occurred in highest VTE risk (≥5 scores) patients, >40 years patients, and in patients with severe COVID-19 symptoms (100%). However, parenteral thromboprophylaxis was prescribed only for 98 (67.12%) patients. Out of 23 patients who developed VTE, 15 didn’t receive prophylaxis, and the remaining 8 received thromboprophylaxis. Unfractionated heparin (UFH) was the most widely used prophylaxis. For patients who developed VTE, the majority of them (86.96%) were given a therapeutic dose of UFH. Conclusion(s): All patients with COVID-19 were at risk of developing VTE. Only one-third received thromboprophylaxis. The incidence of VTE was high and majorly occurred in patients that didn’t receive prophylaxis. 107796 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JUAN CARLOS ZERPA ACOSTA1, Jose Carlos Pachon Mateos2, Enrique Indalecio Pachon Mateos1, Carlos Thiene Cunha Pachon2, Juan Carlos Pachon Mateos1, Tasso Julio Lobo1, Tomar Guillermo Santillana Pena1, Felipe Augusto Ortencio1, Ricardo Cardneiro Amarante1, Maria Zelia Cunha Pachon2 (1) Hospital do Coração – São Paulo; (2) SEMAP – Serviço de Eletrofisiologia, Marca-passo, Desfibriladores, Ressincronizadores e Arritmias Prof. Dr. José Carlos Pachón M. Background: Carotid sinus syndrome (CSS) is a rare condition caused by sudden autonomic nervous system imbalance and Vagal overactivity that may lead to sinus arrest, AV block and syncope with severe symptoms commonly treated with pacemaker (PM). Since 2005 the Cardioneuroablation (CNA) procedure has been applied to aim vagal denervation through endocardial radiofrequency removing the cardio inhibition by ablation. Objective: To study the outcome of CNA controlled by extracardiac vagal stimulation (ECVS) in symptomatic functional CSS. Methods: Prospective, controlled study of 8 patients with symptomatic CSS, positive carotid sinus massage (CSM) and normal atropine response. Biatrial RF-ablation of the neuro endo-myocardial interface guided by filtered endocardial electrograms, fractionation, and 3D anatomical mapping, identifying type-1 AF-Nests (AFN) related to the 4 main ganglionated plexuses with FlexAbility Abbott irrigated catheter(30W/43°C/30s). ECVS(5s) at the right and left jugular foramen and CSM was performed before, during, and at the end of the ablation, to guide the level of denervation defining the higher CNA effect and end of the procedure, analyzing induced pauses and AVB. The endpoint was to eliminate complete pauses and AVB caused by ECVS and CSM. Results: Patients mean age was 58,6 (±11,8). Pre-CNA ECVS and CSM response were sinus pauses (5,4 ± 1,3secs) and transient AVB 3/8. Post-CNA, ECVS, CSM and Atropine responses were completely abolished in 8/100%. There was observed immediate sustained HR increase (59.6 ± 12,7/81,7 ± 5,3bpm) as acute result. In a follow up of 28,1 (±11,8) months there were no syncope recurrence. 1 patient had positive CSM at 6 months FU, without spontaneous syncope and had PM implanted. Conclusion: Bi atrial CNA vagal denervation based on AFN/GP ablation guided by abolishment of the vagal effect by ECVS and CSM response was effective for eliminating syncope recurrence in all the patients with CSS. 107798 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RITA DE CÁSSIA CASTELLI DA ROCHA1, Sérgio Emanuel Kaiser1, Márcia Regina Simas Torres Klein1, Cyro José Morais Martins3, Ricardo Guimarães Fisher2, Maria de Lourdes Guimarães Rodrigues1, Débora Valença1, Carolina Vicente Teófilo2, Carolina de Castelli da Rocha Carneiro3 (1) CLINEX – UERJ; (2) FACULDADE ODONTOLOGIA- UERJ; (3) HUPE Epidemiological studies suggest that chronic periodontitis (PD) associates with an increased risk of developing cardiovascular disease, whereas both share a link through inflammatory mediators. Arterial hypertension is a major risk factor for cardiovascular diseases, and it is believed that periodontal disease may favour its development. The relationship between PD and inflammation has been studied through biomarkers and assessment of endothelial function. Several conditions associated with subclinical inflammation seem to promote subtle changes in left ventricular systolic function, that can be detected by speckle tracking technology coupled with echocardiography. This study sought to explore, among hypertensive (HT) and normotensive (NT) individuals, the possible associations between severe chronic PD, inflammatory biomarkers, carotid intimal-medial thickness, and left ventricular systolic and diastolic function. The study consisted of a cross-sectional analysis with control for potential confounding factors, of 88 non-smoking, non-diabetic individuals of both sexes, among which, there were 50 AH under pharmacological treatment, and 38 NT. All underwent dental evaluation with a probe bag protocol aimed at identifying severe periodontitis, as well as laboratory tests (lipid profile, glucose, high-sensitivity C-reactive protei (HsCRP), interleukin-6 and tumor necrosis. They were broken down into 4 groups:AH/no PD; G3(N = 23):NT+PD; G4(N = 15):NT/no PD. Results: Mean age was 49.94 ± 7.32 (Range 35–60) years, 43% women; 39% non-white. Blood pressure (BP) mmHg: G1:136/82; G2:136/79; G3:125/79; G4:131/76 (p < 0.004 for systolic BP G3 vs G1). Mean BP was significantly higher in hypertensives (p < 0.01) regardless of severe PD. HsCRP (mg/dL) was significantly lower in G4 (p < 0.003); Intimal-medial thickness (IMT) was not significantly different among groups. Parameters of diastolic function did not differ significantly among groups regardless of the presence or absence of hypertension or PD. Global longitudinal strain (GLS) was also similar among groups; G1:–21.11 ± 2.24 G2:–21.33 ± 2.67 G3:–20.80 ± 3.17 G4:–21 ± 2.82 (p = 0.94). There were no significant differences in proportion of GLS ≥ –18 among the 4 groups. Since all HT patients were treated, no correlation systolic or diastolic BP and GLS was found. In conclusion, severe chronic PD was not independently associated with IMT, diastolic function nor with subclinical left ventricular dysfunction in the sample studied. 107816 Modality: E-Poster Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE MARIA CAROLINA GUIDO1, Priscila Oliveira de Carvalho1, Natalia de Menezes Lopes1, Aline de Oliveira Silva1, Leonardo Jensen1, Roberto Kalil-Filho1, Lygia da Veiga Pereira2, Francisco Rafael Martins Laurindo1, Raul Cavalcante Maranhão1 (1) Instituto do Coração – HCFMUSP; (2) Instituto de Biociências – USP Introduction: Patients with Marfan syndrome (MFS), a disease caused by mutation of the fibrillin-1 gene, are vulnerable to left ventricular (LV) cardiomyopathy. MicroRNA-1 (miR-1) has involved in cardiomyopathy, but the role of miR-1 in MFS is unknown. Methotrexate (MTX), a chemotherapeutic agent, when associated with lipid nanoparticles (LDE) that mimic LDL composition, markedly increased cell uptake, which enhances the pharmacologic properties and diminishes the toxicity of the drug. LDEMTX showed powerful anti-inflammatory and anti-proliferative effects on rheumatoid arthritis and atherosclerosis rabbit models. In rats submitted to acute myocardial infarction, LDEMTX contributed to the reduction of LV remodeling. Objective: To investigate the effect of LDEMTX on miR-1 expression in the development of LV cardiomyopathy in MFS mice. Methods: MFS and wild-type (WT) mice were allocated to the following groups: WT and MFS, both untreated; MFS-MTX, MFS treated with commercial MTX; MFS-LDEMTX, MFS treated with LDE-MTX. The treatment occurred weekly at a dose of 1 mg/kg ip, between the 3rd and 6th month of life. After 12 weeks, the animals were submitted to echocardiography, morphometry and, miR-1 and proteins expressions of LV. Results: LDEMTX did not improve the LV diastolic dysfunction in MFS mice. LDEMTX reduced LV hypertrophy by decreasing the interventricular septum and posterior wall thickness and the myocytes diameter. Collagen volume fraction in subendocardial, interstitial and papillary muscle areas was also diminished. The protein expression of caspase 3 and hypoxia-inducible factor 2α were lower, whereas VEGF and angiopoietin 1/2 were higher in LDEMTX group. Compared to MFS and MFS-MTX groups, MFS-LDEMTX increased miR-1 expression. The expression of miR-1 had a negative correlation with pro-apoptotic (r2 = 0.48; p < 0.01), cellular hypoxia (r2 = 0.45; p < 0.01), fibrosis (r2 = 0.40; p < 0.01) and myocyte hypertrophy (r2 = 0.24; p < 0.05) markers; while had positive correlation with pro-angiogenic marker (r2 = 0.50; p < 0.01). Conclusion: Although LDEMTX had no effect on diastolic dysfunction, LDEMTX treatment improved cellular changes in LV cardiomyopathy in MFS mice. LDEMTX had beneficial effects on miR-1 modulation by increasing angiogenesis and decreasing apoptosis, cell hypoxia, fibrosis and hypertrophy of LV. This is the first study that shows therapeutic efficacy in the cardiomyopathy of MFS mice, under the modulation of miR-1. 107826 Modality: E-Poster Researcher – Non-case Report Category: DYSLIPIDEMIA FABIANA CORDEIRO JULIANI1, Fátima Rodrigues Freitas1, Viviane Zorzanelli Rocha1, Márcio Hiroshi Miname1, Ana Paula Chacra Marte1, Wilson Salgado1, Raul Dias Santos1, Raul Cavalcante Maranhão1 (1) Heart Institute of the Medical School, University of Sao Paulo – Brazil Introduction: Familial hypercholesterolemia (FH) is a monogenic disease characterized by elevated LDL-C from birth and increased risk of premature coronary artery disease (CAD). Accumulation of LDL-C in plasma may impair anti-atherosclerotic actions attributed to HDL. In addition to HDL particle size, metabolic aspects related to the antiatherosclerotic protection of HDL should be considered beyond simple determination of HDL-C, such as antioxidant action of HDL-associated paraoxonase 1 (PON1) that protects LDL and other lipoproteins from oxidative stress. Aim: To assess whether PON1 activity and HDL particle diameter are altered in individuals with FH with or without subclinical CAD. Methods: Twenty patients with genetic diagnosis of heterozygous FH and subclinical CAD documented by coronary CT angiography and 20 patients with heterozygous FH without subclinical CAD, matched by age, sex and BMI were enrolled. After 30-day interruption of lipid-lowering drugs, plasma lipids and apolipoproteins (apo) B and A-I were determined by commercial kits. LDL-C was calculated using the Friedewald formula. PON1 activity was determined by spectrophotometry. HDL diameter was determined by laser light scattering method. Results: LDL-C, non-HDL-C and apo B were equally high. There were no differences in triglycerides, HDL-C or apo A-I. PON1 activity and HDL diameter also did not differ between the two groups. Conclusion: Among patients with FH, the presence of subclinical CAD was not associated with significant differences in plasma lipids and apo’s and in either HDL size or activity of PON1. In the quest for factors not related to high LDL-C that can facilitate or protect against the development of atherosclerosis in FH patients, other defects of HDL functions should be investigated. 107879 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING NIKOLAI NELASOV1, Dmitry Safonov1, Nikolai Nelasov1, Alexander Zubov2, Maria Ovrulova1, Maxim Morgunov1, Roman Sidorov1, Olga Eroshenko1, Anna Nechaeva1, Georgiy Chudinov1, Anna Bazilevich1, Elizaveta Palenaja2 (1) Rostov State Medical University, Russian Federation; (2) Donetsk National Medical University, Ukraine Introduction: Analysis of left ventricular myocardial strain parameters during stress-Echo showed promising results in detection transient myocardial ischemia. Various echocardiographic modalities of strain analysis can be used: speckle tracking longitudinal (Ls), circumferential (Cs), radial (Rs), area (As) strain and tissue Doppler longitudinal strain (TDI Ls). However, it is still debatable which of these modalities better detects stress-induced ischemia in regions of vascularization of obstructed coronary arteries. Objectives. The objectives of this study were: 1) to explore which of the methods (Ls, Cs, Rs, As or TDI Ls) can more frequently reveal regional transitory deformation disorders and identify coronary arteries responsible for induction of ischemia in patients with stable obstructive coronary artery disease in dynamics of left ventricular 4D stress-Echo with adenosine triphosphate; 2) to compare the efficacy of conventional method of visual evaluation of local contractility and above mentioned strain modalities in detection of ischemia-associated obstructed coronary arteries. Methods: 32 patients (male 29, mean age 58,2 years (95% CI: 58,2–60,2 years) with stable angiographically documented coronary artery disease (single vessel obstructive disease was detected in 6, multi vessel – in 26 cases) underwent left ventricular 4D stress-Echo with adenosine triphosphate. Criterion of strain impairment during stress-test was an increase in values by 5% in at least 2 adjacent myocardial segments using Ls, Cs and TDI Ls methods, by 7% using As method, and decrease in strain value by 10% using Ra modality. Results: During stress-test Ls revealed appearance/increase of strain disorders in the regions of vascularization of obstructed coronary arteries in 71.9%, Cs – in 65.6%, Rs – in 59.4%, As – in 75.0% and TDI Ls – in 62.5% of patients, while visual assessment of LV myocardial contractility revealed impairment of contractility in the territories of vascularization of obstructed coronary arteries only in 31.2% of patients (differences with strain methods in all cases were statistically significant). Conclusion: All methods of regional strain analysis (Ls, Cs, Rs, As, TDI Ls) can identify coronary arteries responsible for induction of ischemia during 4D stress-Echo with adenosine triphosphate in patients with stable coronary artery disease more effectively then visual evaluation of myocardial contractility; the best results can be obtained by applying As. 107881 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO DE PADUA MANSUR1, Carlos Henrique Del Carlo1, José Antonio Ramos Neto1, André Barbosa de Abreu1, Airton Roberto Scipioni1, Antonio Carlos Pereira Barretto1 (1) Insituto do Coração – HC FMUSP Background: Congestive heart failure (CHF) is one of the leading causes of death from cardiovascular disease and years lived with disability. Studies showed that women had better survival than men despite higher hospitalizations in women. However, little is known about differences in mortality and predictors of death in women and men with heart failure with preserved (HFpEF), mildly reduced (HFmrEF), and reduced ejection fraction (HFrEF). Methods: From February 2017 to September 2020, we analyzed the mortality and the predictors of death in women and men with CHF (Framingham criteria). Baseline data included clinical characteristics and echocardiographic findings. Statistical analyses were performed with the Kaplan-Meier (K-M) method and the Cox proportional hazards methods to analyze death rates and search for predictors of death for women and men. Results: We studied 12,015 patients, mean of 63.8 ± 14.3 years, 6637 (55%) males. Females were older (64.9 ± 14.8 vs. 62.8 ± 13.8 years; p < 0.0001), had a higher baseline mean left ventricular ejection fraction (LVEF) (49.8 ± 18.9% vs. 42.6 ± 15.4%; p < 0.001), and a lower left ventricular diastolic diameter (LVDD) (54.1 ± 9.0 vs. 60.1 ± 9.6 mm; p < 0.001). Over a 3-years follow-up period, 1543 (23.2%) men and 1051 (19.5%) women of the cohort died (K-M: log-rank p < 0.0001). Cumulative incidence of death was higher in men (K-M: log-rank p = 0.0002) with HFrEF but similar for HFmrEF and HFpEF (Figure). Cox regression for death adjusted for age, ischemic, idiopathic, hypertension, Chagas, valve, previous myocardial infarction, diabetes, previous stroke, chronic kidney disease (CKD), atrial fibrillation, and LVEF showed that CKD, previous stroke, and diabetes were the main predictors of death for all phenotypes of LVEF in women and men. Conclusion: Women had a better prognosis than men in HFrEF but similar mortality for HFmrEF and HFpEF. Control of diabetes and preventing stroke and CKD could significantly reduce the death rate in women and men with all CHF phenotypes. 107882 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO DE PADUA MANSUR1, Carlos Henrique Del Carlo1, José Antonio Ramos Neto1, André Barbosa de Abreu1, Airton Roberto Scipioni1, Antonio Carlos Pereira Barretto1 (1) Insituto do Coração – HC FMUSP Background: Congestive heart failure (CHF) is one of the leading causes of death from cardiovascular disease and years lived with disability. Studies showed that women had better survival than men despite higher hospitalizations in women. However, there is evidence of a gender-associated risk of dying of heart failure of different types of cardiomyopathies (CMP). Purpose: To analyze the mortality of CHF due to different types of CMP in women and men. Methods: From February 2017 to September 2020, we analyzed the mortality and the predictors of death in women and men with CHF (Framingham criteria) in five types of CMP (ischemic, idiopathic, hypertensive, Chagas, and valve disease). Baseline data included clinical characteristics and echocardiographic findings. Statistical analyses used the Kaplan-Meier (K-M) method and the Cox proportional hazards methods to analyze death rates and search for predictors of death for women and men for each CMP. Results: We studied 12,015 patients, mean of 63.8 ± 14.3 years, 6637 (55%) males. For all patients, death occurred in 27.5%, 15.3%, 17.5%, 40.1%, and 24.4%, respectively, for ischemic, idiopathic, hypertensive, Chagas, and valve CMPs (p < 0.0001). HFrEF was more prevalent in idiopathic (51.2%) and Chagas (49.9%), and for HFpEF valve disease (80.4%), hypertensive (51.9%), and ischemic (44.4%). Compared to men, women were older, had a higher baseline mean left ventricular ejection fraction (LVEF), and a lower left ventricular diastolic diameter (LVDD) for all five types of CMP (p < 0,001). Over a 3-years follow-up period, the cumulative incidence of death was higher in men with ischemic CMP (38% vs. 31%; p = 0.037) and Chagas CMP (48% vs. 38%; p < 0,001) but similar for idiopathic, hypertensive, and valve disease CMP (Figure). Cox regression analysis for death, for each CMP, adjusted for confounders such as age, sex, previous myocardial infarction, diabetes, previous stroke, chronic kidney disease (CKD), atrial fibrillation (AF), any cardiac surgery, cardiac pacemakers, and LVEF showed that men were an independent predictor of death only for Chagas CMP (HR = 1,28; 95%CL: 1.08–1.43; p = 0.009). Conclusion: Men were at a higher risk of death in ischemic and Chagas CMP but not for idiopathic, hypertensive, and valve CMP. The other main predictors of death were similar for all types of CMP, namely CKD, stroke, diabetes, and AF. 110849 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION MICHAŁ LEWANDOWSKI1 (1) National Institute of Cardiology, Warsaw, Poland Introduction: Sudden cardiac death (SCD) constitutes a major clinical and public health problem, whose death burden is comparable to the current worldwide pandemic. The presented abstract and project encompasses the following topics: available rescue systems, wearable electrocardiograms (ECG), detection and transmission technology, and a newly developed fuzzy logic algorithm (FA) for heart rhythm classification which is state-of-the art in the field of SCD prevention. Project “PROTECTOR”, the Polish Rapid Transtelephonic ECG System to Obtain Resuscitation for development of a rapid rescue system is presented. Methods: From a database containing RR interval series of the recorded arrhythmia events and controls, stored in the defibrillator memory and archived on PC during systematic control visits, RR data was chosen consecutively on the basis of full data availability, i.e., RR interval series with simultaneous intracardiac electrogram (IEGM) prints. Then, the heart rhythm classification of the recorded event was assigned into one of the six diagnostic categories of the developed algorithm. The total number of RR recordings evaluated for statistical analysis came to 298 (obtained from 183 pts). Sensitivity and specificity of the proposed algorithm were calculated on the basis of the widely accepted criteria. If a lethal arrhythmia is detected on the basis of FA, the system produces an alarm signal audible for bystanders and transmits the alarm message along with location to the emergency medical center. Phone guided resuscitation can be started immediately because an automated external defibrillator (AED) localization map is available. An automatic, very fast diagnosis is a unique feature of the PROTECTOR prototype. Results: The sensitivity and specificity of the tested algorithm were 100% and 97.8%, respectively. Internet data transfer opens potential application of the presented heart rhythm classification in telemedicine. The presented methodology is completely new ECG-based technique in the diagnosis and treatment of cardiac patients. Conclusions: The rapid detection of SCA is based on a processor characterized by 100% sensitivity (as measured in the pilot studies). An integrated circuit which implements FA has already been designed and a diagnosis is made within few seconds, which is extremely important in ischemic brain damage prophylaxis. This algorithm could be implemented in many mobile devices: smartphones, tablets etc. 107971 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY NATHALIE JEANNE MAGIOLI BRAVO-VALENZUELA1, Alberto Borges Peixoto3, Mattar, Rosiane2, Antonio Fernandes Moron2, David Baptista da Silva Pares2, Gabriele Tonni4, Edward Araujo Júnior2 (1) FEDERAL UNIVERSITY OF RIO DE JANEIRO-RJ, Brazil; (2) FEDERAL UNIVERSITY OF SAO PAULO-SP, Brazil; (3) Federal University of Triangulo Mineiro-MG, Brazil; (4) Prenatal Diagnostic Service, Department of Obstetrics and Gynecology, Istituto di Ricerca a Carattere Clinico Scientifico (IRCCS), AUSL Reggio Emilia, Italy ABSTRACT Purpose: To establish reference values for the systolic-to-diastolic duration ratio (SDR) of the left ventricle (LV) using spectral Doppler, as well as for the SDR’ of the interventricular septum (SEP), LV, and right ventricles (RV) using tissue Doppler of the fetal heart. Methods: This prospective and cross-sectional study evaluated 374 low-risk singleton pregnancies from 20 to 36+6 weeks of gestation. The ventricular filling time (FT) was obtained from LV inflow using spectral Doppler. Tissue Doppler was used to assess the FT of each ventricle by placing the cursor at the atrioventricular junction marked by the mitral and tricuspid valves, respectively. SDR was calculated as the sum of the isovolumic contraction time (ICT) and the ejection time (ET) divided by the sum of the isovolumic relaxation time (IRT) and the ventricular FT. We used regression analysis to obtain the best-fit model polynomial equation for the parameters. The concordance correlation coefficient (CCC) was used to assess intra- and inter-observer reproducibility. Results: SDR and SDR’ LV showed a progressive decrease with gestational age (GA); the SDR’ RV and SDR’ SEP did not show a significant decrease with advancing GA. The SDR LV (r = 0.29, p < 0.0001), SDR’ RV (r = 0.21, p < 0.0001), SDR’ LV (r = 0.20, p = 0.0001), and SDR’ SEP (r = 0.25, p < 0.0001) showed a significant weak positive correlation with fetal heart rate. The inter-observer SDR’ SEP measurements demonstrated poor reproducibility (CCC: 0.50), whereas intra-observer SRD LV measurements demonstrated moderate reproducibility (CCC: 0.78). Conclusions: Reference values for SDR SEP, LV, and RV using spectral and tissue Doppler of fetal heart were established between 20 and 36+6 weeks of gestation. 108035 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogerio Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Objectives: The novel coronavirus (SARS-CoV-2) may predispose patients to arterial or venous thrombosis due to excessive inflammation, platelet activation, endothelial dysfunction, and blood stasis. Pulmonary embolism as one of the most serious manifestations of this systemic framework worries those who are dealing with COVID-19 daily. The need for isolation and the home office can favor the development of risk factors for PE such as a sedentary lifestyle, immobility, obesity and smoking. The present study aimed to assess the influence of COVID-19 on hospitalizations, mortality rate and length of stay for pulmonary embolism in Brazil. Method: We used the database from the Department of Informatics of the Brazilian Health System (DATASUS), through the website https://datasus.saude.gov.br/. We evaluated the number of hospitalizations, deaths and length of stay of patients for PE in Brazil and their relationship with the beginning of the COVID-19 pandemic in Brazil (March 2020) based on data from January 2018 to December 2021. Results: From January 2018 to December 2021, 38902 patients were hospitalized for PE in Brazil, 23706 (60.9%) were female and 15196 (39.1%) were male. There was a slight increase in the average number of hospitalizations, but there was no statistical difference in the average total number of hospitalizations comparing 2018–19 with 2020–21: 9655 ± 624 and 9797 ± 201 (p = 0.804). The number of deaths and mortality rate also did not show important differences for the same period, with 1684 ± 66 deaths and a mortality rate of 17.5 ± 0.43 for 2018–19 and 1805 ± 142 and a mortality rate of 18.4 ± 1.1 for 2020–21 (p = 0.428 and p = 0.415 respectively). The mean hospital stay was similar, with 9.1 ± 0.3 days in 2018–19 and 8.5 ± 0 days in 2020–21 (p = 0.205). Regarding the age group, the number of hospitalizations is higher between 60–69 years old with 7495 cases and an annual average of 1874 ± 59 and the group over 80 years old with the highest mortality rate 34.7 ± 1.5. Conclusion: In the context of the COVID-19 pandemic, the number of hospitalizations due to PE remained stable in the period studied from 2018 to 2021, with a predominance of female patients (60.9%). The number of deaths and mortality rates also remained stable, as well as the average length of hospital stay. Advanced age was an important factor for both hospitalization and mortality. 108036 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogério Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Objective: Cardiovascular diseases are increasingly becoming a health problem in Brazil and worldwide. They are related to important acute complications such as stroke, which have high morbidity and mortality, especially when identified late. Taking into account the difficulty of the population’s access to health services in brazilian pandemic scenario, the present study aimed to assess the impact of COVID-19 pandemic and vaccination on stroke deaths in the elderly population in Brazil. Method: Using data from the Civil Registry Information Center in Brazil, through the website https://transparencia.registrocivil.org.br/especial-covid, we evaluated the number of deaths from stroke in Brazil in the elderly (age above 60 years) and its relationship to the COVID-19 pandemic from January 2019 to December 2021. Results: From January 2019 to December 2021, 239771 patients above 60 years old died of stroke in Brazil, 120107 (50.1%) were male and 119664 (49.9%) female. There was a slight decrease in stroke deaths in the comparison between 2019 and 2020, from 79806 to 79435 with a further increase in 2020 to 80530. The age group with the highest number of deaths was 80–89 with an average of 25807 ± 519, followed by 70–79 with an average of 25180 ± 1012. When we compare the monthly average of deaths and the beginning of the pandemic in Brazil (March 2020), the average was: before 656 ± 44 and after 673 ± 23 (p = 0.27). We also performed an analysis with the start of vaccination in Brazil in January 2021 and found a mean number of monthly deaths of 673 ± 23 before and 671 ± 46 after (p = 0.89). Conclusion: In the context of the Brazilian COVID-19 pandemic scenario, the number of deaths from stroke in the elderly population fell slightly from 2019 to 2020, with a new increase in 2021. The number of deaths remained similar for the years studied, with the age group of 80–89 years old as the highest number of cases followed by the 70–79 group. Neither the COVID-19 pandemic nor vaccination changed the monthly average deaths from stroke in the Brazilian elderly population. 108037 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogerio Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Objectives: The cardiovascular diseases are an important healthcare problem in Brazil and worldwide. They are related to acute complications such as stroke, which have high morbidity and mortality, especially when identified late. The difficulty of the population’s access to health services in brazilian pandemic scenario associated to isolation and home office can favor the development of traditional risk factors for stroke, most often neglected by young people, as sedentary lifestyle, obesity, smoking and poor nutrition. The present study aimed to assess the impact of COVID-19 on stroke deaths in young people in Brazil. Method: We used data from the “Portal da Transparência”, a platform managed by the National Association of Registrars of Natural Persons (Arpen-Brazil), through the website https://transparencia.registrocivil.org.br/especial-covid. We evaluated the number of deaths from stroke in Brazil in young people (aged 20 to 59 years) and its relationship with the beginning of the COVID-19 pandemic in Brazil (March 2020) analyzing data from January 2019 to December 2021. Results: From January 2019 to December 2021, 57,965 patients between 20 and 59 years old died from stroke, 33,764 (58.2%) were male. There was an increase in the number of total deaths over the years studied from 17506 to 19042 and 21417. The average monthly deaths in the period before the pandemic in Brazil was 1451 ± 80, in contrast to 1711 ± 117 after the beginning of the pandemic (p < 0.001). The age group with the highest number of deaths was 50–59 with a mean of 10246 ± 788, followed by 40–49 with a mean of 5215 ± 488. Conclusion: According to official data on deaths from strokeI in young adults between 20 and 59 years old, there was a progressive increase from 2019 to 2021, with a predominance of male patients. The COVID-19 pandemic contributed to this increase significantly. Age remains an important risk factor and older age groups continue to be the most affected. 108038 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogerio Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Objectives: Cardiovascular diseases are one of the main causes of death in Brazil and worldwide, and AMI is its main exponent. During the pandemic of the new coronavirus, health systems had to adapt to adequate care of these patients. The need for isolation and home office can favor the development of traditional risk factors for AMI, most often neglected by young people, such as a sedentary lifestyle, obesity, smoking, excessive consumption of alcohol, stress and poor nutrition. In addition, and taking into account the difficulty of the population’s access to health services in the Brazilian pandemic scenario, the present study aimed to evaluate the impact of COVID-19 on deaths from AMI in young people in Brazil. Method: We used data from the “Portal da Transparência”, a platform managed by the National Association of Registrars of Natural Persons (Arpen-Brazil), through the website https://transparencia.registrocivil.org.br/especial-covid. We evaluated the number of deaths from AMI in Brazil in young people (aged 20 to 59 years) and its relationship with the beginning of the COVID-19 pandemic in Brazil (March 2020) with data from January 2019 to December 2021. Results: From January 2019 to December 2021, 64,512 patients between 20 and 59 years old died from AMI, 44,940 (69.7%) were male. There was an increase in the number of total deaths over the years studied from 20075 to 20706 and 23731. The average monthly deaths in the period before the pandemic in Brazil was 1680 ± 123, in contrast to 1862 ± 181 after the beginning of the pandemic (p = 0.001). The age group with the highest number of deaths was 50–59 with a mean of 13568 ± 766, followed by 40–49 with a mean of 16710 ± 533. Conclusion: According to official data on deaths from AMI in young adults between 20 and 59 years old, there was a progressive increase from 2019 to 2021, with a predominance of male patients. The COVID-19 pandemic contributed to this increase significantly. Age remains an important risk factor and older age groups continue to be the most affected. 108078 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT WALKIRIA SAMUEL AVILA1, Daniel VInicius Rodrigues Pinto1, Vera Maria Cury Salemi1, Walkiria Samuel Avila1 (1) Instituto do Coração do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo; (2) Instituto do Coração do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo; (3) Instituto do Coração do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo; (4) Instituto do Coração do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo Background: Non-compaction cardiomyopathy (NCC) is a genetic congenital disease, with adverse clinical manifestations. The circulatory overload and hypercoagulability status of pregnancy, both can lead to serious complications. Purpose: To study the maternal and fetal outcomes of pregnant women with NCC. Methods: Out of 495 pregnant women with heart disease studied, four of them had a diagnosis of NCC confirmed by echocardiography and/or cardiac magnetic resonance exams. Before pregnancy, the symptoms were angina pectoris, syncope, and three cases with symptomatic arrhythmias, two of them underwent radiofrequency ablation. In the multidisciplinary follow-up, the therapy management was adjusted for pregnancy. Results: The Table below presents the characteristics of patients and the maternal-fetal evolution. There were no deaths, but three patients required hospitalization for treatment of heart failure and cardiac arrhythmias, right away emergency cesarean section resulting premature babies. In the follow-up-12months, no changes were observed in the clinical or myocardium remodeling of NCC patients. The investigation of newborns did not identify any case of NCC. Conclusions: The heterogeneity of NCC expression and the proportion of maternal and fetal complications, observed in this series, discourage pregnancy in patients with NCC. However, if pregnancy occurs, it should be followed into a tertiary hospital with a multidisciplinary team on standby. 108095 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PHILIP MOONS1, Liesbet Van Bulck1, Koen Luyckx1 (1) KU Leuven – University of Leuven, Belgium Background: Living with congenital heart disease (CHD) and the required invasive treatments are believed to have an impact on the psychological health of afflicted individuals. Therefore, mental health in persons with CHD is an area of concern. The epidemiology of depression and anxiety in CHD, however, is not fully understood and literature seems to report contradictory findings. Objective: We aimed (i) to estimate the proportion of depression and anxiety in adolescents and adults with CHD around the globe; and (ii) to explore differences in depression and anxiety rates between people with CHD and the general population. Methods: We conducted a systematic literature review and meta-analysis, registered in PROSPERO (CRD42021228395). Searches were performed in PubMed, Embase, Cinahl and Web of Science from their inception to March 6, 2021. We identified 91 studies, which enrolled 180,176 patients in total. A random effects meta-analysis of single proportions was conducted according to the DerSimonian-Laird method. Hedges’ gu (g) was calculated to compare the level of depression and anxiety with the general population. Results: The pooled estimated proportion showed that 21.1% of the people with CHD had ≥mild depressive symptoms and 10.3% had ≥moderate depressive symptoms. When looking at the clinical diagnosis of depression, 10.3% had actual depression, and 16.1% had a lifetime episode of depression. For anxiety, 42.7% had ≥mild anxiety symptoms and 21.9% had ≥moderate anxiety symptoms. A clinical anxiety disorder was found in 12.7%, and 23.3% had a lifetime episode of anxiety disorder. The mean scores on questionnaires assessing symptoms of depression (g = 0.215; 95%CI = 0.062–0.368) or anxiety (g = 0.292; 95%CI = 0.165–0.419) were significantly higher in CHD than in the general population. However, this higher symptom score was not reflected in a significantly higher proportion of CHD patients having ≥mild depressive symptoms (p = 0.654), ≥moderate depressive symptoms (p = 0.869), ≥mild anxiety symptoms (p = 0.055) or ≥moderate anxiety symptoms (p = 0.287), when using the predefined cut-off scores. Conclusions: This meta-analysis showed that there is a high proportion of depression and anxiety in persons with CHD. Although patients with CHD had higher scores of depressive and anxiety symptoms than people from the general population, the prevalence of depressive or anxiety symptomatology was not higher in CHD. 111457 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE MAURO RICARDO NUNES PONTES1, Mario Borba2, Lucélia B. N. C. Magalhães3, Alvaro Avezum4, Emilio Moriguchi5, Roberto Esporcate6, Sérgio Lívio Menezes Couceiro7, Hermilo Borba Griz8, Fernando A. Lucchese9 (1) Nucleo de Pesquisa Clinica do RS; (2) Cardioclinica do Vale, Lajeado; (3) UniFTC-Faculdade de Ciência e Tecnologia; (4) Hospital Alemão Osvaldo Cruz; (5) Hospital de Clínicas de Porto Alegre; (6) Faculdade de Ciências Médicas, UERJ; (7) Universidade Federal Fluminense; (8) Hospital Santa Joana, Recife; (9) Hospital São Francisco, Santa Casa de Porto Alegre Purpose: Burnout among physicians is frequent and has several consequences for physician and patient. We hypothesized that high spirituality protect against burnout. Methods: A survey with cardiologists, Aug 2018, asked about Burnout, demographics, spirituality, religiosity, job characteristics. We measured Burnout by 3 validated questions (three MBI -Maslach Burnout Inventory- domains). Subjects were Burnout + (B+) if they were in the top 3 scores (“>once a week”) on emotional exhaustion and/or depersonalization. Demographics, Spirituality score, DUREL religion index, job aspects, were correlated with B+ or B–. We evaluated consequences of burnout (suicidal ideation, stress, anxiety, depression measured by the DASS-21 form). Multivariate models evaluated the adjusted impact of spirituality over burnout and its consequences. Statistical analysis with SPSS version 23. Results: 1000 survey forms, 40.5% response rate; 39 excluded (4 incomplete, 35 graduated £2y); final sample, 375 cardiologists; age 48.8 ± 12.6y, male 57.3%. Burnout rate was 34.6%. Variables associated with Burnout in bivariate analysis: civil state (divorced/single 28.9% B+, married 6.8%, P = 0.020), years from graduation (B+, 20.3 ± 13.1y; B–, 25.3 ± 12.5y; P < 0.001), age (B+, 45.4 ± 13.1y; B–, 50.0 ± 12.4y; P = 0.001), duty hours/week (Q1 to Q4; 24.0%, 27.8%, 46.5%, and 60.9%; P < 0.001), and night shifts/week (0 to 3; 30.7%, 38.2%, 41.7%, and 71.4%; P = 0.001). Gender (P = 0.295), Spirituality score (P = 0.099), DUREL (P > 0.05 for all 3 domains), postgrad (P = 0.701) and subspecialty (P = 0.668) not associated with B+. Consequences of Burnout: suicidal ideation (B+, Yes 10.9%; B– Yes 1.3%; P < 0.001), DASS Global score (B+, 18.1 ± 11.7; B–, 8.1 ± 6.8, P < 0.001), and DASS subscales of Anxiety, Stress, and Depression (P < 0.001 for all) were all associated with B+. In a multivariate regression model (Table 1), Number of Duty Hours per Week was associated with Burnout (OR = 1.79, 95% CI 1.34–2.41, P < 0.001), while Spirituality Score (but not Religiosity by DUREL index) protected against Burnout (OR = 0.87, 95% CI 0.77–0.98, P = 0.043). Conclusions: High number of duty hours per week increases Burnout among Brazillian cardiologists, while high Spirituality protects them against Burnout. 108248 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION EDYTA SMOLIS-BAK1, Ilona Kowalik1, Jurand Sloniewicz1, Ewa Rydzewska1, Iwona Korzeniowska-Kubacka1, Jadwiga Wolszakiewicz1, Rafal Dabrowski1, Edyta Smolis-Bak1 (1) National Institute of Cardiology Background: Regular physical activity is crucial for treatment and prevention of cardiovascular diseases. Adaptive changes in response to regular physical exertion show individual variability, which depends on a number of internal and external factors. The aim of the study was to assess the effectiveness of early post-hospital rehabilitation and to analyze the factors which may influence the exercise tolerance in patients with coronary artery disease (CAD) treated with PCI and CABG. Material and methods: The study included 334 patients (pts), men 75.1%, with CAD, treated with PCI (87.4%) or CABG (12.6%). Mean age of the study group was 61.1 ± 10.9 years, mean BMI: 27.6 ± 4.1 kg/m2, mean LVEF: 56.1 ± 7.2%. The main risk factors for CAD were: dyslipidemias: 76%, hypertension: 64.7%, smoking: 28.7%, diabetes: 22.5%. The rehabilitation procedures lasted for 6–8 weeks. Pts participated in endurance training and general conditioning exercises with elements of resistance trainings. Before and after rehabilitation, all patients underwent a symptom-limited exercise test (ETT). In order to identify factors influencing the increase in tolerance of training loads, patients were divided into 2 groups: group 1 (198 pts, 59.3%) – pts with the training load which increased ≥50%, group 2 (136 pts, 40.7%) – pts with training loads which increased less, by <50%. Results: After rehabilitation, a significant improvement in the ETT parameters was observed: 5.7 ± 1.3 vs. 6.8 ± 1.7 METs, p < 0.001; the resulting load predicted for age: 81.5 ± 22.5 vs. 93.7 ± 22.89%, p < 0.001; tests duration: 352 ± 106 vs. 436 ± 136 sec., p < 0.001 and in the training loads obtained: 46.7 ± 10.9 vs. 71.4 ± 22.4 Watts, p < 0.001. On the other hand, after rehabilitation, a significant increase in the number of pts with supraventricular (24.2% vs 34.7%, p < 0.001) and ventricular (34.2% vs 40.5%, p < 0.02) arrhythmias was observed. There was no correlation between the increase in training load tolerance (Watt) and the METs increase in the final ETT. The increase in the tolerance of training loads of ≥50% during rehabilitation was significantly influenced by: younger age, p < 0.001, gender (men), p < 0.001, EF (>50%), p < 0.003, absence of diabetes, p < 0.003, lower initial values of training loads, p < 0.003. The increase in workload (MET > 50%) in the final ETT was significantly influenced by age (younger patients), BMI (thinner subjects) and lower workload (MET) in the baseline test. Other factors: smoking, hypertension. 108254 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT EDYTA SMOLIS-BAK1, Edyta Smolis-Bak1, Zbigniew Lew- Starowicz2, Monika Szymanska2, Ilona Kowalik1, Ryszard Piotrowicz1, Eliza Noszczak1, Rafal Dabrowski1 (1) National Institute of Cardiology, Warsaw, Poland; (2) Postgraduate Medical Education Center, Warsaw, Poland Background: Data show that cardiological patients (pts), after achieving an improvement in the functioning of the cardiovascular system, begin to expect improvement also in the field of sexual health, which is important in partner relationships, mental health and quality of life. The aim of the study was to analyze the occurrence of sexual disorders and to assess the impact of sexual rehabilitation and physiotherapeutic education on sexuality in pts after left ventricular assist device (LVAD) implantation. Material and methods: The study included 50 men after LVAD implantation, mean age 51.6 ± 12.8 years. 18 pts did not complete the study (heart transplant –5 pts, resignation –10 pts, death –3 pts). The pts were assessed by a sexologist in terms of the quality of sexual life and erectile dysfunction. All pts received therapeutic interventions: sexological counseling, education (positions during intercourse, safety recommendations) and physical activity. Additionally, pharmacological treatment of erectile dysfunction was started in 15 pts. Upon entry into the study, the quality of sexual life, sexual needs (Mell-Krat scale) and sexual function (International Index of Erectile Function, IIEF-5) were assessed retrospectively (before the onset of the disease) and actually. Additionally, exercise tolerance was assessed using the 6-minute walk test (6-MWT). The tests were repeated after 6 months. Results: After the implementation of sexual and physical activity education, significant improvement in exercise tolerance assessed by 6-MWT [m] was observed: 350 (310–445) vs. 450 (400–500), p < 0.001. The quality of sex life in the whole group significantly worsened after LVAD implantation compared to the period before the onset of the disease (40 [40–41] vs 33.5 [12–40] vs 34 [26–40]). Mild erectile dysfunction was observed in all the pts, both before onset of the disease, after LVAD implantation and after 6 months. The number of pts with sexual dysfunction increased significantly after LVAD implantation (9.4% vs. 53.1%) and remained at a similar level for 6 months. There was a significant reduction in the frequency of sexual intercourse after LVAD implantation and a significant increase in it after pharmacological treatment, but its level did not reach the level before the disease (3 [2–4] vs 1.5 [0–2.5] vs 2 [1.0–2.5]). The improvement in the quality of sexual life and the reduction of erectile dysfunction were significantly dependent on age and LVEF – bett. 108346 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LUISA CAMPOS CALDEIRA BRANT1, Pedro Cisalpino Pinheiro1, Luiz Guilherme Passaglia1, Eduardo David Gomes de Sousa2, Carla Valença Daher2, Roberta Maria Leite Costa2, Antonio Luiz Pinho Ribeiro1, Bruno Ramos Nascimento1 (1) Faculdade de Medicina e Hospital das Clínicas, Universidade Federal de Minas Gerais, UFMG, Brazil; (2) Department of Specialized and Thematic Healthcare, Ministry of Health, Brasilia, Brazil Introduction: COVID-19 has impacted cardiovascular (CV) mortality worldwide, possibly due to avoidance of healthcare, health system collapse, and the direct CV effects. Data from Brazil are scarce and restricted to some locations or early phases of the pandemic. Objective: To evaluate the impact of COVID-19 pandemic on mortality for CV diseases (CVD), stratified by age, sex and region of Brazil, comparing underlying (UC) and multiple causes (MC) of death. Methods: Ecological, time series study analysing the age-standardized death rates for CVD, from epidemiological week (EW) 10/2020 to EW 11/2021, using data from the Mortality Information System (SIM-SUS). CVD was defined as in Chapter IX, ICD-10, and considered if reported as UC or MC of death (UC + other causes reported in any line of death certificate), in separate analyses. Observed was compared to expected data (mean for the same EW of 2017–2019). Risk ratios (RiR) were analysed and 95% confidence intervals were calculated. Results: Age-standardized mortality rate for CVD as UC of death in the pandemic was 136.8 (95%CI 136.3–137.3)/100,000 inhabitants, 7% (RiR = 0.93; 95%CI 0.93–0.93) lower than expected (147.1; 95%CI 146.6–147.6)/100,000. There was an increase in out-of-hospital mortality (6%) (RiR = 1.06; 95%CI 1.05–1.07) and in deaths with ill-defined CVD causes (49%) (RiR = 1.49; 95%CI 1.47–1.50), particularly in the out-of-hospital setting (58%). The increase in out-of-hospital deaths was more pronounced in the North (RiR = 1.27; 95%CI 1.25–1.28) and Northeast (RiR 1.18; 95% CI 1.17–1.18) regions, both with less structured health systems. Conversely, in MC of death analysis, there was a 4% increase in CV mortality (observed: 225.1 (CI95% 224.5–225.8), expected: 217.2 (95%CI 216.5–217.8)/100,000, RiR: 1.04 (95%CI 1.03–1.04), noticeably in the out-of-hospital setting (11%). In MC of death analysis, the steeper increasing trends also ocurred in the North region (RiR = 1.13; 95%CI 1.12–1.15), and in individuals >60 years (RiR = 1.05; 95%CI 1.05–1.06), and among men (RiR: 1.06; 95%CI 1.05–1.06) vs. women (RiR: 1.02; 95% CI 1.01–1.02). Conclusions: During the pandemic, mortality rates for CVD as UC of death reduced in Brazil, contrasting with an increase when MC were considered. Higher out-of-hospital mortality – resulting from social distancing and overwhelmed health systems – exaberbation of existing CVD by COVID-19, and competing causes of death may have accounted for this pattern. 108258 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION EDYTA SMOLIS-BAK1, Kamil Szczesniak1, Ilona Kowalik1, Grzegorz Skorupski1, Lukasz Lasecki1, Jerzy Osak1, Magdalena Furmanek1, Hanna Szwed1, Rafal Dabrowski1 (1) National Institute of Cardiology, Warsaw, Poland Balance and coordination disorders are factors provoking falls in the elderly. Their fast and precise identification is therefore of vital importance. The aim of this work was to assess the risk of falls and the influence of early hospital rehabilitation on the balance and coordination parameters in patients after cardiac surgery. Material and methodology: The study was conducted on 207 patients: 44 women and 163 men aged 18–87 (mean 60.2 ± 14.0), who participated in comprehensive cardiac rehabilitation following cardiac surgery. The risk of falls and patients’ functional status were tested twice with the use of the FallSkip device: after the initial mobilization and before the discharge from the ward. Results: After rehabilitation, improvement was noted in the whole group in all analysed functional parameters and in the reduction of the risk of falls (p < 0.0001). A significantly higher risk of falls was observed in women in comparison with men (p < 0.001), and in individuals aged >70 in comparison with patients aged <70 (p < 0.001). There were no differences observed in the risk of falls of patients in the groups with the BMI <30 and >30, both before and after rehabilitation. After rehabilitation the risk of falls decreased significantly in men (p < 0.001) and in individuals aged <70, (p < 0.001), as well as in patients with the BMI <30, p < 0.001, and >30, p < 0.012. There was a tendency for better results in men in all tested parameters. Conclusions: The proposed comprehensive rehabilitation model after cardiac surgery significantly improved the effectiveness of gait, time of reaction in response to audio stimuli, increased muscular strength of lower limbs and significantly reduced the risk of falls. The FallSkip equipment is useful in the assessment of the risk of fall and of the level of functional parameters in patients after cardiac surgery. 108351 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LUISA CAMPOS CALDEIRA BRANT1, Luiz Guilherme Passaglia1, Pedro Cisalpino Pinheiro1, Eduardo David Gomes de Sousa2, Carla Valença Daher2, Roberta Maria Leite Costa2, Antonio Luiz Pinho Ribeiro1, Bruno Ramos Nascimento1 (1) Faculdade de Medicina e Hospital das Clínicas, Universidade Federal de Minas Gerais, UFMG, Brazil; (2) Department of Specialized and Thematic Healthcare, Ministry of Health, Brasilia, Brazil Introduction: The COVID-19 pandemic impacted hospitalizations for other causes due to change in populations’ behaviour, as a result of fear or adherence to social distancing, or overwhelmed hospitals. Low and middle-income countries may have higher negative impact because of less resilient health systems. Previous studies in Brazil showed a reduction in hospitalizations for cardiovascular diseases (CVD). However, they were restricted to some cities or the first three months of the pandemic. Purpose: Evaluate the impact of COVID-19 pandemic in the number and severity of hospitalizations for CVD in the Brazilian public and universal health system during 2020 and 2021, considering differentials by age, sex, region of the country. Methods: Ecological, time series study analysing the number of hospitalizations, number and proportion of intensive care unit (ICU) admissions and in-hospital deaths for CVD, from epidemiological week (EW) 10/2020 (first confirmed COVID-19 case) to EW 21/2021, using data from the Hospitalization Information System (SIH-SUS). CVD was defined as in Chapter IX from ICD-10. Observed data was compared to the mean for the same EW of 2017–2019. Risk ratios (RiR) were analysed and 95% confidence intervals were calculated. Results: A 16% (RiR 0.84; 95%CI 0.83–0.84) reduction in hospitalizations for CVD was observed in the pandemic period (n = 953,025). However, higher proportion of ICU admissions (RiR 1.09; 95%CI 1.08–1.09), and in-hospital deaths (RiR 1.14; 95%CI 1.14–1.15) revealed greater severity of hospitalized individuals, which may have ocurred due to delayed admissions, or disrupted pathways of care. Two peaks of reductions in hospitalizations were observed, possibly resulting from different drivers: the first, at the beginning of the pandemic, may have resulted from deferred hospitalized treatment, while the second may represent competing demands with COVID-19, resulting in even higher proportion of deaths (Figures 1 and 2). Women, older individuals and those living in the least developed North and Northeast regions were mostly negatively impacted, exacerbating vulnerabilities in CVD care. Conclusions: The COVID-19 pandemic disrupted CVD care in Brazil. Health policies must address hospitalizations by CVD during future pandemics, including reorganizing pathways of care, public campaigns about symptoms that require hospitalization, and greater availability of ICU beds, besides a plan to face the rebound effect for deferred procedures. 108357 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARCOS VINICIUS COELHO DUTRA1, CARLOS ALBERTO KENJI NAKASHIMA1, LILIAN BELINASO1, VIVIANE DE SA PEREIRA1, RICARDO PHILIPE ZAGO1, ANTONIO DEJAIR ACOSTA PAZZINI1, VANESSA DELMIRO DOS SANTOS1, CASSIO PERFETE1, ERASMO SIQUEIRA1, DALTON BERTOLIM PRECOMA1 (1) Hospital Angelina Caron Background: Early reperfusion of the ischemic myocardium is the most important advance in the last 30 years when it comes to acute myocardial infarction (AMI) with ST-segment elevation (STEMI), which can be in the mechanical form by angioplasty, or pharmacologically with use of thrombolytic, respecting the indication of each one. Antiplatelet therapy, from the studies with acetylsalicylic acid as in the Isis-2 study, demonstrating a 23% reduction in mortality when used alone in acute coronary syndrome (ACS), the addition of low molecular weight heparin, to more recent publications with the new adenosine diphosphate blockers and thrombin receptor blockers, corroborating the reduction of outcomes in the acute scenario of post-angioplasty AMI (thrombosis reduction), and making dual antiplatelet aggregation (DAPT) an essential part of the treatment of ACS. Few trials provide us with information comparing whether such medications, used as indicated in STEMI, were able to maintain the patency of the affected vessel pre-coronary angiography. Objective: To compare, between the two groups analyzed (pre-treatment with clopidogrel vs. ticagrelor) the patency of the culprit coronary artery assessed by TIMI angiographic flow analysis by coronary angiography. Methods: A total of 3287 medical records of patients admitted to a chest pain unit of a tertiary hospital in the metropolitan region of Curitiba – Paraná were analyzed from March 1, 2019 to March 1, 2021. Of the 3287 patients initially evaluated, 384 patients were selected with a diagnosis of STEMI, 16 patients were excluded due to lack of data in their medical records and 334 patients were analyzed until hospital discharge. Results: Of all the cases that used ticagrelor, 65.1% had occluded vessels and of the cases of clopidogrel, 65.8% of the affected vessels remained occluded. Therefore, no significant difference was found between clopidogrel and ticagrelor regarding the probability of being occluded (p = 0.585). Conclusion: We can conclude that in the analyzed sample, there was no statistical difference between the use of clopidogrel and ticagrelor in the patency of the vessel affected by the occlusion. 108384 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY RAFAEL ALEXANDRE MENEGUZ MORENO1, Nisia Lira Gomes1, Alfredo Nunes Ferreira-Neto1, Auristela IO Ramos1, Zilda Meneghelo1, J. Italo Franca1, Amanda GMR Sousa1, Sérgio Luiz Navarro Braga1, Dimytri Siqueira1, J. Ribamar Costa Jr.1 (1) Instituto Dante Pazzanese de Cardiologia; (2) Universidade Federal de Sergipe; (3) Hospital Primavera Background: Mitral valve stenosis (MVS) is one of the most common structural heart diseases in developing countries, primarily due to rheumatic disease. Percutaneous mitral balloon valvuloplasty (PMBV) has been, since its introduction in 1984, the preferred option of treatment for such disease. However, restenosis is presented with an approximate incidence of 20%. Echocardiographic scoring of the mitral apparatus has been the main tool used to indicate and foresee the possible result of the procedure. The objective of this study was to enlight risk factors of mitral valvular restenosis in a significant number of patients submitted to percutaneous mitral balloon commissurotomy for the treatment of mitral stenosis (MS), particularly when secondary to rheumatic heart disease. Methods: This study reports the vast experience of a single center high volume tertiary institution where 1.794 consecutive patients were treated with PMBC between 1987 and 2011. The primary endpoint was to determine the independent predictors of this untoward event, defined as loss of over 50% of the original increase in maximum valve area (MVA) or MVA < 1.5 cm2. Results: Mitral valve restenosis was observed in 26% of the cases (n = 483). Mean population age was 36 years old, with most patients being female (87%). Mean follow up duration was 4.8 years. At multivariate analysis independent pre-procedural predictors of restenosis were: left atrial diameter (HR: 1.03, 95% ci: 1.01–1.04, p < 0.01), pre procedure maximum gradient (HR: 1.01, 95% ci: 1.00–1.03, p = 0.02) and higher wilkins scores (HR: 1.37, 95% ci: 1.13–1.66, p < 0.01). Conclusion: In the very long term follow-up, mitral valve restenosis was observed in a quarter of the population undergoing PMBC. Preprocedure echocardiographic findings, including left atrial diameter, maximum valve gradient and high Wilkins scores were found to be the only independent predictors of this deleterious event. 108388 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LILIAN BELINASO1, Camila Richter1, Carlos Gustavo Marmanillo1, Carlos Alberto Kenji Nakashima1, Erasmo Junior Toledo Siqueira1, Dalton Bertolim Précoma1, Marcos Vinicius Coelho Dutra1, Viviane de Sá Pereira1, Luan Gabriel Paese1, Sérgio Antonio López1, Rômulo de Lima Moreno1, Rodney de Oliveira1 (1) Hospital Angelina Caron – HAC Background: As the prevalent kidney transplant population grows, there is an increasing need to quantify the risk of medical conditions to minimize complications. Bleeding events are a cause of hospitalization and contribute to the morbimortality of these patients. Objective: To evaluate perioperative bleeding complications due to the use of dual antiplatelet therapy (DAPT) in kidney transplantation patients. Methodology: Patients who underwent kidney transplantation between January 2019 and May 2021 were included (n = 372) and divided into 3 groups: control group without antiplatelet therapy (n = 230); patients with perioperative antiplatelet therapy only with acetylsalicylic acid – ASA (n = 123); and patients with perioperative DAPT – ASA and clopidogrel (n = 19). The primary outcome was the rate of bleeding in patients on antiplatelet therapy compared with patients without antiplatelet therapy. Secondary outcomes included location and timing of bleeding, post-bleeding acute myocardial infarction, perioperative transfusion requirement, surgical reintervention, renal explantation, and mortality. Results: Patients with DAPT were a mean age of 59.63 ± 6.94, were more insulin-dependent diabetes and they were all hypertensive (P > 0,002), significantly higher data compared to the other groups. These patients bled significantly more (P > 0.005), mainly at the surgical site (64,7%); 35.3% of the patients required surgical reintervention (p = 0.0013). In this study, most bleeding events (73.6%) occurred within one week after transplantation and 80% required blood transfusion (p = 0,0001). Conclusion: Kidney transplantation can be safely performed without interrupting perioperative ASA therapy and prophylactic anticoagulation. There is an increased risk of bleeding requiring blood transfusion and surgical reintervention when the patient is on DAPT, but without an increase in acute myocardial infarction, renal explantation and short-term mortality. 108408 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY EDER ANDERSON RODRIGUES1, Camila Moreno Rosa1, Dijon Henrique Salome De Campos1, Felipe César Damatto1, Gilson Masahiro Murata2, Lidiane Moreira de Souza1, Amanda Bergamo Gonçalves de Castro Rêgo1, Leiliane Rodrigues dos Santos Oliveira1, Patrícia Aparecida Borin1, Katashi Okoshi1, Marina Politi Okoshi1 (1) Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil; (2) 2 LIM29, Division of Nephrology, University of Sao Paulo Medical School, Sao Paulo Brazil Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have beneficial effects on the cardiovascular system in diabetes mellitus (DM) patients. However, as most clinical trials were performed in type 2 DM, the effects of SGLT2 inhibitors in patients with type 1 DM still need further clarification. In this study, we evaluated the effects of long-term treatment with the SGLT2 inhibitor dapagliflozin on cardiac remodeling, myocardial function, and energy metabolism in rats with type 1 DM. Methods: Male Wistar rats were divided into three groups: control (C, n = 15); DM (n = 15); and DM treated with dapagliflozin (DM+DAPA, n = 15) for 30 weeks. DM was induced by streptozotocin; DAPA was added to the rat chow (5 mg/kg/day). Cardiac performance was evaluated by echocardiogram and myocardial function in isolated left ventricular (LV) papillary muscle preparations. Myocardial energy metabolism enzyme activities were evaluated by spectrophotometry. Statistical analyzes: ANOVA and Tukey or Kruskal-Wallis and Dunn. Results: DM+DAPA had lower glycemia than DM [C 112 (108–116); DM 531 (522–535)*; DM+DAPA 267 (179–339) mg/dL; p < 0.05 vs C and DM+DAPA]. Echocardiogram showed that DM and DM+DAPA had left atrium and left ventricle dilatation with systolic and diastolic dysfunction; in DM+DAPA, the changes were attenuated in relation to DM. Developed tension and +dT/dt were higher in DM+DAPA than DM in basal condition. After inotropic stimulation with post-pause contraction, extracellular calcium concentration elevation, and isoproterenol addition to the nutrient solution, +dT/dt and –dT/dt were higher in DM+DAPA than DM. Hexokinase, phosphofructokinase, and pyruvate kinase activity was lower in DM than the C. Phosphofructokinase and pyruvate kinase activity was higher in DM+DAPA than DM. Conclusion: Long-term dapagliflozin treatment attenuates cardiac remodeling and myocardial dysfunction and preserves hexokinase, phosphofructokinase and pyruvate kinase activity in rats with type 1 diabetes mellitus. 108797 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MARCELA MATTOS SIMÕES1, Angélica Navarro de Oliveira3, Ricardo Simões1, Karina Braga Gomes2, Bruno de Almeida Rezende4, Marcus Vinícius Bolivar Malachias3 (1) Post-Graduate Program in Health Sciences – Faculty of Medical Sciences of Minas Gerais-MG, Belo Horizonte/MG, Brazil; (2) Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte/MG, Brazil; (3) Institute of Hypertension of Minas Gerais, Belo Horizonte/MG, Brazil,; (4) Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte/MG, Brazil Background: Psoriasis is an inflammatory skin disease that can have systemic consequences such as cardiovascular disease (CVD). Objective: To evaluate commonly recommended clinical risk scores and their associations with pulse wave velocity (PWV) and intima-media thickness (IMT), recognized subclinical cardiovascular (CV) biomarkers, in individuals with psoriasis without apparent CVD. Methods: This is a cross-sectional observational pilot study involving 44 male subjects, 11 of whom had moderate to severe psoriasis, according to the Psoriasis Area Severity Index (PASI) > 10 and the Dermatological Quality of Life Index (DLQI) > 10, and 33 healthy subjects (control group). Groups were assessed using the Global Risk Score (GRS), Atherosclerosis Cardiovascular Disease (ASCVD) by Pooled Cohort Equations and the SCORE system, in addition to measurements of PWV and IMT. Comparisons between groups and associations between clinical risk scores and biomarkers measurements were performed considering a significance level of 5%. Results: The psoriasis group (PG) had a higher CV risk estimated by GRS (p = 0.001) and ASCVD (p = 0.025), but not by SCORE (p = 0.289), as well as higher PWV (p = 0.033) compared to the group control (GC). ASCVD (p = 0.014) and SCORE (p = 0.009) were associated with PWV in the psoriasis group. GRS, ASCVD and SCORE showed a positive association with PWV in relation to the total sample. Clinical risk scores showed no significant association with carotid IMT in the psoriasis group. Conclusion: Patients with psoriasis without apparent CVD have a higher cardiovascular risk when evaluated by clinical scores GRS and ASCVD. ASCD and SCORE were associated with subclinical findings such as higher PWV. 108731 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS LEANDRO BRASIL REGO1, Leandro Brasil Rego1, Rodrigo Villar2, Danilo Salles Bocalini3, Gustavo Allegretti João4, Ruth Caldeira Melo1, Bruna Trindade Souza1, Francisco Luciano Pontes Júnior1 (1) Laboratório de Fisiologia do Exercício e Envelhecimento, Escola de Artes, Ciências e Humanidades da Universidade de São Paulo, São Paulo, Brazil; (2) Cardiorespiratory & Physiology of Exercise Research Laboratory, Faculty of Kinesiology and Recreation and Management, University of Manitoba, Winnipeg, Manitoba, Canada; (3) Laboratório de Fisiologia experimental e Bioquimica, Centro de Educação física e Desporto, Universidade Federal do Espirito Santo, Vitoria, Brazil; (4) Laboratório de Fisiologia do Exercício, Faculdades Metropolitanas Unidas, São Paulo, Brazil. São Paulo, Bra Introduction: Aging is associated with a decline in cardiorespiratory and musculoskeletal systems that may compromise maximum aerobic power and muscle strength, fundamental aspects for the maintenance of functional capacity in the elderly. Objective: To verify the correlation between maximal aerobic power, isometric muscle strength, and functional capacity in elderly women. Methods: The sample consisted of 30 elderly women aged 60 years or more (X = 65.4 SD = 2.8). To determine the maximal aerobic power, we performed ergospirometric test (gas analyzer Metalyzer II, Cortex®, Germany) on a treadmill (centurion® 300) with constant 1% inclination, initial speed of 4 km/h and 1 km/h increases every minute. The maximum strength was evaluated in lower and upper limbs by isometric contraction in a portable dynamometer (Lafayette Instrument Company, USA). The functional capacity was evaluated by the tests: TUG- (Timed Up and Go), TC 10 m (10 m walk) and SLC (Sit and Stand Up Chair). In all tests 3 measurements were taken and the mean was computed. In the statistical analysis, significance was set at 5% and the assumptions of normality, homogeneity, and sphericity were confirmed with the Shapiro-Wilk, Levene, and Mauchly tests, respectively, and parametric techniques were used. Results: There was a positive and strong correlation between peak VO2 (mL/kg/min) vs. lower limb strength (r = 0.62; p = < 0.001); negative and strong correlation between peak VO2 (mL/kg/min) vs. TUG (r = –0.63; p = < 0.001); moderate and negative correlations between peak VO2 (mL/kg/min) vs. TC10 m (r = –0.49 p = 0.005); peak VO2 (mL/kg/min) vs. SLC (r = –0.52 p = 0.003) and between lower limb strength vs. TUG (r = –0.50; p = 0.005); TC10 m (r = –0.50; p = 0.005); SLC (r = –0.48; p = 0.006). The multiple linear regression showed an R2 49.8% of the result of the dependent variable aerobic power and the following values of the independent variables lower limb strength (B = 0.25; p = 0.012) and “TUG” test (B = –2.161; p = 0.008). Conclusions: It was demonstrated a positive correlation of aerobic power with isometric muscle strength (lower limb) and a negative correlation of these variables with performance in functional tests. Furthermore, isometric muscle strength (lower limb) and the TUG test were important predictors of maximal aerobic power. 108420 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GILBERTO ANDRADE TAVARES1, Gilberto Andrade Tavares1, Joathan Borges Ribeiro5, Marcos Antonio Almeida-Santos4, Antônio Carlos Sobral Sousa1, José Augusto Soares Barreto-Filho1 (1) Postgraduate Program in Health Sciences, Federal University of Sergipe; (2) Federal University of Sergipe; (3) Rede D’Or São Luiz, Hospital São Lucas, Division of Cardiology; (4) Tiradentes University; (5) Postgraduate Program in Adult Health Nursing, University of São Paulo; (6) Hospital Sírio-Libanês Introduction: In Brazil, the Unified Health System (UHS) regulates public health care and has in the Family Health Strategy (FHS) the main strategy of primary care. In 2010, the American Heart Association (AHA) proposed to check seven cardiovascular health (CVH) metrics, with goal of reducing CVD deaths in the U.S. by 20% until 2020. The results of the FHS regarding the CVH of the Brazilian population are not known. Objective: Evaluate the control of CVH among adult patients assisted by the FHS in the municipality of Aracaju, Sergipe, Brazil. METHODS A cross-sectional study was conducted using the 7 metrics in CVH among patients treated by the FHS. Those metrics being graded at the “ideal”, “intermediate” and “poor” level. The variable “CVH control” was dichotomized into “Controlled” (>five metrics at the ideal level) and “Uncontrolled” (<five metrics at the ideal level). Results: In our study, 32.5% were at ideal level. The majority of the sample (62.75%) were at intermediate level. At the poor level, 4.5% was found in the sample. In the multivariate analysis, after adjustments, <45 years of age (RRa 1.61 (IC 95% 1.15–2.28)), females (RRa 2.07 (IC 95% 1.20–3.60)) and following health guidance from family members and neighbors (RRa 1.28 (IC 95% 1.15–2.28)) were associated with “Controlled” CVH. Having a higher number of children reduces the chance of having “Controlled” CVH by up to 9% (RRa 0.91 (IC 95% 0.84–0.95)). Conclusions: We demonstrated that, only 32.5% of the patients had controlled CVH; the adjusted multivariate analysis showed that being under 45 years of age, female and sharing decisions about their health with neighbors and family members are associated with “Controlled” CVH. Having more children reduces this association. 108421 Modality: E-Poster Researcher – Non-case Report Category: NURSING CAROLINA DE ARAÚJO MEDEIROS1, Maria Beatriz Araújo Silva3, André Luiz Sá de Oliveira4, Maria das Neves Dantas da Silveira Barros1, Maria Elisa Lucena Sales de Melo Assunção1, Maria da Glória Aureliano de Melo Cavalcanti1, Tayne Fernanda Lemos da Silva1, Gênova Maria de Azevedo Oliveira5, Cristina de Fátima Velloso Carrazzone1, Zulma Maria de Medeiros2, Sílvia Marinho Martins1, Wilson de Oliveira Júnior1 (1) Ambulatório de Doença de Chagas e Insuficiência Cardíaca-PROCAPE-UPE; (2) Programa de Pós-graduação em Ciências da Saúde – FCM-UPE; (3) Faculdade de Enfermagem Nossa Senhora das Graças – FENSG-UPE; (4) Instituto Aggeu Magalhães, Fundação Oswaldo Cruz – Recife –PE; (5) Secretaria Estadual de Saúde de Pernambuco – PROGRAMA SANAR-SES-PE Introduction: Chronic Chagas disease (CCD), caused by Trypanosoma cruzi. In Brazil, the Northeast Region is one of the endemic areas for CCD. The geoprocessing allows the identification of patterns and trends in spatio-temporal distributions. Objective: To analyze the spatial distribution of CCD in terms of clinical stages in the reference service in the Northeast region of Brazil. Methods: Ecological population-based approach, the unit of analysis being the municipalities where the population with CCD from 2016 to 2018. The State of Pernambuco is composed of 184 municipalities and 05 mesoregions: Metropolitan Region, Zona da Mata, Agreste, Sertão and Sertão do São Francisco. The classification used the Clinical stages listed in the “I Latin American Directive for the Diagnosis and Treatment of Chagas Heart Disease” (A, B1,B2,C and D). The analyzed indicator was the average annual rate of occurrence. Results: The 801 chronic cases, the mean age was 62 ± 12.0 years, with a predominance of females (60%). As for the clinical stages classified as A: 22.5%; B1: 43.5%; B2: 8.5% and C: 25.5%. The mean CCD occurrence rates in terms of clinical stages, the respective standard deviation (SD): A: mean = 0.76; SD = 2.1 in Zona da Mata and Sertão; B1: mean = 1.6; SD = 4.4 in Zona da Mata and Sertão; B2: mean = 0.23; SD = 0.82 in Zona da Mata, Sertão and Sertão do São Francisco;C: mean = 0.67; SD = 1.27 in Zona da Mata, Agreste Sertão and Sertão do São Francisco as shown in the figure. Conclusions: The spatial distribution of CCD showed spatial heterogeneit, there were clusters in two mesoregions in stages A and B1, while in stages B2 and C in 4 mesoregions indicating the persistence of CCD in these areas, requiring priority health surveillance actions and strengthening of the assistance decentralization. 108422 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION GILBERTO ANDRADE TAVARES1, Matheus Henrique Costa Xavier2, Filipe Euclides Gobatto2, Iara Victoria dos Santos Moura2, Virna Anfrizio Souza2, Wictor Hugo De Souza Silva2, Eleonora Ferraris de Gaspare5, Gledson de Carvalho Santos4, Fabio Batista Santos4, Virgílio Antônio Cardoso Faro4, José Augusto Soares Barreto-Filho1 (1) Postgraduate Program in Health Sciences, Federal University of Sergipe; (2) Federal University of Sergipe; (3) Rede D’Or São Luiz, Hospital São Lucas, Division of Cardiology; (4) Tiradentes University; (5) Parma University Introduction: The Cardiovascular Diseases have been the main cause of death in the world with a slow decrease in mortality rates in most countries. In 2010, The American Heart Association (AHA) defined 7 metrics for a Cardiovascular Health in order to reduce Cardiovascular mortality by 20%. The Mobile Health Tools are able to support shared clinical decision making, telemonitoring feedback and improve patient’s adherence to medication regimen. Objective: Demonstrate the development and applicability of the application “Cardiovascular Health” for mobile phones according to the parameters defined by The AHA. Method: The method chosen is the User Centered Design, Dart programming language, Flutter framework and Firebase database. The application was developed using Scrum, with 2-week sprints in which tasks were divided into small deliverables in order to avoid rework and make the process more agile. The research team monitored the project closely and decided about what was done by the group. Results: Each parameter considered ideal, evaluated as “Good”, following those requirements, will provide the patient 1 mark. If the participant scores between 5 and 7 marks, will be classified as “Good” Cardiovascular Health; if the score is between 3 and 4 marks, the classification will be “Can be improved”; and if the score is null or under 2 marks, it will be defined as “Needs to be improved”. Hence, he will have his “Cardiovascular Health” classified, generating PDF reports. Conclusion: The “Cardiovascular Health” application comprises all the components to measure Cardiovascular Health and will be able to provide physicians and other healthcare workers with better decision making about Cardiovascular Health of the treated population. 109090 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ROMERO HENRIQUE DE ALMEIDA BARBOSA1, Emerson Silva de Jesus1, Eldys Myler Santos Marinho1, Johnnatas Mikael Lopes1 (1) Universidade Federal do Vale do São Francisco Background: Medication adherence is imperative for adequate care of chronic cardiac health conditions due to their disabling and death potential. However, this self-care characteristic does not develop without professional support and may not be homogeneous in the entire population. Objective: To identify the existence of differences in medication adherence in people with a history of acute cardiac events. Methods: Cross-sectional population-based study of the National Health Survey (NHS) in Brazil in 2019. The sample with 90846 interviews in people over 15 years old. Diagnosis of heart disease was given by the presence of heart failure, arrhythmia, angina, history of myocardial infarction. The independent variables were coronary artery bypass graft surgery (yes/no) and myocardial infarction (yes/no). Medication adherence due to cardiac condition was treated as an outcome. Data analyzed by stratifying the prevalence of the outcome and estimating the prevalence ratio, taking into account the complex sampling of participants and a confidence interval of 95%. Results: We identified 4732 (5.1%;4.8–5.3) individuals with some type of heart disease in the NHS, of which 1383 (29.2%;27.0–31.4) reported at least one episode of myocardial infarction. Revascularization was performed in 1483 (28.9%;26.8–31.0) of individuals with heart disease. Approximately one-third of participants with heart disease do not adhere to medication regularly [3284 (31.4%:29.1–33.7)]. However, it is possible to state that individuals who underwent revascularization procedure [1297 (87.9%;85.4–90.0)] have more medication adherence than those who did not undergo. The occurrence of acute myocardial infarction increases the probability of greater adherence to medication by 13.3% in those who underwent revascularization and 27.5% in those who had an infarction and did not undergo revascularization. In relation to individuals with heart disease and without infarction and revascularization events, the probability of adherence is 59.10%. Conclusion: Acute and harmful cardiac events such as myocardial infarction and revascularization seem to be associated with greater medication adherence in this population. This refers to a scenario of low self-care supported by monitoring of cardiac conditions, requiring greater attention and the proposition of mitigating strategies on the part of health professionals. 109088 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH FARID SAMAAN1, Louis Nakayama Ohe1, Lívia Gâmbaro1, Renata Viana1, Emmanuel Almeida Burdmann2 (1) Instituto Dante Pazzanese de Cardiologia; (2) Universidade de São Paulo Introduction: Chronic kidney disease and acute kidney injury (AKI) are important complications of heart diseases. In developing countries, epidemiological and cost information on the interaction of these conditions are scarce. Objectives: To determine the prevalence, costs and outcomes of patients admitted for acute coronary syndrome (ACS) with renal dysfunction and AKI. Methods: The study was based on a prospective database analysis of patients admitted for ACS to a Brazilian public hospital specialized in cardiology between 7/16/2018 and 12/31/2019. Renal dysfunction was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m² at hospital admission. Community-acquired and hospital-acquired AKI were defined as a fall and an increase of ≥0.3 mg/dl in serum creatinine from baseline, respectively. Results: 1295 of the 1620 patients had a confirmed diagnosis of ACS (median age 64.2 [56.5–70.6] years, 65.4% male, 82.7% had hypertension, 45.5% diabetes and 22.6% renal dysfunction). The imaging diagnosis of ACS was coronary angiography in 84.3% and the treatment was performed by angioplasty, only clinically and by myocardial revascularization in 47.3%, 40.0% and 12.7%, respectively. Hospital- and community-acquired AKI occurred in 43.9% and 2.3% of patients, respectively. Compared with patients admitted with eGFR ≥60 ml/min/1.73 m², those with eGFR <60 were older (70.6 vs. 62.5 years, p < 0.001), had a higher prevalence of hypertension (92.1% vs. 80.0%, p < 0.001) and diabetes mellitus (60.6% vs.54.5%, p = 0.010), had a higher incidence of AKI (65.0% vs. 51.4%, p < 0.001) and higher: median amount reimbursed for hospitalization (1,344 [366–2,103] vs. 1,334 [290–2,018] dollars, p = 0.034), median length of stay (5 [3–10] vs. 4 [2–7] days, p < 0.001), death within 30 days (4.1% vs. 1.4%, p = 0.004) and death within 12 months (9.2% vs. 2.9%, p < 0.001). Patients with AKI, compared to those without this condition, were older (65.6 vs.63.3 years, p = 0.008), had lower eGFR on admission (78.1 ml/min/1.73 m² vs.86.1 ml/min/1.73 m², p < 0.001) and greater: median amount reimbursed for hospitalization (1,334 [301–1,865] vs. 1,724 [973–2,549] dollars, p < 0.001), median length of stay (6 [4–13] vs. 3 [2–5] days, p < 0.001) and death within 12 months (4.8% vs. 2.3%, p = 0.032). Conclusions: In patients with ACS at a cardiology referral hospital, renal dysfunction on admission and AKI during hospitalization were frequent and associated with worse clinical and economic outcomes. 108437 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DIANE XAVIER DE ÁVILA1, Gustavo Rodolfo Moreira1, Angelo Michele Di Candia1, Victoria Depes Scaramussa1, Najla Cassibi Cavaliere1, Fernanda Turque Martins1, Maisa Passos Vieira1, Humberto Villacorta1 (1) Universidade Federal Fluminense Background: Natriuretic peptides (NP) are the gold standard biomarkers in HF. However, new biomarkers have emerged, with additional effects to NP in relation to prognosis. Growth differentiation factor-15 (GDF-15) is a marker of oxidative stress and inflammation that may contribute to the prognosis of patients with HF. Objectives: We sought to evaluate the distribution of GDF-15 values, the characteristics of patients with high values and the relationship between their values and NT-proBNP in patients with chronic HF. Methods: This was a cross-sectional study of patients with chronic HF who were on guideline-recommended medical therapies. Patients with signs and symptoms of HF and LVEF <50% were included. The dosage of NT-proBNP was performed using the Elecsys® system (Roche, Basel, Switzerland) and GDF-15 by the sandwich immunoassay method with monoclonal antibodies (Elecsys®, Roche, Basel, Switzerland). Patients were grouped according to GDF-15 values above and below the median. Correlation analysis was performed between GDF-15 and other non-normal continuous variables, using the Spearman method. Results: A total of 67 patients were included. Etiology was predominantly non-ischemic, 61.2% were male, with mean age of 61 ± 13 years. The median GDF-15 values were 1413 pg/mL (interquartile range 1044–2554) and NT-proBNP was 759 pg/mL (168–2354). Patients with GDF-15 values above the median had higher creatinine values (1.32 mg/dL [0.85–1.71] vs 0.91 [0.78–1.08], p < 0.001), higher NT-proBNP values (2071 pg/mL [508–5350] vs 463 [87–962], p = 0.001) and lower LV ejection fraction (30% [27–43] vs 45% [30–54], p = 0.08. There was a positive correlation between GDF-15 and NT-proBNP (r = 0.46, p < 0.001), creatinine (r = 0.56, p < 0.001) and left atrial volume (r = 0.32, p = 0.037). Conclusion: Even after adequate treatment, some patients with chronic HF had high levels of GDF-15. GDF-15 identified patients with greater severity. GDF-15 may be a useful tool in the management of HF patients. 108438 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ANGELO MICHELE DI CANDIA1, Diane Xavier de Ávila1, Gustavo Rodolfo Moreira1, Humberto Villacorta1 (1) Universidade Federal Fluminense Background: Growth differentiation factor-15 (GDF-15) is a marker of oxidative stress and inflammation and is increased in a number of cardiovascular disorders. Objectives: We sought to assess the relationship of GDF-15 with severity parameters in patients hospitalized with atrial fibrillation (AF). Methods: Fifty hospitalized patients with a primary or secondary diagnosis of AF from two hospitals were included. GDF-15 was measured at baseline, as well as cardiac and inflammatory biomarkers. A comparison of baseline characteristics was performed in groups with high (above median) and low (below median) GDF-15 values. Correlations were made between the GDF-15 and other variables, using the Spearman method. Results: Twenty-nine (58%) patients were male and the mean age was 68.5 ± 17.3 years. Twenty-eight (56%) had permanent AF and 18 (36%) had HF with reduced ejection fraction (LVEF <40%, HFrEF). The median GDF-15 was 2724 pg/mL (interquartile range 1116–5139). Patients with high GDF-15 values were older (77.5 ± 9.7 vs 59 ± 18.5 years, p = 0.0001) and more likely to have hypertension (92.3% vs 70%, p = 0.018) and permanent AF (69.2% vs 37.5%, p = 0.016). They also had higher NT-proBNP values (4006 pg/mL [2156–7023] vs 816 [174–3814], p = 0.0015), creatinine (1.2 [0.95–1.7] vs 1, 0 [0.75–1.15], p = 0.017) and C-reactive protein (3.1 [1.55–7.32] vs 1.25 [0.55–2.45], p = 0.008). GDF-15 was higher in patients with HF (n = 34 [68%]) vs non-HF (3126 [1737–6457] vs 774 [565–4074], p = 0.01). Patients with HFrEF had higher levels of GDF-15 as compared with those with LVEF >40% (3019 [2012–7299] vs 1533 [784–4674], p = 0.05). All risk scores were higher in patients with high GDF-15, namely CHA2DS2-VASC (4.5 ± 1.6 vs 2.9 ± 2.3, p = 0.008), HAS-BLED (2.4 ± 1.3 vs 1.3 ± 1.1, p = 0.0029), ORBIT (3.5 ± 1.5 vs 1.7 ± 1.5, p = 0.0003) and MAGGIC (20.8 ± 12.3 vs 11 ± 11.2, p = 0.006). There was a direct correlation between GDF-15 and NT-proBNP (r = 0.5, p = 0.002), D-dimer (r = 0.52, p = 0.002) and age (r = 0.43, p = 0.002). Conclusion: GDF-15 values were associated with parameters of greater severity in AF. GDF-15 may be useful as a prognostic factor and may possibly aid in the indication of anticoagulation in AF. 108439 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GUSTAVO RODOLFO MOREIRA1, Diane Xavier de Ávila1, Angelo Michele Di Candia1, Victoria Depes Scaramussa1, Najla Cassibi Cavaliere1, Maisa Passos Vieira1, Fernanda Turque Martins1, Humberto Villacorta1 (1) Universidade Federal Fluminense Background: Patients with heart failure (HF) often have altered renal function. Urinary sodium is a marker of diuretic resistance and is associated with a worse prognosis in HF. Growth differentiation factor-15 (GDF-15) is a marker of oxidative stress and inflammation and is a prognostic predictor in HF. Objectives: We sought to assess the relationship of GDF-15 with renal function parameters and with urinary sodium in patients with chronic HF. Methods: We undertook a cross-sectional study of patients with HF from specialized outpatient clinic. Patients with signs and symptoms of HF and LVEF <50% were included. An echocardiogram was performed and blood samples were collected, which were frozen for the final study, where the performance of GDF-15 in the prediction of renal outcomes in HF will be evaluated. The dosage of NT-proBNP was performed using the Elecsys® system (Roche, Basel, Switzerland) and GDF-15 by the sandwich immunoassay method with monoclonal antibodies (Elecsys®, Roche, Basel, Switzerland). Analysis of the relationship between GDF-15 and baseline renal parameters was performed. Correlation analysis was performed between GDF-15 and continuous variables, using the Spearman method. Results: Sixty-seven patients were included. The etiologies of HF were hypertension, diabetes mellitus, alcoholic cardiomyopathy and idiopathic cardiomyopathy. Forty-one (61.2%) individuals were male, with a mean age of 61 ± 13 years. Median GDF-15 values were 1413 pg/mL (interquartile range 1044–2554). Patients with GDF-15 values above the median had lower urinary sodium values (88 mEq/L [53–121] vs 112 [76–171], p = 0.06), lower glomerular filtration rate (GFR) (63.9 ± 29.1 vs 91.2 ± 25 mL/min/1.73 m2, p = 0.001) and higher creatinine levels (1.32 mg/dL [0.85–1.71] vs 0 .91 [0.78–1.08], p < 0.001). There was no significant difference in relation to urinary albumin (21 mg/L [7–126] vs 15.1 [5.8–41], p = 0.30). The urinary albumin/creatinine ratio was higher in the group above the median, but did not reach statistical significance (38 mg/g [11–110] vs 12.5 [4.9–29], p = 0.13). There was a direct correlation between GDF-15 and creatinine (r = 0.56, p < 0.001) and an inverse correlation with urinary sodium (r = –0.39, p = 0.005) and with GFR (r = –0.55, p < 0.001). Conclusion: GDF-15 correlated with lower urinary sodium levels and worse kidney function in patients with chronic HF. Future studies should address whether GDF-15 is a predictor of worsening renal function overtime. 108440 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM DIANE XAVIER DE ÁVILA1, Ana Luiza Carraro de Souza2, Gabriel Alverca Meyas2, Mayara Cristina Villela Santos2, Jonatas da Costa Mendonça2, Luciene Maria Mendes da Costa2, Beatriz de Paula Sousa2, Maria Victoria Borges de Oliveira2, Julia Correia Cardoso Guimarães2, Julie Xavier de Ávila Guedes3, Ulisses Oliveira de Melo1, Humberto Villacorta2 (1) Hospital Municipal Che Guevara; (2) Universidade Federal Fluminense; (3) Universidade Estácio de Sá Background: Cardiovascular risk factors are prognostic factors in COVID-19 and have been scarcely studied in Brazil. Objectives: To assess the impact of cardiovascular risk factors on the outcomes in patients admitted for COVID-19. Methods: From July 2020 to February 2021, 200 patients from two public hospitals were enrolled. Patients were included if they had: typical symptoms or signs of COVID-19, a positive real-time polymerase chain reaction test (RT-PCR) for COVID-19, and age above 18 years old. This was a prospective, observational, longitudinal study. Data was collected within 24 hours from admission. The primary endpoint was a combination of hospital mortality, mechanical ventilation, hemodialysis or hospital length of stay >28 days. Results: There were 98 (49%) events during the hospital course and 72 (36%) died in the hospital. Patients with a primary endpoint were older and more likely to have a history of hypertension, diabetes, chronic obstructive pulmonary disease (COPD) and chronic kidney disease (CKD). Vital signs at admission associated with events were diastolic blood pressure, respiratory rate and oxygen saturation in ambient air (O2Sat). Serum creatinine >1.37 mg/dL at admission had sensitivity of 51.6 and specificity of 82% to predict the primary endpoint, with an area under the curve (AUC) of 0.68. In multivariate analysis, age, diabetes, CKD, and COPD were independent predictors of the primary endpoint. Age and CKD were independent predictors of in-hospital mortality. Conclusion: Cardiovascular risk factors, such as diabetes and CKD are related to a worse prognosis in patients hospitalized with COVID-19 in Brazil. 108452 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY EDUARDO ARRAIS ROCHA1, Luís Gustavo Bastos Pinho1, Juvêncio Santos Nobre1, Maria Eduarda Quidute Arrais Rocha3, Marcela Sobreira Kubrusly2, FernandaPimentel Arrais Maia4, Eduardo Augusto Quidute Arrais Rocha2, Maria Camila Timbó Rocha3, Pedro Barbosa Duarte Vidal1, Vitor Olímpio Coimbra2, Bruna Sobreira Kubrusly1, Ana Rosa Pinto Quidute1 (1) Universidade Federal do Ceará; (2) Universidade Unichristus; (3) Universidade de Fortaleza – UNIFOR; (4) Universidade Federal do Ceará – Campos Sobral Introduction: The remote monitoring (RM) of Implantable electronic cardiac devices (IECD) has become a common follow-up modality in many countries given its effectiveness, safety, facilities for the patient and possibilities of early interventions. In Brazil, however, this form of follow-up is an exception. This work aims to demonstrate the findings, evolution and peculiarities of IECD follow-up in a tertiary center in Brazil. Methods: This is a cohort, prospective study, involving 119 patients, followed in room office visit at 6 months interval with daily RM, with mean age 72 ± 14.2, ejection fraction 55% (34.5/57%), 57.1% in functional class ≥II, 30.2% with pacemakers (PM), 42.8% with defibrillators (D), 3.3% with biventricular pacemaker (CRT) and 22.7% with CRT-D. The logistic regression was used to obtain evidences that the RM is associated with the following outcomes: immediate changes in therapy, elective changes in therapy and avoidance of hospitalizations. Results: Events were detected in 63.9% of the cases in 29.5 ± 23 months of follow-up; 16% presented more than 6 events; 36.1% no events and 27.7% had more than one event detected. The most common events were: 18.5% with alterations in the electrode/battery or in the thoracic impedance parameters; 33.6% with ventricular arrhythmia and 44.5% with supra-ventricular arrhythmias. It was observed that 86.5% of patients and 91.6% of physicians reported feeling secure with this method of follow-up. The outcomes found were: 23.5% needed immediate changes in therapy, while 44.5% needed elective changes. The RM was important to the beginning or maintaining of the anticoagulation in 16.9% and avoided hospitalization in 19.3%. Through the use of logistic regression, there are evidences that the RM had an statistically significant impact in the elective changes of conduct (p = 0.03), immediate changes of conduct (p = 0.007) and reduced hospitalization (p = 0.04). Conclusion: The RM was considered effective and safe in the following-up of patients with IECD in some regions of Brazil. This method of follow-up allowed for many early interventions or electives ones which made therapeutic handling and increased safety of the patients. 108459 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING JAIME AFONSO SOUSA NETTO1, Arise Garcia de Siqueira Galil1, Andre Pereira Duque Estrada2, Marcus Gomes Bastos1 (1) Universidade Federal de Juiz de Fora-UFJF; (2) Universidade Federal Fluminense-UFF Background: Half of the patients with heart failure (HF) have diastolic dysfunction (DD) and a preserved left ventricular ejection fraction (LVEF). The indexed left atrial volume (ILAV) is one of the main components in the assessment of DD. However, there are controversies regarding the use of the left atrial strain (LAE) to assess DD. Objectives: To evaluate the use of LAE in the identification of indeterminate diastolic dysfunction (IDD) not detected by ILAV, in patients with chronic kidney disease (CKD) stages 3B to 5 in conservative treatment and LVEF. Methods: Prospective cross-sectional study that evaluated 114 patients with CKD stages 3B to 5 under conservative treatment and who presented LVEF through transthoracic echocardiography (TTE). The patients were divided into two groups, with and without left ventricular hypertrophy (LVH). The analysis of the strain of the left atrium was evaluated by the speckle tracking method from the apical section of 2 and 4 chambers, evaluating the deformation curves of the left atrium (LA). Results: LAE increased the detection of DD in the group with LVH (p < 0.01). The mean glomerular filtration rate (GFR) (in mL/min/1.73 m2) in patients with and without LVH was 32.6 ± 11.9 and 40.4 ± 13.9, respectively (p < 0.05). The LAE found was lower in the LVH group 20.3 ± 5.11 vs 26.1 ± 9.1, (p < 0.05). Conclusion: In patients with non-dialysis CKD stages 3B to 5 with LVH and LVEF, in the studied sample, the use of LAE potentially increases the identification of indeterminate cases of DD. 108717 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO DE PADUA MANSUR1, Carlos Henrique Del Carlo1, José Antonio Ramos Neto1, André Barbosa de Abreu1, Airton Roberto Scipioni1, Antonio Carlos Pereira Barretto1 (1) Insituto do Coração – HC FMUSP Background: Chronic Chagas cardiomyopathy (CCC) is one of the leading causes of congestive heart failure (CHF) in Brazil and carries high morbidity and mortality. Type 2 diabetes mellitus (T2DM) is associated with a higher cardiovascular risk in women than men. This study aimed to analyze the influence of T2DM on CHF mortality from CCC in women and men. Methods: From February 2017 to September 2020, we followed CCC’s cohort of outpatients with CHF (Framingham criteria). Specific serological tests diagnosed Chagas disease. Baseline data included clinical features and echocardiographic findings. Statistical analyzes used the Kaplan-Meier method for time-to-event data and Cox proportional hazards methods to look for predictors of death. Results: We studied 733 patients mean age of 61.4 ± 12.3 years, 381 (52%) were male. T2DM was present in 46 (15%) women and 40 (12%) men. Women with T2DM, compared to men with T2DM, had a higher mean age (62.7 ± 12 vs. 59.7 ± 12.5 years; p < 0.001), had a higher mean left ventricular ejection fraction (LVEF) (42.8 ± 14.3% vs. 37.3 ± 14.3%; p < 0.001) and a smaller left ventricular diastolic diameter (58.2 ± 8.5 vs. 61.9 ± 8 .6 mm; p < 0.001). Over a 3-year follow-up period, 26 (65%) men and 30 (65.2%) women with T2DM died, and mortality were higher in T2DM patients compared to nondiabetics (Women: log-rank p < 0.001; Men: log-rank p < 0.001; Figure). Cox regression adjusted for age, previous myocardial infarction, T2DM, previous stroke, chronic kidney disease (CKD), atrial fibrillation (AF), and LVEF showed, in descending order, T2DM (HR = 2). .19), stroke (HR = 1.81), AF (HR = 1.80), CKD (HR = 1.72), and reduced LVEF (HR = 1.29) as the most important predictors of death in women and, in men, stroke (HR =), DM2 (HR = 2.01), CKD (HR = 1.83), AF (HR = 1.58) and reduced LVEF (HR = 1.24). Conclusions: CHF mortality from CCC was similar in women and men with T2DM. T2DM neutralized the protection of women compared to men concerning mortality from CHF. 108719 Modality: E-Poster Researcher – Non-case Report Category: NURSING LEANDRO LOUREIRO BUZATTO1, Tarsila Perez Mota1, Leticia de Lana Pereira1, Mayumi Alves Hayafugi1, Daisa de Mesquita Escobosa1, Marina Barros de Melo1, Carolina Ivo de Araujo1, Carla Manuela Pereira de Araujo1, Marcelo Franken1 (1) Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein Introduction: The adaptations and changes in the lifestyle of patients with heart failure (HF) is the main reason why the quality of life of these individuals becomes fragile. Studies show the high prevalence of depression and associate this condition directly with HF. The relationship between changes in quality of life, the occurrence of depression and the presence of HF result in unfavorable outcomes for the patient, justifying the performance of the present study. Objective: Identify factors associated with quality of life and depression in patients with heart failure. Method: This is a descriptive exploratory cross-sectional study, in patients aged over 18 years, hospitalized with a diagnosis of systolic HF and who had a recent hospitalization between the period March 2017 to December 2020. Patient Health Questionnaire (PHQ2) and Kansas City Cardiomyopathy Questionnaire (KCCQ-12) were administered 30 days after discharge by telephone contact. The analysis considered the relationship between the scores of both questionnaires and the following variables: gender, diagnosis, functional class, previous atrial fibrillation, previous diabetes, previous acute myocardial infarction and previous arterial hypertension. Conclusions: Female patients with heart failure showed a greater association with the depression outcome (p = 0.001) and the reduction in quality of life showed a decline once associated with diagnosis, functional class and age. 108484 Modality: E-Poster Researcher – Non-case Report Category: NURSING EWA-LENA BRATT1, Mariela Acuna Mora1, Carina Sparud-Lundin1, Sandra Skogby1, Åsa Burström2, Markus Saarijärvi1, Katrina Hanseus3, Annika Rydberg4, Shalan Fadl5, Eva Fernlund6, Kalliopi Kazamia7, Philip Moons8 (1) Institute of health and care science, University of Gothenburg, Sweden; (2) Karolinska Institute, Department of Neurobiology, Care Sciences and Society, Stockholm, Sweden; (3) Department of Pediatric Cardiology, Children’s Heart Center, Skane University Hospital, Lund, Sweden; (4) Department of Clinical Sciences, Umeå University, Umeå, Sweden; (5) Department of Paediatrics, Örebro University Hospital, Örebro, Sweden; (6) Department of Clinical and Experimental Medicine, Linköping University, Division of Pediatrics, Crown Princess Victoria Children’s Hospital, Linköping University Hospital, Linköping, Sweden; (7) Children’s Heart Center Stockholm-Uppsala, Karolinska University Hospital and Akademiska University Hospital, Sweden; (8) KU Leuven, Department of Public Health and Primary Care, Leuven, Belgium Introduction: Adolescents with congenital heart disease (CHD) need to acquire knowledge about their heart disease and treatment. This is fundamental for them to implement health promoting behaviors and know how to manage their CHD. Amongst other things, transition programs aim to equip adolescents with this knowledge. However, evidence on the effectiveness of such interventions is limited. Objective: To investigate if a transition program is effective in improving disease-related knowledge of adolescents with CHD. Methods: The STEPSTONES (Swedish Transition Effects Project Supporting Teenagers with chrONic mEdical conditionS) transition program is an 8 components intervention. The central component was a transition coordinator (specialized nurse) who had three consultations with the participants during a 2y period. A randomized controlled trial (RCT) was conducted in two CHD centers in Sweden. Participants were randomly assigned to the intervention (IG; n = 70) or control group (CG; n = 69). Knowledge was measured at the age of 16 y (T0; baseline), 17y (T1) and 18.5y (T2) using the Knowledge Scale for Adults with Congenitally Malformed Hearts questionnaire, psychometric evidence supported its validity and reliability. The total score ranges from 0–7, with higher scores denoting a higher CHD-related knowledge. Change in score between T0 and T2 was analyzed using Fisher’s non-parametric permutation test unadjusted between the two groups. Analyses were performed on the Full Analysis Set (FAS). Results: In total, 114 participants were included in the FAS, of which 54 were in the IG and 60 in the CG. At baseline, the mean knowledge score in the IG was significantly lower (3.7 ± 1.6) than in the CG (4.4 ± 1.5) (p = 0.03). At T2, the knowledge score was significantly higher in the IG (5.0 ± 1.3) than in the CG (4.5 ± 1.5) (p = 0.045). In line with this, the change over time (T0–T2; 2.5y) was significantly different between the two groups (Mean difference = 1.22; 95%CI = 0.60–1.82; p = 0.0002). The effect size was 0.74, indicating a moderately large effect. Conclusion: This RCT showed that the STEPSTONES transition program was effective in increasing disease-related knowledge in adolescents with CHD, which is an important aspect in preparing them for the transition to adulthood and the transfer to adult care. 108485 Modality: E-Poster Researcher – Non-case Report Category: CARDIO-ONCOLOGY DIEGO RAFAEL FREITAS BERENGUER1, Gustavo Freitas Alves de Arruda1, Mayara Laís Coêlho Dourado1, Felipe Alves Mourato2, Monica de Moraes Chaves Becker1, Roberto de Oliveira Buril1, Brivaldo Markman Filho1, Simone Cristina Soares Brandão1 (1) Universidade Federal de Pernambuco (UFPE); (2) Real Hospital Português de Beneficiência em Pernambuco Objective: To evaluate the behavior of the standardized uptake value (SUV) of the radiopharmaceutical 18F-FDG before, during and after chemotherapy (QT) at different cardiac sites, as well as to measure the degree of reproducibility for the method in the context of cancer treatment follow-up. Materials and Methods: Retrospective cohort including lymphoma patients who underwent 18F-FDG PET/CT before, during and/or after chemotherapy. The uptake behavior through the mean and maximum SUVs was evaluated in four cardiac sites and in control sites in the aorta and liver. Twenty exams were randomized for reproducibility analysis by two examiners who were blinded to each other’s results. Each one of them did the evaluation in a second moment to elucidate the intra-observer reproducibility. Results: A significant increase in SUVs was observed in all cardiac sites in the interim and final moments (post-terminus of QT) when compared with pre-QT SUVs. The left ventricular (LV) free wall was the cardiac region with the greatest increase in 18F-FDG uptake (Figure). As for the reproducibility, it was possible to verify substantial results for the reliability of the measurement of SUVs, both intra and inter-observer (Figure). Conclusions: 18F-FDG Cardiac uptake increased along QT, with the LV free wall being the site with the greatest increase. The reproducibility analysis showed high intra- and inter-observer correlation values. 108487 Modality: E-Poster Researcher – Non-case Report Category: NUTRITION RAMPHUL YOGESHWAREE1, Chan Sun Marie France1, Cheeneebash Jayrani1 (1) University of Mauritius, Faculty of Medicine and Health Sciences Introduction: In Mauritius, a rapidly developing island in the Indian Ocean, 89% of the deaths are due to non-communicable diseases, out of which 33% are caused by Cardiovascular Diseases. With the prevalence of obesity which has increased from 10.3% in 2004 to 19.1% in 2015, cardiovascular complications linked to obesity are expected to increase, based on the well-established relationship between obesity and cardiovascular diseases. Objective: This study investigated the perception on obesity of Mauritian police officers for tailor-made health interventions. No such study had previously been conducted in Mauritius. Methods: This cross-sectional study was carried out using a pre-tested survey instrument, based on the Health Belief Model with obesity-related questions pertaining to participants’ perceived susceptibility, severity, barriers, benefits, cues to action and self-efficacy. The study population consisted of random sample police officers, with the calculated sample size being 384. Ethical clearance was obtained from the relevant research ethics committee. Results: There were 384 respondents, consisting of 240 men (62.5%), and 144 women (37.5%) with age span from 18 to 59, 77.3% of the study population being in the age group 26 to 50. The participants’ mean score on the perceived severity scale was 3.8 out of five points (±1.2), while their mean score on the perceived susceptibility scale was 3.0 out of five points (±1.2). A stepwise multiple regression analysis showed that the susceptibility score (dependent variable) and sex, age, ethnicity, socioeconomic position and alcohol (independent variables) were significant at 95% confidence interval. Discussion: The lower mean susceptibility score among women as compared to men and among those who are overweight/obese as compared to those with normal Body Mass Index is of great concern. The findings of this study need to be the basis for the development of customized health interventions for the primary prevention of cardiovascular diseases. The vulnerability of women to cardiovascular diseases needs to be emphasized to make women more cautious on their health. Conclusions: In light of the findings of this study, we recommend primary prevention with integrated approach for promotion of healthy eating and physical activities, with a particular focus on women who need to be sensitized on their vulnerability to cardiovascular diseases. 108490 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BRUNO REZNIK WAJSBROT1, Ana Luiza Ferreira Sales1, Andre Luis Sales Feitosa1, Carolina Pereira de Barros1, Felipe Neves de Albuquerque1, Marcelo Imbroinise Bittencourt1, Pedro Pimenta de Mello Spineti1, Roberto Esporcatte1, Denilson Campos de Albuquerque1, Ricardo Mourilhe-Rocha1 (1) State University of Rio de Janeiro – Pedro Ernesto University Hospital Introduction: Right Ventricle (RV) failure is found in as many as fifty percent of heart failure (HF) patients. Echocardiographic parameters are studied with the aim of refining prognosis of HF, but isolated prognostic impact of the “forgotten ventricle” is not often studied. Purpose: The objective of this study is to define echocardiographic parameters associated with post-discharge mortality after admission for ADHF. Methods: This is a study in a single center, transversal, cohort of hospitalized patients with ADHF, aged >18 years, conducted at a University Hospital in Brazil. Data was collected from September 2019 to September 2021. Patients in renal replacement therapy before admission or with cancer in palliative care were excluded. Continuous variables were analyzed with Mann-Whitney U test, categorical variables with log-Rank test. Analysis was made using SPSS software. Results: A total of 200 patients were enrolled. The mean age was 62 years (SD ± 14.5), 65.9% male, 63% with hypertension, 29.1% with diabetes and 32.8% with ischemic heart disease (IHD). Three month and a year mortality was respectively 8.4% and 39.5%. HFrEF was observed in 67.2%, 14.4% HFmrEF and 18.4% HFpEF. Severe mitral regurgitation (MR) was observed in 22.5% RV dysfunction in 47%. Median ejection fraction, end-systolic (ESD), end-diastolic diameter (EDD) and systolic pulmonary arterial pressure (sPAP) was respectively 33% (24–45.5), 49 mm (38.25–56), 59 mm (52–65) and 44.5 mmHg (32.75–57.25). Only RV dysfunction was associated with reduced long-term mortality (p = 0.038). Average follow-up time was 459 days. Conclusion: RV disfunction has an isolated prognostic impact on long term survival after admission for ADHF. Trials that focus on therapies for RV function might reduce mortality for HF patients. 108496 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY ANTONIO ALCEU DOS SANTOS1, Gilmara Silveira da Silva2, Flávia Cortez Colósimo Bastos2, José Francisco Baumgratz2, José Pedro da Silva2, Luciana da Fonseca da Silva2, Rodrigo Moreira Castro2, Rodrigo Freire Bezerra2, Carlos Eduardo Panfilio1, Isabel Cristina Céspedes1 (1) Escola Paulista de Medicina – Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil; (2) A Beneficência Portuguesa de São Paulo Hospital, São Paulo, Brazil (BP-SP) Introduction: Allogeneic blood is an increasingly scarce resource in blood banks around the world due to its increasing demand [1]. Cardiac surgeries represent an excessive consumption of hemocomponents [2]. Plasma transfusions are associated with more risks than benefits [3,4]. The rational use of allogeneic blood has become a worldwide necessity, aiming to reduce morbidity and mortality and hospital costs [5–7]. Hence the importance of scientific educational measures, encouraging procedures, techniques, and drugs to reduce allogeneic blood transfusions in coronary artery bypass graft surgery (CABG). Objective: To verify whether clinical and surgical strategies for conserving the patient’s own blood are effective in reducing plasma transfusions and their complications in CABG. Methods: Retrospective cohort study of patients undergoing CABG. A total of 4923 patients were studied from a database, 3010 patients from the year 2010 (group 1) and another 1913 patients from the year 2012 (group 2). For one year and six months, biweekly multidisciplinary meetings were held for scientific updates on transfusion practices, focusing on learning and implementing patient blood conservation strategies, based on two main pillars: 1. Reduction of blood loss (meticulous hemostasis and optimal use of antifibrinolytics); 2. Optimization of physiological tolerance to anemia (supplementary oxygen therapy, more restrictive transfusion management, treating anemia). After these educational measures to conserve the patient’s blood, a new data collection was carried out for a comparative analysis of the variables of group 1 and 2. Results: A total of 377 patients received plasma transfusions, with a significant reduction from 13.1% (282 patients) in group 1 to 6.7% (95 patients) in group 2, p < 0.001. There was also a significant reduction in infection (mediastinitis) from 3.0% (group 1) to 1.4% (group 2), p < 0.05. There was no difference in the total length of hospital stay and intensive care unit stay in the two groups. The mortality expected by the Euroscore (2.35 vs 2.44, p = 0.215, groups 1 and 2, respectively) in the preoperative period of the patients showed no significant difference in both groups. Conclusion: Patient blood conservation strategies focused on reducing blood loss and optimizing physiological tolerance to anemia resulted in reduced plasma transfusions and postoperative mediastinitis-like complications in patients undergoing coronary artery bypass graft surgery. 108503 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogerio Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Introduction: Coronary artery disease represents one of the main causes of death in Brazil and worldwide. Rapid diagnosis is important for effective treatment. The difficulty of accessing referral medical centers during the COVID 19 pandemic influenced the management of these diseases. Coronary angioplasty (CA) as an option for the management of patients with coronary disease was also impacted during this period. The present study aimed to evaluate the influence of COVID-19 on coronary angioplasties in Brazil. Method: We used the database from the Department of Informatics of the Brazilian Health System (DATASUS) through the website https://datasus.saude.gov.br/. We evaluated the number of hospitalizations, deaths, costs and length of stay of patients undergoing CA in Brazil and their relationship with the beginning of the COVID-19 pandemic in Brazil (in 2020) based on data from 2018 to 2021. Results: From January 2018 to December 2021, 302572 patients were hospitalized for CA with stent in Brazil with an annual mean of 75643 ± 4742. Meanwhile, 43870 were hospitalized for primary angioplasty with an annual mean of 10968 ± 278. There was an 8.5% decrease in CA averages for the years before the beginning of the pandemic compared to after the beginning of the pandemic, from 79022 ± 4214 to 72264 ± 2008 (p = 0.177). The mean number of primary angioplasty also decreased (1.8%) from 11067 ± 287 to 10868 ± 332 (p = 0.587). Despite the decrease in the number of angioplasties, there was a 13% increase in the mortality rate in this period in CA from 4.3 ± 0 to 4.9 ± 0 (p = 0.013). In cases of primary angioplasty, there was a 4.5% decrease from 6.8 ± 0.1 to 6.5 ± 0.2 (p = 0.200). The mean hospital stay was 8.5% shorter in CA (p = 0.069) and 8% (p = 0.057) in primary angioplasty. Regarding the costs (in brazilian reais) per hospitalization, there was a slight increase both in CA with 1.6% (p = 0.137) and primary angioplasty with 1.3% (p = 0.133). Conclusion: In the context of the COVID-19 pandemic, hospitalizations for CA and primary angioplasty decreased by 8.5% and 1.8%, respectively, comparing the period before the beginning of the COVID-19 pandemic and after. However, we observed a 13% increase in CA mortality rates over the same period while a 4.5% decrease in primary angioplasty rates. The average hospital stay decreased by about 8% for both PTCA and primary angioplasty while costs increased slightly in both cases. 108501 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREO DO CARMO FILHO1, Rogério Gomes Fleury1 (1) Hospital Universitário Gaffrée e Guinle – UNIRIO/EBSERH Introduction: Coronary artery disease represents one of the main causes of death in Brazil and in the world, whose diagnosis and treatment often require speed and appropriate technologies. The difficulty of accessing health systems during the COVID19 pandemic influenced this entire process. Myocardial revascularization surgery (CABG), one of the options for the management of patients with coronary artery disease, was also impacted during this period. The present study aimed to assess the influence of COVID-19 on (CABG) in Brazil. Method: We used the database from the Department of Informatics of the Brazilian Health System (DATASUS) through the website https://datasus.saude.gov.br/. We evaluated the number of hospitalizations, deaths, costs and length of stay of patients undergoing CABG using cardiopulmonary bypass (CPB) in Brazil and their relationship with the beginning of the COVID-19 pandemic in Brazil (2020) based on data from 2018 to 2021. Results: From January 2018 to December 2021, 65827 patients were hospitalized for CABG with CPB in Brazil. There was a decrease in these surgeries from 18987 in 2018 and 19272 in 2019 to 14641 and 12927 in 2020 and 2021 respectively. Surgery averages for the years before the beginning of the pandemic dropped 27.9% compared to after the beginning of the pandemic, from 19130 ± 201 to 13784 ± 1211 (p = 0.025). Despite the drop in the number of surgeries, there was an 8.8% increase in the mortality rate in this period, from 11.9 ± 1.3 to 12.9 ± 0.6 (p = 0.405). The mean hospital stay was slightly shorter (6.5%), with 25.9 ± 0.6 days in 2018–19 and 24.2 ± 0 days in 2020–21 (p = 0.064). Regarding costs (in brazilian reais) per hospitalization, there was a slight increase (1.2%) from 26332 ± 353 to 26655 ± 131. (p = 0.350). Conclusion: In the context of the COVID-19 pandemic, hospitalizations for CABG with CPB decreased by almost 30% when compared to the period before the beginning of the COVID19 pandemic and after. However, we observed an 8% increase in mortality rates in the same period associated with a slight increase in hospitalization costs. The length of hospital stay remained similar. 108521 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARCOS DANILLO PEIXOTO OLIVEIRA1, Lélio Lemos Pinto Neto1, Ednelson Navarro2, Adriano Caixeta1 (1) Universidade Federal de São Paulo, UNIFESP; (2) Hospital Regional do Vale do Praíba The ANGIE (Anatomical sNuffbox for Coronary anGiography and IntervEntions) randomized study compared efficacy and safety between distal transradial (dTRA) and right conventional transradial (cTRA) approaches, observing an association of dTRA with a 2-fold lower risk of occlusion of the proximal radial artery on Doppler ultrasound at 60 days. The dTRA, however, resulted in a 4-fold higher access crossover rate (21.8% vs 5.5%), mainly due to failure to insert the guidewire, as well as longer times to obtain access and perform the procedure. (dTRA vs cTRA: 120 vs 75s and 14 vs 11min, respectively), in addition to a higher radiation area-dose product (~10%). It is therefore questioned whether such limitations associated with the dTRA could impact its incorporation into the primary PCI, which must be performed in a timely manner and by experienced operators. default approach for Coronary Angiography and IntervenTIONs, eventosclinicos.gov.br Identifier: RBR-7nzxkm), among 3,991 patients consecutively submitted to coronary angiography (89%) and/or PCI (60.3%) via dTRA (80.1% by dTRA right), 917 (23%) were due to STEMI. In this subgroup, there were only 17 (2%) access crossovers (failure to insert the sheath wire), 4 of them made possible by contralateral dTRA. Thus, despite the retrospective nature and potential selection biases not documented in this series, it seems to us to be feasible and safe to incorporate dTRA as the route of choice for primary PCI in patients with STEMI. Large randomized trials are still needed and expected to assess the limitations and advantages of this potentially disruptive technique in such a challenging scenario. 108540 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY ISABEL CRISTINA SILVA SOUSA1, Brunnella Alcantara Chagas de Freitas1, Kelvin Oliveira Rocha1, Luciana Moreira Lima1 (1) Universidade Federal de Viçosa – UFV Introduction: Mental stress, represented by disorders such as anxiety and depression, has figured in the current scenario, along with modifiable and non-modifiable risk factors, as a risk factor for cardiovascular diseases (CVD). Objective: To describe the global cardiovascular risk and mental status of students and employees at a public university. Methods: This is an observational, descriptive study, whose sample consisted of students, teachers, and staff, aged 18 or over, of both sexes. Data collection was conducted during the months of August and September 2021, using an online questionnaire contemplating the data necessary to calculate the global cardiovascular risk by the Framingham Score and by three instruments of screening of the metal status: Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI) and Self-Reporting Questionnaire (SQR-20). Results: A total of 247 individuals participated in the study, 171 (69.2%) were female and 76 (30.8%) were male. Regarding age, 120 (48.6%) were under 30 years old, 118 (47.8%) were between 30 and 60 and 9 (3.6%) were over 60 years old. There were 157 (63.3%) students, 49 (19.8) professors and 41 (16.6%) staffs. Female participants had a lower 10-year risk of developing CVD (p < 0.001), higher scores for anxiety (p < 0.001), depression (p < 0.001), and mental distress (p < 0.001) when compared to male participants. In the stratification by age group, dichotomizing by gender, individuals younger than 30 also had a lower risk of developing CVD in 10 years (p < 0.001), higher anxiety scores (p = 0.003), depression (p = 0.007), and mental distress (p = 0.004) when compared with participants aged 30 years or older, regardless of gender. The parameters age, body mass index, total cholesterol, LDL, and triglycerides were significantly higher in professors and staff when compared to students (p < 0.01). However, significantly higher scores of depression, anxiety, and mental distress were observed in students when compared to staff and professors (p < 0.001). In addition, the group of professors had a higher risk of CVD in 10 years than staff and students (p < 0.001). Conclusions: Women have higher scores for mental health disorders compared to men. The younger the age, the higher the scores for mood disorders, anxiety, and mental distress, regardless of sex. The 10-year CVD risk was not correlated with mental health markers in the sample studied. 108584 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MIGUEL MEIRA E CRUZ1, Cristina Salles2, David Gozal4, José Fausto Pinto5, Isabel Rocha3 (1) Centro Europeu do Sono; Sleep Unit, Centro Cardiovascular da Universidade de Lisboa, Lisbon, Portugal; (2) International Center on Clinical Sleep Medicine and Research, Bahiana School of Medicine and Public Health, Salvador, Brazil; (3) Cardiovascular Autonomic Function Lab, Centro Cardiovascular da Universidade de Lisboa, Lisboa, Portugal; (4) University of Missouri, Columbia, United States; (5) Dpt Coração e Vasos, Centro Hospitalar Universitário de Lisboa Norte, CAML, CCUL, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Introduction: While the associations between Sleep Apnea and cardiovascular and metabolic dysfunction are now well established, the interactions between insomnia and cardiometabolic disturbances are less well understood. Notwithstanding, when both sleep disorders occur as in COMISA patients, it is possible that the morbid consequences will be exacerbated, albeit while displaying age and sex dependencies. We therefore examined 3 groups of COMISA patients of markedly different ages to assess whether the presence of COMISA interacts differentially with cardiovascular risk during the lifespan. Methodology: A total of 850 patients were enrolled in this study: Pediatric (n = 50; ages ranging from 9 to 19 years), Adult (n = 685; 31–60 years) and Elderly (n = 115; ≥65 years). Obstructive Sleep Apnea and Insomnia were assessed with validated age-adjusted questionnaires for each group and high-risk COMISA was established when high risk for OSA as well as high risk for Insomnia were present. Results: Among the Pediatric cohort (58% males), 9 (18%) subjects were high-risk for COMISA (age was 13.6 ± 3.3 years; BMI of 23.0 ± 6.9 Kg/m2). Neither hypertension nor CVD were present in this age group; 28% were obese (BMI >95% for age), 11.1% consumed alcohol regularly and 54.4% were sedentary (no physical activity). In the Adult cohort, 25.3% were high-risk for COMISA (49.8 ± 13.8 years; BMI of 31 ± 6.3 Kg/m2). 76.5% were hypertensive or reporting cardiovascular disease, 13.3% were diabetic and 60.3% were sedentary, 8.4% were cigarette smokers, and 46.9% consumed alcohol regularly. Of the Elderly cohort, 22.6% of patients (11 males, 42.3%) were with high risk for COMISA. Their mean age was 70.6 ± 6.6 years and BMI of 28.7 ± 5.1 Kg/m2, with 61.5% being hypertensive or reporting the presence of cardiovascular disease, 21% were diabetic, 65,4% were sedentary and 7.7% were cigarrete smokers. Conclusions: The presence of high risk COMISA is frequent across all ages and may confer increased risk for cardiovascular and metabolic disorders, particularly after reaching adulthood and beyond. It is likely that both the sedentary lifestyle, and alcohol consumption may contribute to this risk, but the presence of COMISA may add an additional risk factor for cardiometabolic complications. Further research is needed to explore how the concurrent presence of Sleep Apnea and Insomnia exacerbates cardiovascular and metabolic risk in larger clinical populations subjected to more precise diagnostic methods. 108589 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION SÉRGIO LUIZ ZIMMERMANN1, Sérgio Luiz Zimmermann1, Leonardo Campanelli Steinhausen2, Bruno Luiz Mueller2 (1) Hospital Santa Isabel, Blumenau; (2) Universidade Regional de Blumenau Background: Coronary angiography is the most accurate method for diagnosing coronary heart disease, but it envolves exposure to radiocontrast agents and radiation, which can increase morbidity and mortality. Thus, it is necessary to study additional markers to identify of coronary artery disease (CAD), such as the diagonal ear lobe crease (DELC). Objectives: To determine DELC’s diagnostic properties in the identification of coronary disease and its association with comorbidities. Methods: A cross-sectional study with 278 patients over 18 years old, who underwent coronary angiography performed by at least 1 of 2 interventional cardiologists. The presence and absence of PLL and associated comorbidities was determined by two trained examiners. Results: PLL was only associated with older age (p < 0.05). PLL had a sensitivity of 76.2%, a specificity of 17.6% in the identification of CAD and an overall accuracy of 58.3%. Conclusion: We concluded that the diagnostic accuracy of PLL was lower when compared to the scientific literature, acting as a low quality marker for the identification of patients with CAD. In our study, there was no association between PLL and CAD. 108592 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DR OSUNKWO DAMARIS AMARACHUKWU1, Dan-Nwafor Chioma3, Chukwuma David Umeokonkwo3, Patrick Nguku2, Emmanuel Nna4 (1) Department of Internal Medicine, National Hospital Abuja; (2) African Field Epidemiology Network Nigeria Country Office; (3) Nigeria Field Epidemiology and Laboratory Training Program, Abuja, Nigeria; (4) Safety Molecular Pathology Lab, The Molecular Pathology Institute, Enugu Background: Hypertension is on the increase in sub-Saharan Africa and it is one of the risk factors for cardiovascular morbidity and mortality. Prisoners are at a greater risk for hypertension because of prevalent tobacco use, unhealthy diet, and incarceration-induced stress. We investigated the prevalence and the risk factors associated with hypertension among Kuje Prison inmates in the Federal Capital Territory (FCT), Nigeria. Materials & Methods: A cross-sectional study was conducted among 180 male prisoners. WHO’s Stepwise approach to surveillance of chronic disease risk factors was used to conduct the study. Data collected at the various steps included: demographic and behavioral risk factors (Step 1); Blood pressure and anthropometric measurements (step 2) and Random blood sugar (Step 3). Body mass index (BMI) was determined. Data were analyzed using Epi-Info version 7. Odds of hypertension among participants with risk factors were calculated, p-value less than 0.05 was considered significant. Results: The mean systolic blood pressure was 131 ± 15 mmHg, diastolic 81 ± 12 mmHg, BMI 24.8 ± 3.6 and RBS 97.1 ± 21.8 mg/dl. Overall, 60 (33.3%) were overweight, 13 (7.2%) obese, 65 (36.1%) were prehypertensive, 57 (31.6%) were hypertensive and 1(0.6%) diabetic. The risk for elevated systolic BP was significantly higher among respondents that has first degree relative with hypertension (OR = 5.6; CI = 1.6–19.9), ≥45years (OR = 7.5: CI = 1.6–35.9), insufficient physical activity (OR = 4.3; CI = 1.5–12.5; P = 0.007) and increased BMI (OR = 9.1; CI = 2.7–31.1). Conclusion: There is a high prevalence of hypertension among prison inmates in FCT, age >45 years, insufficient physical activity, family history of hypertension, and obesity were found to be independent risk factors for hypertension. 108634 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JULIANO AFONSINO JORGE1, Murilo Foppa1, Fábio Tremea Cichelero1, Deniz Martinez1, Marcelo Balbinot Lucca1, Geórgia Pante3, Flávio Danni Fuchs1, Sandra Costa Fuchs1 (1) Graduate Studies Program in Cardiology, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; (2) Division of Cardiology, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil; (3) School of Medicine, UFRGS, Porto Alegre, RS, Brazil Obstructive sleep apnea (OSA) has been associated with left ventricular (LV) diastolic dysfunction. Treating HTN with diuretics could reduce fluid retention and rostral fluid shift in patients with OSA, leading to changes in LV diastolic parameters. We compared the effects of diuretics or amlodipine on echocardiographic LV diastolic parameters. Methods: Patients with HTN and an apnea-hypopnea index between 10 and 40 events/hour were randomized to receive chlorthalidone plus amiloride (25 mg/5 mg) daily or amlodipine (10 mg/daily) for eight weeks. The changes on echocardiographic LV diastolic function parameters were the primary outcome. Results: 62 participants completed the study. Systolic and diastolic BP (24h) decreased without between-treatment differences at the end of study. Nighttime SBP dipping was higher in the diuretic group than the amlodipine group (P = 0.01). The following deltas were found between diuretic and amlodipine groups, respectively: septal E/e‘ ratio, –0.20 ± 0.36 vs. 0.08 ± 0.36 (P = 0.6); lateral E/e‘ ratio –0.14 ± 0.22 vs. 0.66 ± 0.38 (P = 0.07); and average E/e‘ ratio –0.19 ± 0.21 vs. 0.43 ± 0.32 (P = 0.1). Conclusion: Patients with OSA and HTN treated with diuretics or amlodipine showed similar reductions in BP and LV structural measurements. The trend for the association of diuretics to have a greater effect on diastolic parameters and nocturnal SBP dipping suggest it can have beneficial cardiac effects in the long-term management of BP. 108659 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES SANDRO GONÇALVES DE LIMA1, José Arthur Viana de Oliveira Pimentel2, Manuella de Amorim Silva2, Maria Eduarda Cavalcanti Tompson2, Andréa Bezerra de Melo da Silveira Lordsleem1 (1) Hospital das Clínicas – Universidade Federal de Pernambuco – UFPE; (2) Centro Universitário Maurício de Nassau – UNINASSAU Introduction: Cardiovascular diseases are a major cause of death in patients with advanced chronic kidney disease (CKD). Valve calcification (VC), mitral and aortic, associated with significant valve disease is a predictor of cardiovascular mortality, coronary artery disease and arrhythmias in these patients. Objectives: To assess the frequency of heart valve disease, its progression, and associated factors in patients with CKD. Methods: A total of 568 medical records of patients treated between 2007 and 2021 at the cardiology-renal outpatient clinic at a university hospital were analyzed, of which 347 were included due to the presence of CKD and heart valve disease. The explanatory variables analyzed were: age, gender, VC, glomerular filtration rate (GFR), dyslipidemia, coronary artery disease, systemic arterial hypertension, diabetes mellitus, C-reactive protein, secondary hyperparathyroidism and laboratory data on mineral and bone metabolism. To assess the association between categorical variables, the Pearson’s chi-square test or Fisher’s exact test was used. Results: 50.7% were female. The most frequent risk factor was systemic arterial hypertension (83.9%) and 25.1% of patients were diabetic. Mitral valve disease was observed in 81.6%, followed by aortic valve disease (66%). Regurgitant lesions were the most frequent: mitral (75%) and aortic (40.2%). Around half (48.1%) of those with heart valve disease were in the 30–39 age group, confirming the precocity of heart valve disease in CKD. A progression of heart valve disease was observed in 122 patients (35.2%). Valve calcification was significantly associated only with the progression of aortic valve disease (p = 0.01). The variables significantly associated with mitral valve disease were secondary hyperparathyroidism (p = 0.03), GFR (p = 0.02) and total cholesterol (p = 0.02). None of the variables analyzed were significantly associated with aortic and pulmonary valve disease. Secondary hyperparathyroidism (p = 0.019) was also significantly associated with tricuspid valve disease, as well as altered levels of triglycerides (p = 0.017). Conclusion: The frequency of heart valve disease, particularly mitral and aortic, is high in patients with CKD. The factors significantly associated with heart valve disease were: secondary hyperparathyroidism, GFR, total cholesterol and altered triglyceride levels. The progression of valve disease was observed in 35% of patients and was significantly influenced by VC. 108733 Modality: E-Poster Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE MIGUEL MEIRA E CRUZ1, Joana Belo2, Mariana Picado3, Nuno Canha5, Carla Viegas2, Susana Marta Almeida4 (1) Centro Europeu do Sono; Sleep Unit, Centro Cardiovascular da Universidade de Lisboa, Lisbon, Portugal; (2) H&TRC- Health & Technology Research Center, ESTeSL- Escola Superior de Tecnologia da Saúde, Instituto Politecnico de, Lisboa, Portugal; (3) Instituto Português de Oncologia de Lisboa, Lisbon, Portugal; (4) Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; (5) Centre for Environmental and Marine Studies, Department of Environment and Planning, University of Aveiro, Aveiro, Portugal; (6) NOVA National School of Public Health, Public Health Research Centre, Universidade NOVA de, Lisboa, Portugal; (7) Comprehensive Health Research Center (CHRC), Portugal Introduction: Inadequate sleep and poor air quality are both associated to an increased cardiovascular risk. Sleep quality and structure are also vulnerable to environmental influences. Typically, sleeping environments have low ventilation rates, leading to pollutants accumulation. Though during nighttime and early morning there is a circadian propensity to cardiorespiratory events, environmental factors which may aggravate this risk should be taken into account. The aim of this study is to test the associations between ais pollutants, specifically CO and CO2 and heart rate dynamics as an indicator of cardiovascular autonomic function. Methodology: Ten men followed specific inclusion criteria: age between 25 and 40 years old, healthy individuals, non-smoking, without children below five years and without sleeping, cardiac and respiratory problems and whose households are within Lisbon area, were recruited. An unattended polysomnography (PSG) was performed during 2 weeknights in a row. The second night PSG’s results were used in order to minimize the first night effect. For the propose of this study the data related to heart rate (HR) were collected, namely HR acceleration index (HR Acc index), mean of HR (HRmean) and minimum of HR (HRmin). IAQ monitoring was based on a comprehensive multi-pollutant assessment where chemical, in particular carbon dioxide (CO) and carbon monoxide (CO), were assessed through real time instruments. Non-parametric statistics were applied, namely Spearman correlations, to analyze potential associations between sleep and environmental parameters. The level of significance considered was α = 0.05. Results: The mean age was 33.9 ± 5.20 years. HR and IAQ parameters showed a moderately positive correlation between CO exposure and HR Acc index (rs = .635) and HRmin (rs = .0,667) and a highly positive correlated between CO exposure and HRmean (rs = .0,71); also a moderately and positive correlation between CO2 exposure and HR Acc index (rs = .668) awas observed as well as a highly positive correlation between CO2 exposure and HRmin (rs = 713) HRmean (rs = .0,794). Conclusions: Results from this preliminar study suggest that high levels of CO and CO2 may increase the HR Acc index, HRmin and HRmean, raising awareness on the possible impact of indoor air quality on cardiovascular autonomic modulation. Further studies are needed to confirm such findings and to establish their clinical relevance within cardiorespiratory health. 108720 Modality: E-Poster Researcher – Non-case Report Category: ANTICOAGULATION CHRISTIAN HENGSTENBERG1, Martin Unverdorben2, Helge Möllmann3, Nicolas M Van Mieghem4, Holger Thiele5, Peter Nordbeck6, Tienush Rassaf7, Raul Moreno8, Roxana Mehran9, Irene Lang1, Roland Veltkamp10, George D Dangas9 (1) Department of Internal Medicine II, Division of Cardiology, Vienna General Hospital, Medical University, Vienna, Austria; (2) Daiichi Sankyo, Inc., Basking Ridge, NJ, USA; (3) Department of Internal Medicine, St. Johannes Hospital, Dortmund, Germany; (4) Department of Cardiology, Erasmus University Medical Center, Thoraxcenter, Rotterdam, the Netherlands; (5) Department of Internal Medicine–Cardiology, Heart Center Leipzig at University of Leipzig, Leipzig; (6) Department of Internal Medicine I, University Hospital Würzburg, Würzburg & Klinikum Links der Weser gGmbH, Bremen, Germany; (7) Clinic for Cardiology and Vascular Medicine, Essen University Hospital, Essen, Germany; (8) Department of Cardiology, Tübingen University Hospital, Tübingen, Germany; (9) Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai Hospital, New York, NY, USA; (10) Division of Brain Sciences, Imperial College London, London, UK; Department of Neurology, Alfried Krupp Krankenhaus, Essen, Germany Introduction: In ENVISAGE-TAVI AF (NCT02943785) edoxaban was noninferior to vitamin K antagonists (VKAs) for the composite endpoint of net adverse clinical events in patients with atrial fibrillation after transcatheter aortic valve replacement (TAVR). Objective: To evaluate the association between baseline patient characteristics and ischemic stroke (IS) incidence. Methods: This on-treatment analysis of ENVISAGE-TAVI AF included patients that received ≥1 dose of the study drug over the period treatment and ≤3 days after interruption or discontinuation. Baseline demographic and clinical characteristics were stratified by IS incidence. Numerical variables were compared using one-way analysis of variance; categorical variables were compared using Fisher’s exact test. Stepwise logistic regression with 30 independent predictors determined patient characteristics associated with first IS event. Results: Of 1377 patients included in the on-treatment analysis, 41 (3.0%) experienced IS during the on-treatment period (edoxaban, n = 19; VKA, n = 22). Most IS events occurred within 6 months of TAVR for both the edoxaban (57.9%) and VKA (68.2%) arms. Differences in baseline demographic and clinical characteristics are shown for patients who did or did not experience IS while receiving treatment (Table). Significantly more patients who experienced IS had a history of systemic embolic events (SEE; P = 0.015). Only a history of SEE was independently associated with IS (P = 0.006). Body mass index (P = 0.052) and history of myocardial infarction (P = 0.074) were numerically, but not statistically, associated with IS. Conclusions: In this on-treatment analysis of the ENVISAGE-TAVI AF trial, few patients experienced IS. Patients with history of SEE may be at increased risk of IS following TAVR. 109072 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CRISTIANE DA CRUZ LAMAS1, Mariah Rodrigues Paulino1, Léo Rodrigo Abrahão dos Santos2, Ingrid Paiva Duarte2, Marcelo Goulart Correia1, José Alfredo de Sousa Moreira1, Letícia Roberto Sabioni1, Fabiana Bergamin Mucillo1, Rafael Quaresma Garrido1, Bruno Zappa1, Stephan Lachtermacher Pacheco1, Andrea Rocha de Lorenzo1 (1) Instituto Nacional de Cardiologia; (2) Universidade do Grande Rio Introduction: Reported nosocomial acquisition of COVID-19 has varied between 5 to 41%. Our aim was to describe the demographic, clinical and laboratory features and outcomes of patients with cardiac disease hospitalized with COVID-19 in a reference cardiology institution in Brazil, comparing the patients with hospital-acquired COVID-19 (HACOVID19) with those who had non-hospital acquired (NHA) COVID-19. Methods: This is an observational retrospective study of consecutive adult patients admitted, between March and September of 2020 with a diagnosis of SARS-CoV-2 infection confirmed by RT-PCR. Data was collected as per the International Severe Acute Respiratory and Emerging Infection Consortium and complemented with a cardiovascular questionnaire. HA COVID-19 was considered when the patient had a negative nasopharyngeal swab on admission and a positive one 14 or more days later. Statistical analysis was performed using the Jamovi 1.6 and R 4.0.1. Results: One hundred twenty-one patients with a confirmed diagnosis of COVID-19 were included. There were 20 patients (16.5%) with HA-COVID19 and 101 (83.5%) with NHA-COVID-19. Patients who developed COVID-19 in hospital had as reasons for admission: decompensated heart failure (35%), acute coronary syndrome (20%), cardiac surgery (CABG, valve replacement or aortic surgery) in 25%, complete AV block, pacemaker dysfunction, stroke and hematologic disease (5% each). The median(IQR) length of stay of HACOVID19 was 29 days (18.5–60.2) vs 16 (7–31)days. Median age was 64 years (61.8–69.3) for HA-COVID-19, and 63 (52–72) for NHACOVID19. There was no difference between patients with HACOVID-19 and those with NHACOVID-19 regarding the presence of heart disease, COPD, obesity, complicated diabetes mellitus, heart valve disease, coronary artery disease, and systemic arterial hypertension. However the 2 groups were significantly different regarding laboratory features (serum creatinine, D-dimer, ferritin, AST). HACOVID patients had more dyslipidemia (15/20 (75%) vs 49/101 (48.5%), P = 0.030] and chronic renal failure [7/20 (35%) vs 15/101 (15%), p = 0.033]. Most importantly, mortality was significantly higher in HACOVID-19[10/20 (50%) vs 19/101 (18.8%), p = 0.003]. Conclusions: Cardiac patients who acquired COVID-19 in hospital had longer hospital stay and higher mortality, which highlights the impact of the pandemic on the outcomes of cardiac patients admitted to hospital for cardiac, non-COVID related reasons. 108800 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES CHARLE ANDRE VILJOEN1, Mahmoud Al-Naili2, Jean Jacques Noubiap3, Alice Jackson4, Karice Hyun5, Andrea Neves6, Clovis Nkoke7, Charles Mondo8, Juliet Nabbaale9, Julian Hoevelmann10 (1) Cape Heart Institute, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; (2) Division of Cardiology, Groote Schuur Hospital, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; (3) Centre for Heart Rhythm Disorders, The University of Adelaide, Adelaide, Australia; (4) British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, G12 8TA, Scotland; (5) School of Health Sciences, Faculty of Medicine and Health, University of Sydney, Westmead 2145, Australia; (6) Gynecology and Obstetrics, Hospital Geral José Macamo, Av. OUA, 1100, Maputo, Mozambique; (7) Department of Internal Medicine, Buea Regional Hospital, Buea, Cameroon; (8) St. Francis Hospital Nsambya, P. O. Box 1799, Nsambya Road, Kampala, Uganda; (9) Uganda Heart Institute, Plot No. 1, Upper Mulago Hill, P. O. Box 37392, Kampala, Uganda; (10) Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Saarland University Hospital, Homburg (Saar), Deutschland Introduction: Cardiac disease is an important cause of maternal mortality worldwide. However, diagnosing heart failure (HF) during pregnancy remains challenging. Patients with HF present with symptoms that are often attributed to the physiological changes of pregnancy. Although the measurement of natriuretic peptides has been recommended as a cost-effective screening test for HF, its value in predicting underlying structural heart disease on echocardiography during pregnancy is unclear. We aimed to evaluate the accuracy of point-of-care (POC) NT-proBNP to predict echocardiographic evidence of structural heart disease in pregnant women. Methods All consecutive consenting pregnant women with symptoms of HF, who underwent echocardiography at a tertiary hospital in Cape Town, South Africa, between March 2021 and March 2022 were compared with asymptomatic pregnant women. Demographic and obstetric data were collected, as well as clinical and echocardiographic parameters. POC NT-proBNP was measured; a receiver operating characteristic (ROC) curve was used to determine the level of NT-proBNP that would have the best predictive value for detecting structural heart disease on echocardiography. Results: We included 121 women with a median age of 31.3 years (IQR 24.9–36.4), gravidity of 3 (2–4), mostly in their third trimester of pregnancy (76.9%). Symptomatic women (66.9%) presented mainly with dyspnoea (90.8%) and peripheral oedema (50%). Overall, the median POC NT-proBNP was 108 pg/ml (60–470) but was not statistically different between symptomatic and asymptomatic participants. However, NT-proBNP levels were significantly elevated in those with left ventricular (LV) dilatation (395 [86–1668] vs 80.5 [60–303], p = 0.001), left atrial enlargement (417 [98–1211] vs 78 [59–167], p < 0.001), LV systolic dysfunction (539 [94–2582] vs 80 [60–323], p < 0.001), diastolic dysfunction (300 [77–1450] vs 80 [60–322], p = 0.038), mitral regurgitation (247 [60–794] vs 84 [60–323], p = 0.027) and pericardial effusion (448 [84–1487] vs 80 [60–303], p < 0.001). An NT-proBNP of <250 pg/ml had the highest negative predictive value (79.3%) to rule out structural heart disease (ROC area 0.66). Conclusion: In this cohort of pregnant women with symptoms HF, POC NT-proBNP identified those with structural heart disease with acceptable discrimination. POC NT-proBNP testing might be particularly useful as a screening test in settings where pregnant women do not readily have access to echocardiography. 108804 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION DANIELE BARRIONUEVO KALLAS1, Lia Kanae Okita Buschinelli1, Eneas Antonio Rocco1, Amanda Barbuio Teixeira1, Luana Talita Diniz Ferreira1, Raquel Yuri Mori1, Bianca Sprovieri Moraes1, Gabriela Macoppi Carreiro1, Gabriela Zanussi Barreto1, Camila dos Santos Arcas1, Felipe Lopes Malafaia1, Patricia Canteruccio Pontes Vianna1 (1) Hospital Samaritano Paulista Introduction: Health Coaching (HC) is an approach for changes in health behaviors and self-care. The British Association for Cardiovascular Prevention lists health behavior change, education, and management of risk factors related to physical activity, diet, weight control, alcohol and tobacco among the 6 central components of cardiac rehabilitation (CR) programs. Thus, interventions based on changes in health behaviors are justified as part of medical care. Objective: To evaluate the impact of a HC Program in lifestyle of patients under CR immediately after and 6 months after the intervention. Methods: Patients in CR of a specialized institution were invited to voluntarily enroll in a HC Program in the dimensions of diet, physical activity, stress management and sleep. The intervention was composed of 6 weekly coaching sessions, with trained health professionals, lasting 1h30, made 100% remote by the telemedicine of the institution, where patients were gathered in groups of up to 4 people, plus the Health Coach. Fantastic Lifestyle Questionnaire (Fantastic) was applied before, immediately after and 6 months after the intervention. Sessions addressed wellness, ambivalence, behavioral change process, thoughts and beliefs, relapse prevention and habit support plan, and patients were invited to create health goals with the Smart framework to a day-to-day practice. Between sessions they received educational materials about the four dimensions. Results: The intervention took place between August 2020 and March 2022. 31 patients completed the program. Results of the Fantastic are at Table 1. Conclusions: Interventions for health behavior changes seem to be promising for patients in CR and seem to last after 6 months. 108810 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RENATA JUNQUEIRA MOLL BERNARDES1, Sérgio Costa Fortier3, Andréa Silvestre de Sousa4, Renato D. Lopes6, André Feldman8, Guilherme D.A.S. Arruda9, Denílson C. de Albuquerque1, Ariane V. S. Macedo7, Olga Ferreira de Souza1, Fernando A. Bozza1, Ronir Raggio Luiz5, Emiliano Medei2 (1) D’Or Institute for Research and Education, Rio de Janeiro, Brazil; (2) National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, Brazil; (3) Patological Anatomy Laboratory, Rede D’Or São Luiz, São Paulo, Brazil; (4) Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; (5) Public Health Studies Institute – IESC, Federal University of Rio de Janeiro, Brazil; (6) Brazilian Clinical Research Institute, São Paulo, Brazil; (7) Hospital São Luiz Jabaquara, São Paulo, Brazil; (8) Hospital São Luiz Anália Franco, São Paulo, Brazil; (9) Hospital São Luiz São Caetano, São Caetano do Sul, Brazil; (10) Hospital Sino Brasileiro, Osasco, Brazil; (11) Hospital Villa Lobos, São Paulo, Brazil; (12) Hospital São Luiz Morumbi, São Paulo, Brazil Background: Cardiovascular comorbidities and immune response dysregulation are associated with 2019 coronavirus disease (COVID-19) severity. Reduced populations of lymphocytes, including T, B, and natural killer cells, and increased neutrophil counts have been detected in patients with COVID-19. We aimed to explore the key immune cell profile and understand its association with disease progression in patients with hypertension hospitalized due to COVID-19. Methods: Immune cell populations in cryopreserved peripheral blood mononuclear cell samples were obtained from 156 hypertensive patients who were included in the BRACE CORONA randomized trial. The primary outcome was progression to severe disease, according to a modified World Health Organization Ordinal Scale for Clinical Improvement, during hospitalization. The probability of progression to severe disease was estimated using a logistic regression model that included clinical variables and immune cell subsets associated with the primary outcome. Results: During hospitalization, 11 (7.1%) patients showed progression to more severe COVID-19; 3 of these patients died. Obesity, diabetes, oxygen saturation, lung involvement on computed tomography (CT) examination, the C-reactive protein concentration, total lymphocyte count, proportions of CD4+ and CD8+ T cells, CD4/CD8 ratio, CD8+ human leukocyte antigen DR isotope (HLA-DR) median fluorescent intensity (MFI), and CD8+ natural killer group 2 member A (NKG2A) MFI on admission were associated with progression to severe COVID-19. According to our predictive model, the risk of progression to severe disease reached 86.2% in the presence of three clinical variables (obesity, diabetes, reduced oxygen saturation, or >50% CT lung involvement) combined with increased CD8+ NKG2A MFI. Conclusions: This study demonstrated that increased CD8+ NKG2A MFI at hospital admission, in combination with some clinical variables, is associated with a high risk of COVID-19 progression in hypertensive patients. These findings reinforce the hypothesis of functional exhaustion of T cells with the increased expression of NKG2A in patients with severe COVID-19, elucidating how severe acute respiratory syndrome coronavirus 2 infection may break down the innate antiviral immune response at an early stage of the disease, with future potential therapeutic implications. 109063 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MIGUEL MEIRA E CRUZ1, Luis Vicente Franco de Oliveira3, Monica Gomes2, Lilian Giannasi2 (1) Centro Europeu do Sono; Sleep Unit, Centro Cardiovascular da Universidade de Lisboa, Lisboa, Portugal; (2) Universidade Estadual de São Paulo – UNESP, São José dos Campos, São Paulo, Brazil; (3) Centro Universitário de Anapolis-UniEvangélica, São Paulo, Brazil Introduction: COMISA refers to the frequent co-occurrence of Insomnia and Sleep Apnea and has been linked to increased cardiovascular risk where autonomic dysfunction play an important role. Therefore, improving cardiovascular autonomic function (CAF) is a clinically relevant target to be achieved in these patients. While mandibular advancement devices (MAD) are recommended as an effective alternative for obstructive sleep apnea control, the benefits of such therapeutic tool in COMISA remains elusive. In this pilot study we aimed to test the null hypothesis that MAD will have no effect on heart rate variability (HRV) as a surrogate of CAF in COMISA adult patients. Methodology: Patients with COMISA, as defined by OSA + Sleep Latency >30min, were treated with PMPositioner oral appliance (OAm) for their sleep disordered breathing and were evaluated after 3 months of OAm therapy, been retrospectively compared regarding major clinical and polysomnographic outcomes. Sleep variables were evaluated by PSG and Heart rate variability (HRV) was accessed and analized by rythmography. Results: 12 COMISA patients (6 males), mean age = 49.7, mean BMI = 25.9, mean neck circumference = 38.9 were enrolled. Insomnia and OSA were confirmed by both clinical criteria (validated questionnaires) and PSG parameters. All patients were treated with a PMPositioner oral appliance for sleep disorder breathing. The AHI was reduced from 22.7 ± 12.7 to 4.0 ± 3.5(p < 0.0002), IDO was reduced from 18.8 ± 14.0 to 3,5 ± 2.0 (p < 0.02), sleep latency reduced from 63.1 ± 49.4 to 21.8 ± 21.4 (p < 0.2), reaching a normal level. The time-domain and frequency–domain parameters were significant for RR interval and both Fast Fourrier Transform and Wavelet spectral method, respectively. Conclusions: The rejection of the null hypothesis allow us to confirm the impact of MAD in HRV of these patients. This is the first study showing a positive and clinically relevant effect of MAD therapy on both respiratory and insomnia related therapeutic outcomes in COMISA together with an improvement of CAF and therefore on cardiovascular risk as a major concern among these patients. 109361 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANNE GERYMAIA OLIVEIRA DE MELO SILVA1, ANNE GERYMAIA OLIVEIRA DE MELO SILVA1, NEILA ANDERS AIDAR2 (1) SECRETARIA DE SAÚDE DO DISTRITO FEDERAL; (2) MEDCOR Introduction: The interest in understanding what accelerates the degradation of cardiovascular function in some individuals, is matched by the need to understand what protects other subjects. Pulse-wave velocity (PWV) is a measurement of arterial stiffness that is an independent predictor of cardiovascular risk. It can be measured simply and noninvasively by modern oscillometric methods. Objectives: To evaluate vascular aging variables and arterial stiffness in adult patients with trisomy 21, treated at the Distrito Federal Down Syndrome Reference Center – Crisdown. Methods: Transversal study of PWV in 28 adults with Down syndrome. The central hemodynamic parameters of blood pressure, pulse wave velocity (PWV), and heart rate-adjusted increase index (Alx@75) were estimated using the oscillometric device Arteris-AOP (Cardio Sistemas Comercial e Industrial Ltda, São Paulo, Brazil), ANVISA 10361059013. The device performs the analysis through a cuff installed on the patients‘ left arm. The software was used for data analysis comparing with a database with the Brazilian population matched by sex, age, weight, and height. Results: Twenty-eight patients with Down Syndrome ranging from 18 to 64 years old, being 18 female and 10 male, were evaluated. For data analysis, they were divided into groups of 18 to 24 years (N = 13), 25 to 34 years (N = 6), and over 35 years (N = 9). There was no statistical difference between the groups regarding mean BMI (30.7 + 4.3 Kg/m2), mean central systolic pressure – CSP (85.6 + 10.5 mmHg), mean central diastolic pressure – CDP (62.3 + 10.1 mmHg), mean cardiac output (3.4 + 0.5 l/min) and mean total cholesterol serum level (171.7 + 35.3 mg/dl). The mean PWV was 5.2 m/s (SD 1.4) and the mean augmentation index was 17.5% (SD 8.5). There was a statistical difference between the groups in relation to pulse wave velocity and augmentation index, which were significantly higher in the group aged over 35 years. Compared with a database of the typical Brazilian population, the level of PWV in adults with Down Syndrome was within the average, even in the group aged over 35 years. Conclusion: The findings suggest that adults with Down syndrome have a pattern of central arterial hypotension. The lack of relationship of PWV, an independent predictor of adult cardiovascular events, with its traditional determinants, including arterial pressure suggests Down syndrome–specific phenomena may alter such relationships in this population. 109071 Modality: E-Poster Researcher – Non-case Report Category: NURSING MIRIAN FIORESI1, Juliana Mitre da Silva1, Cândida Caniçali Primo1, Maria Edla de Oliveira Bringuente1, Bruno Henrique Fiorin1, Karolini Zuqui Nunes1, Lorena Barros Furieri1, Walckiria Garcia Romero Sipolatti1 (1) Programa de Pós-Graduação em Enfermagem, Universidade Federal do Espírito Santo Introduction: The concept of decreased cardiac output is found mainly in the nursing and medical fields. As a nursing phenomenon, it is present in the NANDA-I and ICNP classification systems. Nurses are the professionals who stay the longest in direct care of people, so they commonly identify clinical changes and signs of deterioration of patients‘ health at all levels of health care. Therefore, a data collection instrument can optimize risk prediction in adults and facilitate clinical thinking and nursing care for patients with this condition. Objective: To develop an instrument to assist nursing in the physical examinations of a suspected low cardiac output patients. Methods: This is a methodological study developed in three stages: literature scope review, pilot instrument construction and validation by judges. The instrument was constructed following the principles of Pasquali’s elaboration of instrument. Content and face validation was based on content validity index (CVI) and was considered as inclusion criteria CVI greater than or equal to 0.8. The judging population consisted of nurses with a minimum degree of specialist and minimum experience of two years in the field of nursing cardiology. The items that made up the instrument were evaluated as: 1- Adequate, 2- Needs adequacy and 3- Inadequate, within the criteria of clarity, relevance or representativeness and comprehensiveness. Results: After the validation process, the instrument was composed of the following nine items, called clinical indicators: consciousness status, respiratory status, activity tolerance, fluid volume, gastric status, sensory alteration of cardiac origin, heart rate and rhythm, blood pressure and tissue perfusion. Each item can be scored from 1 to 3 in ascending order of severity and by summing the total points the patient risk can be stratified into: minimum risk (9 to 12 points), intermediate risk (13 to 18 points) and high risk (19 to 27 points). If the patient is tired with minor exertion (eating) and/or presents with chest pain that does not cease at rest and therapy, he/she should be classified as high risk even if the patient has a score below 19 points. Conclusion: An instrument to assist nursing in the assessment was developed and validated to classify the risk of decreased cardiac output in adults that can support nursing care and favor the clinical reasoning of nurses in the nursing process to patients who present signs and symptoms of this phenomenon. 108869 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING POLYANA EVANGELISTA LIMA1, André Maurício Souza Fernandes2, Marta Silva Menezes1, Edmundo José NassriCamara2, Rafael de Castro da Silva2, Heverton Garcia de Oliveira3, Sarah Rodrigues de Assunção Vaz3, Alane Mota dos Santos3, Matheus Pereira Barreira3 (1) Escola Bahiana de Medicina e Saúde Pública; (2) Universidade Federal da Bahia; (3) Universidade Federal do Vale do São Francisco Introduction: Early identification of myocardial damage appears to be important in the approach to patients with Chagas disease (CD). Echocardiography with strain obtained by speckle tracking (STE) and the evaluation of myocardial fibrosis (MF) through cardiac magnetic resonance imaging (CMRI) may be promising diagnostic methods in this regard. Objective: Evaluate myocardial involvement, specifically in the chronic mild cardiac form of CD using strain by STE and MF by CMRI, as well as their correlations. Methods: A cross-sectional study analyzed patients with the chronic mild cardiac form of CD (preserved ejection fraction) who underwent STE strain echocardiography and CMRI. Results: Twenty-one participants were included (female: 62%, age: 54 +/– 5 years). The prevalence of MF by late myocardial enhancement (LME) was 50%. Global longitudinal strain (GLS) was decreased in 17 patients (81%) with a median of 14.1% (interquartile range 12.1–16.3). The average T1 mapping values were high in patients with CD (993 +/– 163 ms). T1 mapping was significantly correlated with GLS (r = 0.634; p = 0.015). Furthermore, the mechanical dispersion index obtained by strain was increased (>55 ms) by 84%, with the largest area under the receiver operating characteristic curve (AUC 0.696; 95% confidence interval 0.412–0.981) for fibrosis discrimination by LME. Conclusion: The present study suggests that myocardial strain and T1 mapping behave as early markers of myocardial damage in mild chronic CD. The mechanical dispersion index was elevated, and it was the parameter that most correlated with myocardial fibrosis by LME. 108876 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANNE GERYMAIA OLIVEIRA DE MELO SILVA1, NEILA ANDERS AIDAR2, MARIA CAROLINA VIANA VALE1 (1) SECRETARIA DE SAÚDE DO DISTRITO FEDERAL; (2) MEDCOR Introduction: Down syndrome continues to be the most common chromosomal condition, with rising prevalence and increased survival. With increased longevity, such individuals are susceptible to differing cardiovascular disorders. Objectives: Describe cardiovascular disorders in adults with Down Syndrome. Methods: Descritive and observational study of 129 adults with cytogenetically and/or clinically proven Down syndrome. For each individual, carers were interviewed to elicit a past history of any medical condition and to elicit any symptoms suggestive of an ongoing medical illness. A standard physical examination was undertaken. Results: 129 adults with Down syndrome participated in this study, sex distribution being 65 (50.3%) males and 64 females. The mean age of the sample population was 29 years; range 18–64 years. The individuals were living in their family homes. The commonest cardiac disorders were cardiac congenital malformation in 43 (33.3%), dyslipidemia in 37 (28.7%) subjects, cardiac arrhythmia in 12 (9.3%) and dysautonomic disorders in 10 (7.7%). The commonest cardiac malformations were valvular diseases in 21 (16.3%) atrioventricular septal defect in 13 (10.0%), interatrial septal defect in 11 (8.5%), interventricular septal defect in 9 (7.0%), patent ductus arteriosus in 9 (7.0%). The mean rate was 74 beats per minute (SD 14), the mean systolic arterial pressure was 118.7 mmHg (SD 21.6), the mean diastolic arterial pressure was 77.2 mmHg (SD 16.2) and the mean oxygen saturation level was 95% (SD 4). The mean body mass index was 30.8 kg/m2 (SD 15.4), overweight and obesity was observed in 67.6% of individuals. The mean cholesterol was 175.1 mg/dl (SD 46.6). Other important clinical disorders was hypothyroidism in 52 (40.3%) individuals, epilepsy in 17 (13.1%) and diabetes mellitus in 6 (4.6%). Screening of obstructive sleep apnea (OSA) was positive in 20 cases (15.5%), being 13 (10.0%) considered severe OSA and 7 (5.4%) moderate OSA. Carotid ultrasound was performed on 26 individuals and measurement of intima-media thickness was normal in all cases. Conclusion: Overweight/obesity, dyslipidemia and cardiac defects were frequently. Considering that Down syndrome presents with chronic hypotension, it is reasonable to propose that the prolonged reduction of arterial distending pressure may contribute to functional preservation of the arteries in patients with Down syndrome. 108881 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING POLYANA EVANGELISTA LIMA1, Rafael de Castro da Silva -Silva1, André Maurício Souza Fernandes2, Marta Silva Menezes1, Edmundo José NassriCamara2, Matheus Pereira Barreira3, Alane Mota dos Santos3, Sarah Rodrigues de Assunção Vaz3, Heverton Garcia de Oliveira3 (1) Escola Bahiana de Medicina e Saúde Pública; (2) Universidade Federal da Bahia; (3) Universidade Federal do Vale do São Francisco Introduction: Chronic Chagas cardiomyopathy presents as chronic myocardial inflammation that causes progressive tissue destruction and extensive fibrosis. Cardiac magnetic resonance (CMR) is the gold standard noninvasive test to assess myocardial fibrosis (MF) using the technique of late myocardial enhancement (LME). CMR has recently been used to perform new evaluations with parametric mapping (T1 and T2). It is possible that the use of the T1 mapping technique generates complementary and even prognostic information in the evolution of patients with CD. Objective: Describe the T1 mapping values obtained with CMR in the evaluation of patients with CD and their correlation with the LME technique. Methods: This cross-sectional study analyzed patients with the chronic mild cardiac form of CD who underwent CMR. Eligible patients underwent CMR to analyze MF by 2 techniques: the LME technique, which was categorized as present/absent for MF, and T1 mapping time, expressed as average +/– standard deviation. Discussion: Sixteen patients composed the study population; 81% female, and the mean age was 54.3 +/– 5.3 years. Mean left ventricular ejection fraction was 65.3% +/– 5.4%. The prevalence of MF in the sample using the LME technique by CMR was 50%. The T1 mapping values were high in patients with CD without ventricular dysfunction, with mean of 993 +/– 163 ms. The medians and interquartile range (IQR) of T1 mapping were: 1033 (IQR 998–1081) in group I and 1010 (IQR 1002–1047) in group II. In the MF group, the times were even higher, as found in other dilated cardiomyopathies. Conclusion: These data reinforce the ability of T1 mapping to identify MF more completely and earlier when compared with LME and suggest that this technique is a marker of early cardiac involvement by CD. 108908 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH EDUARDO PITTHAN1, Vânia Hirakata2, Natanael Alves de Lima1, Thiago Emanuel Rodrigues Novaes1, Pamela Sandri1 (1) Universidade Federal da Fronteira Sul Passo Fundo; (2) Instituto de Cardiologia do Rio Grande do Sul Introduction: In Brazil, the clinical-epidemiological scenario of Heart Failure (HF) has shown significant changes in recent decades. The main factors that have affected the change in profile were the exponential increase in the elderly population and the impact of new therapies. Objective: To evaluate the trend of variation in gross mortality rates in HF in Brazil from 1996 to 2019, comparing with the variation in the growth of the elderly population stratified by age groups. Methods: Refers to an ecological, descriptive and quantitative study that used data available on DATASUS on March 8th 2022 regarding to mortality in the elderly due to HF, in Brazil, from 1996 to 2019, divided into three age groups: 60 to 69 years, 70 to 79 years and 80 years or more. The gross mortality rates in these variables were calculated per 100,000 inhabitants, based on the population variation in Brazil in the years chosen for the study, made available by IBGE in a consultation that happened on March 8th 2022. Results: The analysis of mortality from HF over 80 years, in 1996, shows a rate of 770.2/105 inhabitants and showed a significant drop in 2019, being 324.4/105 inhabitants. Making a drop of 57.9% per 105 inhabitants. On the other hand, in the same period, the population of this age group has increased 172.5%. The analysis of mortality from HF from 60 to 69 years, in 1996, showed a rate of 87.6/105 inhabitants and showed a significant drop in 2019, being 26.6/105 inhabitants. Making a drop of 69.6% per 105 inhabitants. On the other hand, in the same period, the population of this age group has increased 120.7%. In the 70–79 age group, in 1996, it had a mortality rate of 248.1/105 inhabitants and showed a significant drop in 2019, being 79.6/105 inhabitants. Making a drop of 67.9% per 105 inhabitants. On the other hand, in the same period, the population of this age group has increased 121.3%. Conclusion: The results of this study demonstrate downward trends in the mortality rate from HF in the elderly in Brazil in recent decades. In contrast, there has been an exponential increase in the elderly population in all ages studied. 108942 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY LUIZ FERNANDO KUBRUSLY1, Douglas Mesadri Gewehr2, Alexandre Gelás Haddad1, Victor Daniel Falkenbach Tenius1, Fernanda Prehs Izar1, Allan Fernando Giovanini1, Fernando Bermudez Kubrusly3 (1) Mackenzie Evangelical School of Paraná, Curitiba, Paraná, Brazil; (2) Denton Cooley Institute of Research, Science and Technology, Curitiba, Paraná, Brazil; (3) Curitiba Heart Institute, Curitiba, Paraná, Brazil Introduction: The bovine pericardium is a biological tissue widely used as a biomaterial for tissue engineering applications. Glutaraldehyde and formaldehyde are frequently used in these reticulation processes to improve the material’s resistance and preservation. Objective: The objective was to evaluate the impact of long-term storage in 4% formaldehyde on the quantitative expression of immunophenotypic markers of glutaraldehyde-treated bovine pericardium. Methods: Bovine pericardium (BP) patches used were produced and supplied by Braile Biomédica®. We performed a histological and immunohistochemical analysis comparing two patches of BP, one manufactured in 2009, thickening 0.35 mm/measuring 35 cm2, and another manufactured in 2020, thickening 0.36 mm/measuring 99 cm2. BP were fixed in a 10% formalin solution for 24h, embedded in paraffin blocks, trimmed and mounted on histological glasses. Sections were stained with H&E, Weigert and picrosirius red and immunolabeled with vimentin, laminin 5, collagen I and collagen IV using a standardized protocol. Images were captured using light and polarized microscopy and the area of antibody signal was quantified using Image J Software. Results: Histological analysis of the patches showed no autolysis or significant changes. In immunohistochemical analysis, collagen I and IV was diffused throughout the connective tissue of the patches. In 2020 specimen, collagen I occupied an area of 21.36% and collagen IV an area of 24.67%, while in the 2009 specimen, only 15.87% (collagen I) and 12.02% (collagen IV). Laminin was not reacted between the specimens. Immunopositivity for vimentin differed markedly between patches occupying a 54% area in the 2020 patch and a 13% area in the 2009 patch. Conclusion: We observed no expressive differences in immunophenotypic expression between 2009 and 2020 bovine pericardia, except for the higher expression of the vimentin in the 2020 bovine pericardium patch. 108955 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS EDUARDO PITTHAN1, Vânia Hirakata2, Pamela Sandri1, Guilherme Sommavilla1 (1) Universidade Federal da Fronteira Sul Passo Fundo; (2) Instituto de Cardiologia do Rio Grande do Sul Introduction: Heart failure (HF) presents epidemic characteristics with considerable impact on morbidity and mortality, especially among the elderly. In Brazil, HF is responsible for high mortality rates. Objectives: To evaluate the variation trend of HF gross mortality rates by gender in the elderly in Brazil, comparing with the variability of population growth by age group and gross mortality rate from all causes in recent decades. Methods: An ecological study that used data available on DATASUS on March 2022, regarding mortality by gender in elderly people with HF in Brazil, from 1996 to 2019, stratified into three age groups: 60 to 69 years, 70 to 79 years and >80 years. The gross mortality rates were calculated in these variables per 105 inhabitants, comparing with the variation of the population in Brazil in the years that were studied, made available by the IBGE in a consultation on March 2022. Results: In the analysis of HF mortality in Brazil in men aged 60 to 69 years, there has been a decrease of 67%, falling from 98/105 inhabitants to 32/105 inhabitants. In the same age group, among women, the gross death rate has decreased 72%, falling from 77/105 to 21/105 inhabitants. In contrast, the male and female population aged 60 to 69 has grown 119% and 121%, respectively. In men aged 70 to 79 years, the mortality rate has dropped from 269/105 to 94/105 inhabitants, with a drop of 65%. Moreover, among women in this age group, there has been a drop of 70%, from 229/105 to 68/105 inhabitants. In contrast, the male population grew 112% and the female population 129%. In the age group of more than 80 years, in the same period, male mortality has decreased 54%, falling from 741/105 inhabitants to 339/105 inhabitants. In women, there was a drop of 60%, falling from 785/105 inhabitants to 315/105 inhabitants. In contrast, the male population has grown 151% and the female population, 187%. Conclusion: This study demonstrates trends of a significant decrease in the mortality rate from HF in elderly people of both genders in Brazil in the recent decades. This trend is more pronounced in women aged 60 to 79 years. 108973 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ROMERO HENRIQUE DE ALMEIDA BARBOSA1, Luana Resende Cangussú1, Eduardo Antonio Sartori Alho1, Matheus Rodrigues Lopes1 (1) Universidade Federal do Vale do São Francisco Introduction: Health literacy comprises the cognitive and social skills that determine an individual’s ability to obtain, process, understand and use health and medical information to make decisions that are relevant to their own health. Cardiovascular diseases are the leading causes of death in Brazil and Systemic Arterial Hypertension (SAH) contributes directly or indirectly to 50% of these deaths. Poor health literacy can impact in the management and control of these comorbidities leading to significant losses in quality of life. Objectives: To evaluate the level of health literacy and the quality of life of patients with systemic arterial hypertension users of the Public Health System in a municipality in the Northeast region of Brazil. Methods: Cross-sectional analytical observational study carried out with 105 patients with SAH through the application of the Short Assessment of Health Literacy for Portuguese-speaking Adults (SAHLPA-18), Short Test of Functional Health Literacy in Adults (S-TOFHLA) and the Mini- Quality of Life Questionnaire in Arterial Hypertension (MINICHAL). Results: It was found that about 60% of the interviewed patients did not have adequate health literacy in both tests to measure the level of health literacy. It was found that some factors such as age, economic class and education were associated with inadequate health literacy (p < 0.01). In the assessment of quality of life, using the MINICHAL, 46.7% of patients reported that hypertension interferes with quality of life. It was also possible to show that the time of diagnosis and economic class influenced the quality of life of patients (p < 0.05). Conclusion: A relevant portion of patients with SAH did not have an adequate level of health literacy, which reflects the difficulty in understanding and processing health information and can impact the therapeutic management of the disease. 108986 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY EDGARD FREITAS QUINTELLA1, Leonardo Hadid1, Márcio José da Costa Montenegro1, Luiz Kohn2, Dinaldo Cavalcanti Oliveira5, Gustavo Lycurgo4, Paulo Antonio Marra da Motta4, Maximiliano Otero Lacoste3 (1) Instituto Estadual de Cardiologia Aloysio de Castro (IECAC); (2) Universidade Estadual do Rio de Janeiro, Hospital Pedro Ernesto (HUPE); (3) Hospital Copa Star (Copa Star); (4) Hospital HOME; (5) Universidade Federal de Pernambuco (UFPE) Introduction: The left atrial appendage(LAA)percutaneous closure presents itself as a promising non-pharmacological alternative to anticoagulation. However, due to the anatomical singularity of the LAA, the accurate sizing and selection of the ideal prosthesis becomes a complex task. Objectives: In an attempt to mitigate the risks of inadequate sizing and to enable better standardized choice, this study proposes a new technique for sizing the Amplatzer Cardiac Plug prosthesis(ACP-AMULET)(Abbott Vascular). Methods: The current proposal was based on the consensus between experienced operators, who recommend that sizing should be performed by measuring the area and perimeter of the prosthesis landing zone(LZ), according to the measurements acquired by three-dimensional reconstruction of the LAA neck planes by Computed Tomography Angiography(CTA). In addition, a consolidated table was created from the manufacturer’s recommended data, where the range of the different sizes and oversizing was accounted for to enable anchorage and safe implantation. Hence, after the proper sizing of the LZ measurements, the results are crossed with the table and choice is made with greater precision. Discussion: The rationale for the proposal derives from the irregularity and heterogeneity of the LAA shapes, making the use of bidimensional sizing methods based on the average of the diameters a less accurate decision-making criteria of prosthesis selection. Although TEE is still the most widely used method, it is also subject to biases such as echocardiographer experience and volume status, which may restrict the widespread treatment success when used as a stand-alone decision-making strategy. Moreover, by performing the choice through CTA, it allows a lower dependence of choice during the procedure, avoiding its possible biases. 108992 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS ANGELA SICHINEL1, JOLIANE ALVES DE MORAES ROTILLI,1, LUCI MATSUMURA1, MARILENA INFIESTA ZULIM1, LUCIANE PEREZ DA COSTA1, CLAUDIA GONÇALVES GOUVEIA1, CAMILA SICHINEL SILVA DA CUNHA SOUZA1, MARIA LÚCIA SALAMENE DE OLIVEIRA KROLL1, GABRIELLA P PELLIZZER1, MARCIA MARIA DA COSTA1, MATHEUS PORTOCARRERO PETERLINKAR1, ERIVALDO ELIAS JR1 (1) SAO JULIAO HOSPITAL-HSJ; (2) SAO JULIAO HOSPITAL-HSJ; (3) SAO JULIAO HOSPITAL-HSJ; (4) SAO JULIAO HOSPITAL-HSJ; (5) SAO JULIAO HOSPITAL-HSJ; (6) SAO JULIAO HOSPITAL-HSJ; (7) SAO JULIAO HOSPITAL-HSJ; (8) SAO JULIAO HOSPITAL-HSJ; (10) SAO JULIAO HOSPITAL-HSJ; (11) SAO JULIAO HOSPITAL-HSJ; (12) SAO JULIAO HOSPITAL-HSJ Introduction: The risk factors of falls in the elderly are consequences that occur due to internal or external changes; The internal modifications are those physiologically associated to the aging process, emphasising difficulties walking, lack of muscular strength, movement range restrictions, balance issues and lack of physical exercise; The external ones are those that depend on social or environmental circumstances that represent difficulties to the elder. Objective: To analyze the relationship between gait speed and the occurrence of falls amongst elderly that participate in the MAE Project (Multidisciplinary Assessment in the Elderly). Method: A cross-sectional study was carried out with 109 elderly individuals associated to the MAE Project, 72 of them (66,05%) being female and 37 (33,94%) being male. The data collection took place at the physiotherapy outpatient clinic of São Julião Hospital, from April of 2017 to November of 2019, through application of the gait speed test, in which was measured the time, in seconds, the patient coursed the distance of 20 meters, not taking into account the 5 first meters because it is the acceleration period, nor the 5 last meters which is the deceleration period, and a questionary which contained name, sex and the question: Have you had falls in the last 12 months? Results: The sample was composed by 109 elders. 41 of those (37,6%) reported having falls in the last 12 months. Amongst those, 32 (78,04%) were female and 09 (21,95%) were male. Of the elderly that presented falls, 31 (75,60%) showed reduced gait speed, taking into consideration that the normal gait speed score, which ranges between 1,2 meters per second (m/sec) and 1,4 m/sec. Amongst the elderly with reduced gait speed, the female sex predominated with the number of 25 (80,64%) and average age of 70 years. Conclusion: Gait speed showed significative relationship with the occurrence of falls, being spotted reduced gait speed on more than half of the elders that had falls. However, when analyzing the study sample, in which less than half of the elderly has had falls, it was concluded that active elderly people have better results, that is, with the practice of physical activities, the functional capability increases and so the risk of falls is reduced. 108994 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS EDUARDO PITTHAN1, Vânia Hirakata2, Juarez Barbisan2 (1) Universidade Federal da Fronteira Sul Passo Fundo; (2) Instituto de Cardiologia do Rio Grande do Sul Introduction: The diagnosis of HF in elderly patients in clinical basis is difficult due to comorbidities. The BNP is used as a diagnostic and prognostic tool in HF but is not sufficiently studied in the elderly. Hypothesis: To evaluate the association of plasma BNP levels with diagnostic accuracy of HF and longterm prognostic validation. Methods: Six hundred and thirtyfour consecutive patients presenting with suspected HF in the Emergency Room of a tertiary hospital in southern Brazil participated in this cohort study. All patients underwent BNP measurement (Biosite POCT) as the institution protocol were included between March 2008 and September 2014. Following the Gold Standard for diagnosis of HF consisting of history and physical examination ECG chest Xray echocardiogram Uni and Two Dimensional Color Doppler. The sample was divided into 3 groups: SHF (Systolic Heart Failure) HFPEF (Preserved Ejection Fraction Heart Failure) and NHF (No Heart Failure) and patients were followed for 78 months. The study endpoint was mortality identified by the certificate of death of the Mortality Information Service (SIM). Results: Most patients (59.6%) were female the mean age was 77 ± 8 years 40.5% over 80 years. The majority (46.8%) had a diagnosis of HFPEF and BNP median 335 pg/ml 25% presented SHF and BNP median of 573 pg/ml and 28% did not meet the criteria for NHF and median BNP 45 pg/ml (KruskalWallis’s test p < 0.005). Half of the deaths were caused by HF. In the group with SHF was 79 deaths (49%) with a BNP median of 800 pg/ml and the 80 survivors median 383 pg/ml (p < 0.005). In HFPEF group occurred most deaths that computed 157 deaths (52% of the group) with a median of BNP 380 pg/ml and 140 survived with a median 245 pg/ml (p < 0,005). The NHF group had 40 deaths (22% of the group) with BNP median of 59 pg/ml and 138 survivors median of 36 pg/ml (p < 0,005). Survival analysis in 78 months (6.5 years) was performed by KaplanMeier curve. Two hundred seventh six deaths were recorded in the total group (42%). The group SHF with a 27month mean survival time (MST) the group HFPEF with a 52month MST the NHF group more than 50% survived. Conclusions: The BNP level showed association with the mortality index. BNP is an independent prognostic biomarker for longterm mortality in patients with HF in all ages. 109019 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS EDUARDO PITTHAN1, Vânia Hirakata2, Juarez Barbisan2 (1) Universidade Federal da Fronteira Sul Passo Fundo; (2) Instituto de Cardiologia do Rio Grande do Sul Introduction: Btype natriuretic peptide (BNP) is used as a shortterm biomarker for prognosis in patients with heart failure (HF). The prognostic role for longterm mortality is insufficiently studied. Hypothesis: To validate the BNP test as a biomarker of longterm mortality prognosis in patients with suspected HF, comparing the non-elderly 80 years old. Methods: The sample consisted of 634 patients with suspected HF, attended at the emergency room between March 2008 and September 2014. The efficacy of BNP to identify patients with heart failure and the association between the level of BNP (POCT Biosite) and longterm prognosis were evaluated. The study was divided into three age groups, Elderly (E) 60–79 years (46%), major-elderly ≥80 years (38%) and Non-Elderly <60 years old. Cause of death was identified through a search of death certificates in registry offices, informed by the Brazilian Mortality Information System. Results: Most patients were white (93%), female (64%), with a mean age of 77 (±8.6) years. HF was diagnosed based on a new gold standard that considered the Framingham and Boston criteria, plus echocardiography and ECG. HF was present in 340 patients (53%). Most of these patients (63%, n = 216) had HF with PreservedEjection (HFPEF). In bivariate analysis a BNP > 180 pg/ml was associated with a higher risk of mortality. In multivariate analysis BNP > 180 pg/ml remained associated with increased risk of mortality, with an HR of 3.4 (CI95%: 1.2 to 9.6;p80 years group with HF was 595 pg/ml, with a 27month mean survival time (MST) and 47% mortality rate. The median BNP for the Elderly between 60–79 years group was 369 pg/ml, with a 52month MST and 38% mortality rate. The Non-Elderly group BNP was 222 pg/ml, with an MST >50% and 26% mortality rate. Conclusions: The BNP level showed association with the mortality index. BNP is an independent prognostic biomarker for longterm mortality in patients with HF in all ages. 109029 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION GRACIA LUZ DON1, Eugenia Machbeth1 (1) Hospital San Martín, Paraná, Entre Ríos, Argentina Dyslipidemia has been identified as the risk factor that has the greatest impact of suffering ischemic heart disease. Triglyceride/HDL-cholesterol ratio (TG/HDL-C) was proposed as the best marker of insulin resistance and is considered an atherogenesis marker. It is obtained from triglycerides (mg/dl)/HDL-cholesterol (mg/dl) calculation. The cutoff point of 3.0 correlates with insulin resistance and cardiovascular risk, >3.5 with small and dense LDL-C particles, coronary atherosclerosis and risk of myocardial infarction. The present study was conducted to evaluate the relationship between TG/HDL-C index and cardiovascular diseases that led to hospitalization in the Department of Cardiology of San Martín Hospital. Method Ninety-one patients admitted to the hospital over a four-month period were evaluated according age, gender, TG/HDL-C ratio (triglycerides in mg/dl and HDL-C in mg/dl), BMI, admission blood glucose, coronary lesions and discharge diagnosis Results 30 women, 59 men and two without declaring gender were analyzed. The highest concentration of patients corresponds to ages between 60 and 69. Risk factors of the studied population were: • Hypertension 66% • Diabetes Mellitus 25% • Smoking 20% • Dyslipidemia 19% • Overweight/obesity 67% Variable evaluated -Percentage TGL/HDL Index >3.5 IMC 25–29,9 51% IMC > 30 60% Admission Blood glucose 111–199 mg/dl 61% Admission Blood glucose >200 mg/dl 54% Severe coronary lesions >70% DA: 56% CX 72% CD 72% discharge diagnosis: ischemic heart disease 74% heart failure 48% Previous myocardial infarction 81% history of coronary artery bypass grafting or angioplasty 50% Dyslipidemic patients 52% Total evaluated patients 74% Conclusions TGL/HDL-C index >3/>3.5 is observed in hospitalized patients with high prevalence. This demonstrates the impact of insulin resistance and phenotype B on severe cardiovascular complications such as ischemic heart disease and heart failure. 109036 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY IVANA PICONE BORGES DE ARAGÃO1, Ricardo Trajano Sandoval Peixoto1, Rodrigo Trajano Sandoval Peixoto1, Caio Teixeira dos Santos1, Raul Ferreira de Souza Machado1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Sara Cristine Marques dos Santos1, Edison Carvalho Sandoval Peixoto1 (1) Universidade de Vassouras Background: Mitral balloon valvuloplasty is not Always successful and free from complications. Objectives: To determine the independent risk factors for an unsuccessful procedure, severe mitral regurgitation and major complications in mitral balloon valvuloplasty. Methods: Longitudinal prospective study of 518 mitral balloon valvuloplasties performed between July 6, 1987 and December 31, 2004, on 429 (82.8%) female patients and 89 (17.2%) male patients with a mean age of 37.5 ± 12.8 years. Major complications were considered to be: perforation with cardiac tamponade, stroke and severe mitral regurgitation per procedure. The continuous variables were transformed in categorical variables and the chi-square or Fisher exact tests to compare the categorical variables, and logistic regression and multiple logistic regression were used to identify independente factors for predicting success, incomplete procedure, severe mitral regurgitation and major complications. Results: Success was noted in 452 (94.2%) procedures, with major complications occurring in 22 (4.2%) patients, of which ten were severe mitral regurgitation; there were no per-procedure deaths, with four (0.8%) in-hospital deaths. In the multiple logistic regression, lower age predicted success in the procedure; the only variable that predicted an incomplete procedure was the initial period of the procedure, and a score >11 points predicted severe per-procedure mitral regurgitation. There was no independent predictor of major complications in this study. Conclusions: Success was related to younger patients, an incomplete procedure to the initial period of the procedure and severe per-procedure mitral regurgitation to an echocardiography score >11 points. 109044 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION IVANA PICONE BORGES DE ARAGÃO1, Lívia Liberata Barbosa Bandeira1, Simone Aparecida Simões1, Tatiana Soares Spritzer1, Caio Teixeira dos Santos1, Raul Ferreira de Souza Machado1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Sara Cristine Marques dos Santos1, Vanessa de Freitas Marçolla1 (1) Universidade de Vassouras Cardiovascular disease (CVD) may be clinically different in women compared to men being underdiagnosed and treated. Worldwide, CVD and stroke are the leading causes of death in females reporting 8.6 million deaths/year in the literature. The objective of this study was to identify the self-knowledge (SK) and prevalence (P) of risk factors (RF) for CVD and stroke in female populations of different age groups and work activities: students of basic cycle medical students (group MS), Police Pacifying Units Police (PPU) (group PPU) and government employees (group GE). Methods: Cross-sectional, observational study of P of RF for CVD and stroke in female populations of different ages and labor activities between: group GE-27/09/13 and 10/24/2013; group PPU-10/05/2013 and 10/10/2013; groups MS-06/2016 and 12/2016; through the filling of a similar and anonymous questionnaire with 30 objective questions of quick answers about SK of RF: age, stress level, smoking, hypertension (H), dyslipidemia, sedentary lifestyle, obesity, diabetes. weight, height. pregnancy, menopause, gynecological (G/Y) and cardiological/year (C/Y) consultations. A positive response or ignorance equaled one point. Considered a risk group: women with ~2 points for positive or unknown response. Results: A total of 961 women interviewed were divided into groups MS (total 159), PPU (602) and GE (200), respectively: mean age 20.62, 28.1 and 44.3; high stress 44%, 31%, without report; smoking 3.8%, 7.0%, 16%; H 2.5% (1.3% unaware), 7% (3%), 13% (3%); 76.7% had they cholesterol levels measured (10.0% total cholesterol >200 mg/dl and 33.3% did not know, 62.9% did not know HDL <40 mg/dl), 76.0% (7% and 59%, 87%), 95% (22% E 25%, 62%); 89.9% had measured blood glucose, 76%, 88%; S 45.3%, 53%, 36%; BMI calculated 88.7% (weight and height reported) 12.57% ;?25 and 0.0% ;?30, BMI 51% being 23% ;?25 and 0.0% ;?30 and 49% being 17% ;?25 and 8% ;?30; they did consultations G/Y: 79.9%, 90.0% 98% and C/Y: 7.54% 12% and 33%; score i?2: 98.75%, 97.0%, 74.0%. Conclusion: Most women, in different age groups and work activities, were at risk of developing CVD and stroke due to the high prevalence of RF or their lack of knowledge, after applying a similar questionnaire. It was highlighted the importance of primary prevention and awareness programs. 109046 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY IVANA PICONE BORGES DE ARAGÃO1, Ricardo Trajano Sandoval Peixoto1, Rodrigo Trajano Sandoval Peixoto1, Caio Teixeira dos Santos1, Raul Ferreira de Souza Machado1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Sara Cristine Marques dos Santos1, Edison Carvalho Sandoval Peixoto1 (1) Universidade de Vassouras Objective: To compare the results, in-hospital evolution and cost of 468 percutaneous mitral balloon valvuloplasties (PMBV) with Inoue balloon (IB) and single Balt balloon (SBB). Methods: IB group (IG): 73 procedures and SBB group (BG): 395 performed between 06/1987 and 12/1999. Mean age: IG 37.1 ± 10.1 years and BG 37.3 ± 12.8 (p = 0.71745); 59 women in IG and 327 in BG (0.685255); NYHA functional class in IG and BG, respectively: I in 4 and 4 patients, II in 23 and 87, III in 40 and 265 and IV in 6 and 39 procedures (p = 0.010929). Atrial fibrilation: 7 in IG and 55 BG (p = 0.315511). Echocardiographic score 7.2 ± 1.2 IG and 7.3 ± 1.5 BG (p = 0.958911). Mitral valve área (MVA) Echo pre-PMBV: 0.98 ± 0.19 cm2 IG and 0.94 ± 0.21 BG (p = 0.143954). Results: Within-group comparison IG and BG, respectively: Pre-PMBV: mean pulmonar pressure (MPP) 33.9 ± 13.5 and 38.6 ± 14.3 mmHg (p = 0,007662); mitral gradient (MG) 17.3 ± 6.4 and 19.8 ± 7.0 mmHg (p = 0.013180); MVA Gorlin pre-PMBV 0.90 ± 0.20 and 0.91 ± 0.21 cm2 in BG (p = 0.8228449). Post-PMBV: MPP 25.3 ± 8.6 and 27.2 ± 10.6 mmHg (p = 0.261415); MG 5.9 ± 3.1 and 5.5 ± 3.7 mmHg (p = 0.083664); MVA Gorlin 1.98 ± 0.46 and 2.04 ± 0.40 cm2 (p = 0.419208). Complications: 5 cardiac tamponade in BG: 3 treated by surgery with 2 deaths, 2 with pericardial drenage without death. 1 stroke in BG. Severe mitral regurgitation (MR) 1 patient of each group, treated by surgery. Calculated cost of both technique 2 consecutive years with reuse and price of acquision at current prices demonstrated: IB technique U$1,286,32 and SBB U$309.94 for procedures. Conclusions: Both techniques were efficients. IG less symptomatic; MPP and MG were higher in BG; results post-PMBV were similar. MR were similar. Other complicaion only in BG. Lower cost for material acquisition in BG. 109047 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY IVANA PICONE BORGES DE ARAGÃO1, Ricardo Trajano Sandoval Peixoto1, Rodrigo Trajano Sandoval Peixoto1, Caio Teixeira dos Santos1, Raul Ferreira de Souza Machado1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Sara Cristine Marques dos Santos1, Edison Carvalho Sandoval Peixoto1 (1) Universidade de Vassouras Introduction: Percutaneous mitral balloon valvuloplasty is effective in mitral stenosis. Objectives: To evaluate prior mitral surgical commissurotomy (PMC) and echocardiographic score (ES) in the results and complications of mitral balloon valvuloplasty (MBV). Methods: From 1987 to 2013, 526 procedures with Inoue balloon, double or single Balt balloon technique; 480 without PMC named primary MBV group (PMBVG) and 46 that have been submitted to PMC, the PMC group. The PMCG was older than PMBVG (42.7 ± 12.4 vs 36.9 ± 12.5 years, p = 0.0030). Gender, atrial fibrilation and NYHA functional class were similar. In PMBVG and PMCG, respectively, ES were 7.2 ± 1,4 and 7.7 ± 1.5 points (p = 0.0158) and mitral valve area (MVA) 0.94 ± 0.21 and 1.00 ± 0.22 cm2 (p = 0.0699). Results: Pre-MBV: mean pulmonary artery pressures (MPAP) were 37.8 ± 14.2 and 37.6 ± 14.4 mmHg, p = 0.9515; mean gradient (MG) 19.6 ± 6.9 and 18.3 ± 6.9 mmHg, p = 0.2342; MVA 0.90 ± 0.21 and 0.93 ± 0.19 cm2, p = 0.4092, respectively, whem compare PMBVG and PMCG. Post-MBV: MPAP were 26.8 ± 10.2 and 26.6 ± 10.9 mmHg, p = 0.9062; MG 5.4 ± 3.5 and 6.3 ± 4.2 mmHg, p = 0.1492; MVA 2.04 ± 0.42 and 1.92 ± 0.41 cm2, p = 0.0801, respectively. Mitral regurgitation (MR) were similar pre and post-MBV. Severe MR post-MBV in 10 patients: 8 in PMBVG and 2 in PMCG, p = 0.2048. As there were not found significant diferences, the total group were divided in ES ≤ 8 and >8 groups: Pre-MBV: MPAP 37.5 ± 13.9 and 39.3 ± 16.6 mmHg, p = 0.4041; MG 19.7 ± 6.8 and 18.3 ± 7.3 mmHg, p = 0.1753; MVA 0.90 ± 0.21 and 0.94 ± 0.20 cm2, p = 0.0090 respectively. Post-MBV: MPAP 26.7 ± 10.1 and 28.0 ± 10.6 mmHg, p = 0.3730, MG 5.5 ± 3.6 and 5.5 ± 3.3 mmHg, MVA 2.06 ± 0.42 and 1.90 ± 0.40 cm2, p = 0.0090. Conclusion: The groups with and without prior mitral commissurotomy in MBV were compare and no differences were found in pre- and post-procedure, as mean pulmonary artery pressure, mean mitral gradient, mitral valve area, and mitral regurgitation. Although PMCG was older, with higher ES, its hemodynamics datas were similar. Whem the entire group was divided based on echo scores, those with echo scores >8 had highse MV (p = 0.0090). and smaler mitral valve areas post-valvuloplasty. The valve anatomy were more important than prior commissurotomy. 109049 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY IVANA PICONE BORGES DE ARAGÃO1, Ricardo Trajano Sandoval Peixoto1, Rodrigo Trajano Sandoval Peixoto1, Caio Teixeira dos Santos1, Raul Ferreira de Souza Machado1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Sara Cristine Marques dos Santos1, Edison Carvalho Sandoval Peixoto1 (1) Universidade de Vassouras Introduction: The single balloon (SB) is the less expensive technique to perform mitral balloon valvuloplasty (MBV). Objectives: This study aimed to demonstrate that MBV with the Balt single (BSB) and Inoue ballon, the wordwire accepted technique, had similar outcome and long-term follow-up (FU). Methods: From 1987 to 2013, 526 procedures were performed, being 312 with a FU, 56 (17,9%) with Inoue balloon (IB) and 256 (82,1%) with BSB. The mean FU 156 ± 144 months, p < 0.0001. Univariate analysis (UA) and multivariate Cox analysis (MCA) to determine independent predict variables of survival and event free survival (EFS) of death, cardiac surgery and new MBV, in both techniques groups. Results: In IB and BSB groups there were, respectively: female 42 (75.0%) and 222 (86.7%); mean age 37.3 ± 10.0 (19 to 63) and 38.0 ± 12.6 (13 to 83) years, p = 0.7138; sinus rhythm 51 (91.1%) and 215 (84.0%), p = 0.1754; echo score (ES) 7.6 ± 1.3 (5 to 10) and 7.2 ± 1.5 (4 to 14) points, p = 0.0528; echo mitral valve area (MVA) pre-MBV 0.96 ± 0.18 and 0.93 ± 0.21 cm², p = 0.2265; post-MBV mean MVA (Gorlin) were 2.00 ± 0.52 and 2.02 ± 0.37 cm², p = 0.9554; MBV dilatation área 6,09 ± 0,27 and 7,02 ± 0,30, p < 0,0001. At the end of the FU, there were in IB and BSB groups, respectively: echo MVA 1.71 ± 0.41 and 1.54 ± 0.51 cm², p = 0.0552; new severe mitral regurgitation in 5 (8.9%) and 17 (6.6%) patients, p = 0.5633; new MBV in 1 (1.8%) and 13 (5.1%), p = 0.4779; mitral valve surgery in 3 (5.4%) and 27 (10.4%), p = 0.3456; deaths 2 (3.6%) and 11 (4.3%), p = 1.000; cardiac deaths 1 (1.8%) and 9 (3.5%), p = 1.000; ME 5 (8.9%) and 46 (18.0%), p = 0.1449. In UA and MCA the BSB or IB technique do not predict survival or EFS. The independent risk factors to survival were: age <50 years (p = 0.016, HR = 0.233, 95% IC 0.071–0.764), ES ≤ 8 (p < 0.001, HR = 0.105, 95% IC 0.34–0.327), MBV dilatation area (p < 0.001, HR 16.838, 95% IC 3.353–84.580) and no mitral valve surgery in the FU (p = 0.001, HR0.152, 95% IC 0.050–0.459). Independent risk factors to EFS: no prior commissurotomy (p = 0.012, HR = 0.390, 95% IC 0.187–0.813) and post-MBV MVA ≥ 1.50 cm² (p = 0.001, HR = 7.969, 95% IC 3.413–18.608). Conclusion: In 25 years, survival and EFS were similar in BSB and IB technique. Independent predictors of surviva: age < 50 years, ES ≤ 8 points, MBV dilatation area > 7 mm2 and no mitral valve surgery in the FU. Independent predictors of EFS: no prior commissurotomy and post-MBV MVA ≥ 1.50 cm². 110916 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION SATYENDRA TEWARI1, SOHAM CHAUDHARI2, ANKIT SAHU3, ROOPALI KHANNA4, SUDEEP KUMAR5, NAVEEN GARG6, ADITYA KAPOOR7 (1) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (2) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (3) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (4) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (5) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (6) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India); (7) Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow (India) Introduction: CAD is one of the principle cause of mortality and morbidity. Considerable data suggests involvement of Lipoprotein (a), apolipoprotein B, apolipoprotein A-I, its ratio and abnormal lipids. Objectives: To compare the risk factor profile, clinical presentation, and angiographic severity of CAD in young patients (Age ≤ 45 years) and elderly patients (Age > 45 years). To correlate the level of Lipoprotein (a), Apolipoprotein (a), Apolipoprotein (b) and Apo B/Apo-A1 with the angiographic severity of CAD. Methods: This a retrospective, observational, single-centre study performed at tertiary care hospital. Angiographically proven CAD patients of all age groups were enrolled. Results: In our study, 714 patients (12.49%) belonged to group 1 (age ≤ 45 years) and 5003 patients (87.51%) belonged to group 2 (age > 45 years). Mean value of age(years) of study subjects was 56.92 ± 9.8. Distribution of gender was comparable between age group <=45 and >45 years. (Female:– 17.23% vs 17.49% respectively, Male:– 82.77% vs 82.51% respectively) (p value = 0.863). Proportion of patients with number of vessels involved:– 1 (single vessel) was significantly higher in age group <=45 years as compared to >45 years (1:– 79.97% vs 76.31% respectively). Proportion of patients with number of vessels involved:– 2 (double), 3 (triple) was significantly lower in age group <=45 years as compared to >45 years (2:– 19.33% vs 21.75% respectively, 3:– 0.70% vs 1.94% respectively). (p value = 0.017) There is statistically significant rise in the level of Lp (a) (mg/dL) and Apolipoprotein B/Apolipoprotein A-1 ratio with number of vessels involved in age group <=45 years and >45 years (p value < .05). Conclusion: Elevated Lp(a) is strongly associated with the development of high-risk vulnerable plaques that are prone to rupture. The ratio of Apo B to Apo A-I represents the ratio of total atherogenic to antiatherogenic lipoproteins. We observed a gradual increase in Lp(a) and Apo-B/A1 levels with the increasing number of stenotic vessels and an independent association between Lp(a) and Apo B/Apo A-1 with the severity of CAD after adjusting for other possible confounding factors. Effort should also be made to assess the atherothrombotic risk due to the Lp (a) particles on one hand and to apolipoproteins on the other hand. It is also essential that Lp (a), apo B, and ratio of abo B/apo A as an independent risk factor should be integrated into the treatment guideline. 109096 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH FARID SAMAAN1, Gianna Mastroianni Kirsztajn2, Ricardo Sesso2 (1) Instituto Dante Pazzanese de Cardiologia; (2) Universidade Federal de São Paulo Introduction: Chronic kidney disease (CKD) is common, preventable and silent in its early stages. Therefore, early detection of this condition in the population at risk, through laboratory tests, is essential. Objectives: To estimate the CKD prevalence and perform its risk stratification in a tertiary health service specialized in cardiology. Methods: The study was cross-sectional and based on laboratory records of patients from a public hospital specialized in cardiology. The evaluated tests were serum creatinine and urinary albumin/creatinine ratio (ACR, random sample) performed on an outpatient basis between 01/01/2021 and 12/31/2021. Duplicate exams and patients under 18 years of age were excluded. The estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI creatinine equation. CKD was defined by eGFR <60 ml/min/1.73 m² and classified into the following stages: 3a (45–59 ml/min/1.73 m²), 3b (30–44 ml/min/1.73 m²), 4 (15–29 ml/min/1.73 m²) and 5 (<15 ml/min/1.73 m²). Albuminuria was classified into three levels: A1 (<30 mg/g), A2 (30–300 mg/g) and A3 (>300 mg/g). According to the CKD risk map, individuals with simultaneous creatinine and ACR measurements were stratified into low, moderate, high or very high risk. Results: The sample consisted of 36,651 patients in whom the same number of serum creatinine results and 19,031 ACR results were evaluated (median patients‘ age 72.5 [51.0–73.6] years, 51.3% male). The prevalence of CKD was 30.9% and patients with stages 3a, 3b, 4 and 5 corresponded to 15.3%, 10.2%, 3.6% and 1.7%, respectively. CKD was more frequent in older age groups: 18–29 years (2.5%), 30–44 years (8.4%), 45–59 years (25.5%), 60–74 years (30 .7%) and ≥75 years (56.8%) (p < 0.001). Patients with albuminuria categories A1, A2 and A3 were 71.5%, 22.6% and 5.9%, respectively. ACR ≥ 30 mg/g was not associated with age: 18–29 years (23.3%), 30–44 years (23.4%), 45–69 years (26.0%), 60–74 years (28 .5%) and ≥75 years (36.9%) (p = 0.671). Patients with simultaneous measurements of serum creatinine and ACR were 19,031 and their distribution in the CKD risk categories was: low (52.0%), moderate (23.8%), high (13.1%) and very high risk (11.2%). Conclusions: The results showed that CKD is present in about 30% of the patients assisted in the cardiology institute evaluated. In up to half of the patients, the risk of major outcomes such as hospitalization, need for renal replacement therapy, and death was moderate, high, or very high. 109100 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LUIZ FERNANDO KUBRUSLY1, Douglas Mesadri Gewehr2, Jéssica Ferreira Eduardo1, Izabele Maria Geri1, Allan Fernando Giovanini1, Fernando Bermudez Kubrusly3 (1) Mackenzie Evangelical School of Paraná, Curitiba, Paraná, Brazil; (2) Denton Cooley Institute of Research, Science and Technology, Curitiba, Paraná, Brazil; (3) Curitiba Heart Institute, Curitiba, Paraná, Brazil Introduction: Pulmonary arterial hypertension (PAH) is a high severity vascular disorder with fast progression, which clinical spectrum is related to the reduction of the pulmonary arteries lumen with an increase in pressure and vascular resistance. These changes result from many mechanisms such as medial hypertrophy, concentric intimal fibrosis and necrotizing vasculitis, which are responsible for the remodeling of pulmonary artery wall, the reduction of pulmonary compliance and leads to cardiac muscle overload. Objective: To evaluate the severity of pulmonary arteriopathy in an animal model of Monocrotaline-induced Pulmonary Arterial Hypertension, through histological morphometric analysis of pulmonary vessels. Methodology: Morphometric analysis was performed in Zen 3.2 Software and 50 PAH animal model slides, with random selection of 10 arterioles and 10 pulmonary non-arteriolar vessels for each animal. The gravity was delimited through three pulmonary systems elements (1) medial layer thickness, (2) microvascular muscularization of non-muscle vessels, and (3) obliteration of muscle arterioles and non-muscle vessels. We also assess cardiac overload thought right ventricular freewall thickness and the right ventricular chamber area. Results: There was a gradual onset of pulmonary disease in MCT animals, whose evolution was followed by histological findings of pulmonary arteriopathy, including arterioles’ muscularization, medial layer hypertrophy, concentric and “plexiform-like” laminar and non-laminar neointimal lesions. There was an important increase in the thickness of the tunica media and the degree in microvascular muscularization through the experimental groups compared with the control. There was a significant increase in luminal obliteration of muscle arterioles and non-muscular vessels, measured by the luminal obliteration ratio. The right ventricular hypertrophy (HVD), assessed by measuring the thickness of the right ventricle, had increased significantly on 30-to-37-day groups, represented almost twice the value of the control group. Besides that, there was a significant dilatation of the Right Ventricular Chamber on 30-to-37-day groups. Conclusion: The MCT application proved the effective inducing PAH, being able to generate changes in vessels through the lower exposure group. Furthermore, lungs lesions are intensified over time, and the observation of plexiform lesions were not registered in a similar model. 109135 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ROMERO HENRIQUE DE ALMEIDA BARBOSA1, Emerson Silva de Jesus1, Eldys Myler Santos Marinho1, Johnnatas Mikael Lopes1 (1) Universidade Federal do Vale do São Francisco Background: Heart disease burdens individual health, limiting life activities such as people’s productive capacity and quality of life. In addition to understanding the clinical characteristics of heart diseases, identifying subjects at risk prior to the installation of limitations helps in directing preventive therapeutic and multiprofessional actions. Objective: To develop an algorithm to screen people with heart disease in an outpatient setting for their risk of future functional life activity limitations (LAL). Methods: Cross-sectional study with data from the 2019 National Health Survey in Brazil. The general sample consisted of 90846 interviews, being selected individuals aged 18 years and some type of heart disease diagnosed for more than a year by physicians. The outcome was current usual LAL due to the disease. Independent variables were grouped into cardiac, self-care and social morbidities. The data were analyzed using the CHAID-type decision tree algorithm, and the sample was divided into training (70%) and testing (30%). Sensitivity (S), specificity (SP), positive (PV+) and negative (PV-) predictive values for the outcome were estimated. Results: 4711 (5.3%;95%CI: 5.0–5.6) individuals have a diagnosis of heart disease, of which 4409 (94.4%; 95%CI:93.4–95.3) have been diagnosed for more than one year and mean age of 59.31 (95%CI: 58.31–60.31) years. 2374 (49.2%; 95%CI 46.5–51.8) of those with heart disease reveal LAL. 2830 observations were used to train the algorithm and 1171 to test it. Five attributes remained in the final model, two of morbidity, two of self-care and one of social, generating 13 nodes and a thickness of 3 in the decision tree. An algorithm with S = 81.9%, SP = 43.2%, PV+ = 58.94%, PV– = 70.54% was obtained. In the test, S = 83.8%, SP = 43.6%, PV+ = 60.67% and PV– = 72.04%. Conclusion: The algorithm has a relevant rate of true positives, however it has a low rate of true negatives for LAD. As the damage caused by LAL and its high prevalence are much greater than the damage caused by the provision of care and follow-up, its application in clinical and community contexts in Brazil is justified. 109138 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LUCIANO DE MORAIS PINTO1, Marcelo José Böck1, Vitor Pires Pereira1, Marcelo Leite da Veiga2, Claudia de Melo Bertoncheli dos Santos3, Luciano de Morais-Pinto1 (1) Laboratório de Design Anatômico – Universidade Federal de Santa Maria; (2) Laboratório de Morfofisiologia Experimental – Universidade Federal de Santa Maria; (3) Laboratório de Anatomia Patológica – Hospital Universitário – Universidade Federal de Santa Maria Aortic rupture is relatively common in horses. However, architectural differences have not yet been considered in the pathogenesis of aortic catastrophe in this species. Thus, our objective was to describe the morphological and morphometric aspects of the tunica intima (TI) and media (MT) in the aortic bulb (AB) and ascending aorta (AA). 52 healthy aortas from adult horses were processed according to standard optical and scanning electron microscopy protocol. TI and TM thickness as well as aortic lamellar units were digitally measured. The aorta was pale yellow and had a rigid texture and distinct thicknesses. The AA wall was thicker than the BA, in totality and in the thickness of the layers (Tab.1). The lamellar units were better defined in AA and arranged in interleaved layers of elastic lamellae, smooth muscle cells and collagen fibers, while in BA the trilaminar aspect was not maintained and the muscle tissue bands were arranged in mosaic. The increase in the thickness of the aortic wall in the BA-AA direction suggests that blood flow is clockwise helical. It is assumed that blood pressure was higher in the septal AB for the left AA; from left BA to right AA and from BA to septal AA. As dissection/rupture involves failure of tissue structure, it is expected that a better understanding of the normal tissue architecture of the aorta in horses will contribute to future studies of the pathogenesis involving aortic catastrophes in animals and humans. 109147 Modality: E-Poster Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE SILVIA INES GARCÍA1, Maria Silvina Landa1, Maia Aisicovich1, Mariano Luis Schuman1, Ludmila Soledad Péres Díaz1, Graciela Giardino2, Carlos Jose Pirola1, Silvia Inés García1 (1) Molecular Cardiology Laboratory, Institute of Medical Research-IDIM University of Buenos Aires-CONICET; (2) Experimental Medicine Laboratory, Hospital Aleman, Buenos Aires Cardiac TRH (cTRH) induces cardiac damage and its inhibition attenuates heart injury in different models: SHR and cTRH overexpression (Schuman 2011,2014); Angiotensin II infusion and doxorrubicin cardiotoxicity (Peres Diaz 2018,2020) and LVH induced by obesity or leptin infusion (Aisicovich 2019,2021). We described that cTRH inhibition increase LVEF% and preserve cardiac function after acute MI in rats (Schuman 2021). Histone acetylation (HDACs) modulates gene expression by epigenetic alterations as DNA methylation. VPA, an FDA approved drug for bipolar disease and epilepsy, protects heart against MI injury (Tian 2019). As VPA is an inhibitor of HDACs, we hypothesized that inhibition of HDACs with VPA might attenuate LVH and fibrosis in SHR by the modulation of cTRH. 7 w-old male SHR and WKY received VPA and hearts were used after 10w of treatment. BP, LVH index and cTRH expression significantly increased in SHR. VPA slightly attenuated (p < 0.05) the higher SHR BP, without effect in WKY. LVH index decreased (p < 0.05) only in SHR. LVPWT significantly decreased (p < 0.05) only in SHR. As hypothesized, VPA normalized cTRH mRNA expression in SHR (WKY = C:0.61 ± 0.7vs VPA:0.41 ± 0.97; SHR = C:5.72 + 0.9 vs VPA:0.61 + 0.9, p < 0.05) and TRH IHQ confirmed these results (p < 0.05) which brought a strong reduction in fibrosis in LV, by decrease in BNP and 3-collagen expression in SHR+VPA. This was confirmed by Masson’s an d Sirius Red stainings (p < 0.01). SHR offspring born from VPA-treated parents with a 2-week washout period before mating, wich never received VPA, surprisingly had a significant decrease in hypertrophy (LVW/BW) vs offspring of both SHR untreated parents, showing a transgenerational inheritance. Indeed, SHR showed a decrease in % TRH promoter methylation (Increase in gene expresion) that is significantly reverted in offspring of VPA treated parents (methylation-specific PCR). Consistently, we observed a decrease in cTRH expression (p < 0.05), and therefore, a significant reduction (p < 0.05) in BNP and type 3 Collagen expression, despite the high BP in VPA parents offsprin. To sum up, we described for the first time that VPA treatment modulates cTRH gene and consequently attenuates heart fibrosis and hypertrophy in the SHR, without affecting BP. We show an epigenetic modulation of VPA over cTRH promoter that could be responsible not only of cardiac alterations during VPA treatment, but beyond affecting next generation with results still unknown. 109150 Modality: E-Poster Researcher – Non-case Report Category: NUTRITION RENATA BORBA DE AMORIM OLIVEIRA1, Renata Borba de Amorim Oliveira1, Ingrid Beranger da Costa Pereira1, Ana Paula Menna Barreto1, Monica Feroni de Carvalho1 (1) Centro Multidisciplinar UFRJ-Macaé Obstructive Sleep Apnea Syndrome (OSAS) is a very common disorder of breathing during sleep, characterized by recurrent episodes of total interruption (apnea) and/or partial interruption (hypopnea) of ventilation resulting from collapse of the upper airways. Obesity, systemic arterial hypertension and cardiovascular diseases are risk factors of the disease, being related to a major risk of morbidity and worsening of the already installed disorder. The objective of study was to assess the frequency of obesity by different anthropometric measures of total and central body adiposity in adult individuals with OSAS, and its association with excessive daytime sleepiness and severity of the syndrome. The present study was a prospective observational study, in which adults who underwent polysomnography for the diagnosis of OSAS were evaluated. Were evaluated variables: socio-demographic, sleep-related complaints, degree of daytime sleepiness, which was evaluated by the Epworth Sleepiness Scale developed by Johns (1991) and validated in Brazil by BERTOLAZZI et al. (2009), and also to risk factors for OSAS and anthropometric: weight, height, Body Mass Index (BMI) and Waist Circumference (WC). In the 64 patients evaluated, only 43 subjects (67.2%) were diagnosed with OSAS and entered the study. Regarding the classification of OSAS, 44.2% presented mild degree, 23.2% moderate and 32.6% severe degree. Excessive Daytime Sleepiness (EDS) was present in 72.1% of the patients. The classification by BMI is 11.6% of eutrophy, 18.6% of overweight, 25.6% of obesity grade I, 27.9% of obesity grade II and 16.3% of obesity grade III. Regarding the classification of WC, 62.8% presented a much increased risk. Analyzing the morbidity profile of the studied population, 39.50% had systemic arterial hypertension and 11.60% cardiovascular disease. There was a statistically significant association between obesity and WC (p = 0.001), otherwise was not found between obesity and severity disease (OSAS) (p = 0.76) or with SDE (p = 0.127). To conclude, the studied group presented high prevalence of obesity, arterial hypertension and cardiovascular diseases and changes in weight and in the health condition are important to manage symptoms and aggravation of the OSAS and cardiovascular disease. 109167 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GERARDO GARCIA-RIVAS1, Omar Lozano1, Christian Silva-Platas1, Judith Bernal-Ramírez1, Hugo Alves1, Carlos Jerjes-Sánchez1, Guillermo Torre-Amione1 (1) TECNOLOGICO DE MONTERREY Increasing evidence points to CBD as a promissory therapeutic molecule for diverse inflammatory pathologies, including cardiovascular diseases such as coronary disease and myocarditis. Our study evaluates the application of CBD as a potential therapy for heart failure (HF). Here we assessed the cardioprotective effects of CBD administration in a non-ischemic HF model in mice. Every 3rd day a dose of 1 mg/kg of synthetic CBD was administered via a subcutaneous injection. After four weeks of treatment, the animals were euthanized, and their heart was collected for further analysis. Fibrosis development and myocyte hypertrophy was determined by histopathologic imaging quantification. Additionally, to explore the CBD-antihypertrophic mechanisms, we used cultured cardiomyoblasts to explore the role of cytosolic and mitochondrial oxidative stress and NF-kB pathway intermediates. We developed a multi-center, double-blind, placebo-controlled, randomized trial for the clinical translation, considering the preclinical data. The primary objective of this study is to evaluate the effect of CBD on the prevention of cardiovascular and COVID-19 complications in patients hospitalized for COVID-19. We observed a significant reduction of fibrosis and cell hypertrophy in CBD-treated animals. The expression by qPCR of BNP and collagen1 as a pathological remodeling marker was significantly reduced when CBD was administered in contrast with the HF mice. Pro-inflammatory cytokines expression was observed in IL1b and IL6 expression. At the cellular level, a significant reduction in cell area was observed in CBD treated groups even when exposed to Ang II, achieving sizes comparable to those of the controls. CBD significantly reduced the generation of mitochondrial ROS entities in our hypertrophy model and showed an increase in NF-kB activation that can be reversed by CBD treatment. Also, CBD normalizes the expression of mechanical stress and remodeling markers such as BNP, TGFa, SOD2, IL6, and IL10 involved in inflammation. 23 Non-critical patients hospitalized within the previous 48 hours who tested positive for COVID-19 within seven days and have a prior history of cardiovascular disease (CVD) and significant risk factors for CVD were enrolled. Preliminary results indicate the safety of CBD in patients with COVID-19 infection and cardiovascular disease. 109186 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING NATALIA CARRO1, Graciela María Rousse1, Cabrejos Gustavo1, Matilde Del Campo Contreras1, Leandro Parrilla1, Nudelman Ezequiel Adolfo1, Rubio Edgardo1 (1) Division of Cardiology. Echocardiography Laboratory, Hospital of the Government of the city of Buenos Aires Introduction: Global Longitudinal Strain (GLS) is a sensitive method for the early detection of myocardial damage. The latest AHA classification of Chagas disease (ChD) includes, in stage BI, a population with preserved Ejection Fraction (EF) heterogeneous due to ECG changes and/or segmental wall motion abnormalities. The evaluation of abnormal Segmental Longitudinal Strain (SLS) and its comparison with MRI has not been sufficiently evaluated in this population. Objective: In patients (p) with ChD in Stage BI the aim was1)To evaluate the usefulness of GLS in comparison with p with the indetermined form of ChD or Stage A 2) To compare abnormal SLS with cardiac MRI in detecting the anatomical substrate of the arrhytmia. Methods: Transversal, monocentric study. 55p were included January 2018-March 2020: 17p with ChD in Stage B1 with frequent ventricular arrhythmia, cardiac Echo-Doppler with Longitudinal Strain assessment, and gadolinium-enhanced cardiac MRI (Group B); they were compared with 38 p Stage A of ChD (Group A). The association between Strain and abnormal cardiac MRI in group B patients was analyzed. Results: 2 p were excluded due to inadequate ultrasonic image quality. The p from Group B compared to those from Group A were older, had higher Left Atrial volume, lower MAPSE, lower TDI, lower absolute value of GLS (18.3 +– 2.3% stage B vs 21.81 +– 2.5% stage A, p < 0.001) and similar EF. Through ROC curve between group A and B patients, the best cut-off point GLS was determined, a value of –20.5% with sensitivity of 93% and specificity of 70%, and area under the curve of 0.83 CI 95% (0.71–0.95). A cardiac MRI was performed in 11 out of 16 p; in 6 of them it was abnormal: due to late enhancement (4 p), edema plus enhancement (1 p), and thinning and akinesia (1 p). 5 of the 6 p with pathological cardiac MRI presented abnormal SLS and none of the p with normal cardiac MRI. The association between cardiac MRI alterations and SLS abnormal evaluated by means of the Kappa index was 0.82. Conclusions: In our limited population: 1.GLS was significantly lower in in group B1.2. The accurate association between cardiac MRI and abnormal SLS shows the latter as a useful alternative for the diagnosis of the anatomical substrate of arrhythmia. 109181 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY RITA ELIZABETH IBARRA CASTILLO1, JORGE LUIS ARBAIZA SIMON2, JOSE LUIS LASO BAYAS1 (1) Hospital de Especialidades Carlos Andrade Marín; (2) Hospital Vozandes Quito HVQ Introduction: Cephalic vein cutdown (CVC) and subclavian vein puncture are the most widely used techniques for leads insertion during the implantation of cardiac stimulation devices (CSD). Lately, axillary vein access (AVA) has emerged as an alternative. Many approaches have been described for AVA, including the ultrasound (US) guided technique which seems to be fast and safe. objective: Our purpose was to assess whether the US guided puncture of the axillary vein was as efficient as the CVC for CSD implantation. Methods: This was a multicenter prospective trial. All patients undergoing first pacemaker or implantable cardiac defibrillator implantation were submitted to an US scanning of the site of implantation to determine if the axillary vein was visible. Only those with a clear axillary vein were included. None of the operators had previous experience in US AVA. All procedures were planned as ambulatory. We randomized 76 patients to either US AVA (n = 38) or CVC (n = 38). We included only patients with CSD with ≤2 leads. Primary endpoint was success in introducing all the intended leads through the selected access. Secondary endpoints were: complications during the first week after the implantation, time of fluoroscopy, duration of the procedure, use of local and general anesthesia and need of hospitalization. We used X squared and T-tests to compare results from categorical and continuous variables. Analyses were performed according to the intention-to-treat principle. Results: There were no differences in the demographic characteristics. The mean age of all patients (n = 76) was 70.8 years, with 56.6% (n = 43) being male. In the AVA group, access success was achieved in all the subjects (n = 38), and in 78.9% of patients (n = 30) the vein was gained with US. The mean of attempts to gain the first access was 1.8. In the CVC group, the access success was lower, 76.3% (n = 38, p = 0.002). Regarding the rate of complications, there were no differences (AVA 5.2%, CVC 7.8%; n = 76, p = 0.64). Furthermore, we found no differences in time of X-ray exposure, procedure duration, quantity of local anesthetic used, need of general anesthesia or hospitalization (n = 76, p > 0.05). Conclusion: We found that, in comparison to CVC, US AVA was a more efficient technique, allowing successfully insertion of all leads from a conventional bicameral CSD, without increasing complications or issues of the implantation procedure. Our results are in concordance with other randomized clinical trials. 109233 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MIGUEL MEIRA E CRUZ1, Germaines Escames2, Jose Fernandez Martinez2, Dario Acuña Castroviejo2 (1) Centro Europeu do Sono; Sleep Unit, Centro Cardiovascular da Universidade de Lisboa, Lisbon, Portugal; (2) Centro de Investigación Biomedica, Departamento de Fisiologia, Facultad de Medicina; Instituto de Biotecnologia, Universidad de Granada, Spain Introduction: Cardiovascular diseases (CVD) constitute the leading cause of death in the world, and aging is by far the major risk factor for cardiac dysfunction. Aging involves subclinical inflammatory activation, with NF-kb and NLRP3 inflammasome as the main components of such response. But inflammation also induces a pro-oxidative response, responsible for the oxidative damage to the cell and mitochondria, leading to a bioenergetic failure. In turn, melatonin exerts profound anti-inflammatory and antioxidant properties, with the mitochondria as the main intracellular target. Here, we analyzed the impact of the NRLP3 inflammasome in the myocardial aging and the role of melatonin in preventing it. Methodology: For this study, we used wild-type and NLRP3-deficient mice of 3, 12, and 124 months of age, with and without melatonin treatment. Results: The absence of NLRP3 prevented the age-dependent myocardial failure and mitochondrial impairment, affecting the Bax/Bcl2 ratio. The Nef2-dependent antioxidant response was unaffected by the lack of NLRP3. In wild-type mice, melatonin treatment produced counteract the age-dependent damage, providing similar protective features than the absence of NLRP3. Melatonin also improved mitochondria structure and enhances the Nrf2 antioxidant response. Conclusions: NLRP3 inflammasome-dependent chronic inflammation during ageing is main responsible for myocardial loss of function, causing proapoptotic phenomena, free radicals’ formation and oxidative damage, and mitochondrial bioenergetic deficit, suggesting a molecular target for cardiac protective therapies. Melatonin, which reaches all subcellular compartment of the myocardial, blocks the NLRP3 inflammasome response and maintains the integrity of the heart, doing it a drug of choice for this age-dependent preventive therapy. 109239 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING KLEIBER MARCIANO LIMA BOMFIM1, Alan Alves de Lima Cidrão1, Pedro Braga Linhares Garcia1, Leonardo Miranda Macêdo1, José Doriberto Freitas1, Giovanna Rolim Pinheiro Lima1 (1) HOSPITAL REGIONAL DO SERTÃO CENTRAL Introduction: Stroke is one of the main causes of morbidity and mortality, currently representing the second leading cause of death worldwide. Regarding the etiologies, the cardioembolic type is responsible for 14 to 30% of the ischemic cases and presents higher mortality and worse functional complications. In terms of structural heart diseases, the increase in left atrial volume is an independent factor for stroke, in which the chance of thromboembolism is 20% per year. Objective: To evaluate left atrial volume increase, determined by transthoracic echocardiographic study, as an independent variable for risk of ischemic stroke and transient ischemic attack. Methods: This is a quantitative descriptive, cross-sectional, prospective cohort study conducted between April 2020 and April 2021 in the stroke unit of a tertiary public hospital. Data analysis was performed using the statistical package SPSS, version 22 (IBM – 2017), in which it was possible to perform descriptive analysis to characterize the sample, student’s t test for independent samples, correlation analysis and simple linear regression. The calculation of the left atrial volume was performed by two-dimensional echocardiography in the apical projections of two and four chambers, indexed by the body surface. Results: The study included 308 patients from the State of Ceará who had ischemic stroke and transient ischemic attack. Age ranged from 50 to 98 years (M = 72; SD = 10.8), being 53.7% male and 46.3% female. Most had a diagnosis of ischemic stroke 93.9% and 6.1% transient ischemic attack. Results revealed that the variable “left atrial volume” did not have a normal distribution (K-S (308) = 0.46, P < 0.05; S-W(308) = 0.55, P < 0.05). The Pearson correlation analysis between left atrial volume and diagnosis of ischemic stroke/transient ischemic attack evidenced a positive linear correlation (0.021). Conclusion: The present data demonstrate a positive correlation between increased left atrial volume and the occurrence of ischemic stroke/transient ischemic attack. 110338 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JC PACHON-M1, Enrique I. Pachon-M1, Juan C Zerpa-Acosta2, Carlos TC Pachon2, Tomas G Santillana-P1, Tasso J Lobo2, Felipe A Ortencio2, Ricardo C Amarante1, Juan C Pachon-M1 (1) São Paulo University; (2) São Paulo Heart Hospital; (3) Semap Arrhythmia’s Service Background: Vagal tone predominates most of the time. Therefore, isolated sinus heart rate(HR) is higher than the in-situ sinus rate. Atropine stops vagal action by blocking muscarinic receptors. Cardioneuroablation (CNA) also abolishes vagal action by eliminating the postganglionic vagal neuron, causing HR increase. Knowing previously whether HR will increase with CNA is very important since CNA is not indicated if there is no atropine response. Objective: To assess the correlation between HR and Wenckebach point(PW) determined by the atropine test (AT) and that one resulting from CNA. Methods: 76p, 41 females (54%), 14–67 (37 ± 13.2) years, with reflex functional bradyarrhythmias (neurocardiogenic syncope) or non-reflex (sinus dysfunction, vagal AF or bradytachycardia) submitted to CNA controlled by extracardiac vagal stimulation. AT was performed with IV infusion of 0.04 mg/kg. CNA was performed by ablation of the P-point, the presumed 4 main GPs areas and spare AF-Nests, at least 02 days post-AT. The maximum HR and PW obtained under the atropine and at the end of the CNA were compared. Results: There were significant HR and PW increases with atropine and with CNA. The increases were smaller with CNA probably because CNA also eliminates sympathetic fibers and AT does not. Conclusion: There is a strong correlation between HR and PW determined by atropine and those resulting of CNA. Thus, atropine test can be used as a good predictor of the CNA result. 110339 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JC PACHON-M1, Enrique I Pachon-M1, Carlos TC Pachon2, Tasso J Lobo2, Tomas G Santillana-P1, Juan C Zerpa-A2, Felipe A Ortencio2, Maria Zelia C Pachon3, Ricardo C Amarante1, Juan C Pahon-M1 (1) São Paulo University; (2) São Paulo Heart Hospital; (3) Semap Arrhythmia’s Service Background: Symptomatic reflex and non-reflex functional bradyarrhythmias are related to vagal hyperactivity. Cardioneuroablation[CNA] provides vagal denervation through endocardial RF ablation of the post-ganglionic vagal neurons. Neuro-myocardium interface identification, stepwise denervation control and denervation validation are critical. Several findings have shown that the Arial Fibrillation-Nest[AFN] are intrinsically related to the vagal innervation. Objective: To verify whether CNA based on AFN ablation promotes enough vagal denervation compared to a control group with non-AFN ablation. Method: Prospective, controlled study with 54 p gathered in two groups: AFN group (AFNG-24 p) with functional or reflex bradyarrhythmias treated with AFN ablation, and a control group (CG-30 p) with anomalous bundles, ventricular premature beats, atrial flutter, nodal reentry and atrial tachycardia, treated with conventional ablation (non-AFN ablation). Ablation of AFNG was targeted to AFN detected by fragmentation of the endocardial electrograms and/or by Fractionation Software. Vagal response was evaluated before, during, and post-ablation by 5s non-contact vagal stimulation at the jugular foramen, through the internal jugular veins[ECVS], analyzing longest RR, pauses, and AV block induced by ECVS. All patients had current guidelines arrhythmia ablation indications. Results: There was found a high density of AFN over the P-Point (right half of the left interatrial septum), over the presumed areas of the main 4 ganglionated plexi, over the coronary sinus roof, and over the Waterston’s groove. Pre-ablation ECVS induced sinus pauses, asystole and transient AV block in both groups, showing normal vagal response (p = 0.94). Post-ablation ECVS in the AFNG showed complete elimination of the cardiac vagal response in all cases (Pre/Post-ablation ECVS = p < 0.01), demonstrating acute and complete vagal denervation. In the CG, vagal response remained practically the same as pre-ablation (p = 0.31) showing that non-AFN ablation caused no significant denervation. Conclusion: CNA based on AFN ablation attained an excellent degree of acute vagal denervation while non-AFN ablation did not cause denervation. This fact suggests that AFNs are directly related to vagal innervation and may be a good alternative for CNA. Additionally, ECVS was remarkably valuable for CNA control. 109246 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM PEDRO GABRIEL MELO DE BARROS E SILVA1, RD. Lopes4, RHM. Furtado5, AVS. Macedo1, B. Bronhara1, E. Ramacciotti6, PA. Martins7, AL. De Oliveira7, VS. Nunes7, LEF. Ritt8, AT. Rocha8, L. Tramujas2 (1) Brazilian Clinical Research Institute; (2) IP-HCor; (3) Hospital Samaritano Paulista; (4) Duke Clinical Research Institute, Durham, United States of America; (5) Hospital Israelita Albert Einstein, Sao Paulo, Brazil; (6) Loyola University, Chicago, United States of America; (7) Hospital Estadual Dr Jayme Santos Neves, Serra, Brazil; (8) Cardio Pulmonary Hospital, Salvador, Brazil In the ACTION trial, therapeutic anticoagulation did not show benefit on mortality, days of hospitalization and oxygens therapy at 30 days among patients with COVID19. However, this strategy was associated with higher rate of bleeding and a potential reduction in the rate of clinical thrombotic events. The current analysis evaluated which variables were independently associated with both outcomes in order to help the identification of the risk for thrombotic and hemorrhagic events among patients with COVID19. Methods: A total of 615 patients hospitalized with COVID-19 and elevated D-dimer were randomly assigned to prophylactic anticoagulation (mainly in-hospital heparin) or a therapeutic-dose strategy that used in-hospital rivaroxaban 20 mg/day for stable patients, or full-dose enoxaparin for unstable patients, followed by rivaroxaban through 30 days. One patient withdrew consent and was not included in the analysis. The current analysis tested baseline clinical characteristics and laboratorial exams one by one with independent logistic regressions for the composite of bleeding (major bleeding and clinically relevant nonmajor bleeding) and thrombotic events (venous thromboembolism, myocardial infarction, stroke, systemic embolism, and major adverse limb events). Significant variables (p < 0.05) were selected to adjust several multiple logistic models. Final models were chosen based on Akaike information criterion and therapeutic anticoagulation was included in the final model based on the primary results of the trial. Results: The model for bleeding events showed an accuracy of area under the curve (AUC) of 0.635 while the model for thrombotic events had an AUC of 0.725. Level of respiratory support (especially invasive ventilation) was associated with both outcomes in the multivariable analysis. Beyond respiratory support, level of creatinine and history of coronary disease were also independently associated to the risk of thrombotic events. When the utilization of therapeutic anticoagulation (mainly with rivaroxaban) was included in the multivariable analysis, this variable was strongly associated with higher risk of bleeding (model AUC of 0.718) but was not associated with lower rate of thrombotic events. Conclusion: Variables associated with higher risk of thrombotic events commonly are associated also to bleeding complications. The models developed in ACTION may help the selection of patients with better balance of risk vs. benefit to anticoagulation. 109274 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ALESSANDRO ROCHA MILAN DE SOUZA1, Bernardo Queiroz de Carvalho Souza1, Davi da SIlveira Barroso Alves1, Glenda Borges de Lacerda1, Paulo Henrique Godoy1 (1) Universidade Federal do Estado do Rio de Janeiro – Federal University of the Rio de Janeiro State Introduction: Cerebrovascular diseases (CBVD) and cardiovascular diseases represent the leading cause of death in the world. In Brazil, these diseases correspond to 32% of deaths per year. Objective: To analyze the trend of causes of death due to ischemic CBVD (ICBVD) and hemorrhagic CBVD (HCBVD) in Brazil, from 2000 to 2019. Method: Ecological study, in which the data of deaths due to CBVD, according to ICD 10, were obtained from the Mortality Information System. The codes for CBVD were divided into: ICBVD, HCBVD and CBVD not specified as ischemic nor hemorrhagic (NSCBVD). The age groups in years were divided as 20–39, 40–59, 60–79, and 80 or above. Crude and standardized rates were estimated according to sex and age group. The variation in mortality rates was also estimated, comparing 2000 and 2019. Results: There were 1,766,788 deaths from CBVD in the period. The following distribution by causes was observed: ICBVD – 362,666; HCBVD – 409,079; NSCBVD – 1,009,927. The rates in all causes were higher in males. In the HCBVD higher death rates were observed in younger age and in the ICBVD in the older age. Stability trend was observed in the ICBVD, with a small decrease in females and a small increase in males, and a decrease trend in HCBVD and NSCBVD for both sexes. Conclusion: In general, the trend of deaths from ICBVD and HCBVD seems to be decreasing in Brazil, for the period studied, although this trend is lower in ICBVD. 109281 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CLAUDIO QUERIDO FORTES1, Natália Rodrigues Querido Fortes3, Plínio Resende Jr4, Juliano Carvalho G. de Almeida4, Roberto Muniz Ferreira4, Ana Claudia Pinto de Figueiredo Fontes4, Luiz Felipe de Abreu Guimarães4, João Roquette Fleury da Rocha4, Vlander Gomes da Costa Jr.4, Marina da Costa Carvalheira4, Ronir Raggio Luiz2, Mauro Paes Leme de Sá5 (1) Hospital Universitário Clementino Fraga Filho HUCFF/Universidade Estácio de Sá UNESA; (2) Instituto de Estudos em Saúde Coletiva IESC; (3) Hospital Universitário Antonio Pedro HUAP/Hospital Universitário Clementino Fraga Filho HUCFF; (4) Hospital Universitário Clementino Fraga Filho HUCFF; (5) Hospital Universitário Clementino Fraga Filho HUCFF/Instituto do Coração Edson Saad ICES Background: Infective endocarditis (IE) is one of the most dreaded infectious complications in hemodialysis (HD) patients. Methods: Descriptive analysis of HD and non-HD patients with IE. Results: Of the 505 patients (540 episodes) admitted to university hospital between 1978–2021 with definite IE according to the modified Duke criteria, 54 patients (57 episodes) had undergone HD and 451 (483 episodes) had not. Vascular access for HD was central catheter in 75.4% and 49,1% had arteriovenous fistula but some of them with fistula failure. The mean age of HD patients was not statistically different from that of non-HD patients (47.5 vs 43.3, p 0.117). More female gender (57.9% vs. 34.6%, p = 0.001) was observed in HD patients. Diabetes mellitus was significantly more frequent in the HD-patients (36.8% vs. 6.6%, p < 0.001), while intravenous drug use (0% vs 13.9%, p 0.029) and prosthetic valve (7.0% vs 20.7%, p 0.013) were more commonly in non–HD-patients. The mitral valve was the most affected (50.9% vs 51.1%, p 0.773), followed by aortic valve (38.6% vs 43.1%, p 0.416) and tricuspid valve (19.3% vs 13.3, p 0.212). The proportion of Enterococcus spp. was significantly higher in HD group than in non-HD group (33.3% vs. 5.4%, p < 0.001). Staphylococcus aureus was the second most frequent one (29.8% vs 22.0%, p 0.183). Valve replacement for active IE was less frequently performed among HD patients but without statistical significance (35.1% vs 42.2%, p 0.300). In-hospital mortality was significantly higher in hemodialysis than in non-hemodialysis patients (52.6% vs. 37.7%, p 0.030). Conclusions: IE is a serious complication in HD patients. Enterococcus spp. is the most common causative organism in this group. Mortality is very high and significantly higher than in non-HD patients. 109299 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PEDRO GABRIEL MELO DE BARROS E SILVA1, Renato Delascio Lopes4, Charlene Troiani5, Rodrigo Pinto Pedrosa6, Marcelo Nakazone7, Sérgio Luiz Zimmermann8, Rodrigo Morel9, Ricardo Reinaldo Bergo10, Dalton Bertolim Précoma11, Lucas Tramujas1, Ricardo Pavanello1, Eduardo Ramacciotti12 (1) IP-Hcor; (2) Brazilian Clinical Research Institute; (3) Hospital Samaritano Paulista; (4) Duke Clinical Research Institute, Durham, United States of America; (5) Hospital Regional de Presidente Prudente; (6) PROCAPE; (7) Fundação Faculdade Regional de Medicina São José do Rio Preto; (8) Clínica Procárdio; (9) Hospital Ana Nery; (10) Hospital Santa Lucia – Hospital do Coração de Poços De Caldas; (11) Sociedade Hospitalar Angelina Caron; (12) Loyola University, Chicago, United States of America Background: There is limited contemporary prospective real-world evidence of patients with chronic arterial disease in Latin America. Methods: The Network to control atherothrombosis (NEAT) registry is a national prospective study of patients with known coronary (CAD) and peripheral arterial disease (PAD) in Brazil. A total of 2,015 patients were included among 21 sites from September 2020 to March 2022. The follow-up of all patients was one year by the protocol. Patient characteristics, medications under use and laboratorial data were collected. The primary objective is to assess the utilization of evidence-based therapies (EBT) at baseline. Results: From the total of patients enrolled, 56.6% had isolated CAD, 29.6% had PAD and 13.8% had both diagnoses. The overall mean age was 66.3 (±10.5) years and 65.7% were male patients. The median glomerular filtration rate was 76.4 [57.2–96.1] and 72.3% of the patients had an evaluation of microalbuminuria which was detected in 6.2% of the cases. Regarding EBT, 4.0% were not using any antiplatelet and/or anticoagulant therapy but only 0.9% were using low dose of rivaroxaban (2.5 mg BID); 5.0% were not using statins and 55.6% of the patients were not using high intensity statin therapy; ACE inhibitors or ARBs were used in 76.2% of the overall population while, among patients with isolated CAD, 10.3% were not using betablockers. Among diabetic patients, 67.8% were using metformin and only 12.5% were using SGLT2 inhibitors and/or GLP1 agonists. Regarding the targets for secondary prevention, 33.0% had a body-mass index between 18.5 and 24.9; 44.4% were doing at least 150 minutes of exercise per week; 15.7% continued to smoke; 41.0% had a blood pressure <130 × 80 mmHg; 38.7% and 14.7% had LDL-cholesterol below 70 and 50 mg/dl, respectively. Among diabetic patients, 41.2% had a glycated haemoglobin <7%. Patients with PAD had lower use of EBT and lower percentage of patients on target of risk factors control. Among all cases without use of EBT, the main barrier identified was related to the physician perception that did not consider a formal medical indication of these therapies. Conclusion: Our findings highlight that the contemporary practice still has important gaps in the treatment of patients in secondary prevention, especially among patients with PAD. Populational interventions addressing these gaps have the potential to produce a major impact, reducing the burden of atherothrombotic complications in Brazil. 109312 Modality: E-Poster Researcher – Non-case Report Category: CARDIO-ONCOLOGY FERNANDO PIVATTO JÚNIOR1, Marco Aurélio Lumertz Saffi2, Guilherme Oliveira Magalhães Costa1, Vinícius Henrique Fritsch1, Eduarda Foresti Englert1, Ângela Barreto Santiago Santos2, Géris Mazzutti2, Pedro Emanuel Rubini Liedke2, Andreia Biolo1 (1) Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre-RS, Brazil; (2) Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre-RS, Brazil Introduction: Patients with positive troponin (Tn+) during breast cancer treatment are considered to be at high risk for cardiotoxicity, and cardioprotection with ACE±BB is indicated. Doubts persist about the ideal time to collect Tn. Objective: To compare the incidence of cancer therapy-related cardiac dysfunction (CTRD) in the Tn+/onset of cardioprotection and Tn-/no additional intervention groups. Patients and methods. Prospective cohort including consecutive female patients with HER-2+ early breast cancer who consulted at the institution’s breast cancer outpatient clinic between march/19-march/22. CTRD: drop in LVEF > 10 p.p. to <53% (ASE/EACI). Tn collection was performed together with the lab tests requested by Oncology before the 1st and 2nd cycles of trastuzumab (TTZ), in addition to 3 months after its initiation. Tn+: TnTus ≥ 14 ng/L or TnIus > 15.6 pg/mL. It was not considered as Tn+ if baseline Tn+ without previous cancer treatment or if absence of increase >20% after its beginning. Patients with Tn+ were referred to the institution’s Cardio-Oncology outpatient clinic to begin cardioprotection, as were those with CTRD. Comparison between groups: Fisher’s exact test. P < 0.05 was considered statistically significant. Results: We studied 46 patients, mean age 53.1 ± 13.1 years, 21 (45.7%) in a therapeutic protocol including doxorubicin (ACdd-TH). Regarding risk factors, 21 (45.7%) had a history of smoking, 18 (39.1%) were obese and 15 (32.6%) had hypertension. Of the 138 troponins analyzed, there were 18 (13.0%) Tn+, the majority being detected before the 2nd cycle of TTZ (12/18, 66.7%). Of the total number of patients, 12 (26.1%) had ≥1 Tn+, with the majority (7/12, 58.3%) having only one of the three collected. In the Tn- group (n = 34), only 6 (17.6%) had hypertension and used ACEi/ARB as treatment. The incidence of CTRD was 10.9%, 8.3% in the Tn+ group and 11.8% in the Tn- group (P = 1.0). Conclusions: Although patients with Tn+ had a higher risk of CTRD, there was no difference in the incidence in those with Tn+/onset of cardioprotection in relation to those with Tn-/without additional intervention. This finding suggests that the cardioprotection strategy based on the detection of Tn+ collected together with the lab tests requested by Oncology may have been effective, equating the occurrence of this adverse event between the groups. 109383 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PAULO EDUARDO BALLVÉ BEHR1, Rafael Vianna Behr1, Leonardo Henrique Bertolucci1, Gabrielly Burkhard Vilasfam1, Lara Helena Zortéa1, Luiza Zwan Dutra1, Maiara Both1, Mariana Saadi de Azevedo1, Paulo Ernesto Leães1, Fernando Antônio Lucchese1 (1) Hospital São Francisco – Santa Casa de Misericórdia de Porto Alegre Introduction: Traditional risk factors (RF) are used to predict the probability of cardiovascular events. However, it is still uncertain whether they are able to predict the probability of subclinical atherosclerosis. Therefore, our objective is to evaluate the association between the number of RF and coronary calcification measured by the calcium score (CAC). Methods: Cross-sectional study, including patients seen as outpatients between 2012 and 2020, aged between 45 and 75 years, in primary prevention. To assess coronary calcification, the CAC percentile (PCAC) was used, considering PCAC > 75 as an important calcification. The RF evaluated were: hypertension, diabetes, current smoking, dyslipidemia and family history (FH) for coronary artery disease. Results: 444 patients were included, mean age 59 ± 7 years, 54% female, all Caucasian, 54% hypertensive, 41% dyslipidemic, 9% diabetic, 11% smokers; 59% with FH. Table 1 shows the association between the number of RF and coronary calcification. The higher the number of RF, the higher the prevalence of PCAC > 75 (p < 0.01) and the lower the prevalence of PCAC = 0 (p < 0.01). Compared to patients without RF, the prevalence of PCAC > 75 was 1.86 times higher in patients with 1 risk factor (CI 0.60–5.81), 3.59 times higher with 2 RF (CI 1.20–10.76), 3.89 times higher with 3 or more RF (CI 1.27–11.90). However, even in patients with zero, 1, or 2 RF, significant calcification was observed in 9.1%, 16.9%, and 32.6% of patients, respectively. In multivariate analysis, smoking [PR 1.68 (CI 1.16–2.43)] and FH [PR 1.96 (CI 1.37–2.79)] were independent RF. Conclusion: There was an association between the number of RF and coronary calcification. However, a considerable percentage of patients with none or fewer RF had significant coronary calcification. 109364 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARCOS DANILLO PEIXOTO OLIVEIRA1, Lélio Lemos Pinto Neto1, Ednelson Navarro2, Adriano Caixeta1 (1) Universidade Federal de São Paulo, UNIFESP; (2) Hospital Regional do Vale do Praíba Background: Post-CABG coronary and grafts angiography (CGAG) and interventions (PCI) have historically been performed via classic transfemoral approach. Particularly for those with left internal mammary artery (LIMA) grafts, left standard transradial access (lsTRA) represents a feasible alternative, with significant fewer vascular complications, but it has ergonomic disadvantage for the operator because of the need to bend over the patients, especially in obese ones. Distal transradial access (dTRA) may provide important advantages, including shorter hemostasis and greater patient and operator comfort, mainly for left dTRA (ldTRA). We aim to describe the feasibility and safety of right and left dTRA for post-CABG CGAG and PCI. Material and methods: From February 2019 to February 2022, 151 consecutive post-CABG patients submitted to CGAG and/or PCI via dTRA have been enrolled. Results: Mean patient age was 67.81 years old. Most were male (82.1%) and had chronic coronary syndromes (59.6%). Overall, 40.3% had acute coronary syndromes. Distal RA was successfully punctured in all patients, always without ultrasound guidance. All procedures involving LIMA grafts were done via ipsilateral ldTRA. We had only 9 (6.0%) access site crossovers. Successful dTRA sheath insertion was then achieved in 94% of all patients, mostly (66.9%) via ldTRA and with standard 6Fr sheath (98%). Distal and proximal RA pulses were palpable in all patients at hospital discharge. No major adverse cardiac and cerebrovascular events and no major complications related to dTRA were recorded. Conclusions: dTRA for routine post-CABG CGAG and PCI by experienced transradial operators appears to be feasible. Further randomized and larger trials are needed to assure clinical benefits and safety of this new technique. 109367 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MÁRIO CLAUDIO SOARES STURZENEKERM1, Mauricio Montemezzo2, Dalton Bertolim Précoma2, Paola Gonçalves Moreira de Oliveira1, Bruna Karas1, Ana Carla Dlugosz1, Alice Magro Koscianski1, Larissa Almeida Busnello1, Francielle Nocera Viechineski1, Camilla Mattia Calixto1, Júlia Henneberg Hessman1 (1) Universidade Estadual de Ponta Grossa; (2) Pontifícia Universidade Católica do Paraná Introduction: Cross-sectional studies have widely described the relationship between non-alcoholic fatty liver disease (NAFLD) and atherosclerotic coronary artery disease. A clinical study reported an increased risk for cardiovascular events in NAFLD patients, and this relation was suggested in a retrospective study. However, the role of NAFLD in atherosclerotic disease remains inadequately elucidated. Furthermore, the potential correlation between NAFLD severity and the clinical expression of acute coronary syndrome (ACS) remains undetermined. Purpose: To assess whether there is a correlation between NAFLD severity and ACS severity. Methods: Were selected data of 99 adult patients without previously known coronary artery disease or liver disease, without a history of significant alcohol consumption and other common causes of secondary steatosis, who presented to the emergency room with chest pain between March 2015 and March 2016. The diagnostic criteria for acute myocardial infarction with ST-segment elevation (STEMI) were based on ST-elevation ≥1 mm in ≥2 contiguous leads (2 mm for leads V1 to V3). The acute myocardial infarction without ST-segment elevation (NSTEMI) diagnostic was established in patients who did not meet the criteria for STEMI and who had elevated necrosis markers (creatine kinase-MB isoform and troponin I). Unstable angina (UA) diagnostic was established in patients who did not meet the criteria for STEMI and NSTEMI but had more than three cardiovascular risk factors and typical thoracic pain. The presence of steatosis and its degrees were assessed using ultrasound, and the diagnosis of NAFLD was based on the presence of steatosis and clinical history. Results: UA, NSTEMI and STEMI diagnosis were established in 40, 33 and 26 patients, respectively, and NAFLD was observed in 30%, 66.6% and 76.9% of these patients. The diagnostic of STEMI and NSTEMI were more frequently than UA in NAFLD patients (P < 0.01, p < 0.001). NAFLD diagnosis and its degrees were significantly correlated with the three presentations of ACS (P < 0,001) for both. The STEMI and NSTEMI diagnosis frequency was similar in NAFLD patients. Diabetes, obesity and hypertension were not correlated with NAFLD diagnosis and degree. Conclusion(s): In this study, NAFLD diagnosis and its degrees were significantly correlated with ACS and its severity. Therefore, NAFLD could be considered a potential risk marker for coronary atherosclerotic disease progression and instability. 109382 Modality: E-Poster Researcher – Non-case Report Category: NURSING DALMA ALVES PEREIRA1, Priscila Valverde de Oliveira Vitorino2, Katarinne Lima Moraes3, Vanessa da Silva Carvalho Vila2, Marina Aleixo Diniz Rezende2, Maria Alves Barbosa1, Claudia Regina de Oliveira Zanini1, Virginia Visconde Brasil1 (1) Federal University of Goiás – UFG; (2) Pontifical Catholic University of Goiás – PUC Goiás; (3) Brasília University – UnB Background: Support for self-management of chronic conditions is essential in the development of patient-centered care. The use of integrated care models that focus on the person and not only on the disease represents a viable solution for effective care. The Chronic Care Model is based on the relationship between motivated and informed users, and a proactive and prepared health team. According to this model, support for self-management is associated with significant results, especially in hypertension and diabetes. It includes the Patient Assessment of Care for Chronic Conditions (PACIC) instrument to assess and monitor the integrated care and support for self-management, as perceived by the patient. Aim: To assess the quality of care for people with arterial hypertension and diabetes mellitus treated at a specialized service, according to the Chronic Care Model. Methods: Cross-sectional study, carried out in a reference outpatient clinic of hypertension care in Brazil. Eighty-two people with diabetes and hypertension were evaluated, with a follow-up of at least 5 years. Participants completed the 20-item PACIC, as well as measures of demographic and clinical aspects during the nursing appointment. PACIC has 5 scales and 3 of them assess support for self-management. Higher scores (>3.0) indicate greater involvement in self-management and team support. For analysis, the Mann-Whitney Test, Spearman Test, and a significance level of 0.05 were used. Results: The mean age was 68.98 ± 8.79 years, female (82.93%), and median 4 years of study (IQR 3–8). Most parents (70.73%) did not study. The median time from diagnosis of diabetes was 8 years (IQR 6–12). Less than half of the patients had controlled glycated hemoglobin – HbA1c (34.15%) and blood pressure (36.59%). There was a positive correlation between the time of diabetes diagnosis and the (HbA1c) value. The Overall PACIC mean was 3.4 (IQR 2.8–3.8). The scales scores that map onto self-management support were Patient Activation – 3.0; Problem-Solving/Contextual – 3.0 and Goal Setting/Tailoring – 3.8. Patients with controlled (HbA1c) were significantly different on the Goal Setting/Tailoring scale. Having parents with some schooling was significantly different on both the Overall PACIC and Patient Activation scale. Conclusion: Quality of care was high considering the overall PACIC, but was moderate on scales that assess greater patient participation and involvement in decision-making and treatment plans. 111107 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ROBERTA DA SILVA TEIXEIRA1, Arise Garcia de Siqueira Galil2, Ana Paula Cupertino3, Francisco Cartujano-Barrera3, Fernando Antonio Basile Colugnati2 (1) Instituto Nacional de Cardiologia – INC; (2) Universidade Federal de Juiz de Fora – UFJF; (3) University of Rochester Medical Center – URMC Introduction: E-health technologies such as the Pare de Fumar Conosco software can be effective for smoking treatment. This intervention is a web-based tool for decision-making about quitting smoking, drawn on principles of social cognitive theory, comprising motivational messages, behavioral change support, and pharmacotherapy use. Efficacy and effectiveness evaluations are not enough. Before being adopted in the health system, technologies must be adequately evaluated in the economic sphere. Objective: To evaluate the cost-effectiveness of the Pare de Fumar Conosco software versus the standard of care according to Brazilian Ministry of Health guidelines. Methods: We developed an analytical decision model for engagement in the smoking cessation counseling group and smoking cessation under service providers‘ perspectives and the public health system. We measured costs by primary and secondary sources and a randomized clinical trial for effectiveness. The temporal horizon adopted was one year. The TreeAge Pro Suite 2018 software guided the development of the analytical decision model. The analysis comprised the incremental cost-effectiveness ratio (ICER), deterministic sensitivity analysis, and Monte Carlo probabilistic analysis (1,000 simulations). As the outcomes are intermediary, no willingness to pay threshold was adopted. Results: The software had a lower cost and greater effectiveness than its comparator. ICER for the engagement and smoking cessation were dominant in both perspectives adopted ($ – 464, 125.36 to $ – 58,348.50). Quadrant II (lower cost and higher effectiveness) presented higher percentages (53,6% to 82,5%) on Monte Carlo simulations. Conclusions: Pare de Fumar Conosco software is a cost-saving technology for smoking treatment. This cost-effectiveness analysis provides objective and explicit data that support the decision of health managers in evaluating the adoption of a promising web-based decision tool for smoking cessation. 109401 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT TOMASZ CHWYCZKO1, Edyta Smolis-Bak1, Laura Zalucka1, Agnieszka Segiet-Swiecicka1, Ewa Piotrowicz1, Joanna Was1, Magdalena Niedolistek1, Malgorzata Sobieszczanska-Malek1, Tomasz Zielinski1, Mariusz Kusmierczyk1, Ryszard Piotrowicz1, Rafal Dabrowski1 (1) National Institute of Cardiology, Warsaw, Poland Background: The novel method of comprehensive rehabilitation after LVAD implantation was developed in our institution in order to rehabilitate the growing LVAD patients population. Study group: 47 recent LVAD (32 HeartMate III, 15 HeartWare) recipients (19–68 years, mean 58,7 years, 43 men) participated in the rehabilitation program. 4–5 week program included supervised endurance training on a cycle ergometer (5 times per week). The training was programmed on the basis of the cardiopulmonary exercise test results. Other exercises included: resistance training, general fitness exercises with elements of equivalent and coordination exercises (every day). The hospitalization was followed by 6–8 weeks of individual exercises performed at home. 6-minute walking test (6MWT), cardiopulmonary exercise test (CPET) and prognostic biomarkers of heart failure: NT-proBNP, Galectin-3 and ST2 were investigated at the beginning and at the end of the rehabilitation program. Results: Table 1. An increase of 6MWT distance, higher maximal workload, peak VO2 and upward shift of anaerobic threshold in CPET were observed in all patients. Significant reductions of NTproBNP, ST2 and galectin-3 levels were observed. There were no major adverse events during rehabilitation. Conclusions: Comprehensive novel rehabilitation in LVAD recipients is safe and results in significant improvement of functional tests and biomarkers of heart failure. 109413 Modality: E-Poster Researcher – Non-case Report Category: NUTRITION ELISA ALBERTON HAAS1, Mario Jose Abdalla Saad2, Andrey Santos2, Nicola Vitulo3, Wilson José Fernandes Lemos Junior4, Aline Maria Araújo Martins5, Carolina Raíssa Costa Picossi6, Desiderio Favarato1, Renato Simões Gaspar1, Peter Libby7, Francisco Rafael Martins Laurindo1, Protasio Lemos da Luz1 (1) Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; (2) Department of Internal Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil; (3) Department of Biotechnology, Verona University, Verona, Italy; (4) Faculty of Science and Technology, Libera Università di Bolzano, Bolzano, Italy; (5) Department of Medical Science, University of Brasilia (UnB), Brasilia, Brazil; (6) Institute of Chemistry, University of Sao Paulo, Sao Paulo, SP, Brazil; (7) Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Gut microbiota profile closely relates to cardiovascular diseases through mechanisms including the reported deleterious effects of metabolites, such as trimethylamine-N-oxide (TMAO), highly studied as a diagnostic and therapeutic target. Moderate red wine (RW) consumption is reportedly cardioprotective, but RW effects on gut microbiota and plasma TMAO are not fully understood. We conducted a randomized, crossover, controlled trial involving 42 males, average 60 yr with documented coronary artery disease, comparing 3-week consumption of 250 mL of RW/day, 5 days/week, with an equal period of alcohol abstinence, given adequate washouts. RW consumption significantly remodeled gut microbiota, with a difference in beta diversity. Plasma metabolomic analysis (n = 20) revealed changes after RW consistent with improved redox homeostasis modulation, such as an increase in precursors of riboflavin and ascorbate metabolism. In contrast, plasma TMAO did not differ with the RW intervention and TMAO levels showed a low intra-individual concordance over time. Thus, modulation of the gut microbiota may contribute to the putative cardiovascular benefits of moderate RW consumption. 109418 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ERITO MARQUES DE SOUZA FILHO1, Erito Marques de Souza Filho1, Helena Cramer Veiga Rey2, Rose Frajtag2, Daniela Matos Arrowsmith Cook3, Lucas Nunes Dalbonio de Carvalho1, Antonio Luiz Pinho Ribeiro4, Samuel Santana de Oliveira5, Filipe Silva Neves Nogueira dos Santos5, Jorge Amaral6 (1) Universidade Federal Rural do Rio de Janeiro; (2) Instituto Nacional de Cardiologia; (3) Pró-Cardiaco; (4) Universidade Federal de Minas Gerais; (5) Senai Cimatec; (6) Universidade do Estado do Rio de Janeiro Cardiovascular disease and depression are prevalent. Patients with cardiovascular disease are more depressed than the normal population. Depression is associated with an increased risk of cardiovascular disease (CVD) and death. People with CVD who are also depressed fare worse than patients who are not depressed. Depression’s intensity influences one’s risk of death and other cardiovascular problems. Depression is a multifactorial disease that has a high socio-economic impact. Currently, many patients do not receive adequate treatment or diagnosis. In this context, the present work proposes a Deep Learning model for diagnostic screening of patients in primary care using clinical, laboratory, and sociodemographic data. These data were obtained from a telecardiology project of the Cardiovascular Disease Research Network from 2016 to 2018. Due to the complexity and processing demand, we used a high-performance supercomputer (OGBON – Senai/Cimatec) in the hyperparameter search process, training, and testing the models. We utilized a cross-validation strategy to aim at a better evaluation of the external validation of the model. We designed SMOTE and TOMEK links for data augmentation. We implemented the models in Python and used the Optuna package in searching best parameters. We used the area under the receiver operating characteristic curve, recall, precision, F1-score, and accuracy in models evaluation. Our best model reached, respectively, 0.81, 0.76, 0.88, 0.81, and 0.76. These results indicate that Deep Learning has great potential in terms of screening patients with depression based on data from routine use in primary care. 109428 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RODRIGO DE CARVALHO MOREIRA1, Anny Rodrigues22, Beatriz Leonardo1, Daniel Arabe1, Sandra Wagner1, Beatriz Grinsztejn1, Valdilea Veloso1, Antonio Pacheco3 (1) Evandro Chagas National Institute of Infectious Diseases; (2) Universidade Federal do Rio de Janeiro; (3) Programa de Computação Científica, Oswaldo Cruz Foundation Background: Smoking is highly prevalent in people living with HIV/AIDS (PLHA) producing detrimental effects in different organs and leading to illness. There is limited evidence about pharmacological interventions for treating nicotine dependence in PLHA. We examined if Nicotine replacement therapy (NRT) is an option for smoking cessation and ameliorates vascular health in this specific population. Methods: From December 2019 to October 2021, we prospectively enrolled PLHA who were actively smoking in our center. The primary outcome of interest was to assess the effect of NRT plus counseling on smoking cessation and endothelial function measured by brachial artery flow-mediated dilatation (FMD). Statistical analysis evaluated the change in %FMD (Δ%FMD = %FMD at week12-%FMD at baseline) to test the hypothesis that Δ%FMD would improve among participants who quit smoking compared to those who relapse. To confirm the results, we have run multiple linear regression to account for classical cardiovascular (CV) confounders. Results are presented in medians (interquartile ranges) and percentages. Results: We included 115 participants with median age of 45.5 years (IQR = 36.4–54.8); 22 (20.4%) had hypertension, 9 (8.3%) had diabetes and 30 (27.8%) had dyslipidemia, almost half were smoking 20+ cigarettes/day (41.7%). Individuals were living with HIV for a median of 10.9 years (5.7–17.4) and were on antiretroviral therapy for 8.6 years (3.7–13.6) with median Nadir of CD4 of 307 (153–490.5). Baseline of median brachial artery diameter was 3.6 mm (IQR = 3.2–4.1). Unadjusted analysis showed that years of smoking, younger age and white race were associated with poor %FMD (75th per centile). After 12 weeks 29.6% participants quit smoking. Comparison of Δ%FMF showed that among participants adherent to therapy, there has been an increase in Δ%FMD when compared to those who relapsed (1.11% [0.29–2.93] vs –0.15% [–1.8–0.91], p < 0.001). After adjustment for CV factors, multiple linear regression showed that participants who quit smoking present a mean 2.53 (p = 0.007) points increase in Δ%FMD in comparison to those who continued to smoke. Conclusion: This study provides evidence that a strategy of NRT and counseling is effective for smoking cessation in PLHA and improves their vascular health in a short period of time. This reinforces the importance of the widespread anti-tobacco programs in HIV clinics and the expected impact lowering incidence of future cardiovascular events. 109452 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT TOMASZ CHWYCZKO1, Laura Zalucka1, Agnieszka Segiet-Swiecicka1, Edyta Smolis-Bak1, Ilona Kowalik1, Malgorzata Sobieszczanska-Malek1, Tomasz Zielinski1, Anna Borowiec1, Mariusz Kusmierczyk1, Rafal Dabrowski1, Ryszard Piotrowicz1 (1) National Institute of Cardiology, Warsaw, Poland; (2) National Institute of Oncology, Warsaw, Poland Background: Iron Deficiency (ID, ferritin <100 ng/mL or 100–300 ng/mL with transferrin saturation <20%) is present in up to 70% of LVAD (left ventricular assist device) recipients. It causes severe anemia, impairs exercise tolerance, and may worsen the prognosis. Aim of the Study: To determine, which method of iron supplementation: oral or intravenous, is more efficient in LVAD patients. Methods: In 47 recent LVAD recipients (19–68 years, median age 58.7 yrs, 43 men) iron parameters were investigated. 35 patients (74.5%) were diagnosed with ID and 44 patients (93.6%) had anemia. 27 patients were treated with intravenous iron (ferric carboxymaltose, average dose 1248 mg), 9 pts were treated with oral iron, 11 pts did not receive iron (high ferritin level). Blood morphology and iron management markers: serum iron level, transferrin, ferritin, transferrin saturation – TSAT, were measured before and 3 months after the therapy. Results: Resolution of ID was observed in 19 pts from IV Iron Group (73.1%), and in one patient from Oral Iron Group (12.5%), p = 0.002. 8 pts from no treatment group developed ID. Intravenous repletion was more efficient than oral supplementation in ID resolution: OR = 16.29, 95% CI = 2.25–338.1, p = 0.017. IV treatment was more effective than no treatment strategy in anemia resolution: OR = 18, 95% CI = 3.03–159.4, p = 0.003. Conclusions: The study proves the advantage of intravenous over oral iron treatment in LVAD patients. 109460 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PAULO EDUARDO BALLVÉ BEHR1, Rafael Vianna Behr1, Leonardo Henrique Bertolucci1, Gabrielly Burkhard Vilasfam1, Lara Helena Zortéa1, Luiza Zwan Dutra1, Maiara Both1, Mariana Saadi de Azevedo1, Paulo Ernesto Leães1, Fernando Antônio Lucchese1 (1) Hospital São Francisco – Santa Casa de Misericórdia de Porto Alegre Introduction: It is still uncertain how the family history (FH) for coronary heart disease (CAD) should be assessed: at any age or through the inclusion of an age cutoff. In this work, we evaluated the association between FH at any age and coronary calcium score (CAC). Additionally, we analyzed the influence of the number of family members on the probability of having significant subclinical atherosclerosis. Methods: Cross-sectional study, including patients seen as outpatients between 2012 and 2020, aged between 45 and 84 years, in primary prevention. For FH, parents or siblings were considered, with sudden death, AMI, coronary angioplasty or CABG, at any age. To assess coronary calcification, the CAC percentile (PCAC) was used, with PCAC > 75 being considered important calcification. Results: 509 patients were included, mean age 60 ± 8 years, 54% female, all Caucasian, 55% hypertensive, 36% dyslipidemic, 11% diabetic, 11% smokers; 58% with HF for CAD. The table shows the distribution of PCAC according to FH. Compared to patients without FH, the prevalence of PCAC > 75 was 1.65 times higher in patients with FH (CI 1.20–2.26); 1.59 times higher with a family member with CAD (CI 1.14–2.22); 1.58 times higher with two family members (CI 1.00–2.48); and 2.52 times higher with three or more family members (CI 1.51–4.23). The prevalence of PCAC > 75 with 1 or 2 family members was 31%, while with 3 family members it was 50% (p = 0.053). In addition, the greater the number of family members, the greater the prevalence of PCAC > 0 (p = 0.002). Conclusion: In this study, the presence of FH for CAD was associated with a higher prevalence of significative coronary calcification. In addition, the greater the number of family members, the greater the chance of coronary calcification. 109873 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY SERGIO GUSTAVO TARBINE1, Costantino R Costantini1, Costantino Ortiz Costantini1, Shibata Vinicius1, Marcos Denk1, Rafael Macedo1, Marcio M. Luize1, Everton Cardoso Dombek1 (1) Hospital CArdiologico Costantini Background: The Amplatzer™ and the Watchman™ are dedicated devices for percutaneous left atrial appendage (LAA) occlusion. This is an elective procedure planned to avoid thrombus-embolizaton in patients with atrial fibrillation, unable to use anticoagulation. Objectives: The aim of the study was to describe the initial experience with both devices for percutaneous LAA occlusion. Methods: This is a single-center study of patients undergoing percutaneous LAA occlusion. Inclusion criteria considered a formal contraindication for oral anticoagulation, previous history of stroke due to INR lability, left atrial thrombus in use of NOACs, and patient preference. All procedures were done under general anesthesia and transesophageal echocardiography (TEE) guidance. Transthoracic echocardiography was performed during the first 24hs after the procedure in order to rule out complications. Further follow-up was done with clinical visits and TEE. Results: Between 09–2010 and 10–2021, patients with a mean CHA2DS2-VASC of 4.6 ± 0.8 and Has-bled of 4,5 underwent LAA occlusion with the Amplatzer™ device (24) and the Watchman™ device (8). Both were successfully implanted in 32 patients (100%), 75,1 ± 2,7 yrs old, 75% male, without any procedural stroke or device embolization. TEE showed complete LAA sealing in all patients with no residual leaks. Pericardial effusion needing successful pericardiocentesis in 3 patients. During follow-up, 1 patient had minor retinal embolization and 3 patients died (1: cancer; 2: not related osteomyelitis; 3: chronic renal failure.). Conclusion: In this initial series of patients, both devices showed a good acute and short-term performance considering feasibility and safety regarding the successful implantation rate and the low incidence of complications. 109495 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION WILSON PASCOALINO CAMARGO DE OLIVEIRA1, Fatima Rodrigues Freitas2, Maurício Tavares Costa2, Aline de Oliveira Silva2, Roberto Kalil Filho2, Marco Aurélio Santo3, Raul Cavalcante Maranhão2 (1) Department of Clinical Analysis – FCF-USP; (2) Laboratory of Metabolism and Lipids – InCor; (3) Department of Clinical Analysis – FCF-USP/Discipline of Surgery of the Digestive System at HCFMUSP Introduction: Obesity leads to decrease in HDL-C, which is an independent risk factor for cardiovascular events. Bariatric surgery is an effective treatment for grade III obesity, which leads to decreased LDL-C, triglycerides and increases HDL-C. The impact of bariatric surgery on anti-atherosclerotic functions of HDL, such as transfer of cholesterol from other lipoproteins to HDL was not yet explored. Aim: To evaluate the effects of weight loss by bariatric surgery on the transfer of lipids to HDL and plasma lipids and apolipoproteins (apo) 1 year after bariatric surgery. Methods: Fifteen individuals with grade III obesity (43 ± 6 years, BMI 49 ± 3 kg/m2, 14 women) were evaluated before and 1 year after bariatric surgery. Blood samples were obtained after a 12-hour fast. Lipid transfer to HDL was measured by in vitro assay, using an artificial emulsion labeled with 3H-cholesteryl ester and 14C-cholesterol as lipid donor. Lipids, apo A-I, apo B, glucose, insulin, and C-reactive protein (CRP) were determined by commercial kits. Insulin resistance was estimated by HOMA-IR index. HDL diameter was measured by laser light scattering method. Results: As expected, BMI decreased after surgery (49 ± 6 vs 35 ± 5 kg/m2; p < 0.0001). Total cholesterol (174 ± 43 vs 154 ± 25 mg/dL; p = 0.022), LDL-C (105 ± 34 vs 78 ± 19 mg/dL; p = 0.007), non-HDL-C (128 ± 39 vs 91 ± 19 mg/dL; p < 0.0001), triglycerides (113 ± 39 vs 72 ± 19 mg/dL; p = 0.0012) and apo B (105 ± 27 vs 78 ± 13 mg/dL; p < 0.001) decreased after surgery. On the other hand, HDL-C (46 ± 9 vs 62 ± 10 mg/dL; p < 0.0001), apo A-I (136 ± 24 vs 157 ± 25 mg/dL; p < 0.0029) and diameter of the HDL (8.88 ± 0.39 vs 9.12 ± 0.34 nm; p < 0.05) were increased. CRP (9.77 ± 6.23 vs 1.75 ± 1.90 mg/dL; p < 0.0001) and HOMA-IR index (10.4 ± 12.2 vs 2.07 ± 0.97; p < 0.05) decreased compared to pre-surgical values. Transfer of unesterified (3.64 ± 0.86 vs 3.79 ± 1.05%) and esterified cholesterol (3.42 ± 0.48 vs 3.20 ± 0.52%) to HDL were not changed after surgery. Conclusions: The weight loss 1 year of after bariatric surgery improved plasma lipids, including HDL-C and promoted reduction of insulin resistance and systemic inflammation associated to grade III obesity, and all those changes conceivably contribute to the diminished cardiovascular risk reported in the literature. However, the surgery did not impact the transfer to HDL of both cholesterol forms, which is also involved in atherogenesis. 109526 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY ASSAD MIGUEL SASSINE1, José Carone Filho1, Elisa ito mendes de Andrade1, Ramon Ott Vargas1, José Silva Henrique1, Márcio Luiz Roldi1, Lúcio Pereira Guarçoni1, Dalton Vinicius Menin1, Carlos Alberto Sancio Junior1, Ana Carolina Simões Ramos1, José Carone Junior1, Schariff Moyses2 (1) Hospital Evangelico de Vila Vleha; (2) Instituto de Cardiologia Do Espirito Santo Introduction: Coronary artery bypass grafting (CABG) surgery is an important weapon in the therapeutic arsenal of coronary artery disease. The aim is to perform a safe procedure, with myocardial preservation and a low rate of perioperative complications. The purpose of this study is to evaluate myocardial injury and clinical evolution in patients undergoing CABG in a referral hospital in Espírito Santo state, where intermittent aortic clamping in hypothermia is used as a myocardial protection strategy. Methods: Observational, cross-sectional and prospective study were developed in a referral Hospital in Espirito Santo state. Results: 108 patients were included in the study between April and December 2019. The mean age of patients was 65 years. The mean EuroSCORE II was 2.54 and the mean Society of Thoracic Surgeons (STS) score was 1.54. Most patients were male (62%) and 75.9% had hypertension. Approximately half of patients had diabetes mellitus and dyslipidemia. Less than a fifth of the sample reported prior acute myocardial infarction. Mean cardiopulmonary bypass (CPB) time was 56 minutes and the average period of cross-clamping time was 43 minutes. Cardiac troponin I alterations were associated with death when considered from 6.1 ng/mL on the 1st postoperative day; values >6.1 ng/mL were related to longer CPB and cross- clamping times. Conclusion: The technique of intermittent aortic cross-clamping in CABG surgery proved to be safe, with a low rate of postoperative morbidity and mortality. New studies, including analyzes with a larger sample, should be carried out to better understand this relationship. 109552 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PAULO EDUARDO BALLVÉ BEHR1, Rafael Vianna Behr1, Leonardo Henrique Bertolucci1, Gabrielly Burkhard Vilasfam1, Lara Helena Zortéa1, Luiza Zwan Dutra1, Maiara Both1, Mariana Saadi de Azevedo1, Paulo Ernesto Leães1, Fernando Antônio Lucchese1 (1) Hospital São Francisco – Santa Casa de Misericórdia de Porto Alegre Introduction: The ACC/AHA 10-year Atherosclerotic Cardiovascular Disease Risk Score (ACC/AHA Score) is one of the most used to assess the risk of MI or stroke. However, it was not evaluated whether it is able to identify individuals with greater or lesser coronary calcification. Therefore, our objective is to assess whether the risk categories of the ACC/AHA Score are able to predict coronary calcification. Methods: Cross-sectional study, including patients seen as outpatients between 2012 and 2020, aged between 41 and 69 years, in primary prevention. Patients were divided into the four risk categories of the ACC/AHA Score: low (<5%), borderline (≥5% and <7.5%), intermediate (≥7.5% and <20%) and high (≥20%). Coronary calcification was measured by the Calcium Score (CAC), using absolute values (in Agatston), and CAC ≥ 100 was considered a significative calcification. Results: 518 patients were included, mean age 59 ± 8 years, 52% female, all Caucasian, 54% hypertensive, 59% dyslipidemic, 11% diabetic, 11% smokers. The Table shows the distribution of CAC according to the ACC/AHA risk category. The increase in risk category was associated with a progressively higher prevalence of CAC ≥ 100 (p < 0.01), just as the reduction of category was associated with a higher prevalence of CAC = 0 (p < 0.01). Compared to low-risk patients, the prevalence ratio for CAC ≥ 100 was 2.35 in borderline-risk patients [(CI 1.44–3.82) (p = 0.01)]; 2.88 in intermediate risk [(CI 1.89–4.39) (p < 0.01)] and 4.32 in high risk [(CI 2.73–6.82) (p < 0. 01)]. Conclusion: In a population sample from southern Brazil, we observed that the ACC/AHA Score categories are able to predict the probability of significant subclinical atherosclerosis, assessed by coronary calcification. 109571 Modality: E-Poster Researcher – Non-case Report Category: NURSING ÉRICA SOBRAL GONDIM1, Emiliana Bezerra Gomes1, José Hiago Feitosa de Matos1, Ana Camila Gonçalves Leonel1, Sarah de Lima Pinto1, Amanda da Costa Sousa1, Antônia Elizângela Alves Moreira1, Raynara Augustin Queiroz1, Ana Luiza Rodrigues Santos1, Mariane Ribeiro Lopes1 (1) Universidade Regional do Cariri – URCA Introduction: Critical care units are characterized by the continuous monitoring of physiological parameters essential to the maintenance of life and the immediate conduct of health interventions. Subtle changes in organic functioning variables are indicative of potential risks to homeostasis, which can cause events that culminate in interruption of blood flow to vital organs and cardiorespiratory arrest. Heart rate, the number of beats per minute that determines the effectiveness of blood pumping from the heart to the other organs, is one of the vital parameters in controlling the proper functioning of the cardiovascular system. Although it may indicate a cardiac disorder itself, tachycardia is also present in non-cardiac conditions common in the Intensive Care Unit (ICU), such as delirium, shock, sepsis, anxiety, pain, inadequate management of sedation and respiratory distress. In this context, a nursing guide was developed to approach tachycardias in the ICU, seeking to optimize the time to restore hemodynamic balance and prevent cardiorespiratory arrest. Objective: The present study, therefore, aims to report the experience of an intensive care nurse in the development of an instrument that classifies tachycardias according to intervention priorities and action packages. Method: This is an experience report on the development of nursing technology by the professional, whose 12-year experience in intensive care and immersion in studies on the subject provided subsidies to identify priorities and conduct them properly according to their own professional skills. Results: The elaborate script identified cardiac and non-cardiac causes, directing a direct nursing intervention to each cause and its time of completion according to the required urgency, citing in each intervention the subsequent steps and the professionals who must be activated according to the limits of professional exercise. Conclusions: The development of this technology, based on the technical competence and experience of the professional nurse, followed an approach, although simple, practical and directed to the most prevalent causes in the ICU. The instrument resulting from this perception demonstrates practicality and applicability in routine care and nursing in the prevention of adverse events in intensive care units, optimizing the quality of care provided. 109608 Modality: E-Poster Researcher – Non-case Report Category: NURSING JACQUELINE VAZ ALENCAR1, Caroline Naidon Coelho1, Clarissa G. Rodrigues10, Liliana Fortini Cavalheiro Boll1, Luiza Junqueira Trarbach Lovato2, Renata Póvoas3, Nicole Saldanha de Souza5, Emily Justiniano6, Fernanda Consolim-Colombo7, Katia de Angelis8, Maria Claudia Irigoyen3, Danielle Irigoyen da Costa9 (1) Instituto de Cardiologia da Fundação Universitária de Cardiologia (IC/FUC) – Porto Alegre, RS – Brasil; (2) University of Technology Sydney (UTS) – Austrália, Sidney; (3) Instituto do Coração da FMUSP (InCor) – São Paulo, SP – Brasil; (4) Universidade Nove de Julho (Uninove) – São Paulo, SP – Brasil; (5) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) – Porto Alegre, Rio Grande do Sul – Brasil; (6) Hospital de Clínicas de Porto Alegre (HCPA) – Porto Alegre, RS – Brasil; (7) Universidade Nove de Julho (Uninove) – São Paulo, SP – Brasil; (8) Universidade Federal de São Paulo (Unifesp) – São Paulo, SP – Brasil; (9) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) – Porto Alegre, Rio Grande do Sul – Brasil; (10) Global Research and Innovation Network (GRINN) – Curitiba, Paraná – Brasil Introduction: Multicenter registries that represent the real-world provide important information, but there are few studies describing how to implement it. Objective: To describe the feasibility pilot project of a database on systemic arterial hypertension in children and adolescents in a reference hospital. Methods: Prospective, observational study to document and assess the feasibility of a multicenter registry of systemic arterial hypertension in children and adolescents. 3 steps were performed. Step 1: ethical aspects, contact with the school management, project pitch meeting and team training. Step 2: participants and workflow, variables included, action at school (screening and confirmatory phase). Step 3: evaluation of protocols, variables analyzed. Results: Sample composed of 80 students, average age of 15.73 ± 0.77 years, 77.5% female and 22.5% male. The mean BMI was 23.58 ± 4.11 kg. Students who presented at least two altered measured pressures in the screening phase were: altered BP 42 (52.5%) and normal BP 38 (47.5%). In the Confirmatory Phase: 22 (32.8%) maintained altered BP. These will be monitored at a specialized center. The data was analyzed with the “REDCap” software. After the viability of the Registry, other centers participated through training. Data quality reports were generated for quality control. Conclusions: The description of the methodology makes HASCA possible, enabling other centers to standardize data collection and promote the development of new health technologies, assisting in public policies on systemic arterial hypertension in children and adolescents. 109786 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION FIRMINO HAAG FERREIRA JUNIOR1, Rosa Maria da Costa Simões1, Diany Priscilla Oliveira1, Carolina Vieira1 (1) Hospital Geral de São Mateus Objective: To analyze the accuracy index of electrocardiogram reports performed in Intensive Care, reported by an artificial intelligence platform, compared with reports performed by cardiologists in person. The electrocardiograms were randomly assigned and reported “blind” to the results provided by the artificial intelligence, and then both results were compared. Report: Thirty (30) electrocardiograms of different patients were analyzed, randomly chosen, admitted to an intensive care unit, with a baseline diagnosis of cardiovascular diseases. Among the electrocardiograms analyzed, artificial intelligence was able to accurately diagnose 16 (sixteen) cases of inactive zone/myocardial infarction, 2 (two) cases of ST segment elevation, 2 (two) cases of atrial flutter, 5 (five) cases of atrial fibrillation, 5 (five) cases of diffuse changes in ventricular repolarization, 1 (one) case of left ventricular overload, 1 (one) case of anterior superior divisional hemiblock. Artificial Intelligence was able to identify electrode failures, as it did not consider the report. There was 100% compatibility of the artificial intelligence reports with the reports prepared by the cardiologists in person. Conclusion: The introduction of new technologies such as artificial intelligence producing reports in electrocardiography is of significant importance, due to the reliability of the reports presented, not different when compared with reports prepared by experienced cardiologists in person, being therefore an important tool in helping to physicians in identifying and confirming the diagnosis, especially for non-specialists, often present in emergency services and intensive care units. 109612 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ALEXANDRA CORRÊA GERVAZONI BALBUENA DE LIMA1, Caroline Barreto Cavalcanti1, Paula Fernandes Freitas Lima1, Barbara Cunha Barreto2, Bruno Ramos Carneiro2, Gabriela de Oliveira Silva2, Maria Alice Ramalho Bragatto2, Luís Moreira da Silva de Azevedo Meireles3, Sergio Henrique Rodolpho Ramalho4 (1) North Wing Regional Hospital – Brasilia – Brazil; (2) School of Health Sciences – Brasília – Brazil; (3) MDI Industrial, Salvador – Brazil; (4) Clinical Research Center of the Brasilia of the Dasa Hospitals Network – Brasilia – Brazil Background: Rapid differentiation the cause of dyspnea from other heart or respiratory causes, is very important for choosing an appropriate therapy. Capnography is a non-invasive and accurate method to measure end-tidal carbon dioxide (PETCO2) and can help physicians in some critical situations. Although this is not used in many emergency situations and it is not used routinely in the emergency department, its application is increasing in many emergency situations, such as patients undergoing mechanical ventilation, procedural sedation and analgesia, pulmonary disease, heat failure, shock, metabolic disorder and trauma. Aim: To evaluate the PETCO2 in differentiating heart and/or pulmonary disease from non-heart and non-pulmonary related dyspnea in hospital setting. Methods: This was observational prospective study performed in the North Wing Regional Hospital, Brasilia, Brazil, August 2021–Febuary 2022. 254 Adults, conscious and spontaneous breathing hospitalized individuals were evaluated and divided in four groups of patients: heart disease (heart failure, ischemic heart disease) group 1 (n = 54), pulmonary disease (chronic obstructive pulmonary disease, asthma) group 2 (n = 68), heart and pulmonary disease group 3 (n = 74) and non-heart and non-pulmonary disease group 4 (n = 56). PETCO2 was measured by a portable capnograph, using a nasal catheter, without supplemental oxygen. Results: The groups were similar in age (group 1 62.2 ± 15,7 vs group 2 64.5 ± 18.0 vs group 3 62.0 ± 15.6 vs group 4 66.9 ± 17.4, p = 0.33) and gender (male, group 1 23% vs group 2 26% vs group 3 28% vs group 4 22%, p < 0.82). The PETCO2 was lower and similar in the heart and/or pulmonary groups and higher in non-heart and non-pulmonary group (group 1 29.2 ± 6.9 vs group 28.6 ± 5.7 vs group 3 28.7 ± 5.8 vs group 4 31.2 ± 5.0, p = 0.05). The PETCO2 in non-heart and non-pulmonary disease (area under the curve, 0.63; 95% confidence interval, 0.57–0.71, p = 0.03). Conclusion: The PETCO2 was able to screen individuals without heart and pulmonary disease in treatment of dyspnea in a public hospital. Capnography can be an easy, cost-effective and non-invasive tool to evaluate dyspnea and does not require cooperation of the patient. Other studies are necessary to different heart from pulmonary disease as cause of dyspnea. 109619 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RENATA JUNQUEIRA MOLL BERNARDES1, Julia Barroso1, Andréa Silvestre de Sousa1, Eduardo Schaustz1, João Dario Mattos1, Emiliano Medei1, Olga Ferreira de Souza1, Denilson de Campos Albuquerque1, Ana Cristina Baptista da Silva Figueiredo1, Mariana Tortelly1, Juliana Ferreira1, Gabriel Cordeiro Camargo1 (1) D’Or Institute for Research and Education, Rio de Janeiro, Brazil Background: The pathophysiological mechanisms associated with cardiac symptoms in post-acute COVID-19 are still poorly understood. A high prevalence of myocardial injury, associated with higher in-hospital mortality has been reported and one of the postulated mechanisms is an autoimmune inflammatory response. Cardiovascular magnetic resonance (CMR) is the best non-invasive method for the assessment of myocardial and pericardial inflammation, allowing the detection of edema and fibrosis. AIM To characterize the presence and prevalence of myopericardial inflammation in a population with a high prevalence of cardiac injury, recently discharged from COVID-19 hospitalization. Methods: In this prospective study, 190 hospitalized COVID-19 patients with clinical or laboratory cardiovascular abnormalities were included in a multicentric registry between November 2020 and December 2021. After discharge, all patients were contacted and 48 consented to return for CMR exam. Results: Of 48 included patients, 33 (68.8%) were male, the mean (SD) age was 57.0 (18.9) years, 24 (50%) were hypertensive and 12 (25%) had diabetes. Chronic cardiac disease was reported in 6 (12.5%), asthma in 4 (8.3%), and chronic obstructive pulmonary disease in 3 (6.3%). Thirty-one patients (64.6%) were overweighed and 37 (77.1%) had myocardial injury detected by increased troponin levels. The median (IQR) time interval between hospital admission and CMR was 74 (26–157) days. Myocardial late gadolinium enhancement (LGE) was observed in 20 (41.7%) patients, including 5 with subendocardial, 11 mesocardiac, and 4 with transmural LGE. Patients with transmural and subendocardial LGE were diagnosed with ischemic heart disease (5), hypertrophic myocardiopathy (2), and right ventricular muscular band (1). Fourteen patients (32.6%) had pericardial LGE, and the prevalence of this finding reduced from 41.7% to 30% and 11%, according to the time interval from hospital discharge to the CMR exam (0–3, 3–6, and 6–12 months, respectively). Small pericardial effusion was detected in 11 (25.6%) patients, and the frequency decreased from 37.5% to 10% and 11%. Conclusion: There is a high incidence of post-COVID myopericardial inflammation in patients who presented with cardiac abnormalities during acute illness, particularly myocardial injury. These findings indicate that myocardial injury may be related to late myocardial inflammation and might explain some of the long-term cardiovascular symptoms of COVID-19. 109651 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ANA CARLA DANTAS CAVALCANTI1, Ana Carla Dantas Cavalcanti1, Lyvia da Silva Figueiredo1, Jessica Santos de Souza Leal1, Mellissa Barreto Oliveira Da Silva1, Flavio Luiz Seixas1, Caroline Barboza Braga1, Kelly Maria Augusta Tavares Bentes1, José Paulo de Mello Gomes1, Paula Vanessa Peclat Flores1, Evandro Tinoco Mesquita1 (1) Universidade Federal Fluminense Introduction: Heart failure is a clinical condition associated with morbidity and mortality, and it is necessary to manage the disease, relieve symptoms, prevent hospitalization and reduce mortality. Therefore, the use of mobile applications represents a strategy to optimize care. Objective: To compare self-care, quality of life, and depressive symptoms in patients with chronic heart failure using a mobile app with conventional follow-up. Method: The study was carried out in two phases, the first, a technological development study, and the second, a (pilot) randomized clinical trial. For the application development, the following software was used: Android Studio, Flutter and the Android operating system. Prospective validation was carried out with experts in heart failure through the System Usability Scale (SUS). In the pilot study, patients were divided into two groups. Patients in the intervention group used the application for 30 days, and patients in the control group used the conventional telephone follow-up. Outcomes were assessed using the European Heart Failure Selfcare Behavior Scale, the Minnesota Living with Heart Failure questionnaire, and the Beck Inventory. Data were analyzed using SPSS v.24 and a repeated-measures ANOVA. The study was registered in ReBEC (RBR-2w7wkb) and approved by an Ethics Committee. Results: The “Card.io” application features, as a resource, the sending of notifications and alarms reminding the patient of a certain action defined by the professional. The prospective validation was performed by 39 experts, mostly nurses (89.7%), with a mean age of 33 years, mostly female (82.1%), and 35 (89.7%) residing in Brazil. The application was considered excellent based on the SUS score. The pilot clinical trial was carried out with 42 patients. There were significant differences in the interaction regarding depressive symptoms (p = 0.016), self-care (p = 0.019), and quality of life (p < 0.001). Conclusion: The Card.io application for remote monitoring effectively improves the quality of life of patients and offers an alternative for healthcare professionals, with an innovative and low-cost proposal that can be implemented in healthcare services. 111068 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RICARDO MOURILHE-ROCHA1, Bruno Reznik Wajsbrot1, Marcelo Luiz da Silva Bandeira1, Julia Paulo Mourilhe Rocha1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Eric Costa de Almeida1, Roberta Siuffo Schneider1, Ricardo Mendes Carneiro1, Claudia Lanzillotti Weksler1, Fernando Oswaldo Dias Rangel1, Daniel Xavier de Britto Setta1 (1) HOSPITAL PRÓ-CARDÍACO Background: Measuring the quality of care indicators enable the recognition of weaknesses in health care. Based on this assessment, it will be possible to improve outcomes. Objectives: To evaluate the quality of care based on ventricular dysfunction, length of stay, coronary angioplasty and hospital mortality rates. Materials and Methods: Observational, retrospective, cohort study of 398 patients admitted with a diagnosis of AMI, with and without ST-segment elevation between January 2018 and January 2022; 69.3% male with a mean age of 65 years. Data were analyzed by the SPSS software. Results: They had the following comorbidities: 47.5% dyslipidemia, 77.9% hypertension, 50.5% previous CAD, 41.2% diabetes, 8.5% heart failure, 37.2% stroke, 15.1% coronary artery bypass surgery, 29.6% previous AMI, 10.1% chronic kidney disease, 43.0% smoking, and 10.3% atrial fibrillation. There were 31.2% with STEMI and 68.8% with NSTEMI, with 83.4% in Killip I, 8.4% in II, 3.1% in III, and 5.0% in IV. The median length of stay was 4 days (IQR 3–8) in the overall population, 4 days in NSTEMI and 5 days in STEMI; p = 0.02. The rates of events at discharge in the overall population and subgroups were: hospital mortality = 7.5% (4.7%, NSTEMI and 13.7%, STEMI; p = 0.002), ventricular dysfunction = 34.4% (28.1%, NSTEMI and 49.1%, STEMI; p < 0.001), being mild dysfunction in 15.5%, moderate 12.6% and severe 5.3%. The angioplasty rate was 79.1% (74.4% NSTEMI and 89.5% STEMI; p < 0.001). Conclusions: The mortality rate was higher than predicted by the Killip score. The incidence of ventricular dysfunction was high, but it was not possible to determine how many of these patients presented ventricular dysfunction prior to the event. The length of hospital stay was within the recommended range and the coronary angioplasty rate was also high. 111069 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY ANA INÊS GONZÁLES1, Ana Inês Gonzáles1, Guilherme Michels1, Jackson da Silva Gullo1 (1) Universidade Estácio de Santa Catarina – São José Introduction: Studies on “Long Covid” suggest that long-term persistent clinical manifestations can last even months after infection. In this sense, post-covid rehabilitation programs are essential. Due to the restrictions imposed by the pandemic, carrying out rehabilitation programs in closed environments has become a concern, leaving home rehabilitation as the only option, also called Home Based (HB). Objectives: To verify the benefits of a post-covid-19 cardiopulmonary rehabilitation program in Home Based format. Method: Longitudinal, interventional study, with an accessibility sample, with individuals of both sexes, ≥30 years old, with a clinical diagnosis of COVID-19 infection by the reverse-transcriptase polymerase chain reaction (RT-PCR) method, already completed, and who have remained with cardiopulmonary complications. Patient recruitment occurred publicity on social media and radio. The recruited individuals were submitted to the 30-second sit-and-stand test (30CST) and the 2-minute stationary gait test (2MWT) remotely during the pre- and post-rehabilitation program period. The intervention protocol took place synchronously, in a home-based format, using video calls via Whatsapp app, based on: 1) respiratory kinesiotherapy exercises, 2) aerobic training; 3) resistance training with localized muscle strength exercise, 4) stretching, two days a week for approximately 60 minutes, and individuals performed rehab with the help of tutors. The Borg Subjective Scale was used to monitor the intensity of the exercises, and should remain between 3 and 4 points. Results: Twenty-eight individuals were treated, 12 men (43%) and 16 women (57%), mean age of 50 ± 5 years, who performed 16 ± 1.2 sessions. There was a mean improvement of 8 ± 1.2 elevations (p = 0.002) to 30CST and 10 ± 2 elevations (p = 0.01) of the dominant limb to TME2 in the post-intervention values, with Borg subjective scale to 2MWT falling from 5 to 3 during testing after intervention. Conclusions: A home-based post-covid 19 rehabilitation program promotes improvement in functional capacity, proving to be an applicable and beneficial intervention method for these patients. 110374 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM FERNANDA SAHAR LUCAS VIDAL DOMECG2, Fabricio Braga da Silva1, Marcelo Kalichsztein3, Gustavo Nobre3, José Kezen3, Gabriel Espinosa1, Christiane Prado1, Marcelo Faccio1, Illan Gotlieb3, Ronaldo Leão Lima2 (1) Laboratório de Performance Humana; (2) Universidade Federal do Rio de Janeiro; (3) Casa de Saúde São José Background: Limited exercise capacity (LEC) is a prevalent complaint in COVID-19 survivors. The cardiopulmonary exercise test (CPET) is the gold standard in determining LEC mechanisms. Objectives: To analyze LEC mechanisms in COVID-19 survivors of different clinical severities and compare them with a non-COVID-19 control population. Materials and methods: Cross-sectional analysis of CPET was performed between July/19 and March/21. Patients were divided into 3 groups: mild COVID-19(L-Cov; outpatient treatment); Severe COVID-19(G-Cov; hospital admission) and non-COVID-19(N-Cov; CPET performed before the start of the pandemic). LEC was defined as VO2 <85% of predicted at peak exercise and classified as: cardiocirculatory(CC), ventilatory(VEN), mixed(MIX; a combination of CC and VEN), or aerobic deconditioning(AD). Furthermore, the prevalence of findings suggestive of vascular-pulmonary involvement(Vas-Pul) was compared between the groups. Based on the values of end-tidal CO2 pressure and the ratio between ventilation and CO2 production, patients were classified at the first ventilatory threshold according to the probability of Vas-Pul involvement as non-suspected, suspected, likely, and very likely. Results: 702 patients were included(61.1% men; 52.1 ± 14.3 years), 310(44.2%); 305(43.4%) and 87(12.4%) N-Cov, L-Cov and G-Cov, respectively. Table 1 shows the LEC mechanisms between the groups(χ2 = 3.76; df = 6;p = 0.709). Table 2 shows the probability of Vas-Pul involvement(χ2 = 34.26; df = 6; p < 0.001). In the posthoc analysis, a probable and very likely pattern was higher in the G-Cov group (p < 0.05). Conclusion: AD was the main mechanism of exercise limitation, followed by cardiocirculatory, ventilatory and mixed limitation (CC+Vent.). 110375 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO RAMOS NASCIMENTO1, BRUNO RAMOS NASCIMENTO, Maria Carmo Pereira Nunes1, Nicholas Ollberding2, Allison Hays3, Pranoti Hiremath3, Federico Asch4, Chad League4, Chris Fung5, Laurie LeBouef5, Craig Sable6, Andrea Zawacki Beaton2 (1) Serviço de Cardiologia e Cirurgia Cardiovascular e Centro de Telessaúde do Hospital das Clínicas da UFMG, Belo Horizonte – MG, Brazil; (2) The Heart Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati School of Medicine, Cincinnati – OH, United States; (3) Cardiology, The Johns Hopkins Hospital, Baltimore – MD, United States; (4) The Heart and Vascular, MedStar Washington Hospital Center, Washington – DC, Unites States; (5) Cardiovascular Center, University of Michigan Hospital, Ann Harbor – MI, United States; (6) Cardiology, Children’s National Health System, Washington – DC, United States Introduction: Cardiac involvement impacts prognosis of COVID-19, especially in critically ill patients. We aimed to assess the prognostic value of limited cardiac assessment by bedside triage echocardiography (echo) in patients admitted to emergency departments (ED) in the United States with COVID-19. Methods: Patients admitted in 4 reference US EDs with confirmed COVID-19 underwent triage echo within 72h of symptom onset, with remote interpretation. Clinical and laboratory data, as well as COVID-19 symptoms, were collected. A comprehensive echo protocol, with quantitative assessment, was applied in high-resource units, while low-resource EDs utilized a focused qualitative protocol. Association between echo variables, demographics and clinical data with all-cause mortality and intensive care unit (ICU) admission was assessed; factors significant at p < 0.10 were put into multivariable models. Results: A total of 399 patients were enrolled (137 from low-resource EDs), 41% women, with a mean age of 62 ± 16 years. Mean oxygen saturation on presentation was 92.3 ± 9.2%, and the average number of comorbidities was 2.1 ± 1.7. Compared to survivors, non-survivors were older (68 ± 12 vs. 60 ± 17 years, p < 0.01), had lower oxygen saturation (88.8 ± 12.0% vs. 93.1 ± 8.2%, p < 0.01), were more likely to have a chronic condition (2.6 ± 1.6 vs. 2.0 ± 1.7 comorbidities, p = 0.01) and had lower LV ejection fraction (50.3 ± 19.7 vs. 58.0 ± 13.6, p < 0.01). 101 (25%) patients had moderate/severe LV dysfunction and 131 (33%) had moderate/severe RV dysfunction. Older and lower oxygen saturation were independently associated with death (OR = 2.14 (95%CI 1.06–4.32) and OR = 0.67 (95%CI 0.53–0.85), respectively) and ICU admission (OR = 0.65 (95%CI 0.46–0.94) and OR = 0.48 (95%CI 0.37–0.64)). No echo variables, including LV and RV function, were independent predictors of outcomes. The mortality model comprised of age, sex, count of co-morbid conditions, and oxygen saturation, had a C-statistic = 0.68 and Brier score = 0.14. The inclusion of LV/RV dysfunction, and pericardial effusion did not improve performance (C-statistic = 0.67, Brier score = 0.15). A similar pattern was observed for the ICU admission model. Conclusion: In patients admitted with COVID-19 and undergoing early echo triage, independent predictors of death and ICU admission were age and oxygen saturation. Despite differences in baseline LVEF, no echo variables were independently associated with unfavorable outcomes. 109741 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARCELO SABEDOTTI1, Bibiana Guimarães Maggi2, Gabriel Almeida Krul2, Elisa Rocha Nonemacher2, Rafaela Oliveira Leite2 (1) Unimed Nordeste; (2) Fundação Universidade de Caxias do Sul Introduction: Percutaneous patent foramen ovale (PFO) closure effectively prevents embolic ischemic stroke. However, most studies do not include patients over sixty years of age. This group of patients usually has other causes of ischemic stroke, the main ones including cardiac arrhythmias, small cerebral vessel disease, and atherosclerosis. The present study aims to support the hypothesis that PFO closure is safe in patients over 60 years of age. Objective: To assess the safety of percutaneous PFO closure in patients over sixty years. Methods: A retrospective, single-center cohort study. Patients aged over sixty years who underwent percutaneous PFO closure to prevent recurrence of cerebrovascular events at Hospital Unimed Nordeste do the Rio Grande do Sul, between September 2019 and January 2022 were included. Results: From June 2019 to March 2022, 15 patients were submitted to percutaneous PFO closure (Table 1), with a mean age of 70 ± 7 years, 60% were female. All patients had PFO with ISA, transcranial ultrasound (TU) with more than 100 high-intensity signals (HITS), Holter without atrial fibrillation, and hematological investigation without coagulopathies. As for medications, 53.3% of patients were being treated with Acetylsalicylic Acid (ASA) associated with clopidogrel and 46.7% using anticoagulants being 26% Dabigatran, 13% Apixaban, and 6% Rivaroxaban. Two days before the procedure and three months after, all remained with ASA and clopidogrel. There weren‘t any complications during the procedure. In 30 days, echocardiographic control showed a foramen completely occluded, without a residual shunt. Conclusion: The procedure for closing PFO in patients over 60 years of age is effective and safe. Careful evaluation is important, including shunt quantification by TU, exclusion of coagulopathies, arrhythmias, and atherosclerotic causes of ischemic stroke. A high-risk PFO morphology can also influence the decision to carry out closure. Data from studies in patients over 60 years of age are limited and additional studies are needed to assess the benefits of this treatment as the population grows old. 109758 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ERLON OLIVEIRA DE ABREU SILVA1, Erlon Oliveira de Abreu-Silva1, Angela C. Bersch-Ferreira1, Rachel H. Vieira Machado1, Lucas R. Silva1, Erica R. Ribeiro Sady1, Debora H. Kodama Miyada1, Bernardete Weber1, Aline Marcadenti1 (1) Hcor Research Institute (IP-Hcor), Hcor, São Paulo, São Paulo, Brazil. Background: Differences between sexes regarding therapeutic targets of lipid profile in populations at very high cardiovascular risk are not fully elucidated. Objective: To evaluate the association between sex and therapeutic targets for LDL-cholesterol (LDL-c) and non-HDL-cholesterol (NHDLc) in individuals diagnosed with acute myocardial infarction (AMI). Methods: Cross-sectional analysis with baseline data from a multicenter randomized clinical trial (DICA-NUTS Trial, NCT03728127) conducted among subjects ≥40 years with previous AMI (60 to 180 days). Sociodemographic, clinical and lifestyle data were collected using specific questionnaires. Therapeutic goals of LDL-c (<50 mg/dL) and NHDLc (<80 mg/dL) were defined according to American Heart Association guidelines. Binary logistic regression was used to evaluate potential associations adjusted for confounders. Results: In total, 471 individuals with a mean age of 59.6 ± 9.3 years were evaluated; 72.2% were men, the estimated monthly household income was US$ 180.00, 10.2% were smokers and 61.8% had AMI with ST-segment elevation (STEMI). The prevalence of married individuals was higher among men compared to women (61.5% and 45.8% respectively, P = 0.001), and among women there were higher proportions of high levels of physical activity (35.9% vs. 22.9%, P = 0.003) and from lower social classes (D/E classification) (68.7% vs. 53.2%, P = 0.002). The use of simvastatin was more frequent (52.4%), followed by rosuvastatin (21.4%) and atorvastatin (21%), with no difference between men and women. Among men, 14.4% and 18.2% had LDL-c and NHDL-c concentrations in line with the recommended targets; these prevalences were higher than those observed in women (LDL-c: 6.9% [P = 0.028]; CNHDL: 8.4% [P = 0.007]). After adjusting for age, marital status, social class and physical activity levels, there was an association between male sex and achieving therapeutic targets for NHDL-c (OR 2.04; 95%CI 1.003–4.15), but not for LDL-c (OR 1.84; 95%CI 0.84–4.03). However, being in lower social classes conferred a 74% lower chance of reaching LDL-c targets (OR 0.26; 95%CI 0.07–0.92) and 81% lower chance of reaching NHDL-c targets (OR 0.19; CI95% 0.06–0.59) regardless of age, sex, marital status, and levels of physical activity. Conclusions: There are differences between sexes regarding therapeutic targets for LDL-c and NHDL-c. However, social class appears to be a major determinant. 109784 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT RAFAEL BUARQUE DE MACÊDO GADÊLHA1, Mirela Ávila Litvin1, Lucian Batista de Oliveira1, Cybelle Luza Costa1, Jessica Myrian de Amorim Garcia1, Francisco Bandeira1 (1) Hospital Agamenon Magalhães Introduction: Heart failure (HF) is associated with a higher risk of osteoporotic fractures, with few studies evaluating the factors related to this increase. The trabecular bone score (TBS) is a tool to assess bone microarchitecture, appearing as an independent predictor of fracture risk and gaining greater importance in populations where there is an increased risk of these events even with preserved bone mineral density (BMD). Objectives: To evaluate TBS in elderly patients with HF, comparing it with BMD results and with echocardiographic and body composition parameters. Methodology: Observational, cross-sectional and analytical study involving elderly individuals (≥65 years) diagnosed with HF, followed up in a cardiology service from August 2020 to August 2021. Patients with contact isolation, lipodystrophies or body mass index >37 kg/m² were excluded. All were submitted to dual energy X-ray absorptiometry (DXA) for analysis of body composition, BMD and TBS. Results: Sixty patients were evaluated, with a mean age of 73.4 ± 5.3 years, 51.7% of which were female. The mean left ventricular ejection fraction (LVEF) was 46.5 ± 16.1%, with 40% having reduced LVEF (<40%). BMD showed osteoporosis (T-Score ≤ –2,5) in 38.3% and osteopenia (T-Score between –1.0 and –2.5) in 46.7%, while TBS showed bone degradation (TBS < 1,230) by 35% and partial degradation (TBS between 1.230 and 1.310) by 30%. There was a statistically significant association between some degree of degradation indicated by TBS with lower lumbar spine BMD (0.99 ± 0.22 g/cm² × 1.21 ± 0.24 g/cm²; p < 0.001), femoral neck (0.78 ± 0.15 g/cm² × 0.88 ± 0.08 g/cm²; p < 0.001) and total femur (0.86 ± 0.16 g/cm² × 0.98 ± 0.13 g/cm²; p = 0.006). There was no association between degraded/partially degraded TBS and lower LVEF means (47.49 ± 16.42% × 44.50 ± 15.66%; p = 0.523). Regarding body composition, an association was observed between degraded/partially degraded TBS with lower means of lean mass in the upper limbs (4.47 ± 1.04 kg × 5.16 ± 1.50 kg; p = 0.041). Conclusion: Low BMD and changes in bone microarchitecture are frequent in elderly patients with HF. Degradation of bone microarchitecture was associated with a reduction in appendicular lean mass. 109799 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION FIRMINO HAAG FERREIRA JUNIOR1, Rosa Maria da Costa Simões1, Diany Priscilla Oliveira1, Carolina Vieira1 (1) Hospital Geral de São Mateus Objective: To analyze the accuracy index of electrocardiogram reports performed in Intensive Care, reported by an artificial intelligence platform, compared with reports performed by cardiologists in person. The electrocardiograms were randomly assigned and reported “blind” to the results provided by the artificial intelligence, and then both results were compared. Results: Thirty (30) electrocardiograms of different patients were analyzed, randomly chosen, admitted to an intensive care unit, with a baseline diagnosis of cardiovascular diseases. Among the electrocardiograms analyzed, artificial intelligence was able to accurately diagnose 16 (sixteen) cases of inactive zone/myocardial infarction, 2 (two) cases of ST segment elevation, 2 (two) cases of atrial flutter, 5 (five) cases of atrial fibrillation, 5 (five) cases of diffuse changes in ventricular repolarization, 1 (one) case of left ventricular overload, 1 (one) case of anterior superior divisional hemiblock. Artificial Intelligence was able to identify electrode failures, as it did not consider the report. There was 100% compatibility of the artificial intelligence reports with the reports prepared by the cardiologists in person. Conclusion: The introduction of new technologies such as artificial intelligence producing reports in electrocardiography is of significant importance, due to the reliability of the reports presented, not different when compared with reports prepared by experienced cardiologists in person, being therefore an important tool in helping to physicians in identifying and confirming the diagnosis, especially for non-specialists, often present in emergency services and intensive care units. 109805 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION CLAUDIA FETTER1, Maria Cláudia Irigoyen3, Liliane Appratto de Souza1 (1) Instituto de Cardiologia do Rio Grande do Sul; (2) TECNOPUC/RS; (3) Incor/USP CardioBreath is an application for mobile and tablets available at Google Play and Apple Store (only in Portuguese). This application prescibes, teaches and evaluates slow deep breath for health and performance. The main objective of Cardiobreath is to offer to users an option for practicing respiratory exercises at rates slower than individual spontaneous rate in order to increase vagal modulation, decrease arterial pressure, increase strenght and resistance of respiratory muscles and cardiorespiratory fitness. Together these benefits may represent great achievements in health and performance. Handling CardioBreath App allows the user to be in touch with their own biological signals like respiratory rate and heart rate. For slow breath exercises prescription, some biological information like age, gender, weight, height (and body mass index is calculated inside the app), and including other conditions like smoking, sedentary life style, hypertension, diabetes, obstructive sleep apnea and cardiopaty make a score that drive users to their more reccomendable band of respiratory exercise. Users may chose on position: sit, standing or lying. Audio lessons are available about posture and respiratory technique (victoriuous brath/ujjayi pranayama from yoga). The prescription includes basic, intermedium and advanced exercises in order to identify progression stages. There is the option for the user to chose the respiratory exercise rate (cycles per minutes). The respiratory frequencies of exercise may vary from 15 to 2 cycles per minute using a metronome, either visual as bell sign, or audio lesson recorded. Other option is four frequencies of exercise with respiratory hold (or pause), either in inspiratory or expiratory hold, with metronome, bell or audio lesson. Users may follow their progress by a graphic of exercise respiratory rate X time of exercise, and other functionality available is heart rate before and after the exercises, which are also represented in graphic. And the most easy lesson of the application is available at the first screen, is the 17 minutes guided relaxation based on a specific miofascial and visceral relaxation. CardioBrreath was created in 2018 and a second version is being developed through a grant (Programa doutor Empreendedor/FAPERGS/CNPQ SEBRAE) and will be availabe in June. Any other information may be found at www.cardiobreath.com and includes more functionalities and gamification in order to increase adherence of users. 109813 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION FIRMINO HAAG FERREIRA JUNIOR1, Rosa Maria da Costa Simões1, Diany Priscilla de Oliveira1, Carolina Vieira1 (1) Hospital Geral de São Mateus Objective: To analyze the main diagnoses found through the use of an artificial intelligence platform for electrocardiogram reports performed in adult intensive care. Patients were randomly selected from November 2021 to March 2022. Results: Seventy-one (71) electrocardiograms of randomly chosen patients admitted to an intensive care unit with a baseline diagnosis of cardiovascular disease were analyzed. Among the analyzed electrocardiograms, artificial intelligence was able to accurately elaborate the main diagnoses: 13 (thirteen) patients had electrocardiographic alterations compatible with diffuse alteration of ventricular repolarization; 09 (nine) patients had a diagnosis of acute myocardial infarction with ST segment elevation; 12 (twelve) patients had atrial fibrillation; 7 (seven) patients had a diagnosis of subepicardial ischemia and 01 (one) patient had a diagnosis of atrioventricular block. Of the cases analyzed, 28 patients had an electrocardiogram within the normal range. Artificial intelligence was able to identify electrode failures, as it did not consider the report. There was 100% compatibility of the Artificial Intelligence reports with the clinical diagnoses of the patients. Conclusion: In addition to the precise diagnosis that allowed guiding directed procedures in each case, artificial intelligence was significantly important due to the reliability of the reports presented, helping physicians in the identification and confirmation of the diagnosis, becoming an important tool in the approach of critical patients for physicians not specialists working in intensive care units 110123 Modality: E-Poster Researcher – Non-case Report Category: NURSING JULIANA DE MELO VELLOZO PEREIRA TINOCO1, Ana Carla Dantas Cavalcanti1, Bruna Lins Rocha de Padua1, Beatriz Paiva e Silva de Souza1, Tereza Cristina Felippe Guimarães2, Evandro Tinoco Mesquita1 (1) Universidade Federal Fluminense; (2) Instituto Nacional de Cardiologia Background: Heart failure (HF), a syndrome that requires complex therapeutic regimens, is associated with high rates of hospitalization. Transitional care programs are crucial for encouraging patients to practice self-care and for minimizing preventable readmissions. These interventions are essential in promoting self-care for HF hospitalized patients. However, brazilian clinical evidence on patients from The “Sistema Único de Saúde” is still scarce. Objective: To compare the effect of intervention with transitional care versus conventional hospital follow-up on self-care skills, knowledge of the disease, quality of life and depressive symptoms of patients hospitalized with HF. Methods: A blind, randomized clinical trial of 74 patients with HF admitted to two quaternary-care hospitals in Rio de Janeiro was conducted. Criteria for inclusion: age ≥ 18 years; clinical diagnosis of HF, irrespective of etiology; hospitalization for decompensated HF. Criteria for exclusion: hemodynamic instability; neurological/cognitive impairment reported in medical records; participation in previous studies involving educational interventions; perioperative hospitalization; transfer to another hospital; undergoing preparation for heart transplantation. The intervention group (IG) received transitional-care follow-up in the form of five encounters during hospitalization and weekly post-discharge telephone calls for four weeks. The control group (CG) received conventional hospital follow-up. Outcomes scored: self-care, quality of life, knowledge of the disease and depressive symptoms within 30 days post-discharge. Blinding was achieved by designating separate teams for evaluation, randomization, and intervention. Sample size calculation was based on a pilot study of 70 patients. Data were treated with repeated-measures ANOVA and Fisher’s exact test. Results: Thirty days post-discharge, the scores for IG patients, compared with CG counterparts, were higher for self-care maintenance (74.3 vs. 44.2; p < 0.001), self-care confidence (79.3 vs. 56.4; p < 0.001), knowledge of the disease (41.3 vs. 27.5; p < 0.001), and lower for quality of life (42.1 vs. 64.5; p < 0.001). There was no effect on self-care management skills and depressive symptoms. Conclusion: The transitional-care program improved the outcomes self-care maintenance, self-care confidence, knowledge of the disease and quality of life. Study is registred in Brazilian Registry of Clinical Trials (RBR-2dpc6b). 109845 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES RENÉE SARMENTO DE OLIVEIRA1, Renée Sarmento de Oliveira1, Renata Junqueira Moll Bernardes1, Adriana Soares Xavier de Brito1, Paulo Henrique Rosado de Castro1, Martha Valéria Tavares Pinheiro1, Sergio Salles Xavier2, Otacilio da Cruz Moreira2, Flavia Vernin de Oliveira Terzi1, Andréa Silvestre de Sousa1 (1) Instituto D’or de Pesquisa e Ensino; (2) Instituto Oswaldo Cruz/Fiocruz Background: Chagas cardiomyopathy is the most frequent and potentially severe manifestation of Chagas disease, with the highest morbidity among neglected tropical diseases. Although endemic in Latin America, it has become a global problem due to the migration of individuals to non-endemic areas. Sudden death is the main mechanism of death in chronic Chagas cardiomyopathy, being associated with ventricular tachycardia and may even occur in patients in the early stages of heart disease. In addition to fibrosis, persistent inflammation may be implicated in the genesis of arrhythmias. Strategies that identify patients at greater risk of developing complex ventricular arrhythmias in their different mechanisms of presentation would be able to guide the prophylaxis of sudden death more appropriately in chronic Chagas cardiomyopathy. Recent studies showed promising results using radionuclide imaging for the identification of areas of inflammation in the myocardium of patients with non-ischemic cardiomyopathies, such as sarcoidosis. Objective: To correlate the presence of persistent myocardial inflammation with FDG-18F and DOTATOC-68Ga PET-CT with the severity of ventricular arrhythmias and the presence of parasite in patients with chronic Chagas‘ heart disease. Methods: Two groups were included, totaling 24 patients. Group 1 consisted of patients with sustained ventricular tachycardia who required implantable cardioverter-defibrillator (ICD). Group 2 was the comparison group composed of patients at the same stage of Chagas heart disease (B1, B2 and C) but without complex ventricular arrhythmia. All patients underwent FDG-18F and DOTATOC-68Ga PET-CT, Holter, and polymerase chain reaction for trypanosoma cruzi. Results: The presence of the parasite in chronic Chagas cardiomyopathy was higher in patients from the ICD group (66.7%) compared to group 2 (33.3%). There was no statistical difference between the uptake of FDG-18F and DOTATOC-68Ga by PET-CT in both groups. Physiological increased uptake of FDG-18F may have jeopardized the analysis in some patients. Conclusion: The persistence of chronic parasitemia in Chagas‘ heart disease may be implicated in the genesis of arrythmia, increasing the risk of sustained ventricular arrhythmia; however, larger studies are necessary to investigate this association. PET-CT with FDG-18F and DOTATOC-68Ga are not currently recommended as markers or inflammation in Chagas cardiomyopathy. 111116 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE ADELLE CRISTINE LIMA CARDOZO1 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital; (3) Primavera Hospital Introduction: Despite advances in treatment, chronic coronary syndrome (CCS) persists as an important cause of morbidity and mortality worldwide. There is evidence of the influence of the disposition to forgive in the processes of illness and healing of patients with cardiovascular disease, including myocardial ischemia. Objectives: To evaluate the willingness to forgive in patients with CCS and its association with Spirituality/Religiosity. Methodology: This is an observational, cross-sectional, analytical study, whose sample included patients with CCS assisted at the cardiology outpatient clinics of three hospitals in Sergipe. Two scales were applied: DUREL (Duke Religious Index) and the BMMRS (Brief Multidimensional Measure of Religiosity and Spirituality), whose domain number 3 assesses forgiveness. This domain is composed of three items: I) disposition to self-forgiveness; II) disposition to forgive those who offend us; III) belief in God’s forgiveness. Each item has an ordinal scale from 1 to 4, corresponding to responses: Never, Rarely, Often, and Always, respectively. The level of forgiveness is determined from the sum of the three items and ranges from 3 to 12. Student’s t test was used for comparison between the groups, with the significance level set at 0.05. Results: Fifty-three patients with CCS were included, of which 50.9% were female. The mean age of patients was 62.2 ± 10.5 years. Regarding the clinical profile, 81.1% were hypertensive, 80.0% dyslipidemic and 46.2% had diabetes mellitus. Regarding the religious profile, 65.4% were Catholic, 23.1% Evangelical, 5.8% Spiritualist, 1.9% Umbandaist, 1.9% Atheist, and 1.9% believed in God but did not belong to any religion. The patients‘ religiosity was evaluated using the DUREL, and was divided into organizational, non-organizational, and intrinsic religiosity. Patients with high levels of organizational religiosity, non-organizational religiosity, and intrinsic religiosity showed higher levels of forgiveness compared to patients with low levels of religiosity in these categories (11.1 vs. 9.8; p < 0.05) (10.8 vs. 7.0; p < 0.001) (11.0 vs. 8.7; p = 0.001). Conclusions: The results show that CCS patients who have higher levels of religiosity are more likely to forgive themselves, forgive others, and believe in divine forgiveness when compared to patients with lower levels of religiosity. 109864 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY ELLEN HETTWER MAGEDANZ1, João Carlos Vieira da Costa Guaragna2, Luciano Cabral Albuquerque4, Fernanda Lourega Chieza3, Brenda Gonçalves Donay1, Luize Mancuso Silva1, Jasseane De Borba Sparremberger Vitt1, Luiz Carlos Bodanese1 (1) PUCRS; (2) Hospital Divina Providência; (3) Santa Casa de Misericórdia de Porto Alegre; (4) Hospital São Lucas PUCRS Introduction: Stroke is a complication that causes considerable morbidity and mortality during the heart surgery postoperative period (incidence: 1.3 to 5%; mortality: 13 to 41%). Models for assessing the risk of stroke after heart surgery have been proposed, but most of them do not evaluate postoperative morbidity. Objective: The aim of this study was to develop a risk score for postoperative stroke in patients who undergo heart surgery with cardiopulmonary bypass. Methods: A cohort study was conducted with data from 4,862 patients who underwent surgery from 1996 to 2016. Logistic regression was used to assess relationships between risk factors and stroke. Data from 3,258 patients were used to construct the model. The model’s performance was then validated using data from the remainder of the patients (n = 1,604). The model’s accuracy was tested using the area under the receiver operating characteristic (ROC) curve. Results: The prevalence of stroke during the postoperative period was 3% (n = 149); 59% of the patients who exhibited this outcome were male, 51% were aged ≥ 66 years, and 31.5% of the patients died. The variables that remained as independent predictors of the outcome after multivariate analysis were advanced age, urgent/emergency surgery, peripheral arterial occlusive disease, history of cerebrovascular disease, and cardiopulmonary bypass time ≥110 minutes. The area under the ROC curve was 0.71 (95% confidence interval 0.66–0.75). Conclusion: We were able to develop a risk score for stroke after heart surgery. This score classifies patients as low, medium, high, or very high risk of a surgery-related stroke. 110935 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ELISA ALBERTON HAAS1, Elisa Alberton Haas1, Wilson José Fernandes Lemos Junior2, Laura Treu3, Andrey Santos4, Mário José Abdalla Saad4, Francisco Rafael Martins Laurindo1, Protásio Lemos da Luz1 (1) Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; (2) Faculty of Science and Technology, Libera Università di Bolzano, Bolzano, Italy; (3) Biology Department, University of Padova, Italy; (4) Department of Internal Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil Background: Regular physical activity supports cardiovascular disease (CVD) prevention through mechanisms that include: influences on inflammatory profile, on the autonomic nervous system and endothelial modifications. One potentially beneficial and overlooked mechanism of physical activity is its effects on the gut microbiota. Methods: This was a cross-sectional cohort analysis of 39 participants of a larger cohort (registered at ClinicalTrial.gov, NCT03232099), evaluating their baseline characteristics. Participants had established coronary artery disease (CAD), were male, aged 46–69 years, had BMI < 30, were stable and non-symptomatic. The cohort was divided into two groups: 21 physically active (PA) participants and 18 physically inactive (PI). The gut microbiota was evaluated with 16S amplicons, and the level of self-reported physical activity was calculated in metabolic equivalents (MET)-hour/week score (METs.h/wk). Patients were considered physically active when levels of Mets.h/wk were >5. A 3-day food frequency questionnaire obtained nutritional information regarding total calories, macronutrients and micronutrients. Results: The gut microbiota profile of PA and PI individuals were clearly separated by sparse partial least squares discriminant analysis. The main taxa that differed the PA group were Ruminococcaceae_UCG-014 and Coprococcus_2 eutactus, which are associated with reduced visceral adiposity and with butyrate production, respectively. Meanwhile, nutrient consumption was similar in the two groups. Conclusions: In patients with CAD, physical activity was associated with different and putative beneficial gut microbiota profile. These findings indicate that gut microbiota modifications further support the healthy mechanisms promoted by physical activity to reduce CVD. 109902 Modality: E-Poster Researcher – Non-case Report Category: NURSING ADRIANA DA COSTA COELHO1, Dasymar Martins da Silva Lucas1, Milena Preissler das Neves1, Geovane de Kassio Nunes2, Renata Flavia Abreu da Silva3 (1) Federal Hospital for State Servants – HFSE; (2) Hospital Pro Cardiac; (3) Federal University of the State of Rio de Janeiro – UNIRIO Introduction: The oral anticoagulants are widely prescribed drugs for treatment and prophylaxis of thrombotic diseases, and the antagonists of vitamin K, such as Warfarin, the main protagonists of this pharmaceutical class. Anticoagulation therapy requires specific knowledge from the nursing team for its safe management and training in service is necessary for the improvement of a qualified care, and constitutes a foundation between practice performed by the professionals and their instrumentalization. Objective: Describing the validation of an educational strategy for capacitation of the nursing team on the management of oral anticoagulants. Method: Methodological study with elaboration of tools such as word search puzzles and a checklist to be performed by the nursing team in the administration of oral anticoagulants, that was based on an integrative review. The theoretical validation of the products by experts was made by online questionnaire. The content validity index acceptable must be higher than 0.70. Results: The 16 experts agreed to take part in the research and evaluate the tools. The subject’s profiles were: nurses with professional degrees between 14 and 20 years (43,8%) and working with anticoagulants between 8 and 11 years (37,8%); 8 (50%) of them participated in scientific events about oral anticoagulants in the last 5 years. The Content Validity Index by the experts was 0.83. The suggestions were accepted and modified for the final version of the tools. The statements that showed greater agreement (93,4%) were the ones stating the Content, that included nursing care management. The one that had the minor agreement was the item about the Title clarity (73,4%). Despite the high level of agreement, some experts suggested a few improvements to the word search puzzle, such as: replace the “Stop” sign carried by the character for an “Attention” one, and re-elaborate some sentences. Conclusion: The word search puzzle has been theoretically validated, therefore it should be used as an educational strategy for the patient’s care in use of the oral anticoagulation by the nursing team. 109914 Modality: E-Poster Researcher – Non-case Report Category: PHYSICAL EDUCATION CLAUDIA FETTER1, José Antonio Caçapietra Reteguy1, Juliana Bertoletti1 (1) Instituto de Cardiologia do Rio Grande do Sul Introduction: Sedentary lifestyle presents deleterious effects over human health and this condition may be reverted through the regular practice of physical exercise. Regular exercise practice offers benefits beyond physical health, with evidence to the improvement of psychosocial factors such as anxiety, depression and stress. Gyms are places destined to the practice of physical exercises, although most of it does not perform the follow-up of benefits to physical and mental health. Besides that, the strong appeal to the search of the perfect body very prevalent in this environment may exert a reverse effect of dissatisfaction with body image. Method: This pilot study about the viability of follow up of hemodynamic measures (blood pressure and heart rate) psychosocial factors through the DASS-21 (anxiety, depression, stress) and (brazilian scale of silhouette (body image) in gyms recruited 24 sedentary individuals for 4 weeks (men and women) with two weekly sessions of resistance exercises, and evaluated these psychosocial factors in order to verify the efficacy of the exercises and the viability of the follow up. 24 individuals, 12 women with mean age of 51 years and 12 men with mean age of 45 years took part in this research, assessed in one moment (T1) pre wash out of 4 weeks maintaining the sedentary condition, moment T2 immediately before initiating exercise and moment T3 at the end of the exercise intervention. The instruments used for the assessment were: Depression, Anxiety and Stress Scale (DASS-21) and the Brazilian Scale of Silhouettes for Adults. Data analysis was performed by GEE and significance level of p 0,05. Results: The results of the assessments of T3 were significant in relation to T2 for the variables Body Mass Index (BMI), Systolic Arterial Pressure (SAP) and Diastolic (DAS) and the three variables of DASS 21(anxiety, depression, stress) and relation of actual silhouette to body mass index, both for women and men. Conclusions: These results suggest that the practice of two weekly sessions of resisted exercise for 4 weeks may have a beneficial effect over psychosocial factors and that it is viable and recommended to implement the follow up of these variables in the context of gyms. Besides, the difference between actual silhouette and BMI very much closer at moment T3 suggests that the positive effect of exercise plays an important role over body image. Data found about blood pressure are promising but better understanding is needed. 109930 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION FÁBIO TANIL MONTREZOL1, Daniel Bannel2, Helen Jones2, Alessandra Medeiros1, David Low2 (1) Federal University of Sao Paulo; (2) Liverpool John Moores University Ischemic events can cause damage. However, reperfusion can cause more damage. This cluster of injuries are named ischemia/reperfusion injury (IRI). Studies show that short intermittent periods of ischemia can produce beneficial effects, named ischemic preconditioning (IPC), this strategy can reduce the area and injury severity. In humans, IPC have shown reductions on the deleterious effects of an IRI, including in the myocardium. In animal models, previous IPC can preserve systolic function after an IRI. IPC can be applied only remotely (R-IPC) in humans to preserve the myocardium. Then, resistance exercise can be a potential toll to produce such effect. The present study aimed to compare the effects of R-IPC and resistance exercise in lower limbs on the vascular function after a bout of IRI. 13 participants aged 23.61 ± 2.95 (Young group – YG) and 7 participants aged 62.83 ± 2.63 (Elderly group – EG) were evaluated. The protocol was approved by the Liverpool John Moores University (19/SPS/025). Participants showed at the lab in 3 non-consecutive days with interval of at least 72 hours during the morning. The sessions were randomized. Session structure was: participants rested for 20 minutes, then underwent to a brachial artery flow-mediated dilation analysis (FMD), then received the experimental treatment: Control visit rested for 40 minutes, R-IPC visit, cuffs were placed at the medial part of the thighs then inflated for 5 minutes at a pressure of 220 mmHg and deflated for 5 minutes for 4 times, at the Squatting visit performed 5 minutes of squatting without load with cadence of 20 squats per minute and rested for 5 minutes for 4 times. After, for the IRI, a cuff was placed in the humeral midpoint and inflated at 220 mmHg for 15 minutes, after deflation, the reperfusion occurred for 15 minutes. Then, FMD was repeated. 2-factor analysis of variance was performed, p ≤ 0.05. Results are shown in figure 1. We concluded that IRI can produce temporary vascular dysfunction, such dysfunction can be abolished by R-IPC in young adults and in elderly resistance exercise can abolish deleterious effects of IRI. 109955 Modality: E-Poster Researcher – Non-case Report Category: NURSING JACQUELINE VAZ ALENCAR1, Emily Justiniano6, Nicole Saldanha de Souza5, Liliana Fortini Cavalheiro Boll1, Luiza Junqueira Trarbach Lovato2, Renata Póvoas3, Danielle Dias8, Fernanda Consolim-Colombo4, Katia de Angelis8, Maria Cláudia Irigoyen3 (1) Instituto de Cardiologia da Fundação Universitária de Cardiologia (IC/FUC) – Porto Alegre, RS – Brasil; (2) University of Technology Sydney (UTS) – Austrália, Sidney; (3) Instituto do Coração da FMUSP (InCor) – São Paulo, SP – Brasil; (4) Universidade Nove de Julho (Uninove) – São Paulo, SP – Brasil; (5) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) – Porto Alegre, Rio Grande do Sul – Brasil; (6) Hospital de Clínicas de Porto Alegre (HCPA) – Porto Alegre, RS – Brasil; (8) Universidade Federal de São Paulo (Unifesp) – São Paulo, SP – Brasil Background/Introduction: Hypertension is asymptomatic in childhood and adolescence. We used REDCap to organize the Multicenter Registry of Hypertension in Children and Adolescents (HASCA) survey. Nine Teaching/Health/Research, distributed in different regions, participated in data collection at the national level. Purpose: This study analyzes the national data reported from the REDCap platform and optimizes the obtained results. Methods: This is a Longitudinal observational multicenter study of the registry type using the REDCap platform to implement data from 9 national centers, with children and adolescents aged 7 to 18 years, both genders, from public or private schools, with altered blood pressure indicative of hypertension in any level. On the REDCap platform, the input of the data occurred in different stages: – Screening: form for collecting identification data, anthropometric data, and blood pressure values. – Confirmatory: confirmation of blood pressure values. – Registry: one-year follow-up and systematic data collection. The data were collected electronically and inserted in the REDCap software, a safe methodology. The study follows ethical principles and guidelines for good clinical practice. Results: The HASCA Registry CRF consists of a total of 180 questions. Screening data were collected from July 2018 to December 2019, with 4398 Brazilian children and adolescents, of which 915 had high BP at least twice. Conclusions: It is possible to make a report with HASCA information generated throughout Brazil by Regions, Coordinating Centers, or by Schools to identify where and which children have high blood pressure levels and need to be monitored. 109983 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE GILMARA RIBEIRO SANTOS RODRIGUES1, Fernanda Sampaio Novaes2, Gabriella da Cruz Silva Dias2, Cláudio Luiz Anunciação Santos Junior2, Paula Silva Peixoto2, Luciana Feitosa1 (1) Universidade Federal da Bahia; (2) Escola Bahiana de Medicina e Saúde Pública Introduction: Spirituality as an object of study of science in the health area is a recent approach. It consists of an individual’s strategy to transcend the process of illness, losses and existential emptiness. It is anchored in Brazilian culture and is part of common sense, influencing beliefs, behaviors and worldview. It was considered the assumption that there is a possibility of spirituality influencing the adherence to treatment of people with arterial hypertension and diabetes mellitus. Objectives: To identify whether the representations of people with arterial hypertension and diabetes mellitus about religiosity/spirituality can influence non-adherence to treatment and describe the religious/spiritual practices of these people. Method: Qualitative and quantitative exploratory research, carried out at a philanthropic medical center in the city of Salvador, Bahia, Brazil, in 2019. Participants were individuals over 18 years of age with arterial hypertension and diabetes. Data were collected through semi-structured interviews and the Drawing-Story with Theme procedure. A form consisting of three sections was used, the first with an approach to sociodemographic and clinical characterization data. The second with the guiding questions: (1) How does religiosity/spirituality influence adherence to the treatment of arterial hypertension and diabetes mellitus? (2) What religious/spiritual practices do you use related to DM and AH treatment? (3) What kinds of religious/spiritual practices do you think other people use related to DM and AH? (4) What types of religious/spiritual beliefs can interfere with adherence to treatment for AH and DM? (5) Do you consider that there is any religious/spiritual practice that can contribute to non-adherence to the treatment of AH and DM? And the third contains the guidelines for the design of a themed story. Thematic content analysis was used. Results: The data from the interviews were aggregated into two categories: (1) Spiritual/Religious Practices that Assist in Adherence to Treatment and (2) Treatment Dissociated from Faith. Conclusion: There are several representations about spirituality/religiosity and adherence to the treatment of arterial hypertension and diabetes, however, it is clear that people believe in curing the disease and have hope regardless of the conduct they choose for their treatment. 110030 Modality: E-Poster Researcher – Non-case Report Category: DYSLIPIDEMIA ANA CAROLINA MICHELETTI GOMIDE NOGUEIRA DE SÁ1, Elton Junio Sady Prates2, Pedro Cisalpino Pinheiro3, Deborah Carvalho Malta1 (1) Graduate Program in Nursing, Federal University of Minas Gerais (UFMG). Belo Horizonte, MG, Brazil; (2) School of Nursing, Federal University of Minas Gerais (UFMG). Belo Horizonte, MG, Brazil; (3) Faculty of Medicine, Federal University of Minas Gerais (UFMG). Belo Horizonte, MG, Brazil Introduction: The reference values of laboratory tests are defined by two threshold classes, the reference intervals (RI) derived from a healthy population and the clinical decision limits, in which a medical decision is recommended. Lipid reference limits were established by clinical studies of cardiovascular outcomes in which clinical decision limits were defined. It should be noted that lipid reference values were collected from studies in developed countries and lipid levels are influenced by demographic, environmental, genetic, ethnic, lifestyle factors and chronic diseases. Therefore, even having defined decision limits, it has been encouraged to build lipid RIs specific to the population that will be applied. However, determining RI is a challenge because it requires methodological rigor, such as a representative sample of the population and care in collection and analysis. In Brazil, international IR are used. The National Health Survey (PNS) carried out laboratory tests, thus, it was possible to establish, in an unprecedented way by non-parametric methodology, RI of total cholesterol (TC), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) for Brazilians. Objective: To estimate RI of TC and fractions for Brazilian adults. Methods: Cross-sectional study, with data from the PNS, between 2014–2015, in 2,976 adults. To establish RI, exclusion criteria were applied (pregnancy; smoking; chronic diseases), outliers were removed (Tukey’s method) and partitioned (gender; age; race/color). RI were estimated considering 95% of healthy individuals, the lower limit (LL) corresponded to the 2.5th percentile and the upper limit (UL) to 97.5. Differences were evaluated using the Mann Withney and Kruskal Wallis tests (p ≤ 0.05). Results: HDL RI (mg/dL) were higher in women (29–74) than in men (25–67). Men had lower TC and LDL RI (mg/dL) between 20 and 39 years (115–239; 51–151) compared to 40 and 59 years (131–250; 57–156) and 60 years or older (125–252; 64–156). Women had higher RI for TC and LDL (mg/dL) at age 60 years or older (128.5–264; 59–172) and for HDL (mg/dL), UL were higher between 40 and 59 years (29–75) than at 20 to 39 years old (29–74) and in white race/color (29–75) compared to brown (29–73). Conclusion: Own lipid RI evidence the health conditions of Brazilian adults and can support the adequate identification of dyslipidemias and prevention of cardiovascular disease. 110043 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MARTHA V T PINHEIRO1, Renata Junqueira Moll-Bernardes1, Gabriel Cordeiro Camargo1, Marcelo Teixeira de Holanda2, Luiz Henrique Conde Sangenis2, Andrea Silvestre de Souza2 (1) ID’Or; (2) FIOCRUZ; (3) Instituto Nacional de Cardiologia Background: Chagas disease involves progressive myocardial inflammation leading to myocardial fibrosis, which may predispose to sudden cardiac death. Although focal fibrosis can be detected by late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR), this technique is not sensible to detect diffuse interstitial fibrosis. Objectives: The goal of this study was to assess the prognostic value of extracellular volume (ECV) by CMR in predicting ventricular tachycardia (VT) and sudden death in a 4-year follow-up period in Chagas disease. Methods: This prospective cohort study included patients in the early stages of Chagas disease. Myocardial fibrosis assessment by CMR with measurement of ECV was performed. The patients were followed on an outpatient basis and submitted periodically to electrocardiogram and Holter for a period of 4 years. The combined primary outcome was cardiac sudden death, sustained ventricular tachycardia, or cardioverter-defibrillator (ICD) implantation. Results: A total of 47 patients were included; the mean age was 58,6 + 10,4 and 44,7% were male. The ECV maintained an association with the presence of non-sustained VT, a surrogate outcome, even after adjustments for fibrosis mass and left ventricular ejection fraction assessed by CMR. Over the follow-up, 3 patients died suddenly (6,4%) and 2 (4,3%) had sustained VT. These patients had a mean of 45,2 + 21,1 non-sustained VT events (group 1) versus 2,29 + 1,0 (p = 0,001) in the group which not reached the combined endpoint (group 2). In group 1, the mean ECV value was 29,5 + 3,8 and 26,7 + 3,4 (p = 0,04). Conclusion: ECV could be an early marker of increased risk of ventricular arrhythmia in the early stage of Chagas disease, presenting an independent association with NSVT, as a predictor of adverse outcomes. 110045 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY SERGIO GUSTAVO TARBINE1, Costantino R Costantini1, Costantino O Costantini1, Vinicius Shibata1, Marcos Denk1, Rafael Macedo1, Marcio M. Luize1, Everton CArdoso Dombek1 (1) Hospital Cardiologico Costantini Background: The safety and performance of the Absorb Bioresorbable Vascular Scaffold (BVS) has been previously demonstrated with clinical data. However, these trials included patients with simple lesions. The Absorb III trial demonstrated an excess of adverse events following BVS implantation. Aiming to evaluate clinical outcomes, we analyzed the treatment of real world patients using optimal technique and intravascular image guidance in all cases, at long term follow up from a single center. Methods: Observational retrospective study, in a single Brazilian center, from 12/2014 to 12/2017, including 128 patients treated with BVS implantation. Safety and efficacy outcomes were analyzed in the in-hospital and late follow-up (5 years). Results: All 128 patients completed 4 years 3 months follow-up. Mean age was 58,2 years, 85,9% of the patients were men, and 28,1% were diabetic. Regarding clinical presentation, 54,6% had stable angina or silent ischemia. Intravascular imaging (IVUS-OCT) was used in all cases. Lesion preparation included balloon PTCA, and when necessary Cutting balloon and PTCRA. Device success was achieved in 100% of cases with 99,2% overall procedure success rate (1 case of sub acute thrombosis). Long term major adverse cardiovascular events rate were (including hospital stage): Cardiac death 1,5%, acute stent thrombosis 0,78%, MI 2,34%,TVR 16,4%. Conclusions: The analysis of this cohort of pts, in a real world setting with more complex scenarios, showed so far to be safe and effective at late follow-up using an enhanced technique, including intravascular imaging in all cases. Wether these results are durable beyond 5 years will be reported. Keywords: Percutaneous Coronary Intervention, Absorbable Implants/utilization, Everolimus, Coronary Artery Disease, Clinical Evolution. 110059 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANA KAROLINA QUEIROZ DE SOUZA RICARDO1, Natanael Barbosa dos Santos3, Sara Carolline Gomes de Araújo Lima2, Patrícia Caldas de Oliveira1, Eliel Bezerra da Silva Júnior1, Antonio Fernando Barros Pereira Junior4, Isabela de Angelles Floro Alonso1, André Arthur de Souza Lima3, Marcelo Menezes Malta1, Bibiana Toshie Oniki de Mendonça2, Monteiro Pires Bastos Junior3 (1) Hospital do Coração de Alagoas; (2) Centro Universitário Tiradentes de Alagoas; (3) Centro Universitário Cesmac; (4) Universidade Estadual de Ciências da Saúde de Alagoas Introduction: Cardiovascular diseases are important causes of morbidity and mortality worldwide. In 2019, in Brazil, 95,557 deaths were due to acute myocardial infarction, the main etiology. Treatment systems in STEMI aim to improve care and survival; there is a lack of data on their performance. Objective: To evaluate the impact of the institution of a STEMI care program on mortality in the public health network of Alagoas. Methods: Observational, analytical and cross-sectional study, authorized by the CEP (opinion No. 3,283,780), data tabulated between January 2017 and June 2019, analyzed using the Mann-Kendall test, comparison of factors by regression and multiple logistic regression. Census sample of 712 participants. Results: The mean age was 64.3 (±13.1) years and 58.3% were men and 31.3% of the sample was treated; the main cause of non-treatment was symptomatology >12h in 43.5% of the untreated. The prevalence of risk factors (RF) was: 68.3% hypertensive, 36.5% diabetic and 29.4% smokers. The in-hospital death rate was 5.6%. Temporal trends were observed for treated and untreated groups: at time 1, increasing trend for both treated (p < 0.001) and untreated (p = 0.04); while at time 2, there was no increasing or decreasing trend (p > 0.05); at time 3, there is an increasing trend (p < 0.001) for both groups. At time 4, for treated patients, there is a significant decreasing propensity (p = 0.02). Time 5 reinforces a decreasing trend (p = 0.008). Times 6 and 7, no trend was observed. However, at time 8, there is a decreasing trend in the treated group (p = 0.009). At time 9, there is a significant drop in time (p = 0.003). Conclusion: It is evident that the care network is viable and allows better access to STEMI treatment. Hospital mortality was similar to systems in developed countries. There is a need for population education revealed by the significant increasing trend of pre-hospital time. There was an improvement in care for the treatment of STEMI, demonstrated by the significant downward trend at times 4, 5 and 8. In addition, the data contribute to helping the construction of public health policies, as well as guiding prevention strategies, especially for those with RF. 110062 Modality: E-Poster Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LÍLIAN SOARES DA COSTA1, Valerio Fuks4, Maria gabriela Pimenta dos Santos2, Julia Resende de Oliveira2, David Ferreira de Lima Duarte2, Antonio Carlos Eberienos Assad Filho2, Gabriela Gama Zagni Jardim3, Paola Pugian Jardim4, Andréa Vaospasse Cocco Faria4 (1) Universidade Estacio de Sá/IDOMED, Campus Città e Presidente Vargas e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (2) Universidade Estacio de Sá/IDOMED, Campus Città; (3) Universidade Estacio de Sá/IDOMED, Campus Vista Carioca; (4) Instituto Estadual de Cardiologia Aloysio de Castro/IECAC Introduction: Renal denervation (RDN) has been shown to be effective in reducing BP in treatment-resistant hypertension. Ambulatory blood pressure (ABPM) and central blood pressure (BP) are better predictors for overall cardiovascular risk and mortality than brachial BP. Renal denervation (RDN) has been shown to reduce these pressure parameters, but data on central ambulatory BP and arterial stiffness (AS) analysis after RDN are limited and have to be highlighted. Objective: A systematic review on the role of arterial stiffness (AS) in renal denervation (RDN) treated-resistant hypertensive. Methods: PubMed and Scielo databases, using the descriptors “denervation” AND “hypertension” AND “pulse wave velocity”. Twenty-two citations were identified and being considered. Results: Some studies sought to investigate the effect of RDN in patients with treatment-resistant hypertension according to the established definition and confirmed by 24-h ABPM. These data indicate that RDN may reduce office and 24-h ABPM substantially in patients with moderate treatment-resistant hypertension. Central ambulatory BP is reduced and ambulatory assessed averaged daytime pulse wave velocity (PWV) improved after RDN and total vascular resistance decreased. Multivariate analysis showed that short-term effects on PWV were BP-related, whereas during 6 months follow-up, improvement of PWV becomes BP-unrelated. RDN improves peripheral and central blood pressure as well as AS and, thus, may improve cardiovascular outcome. The BP changes were associated with reductions in peripheral resistance, whereas cardiac output, plasma renin, aldosterone levels and renal function remained unchanged. The observed effects were not explained by an increased intake of antihypertensive medications. RDN did not result in a statistical significant effect on end organ damage 12 months after treatment. The statment that the arterial stiffness beneficial was observed during follw-up, assumed the supposition that an improvement of arterial mechanical properties coul be related to a reduced sympathetic arterial drive. Conclusion: Extended assessment of AS can help improve patient preselection for renal sympathetic denervation and identify which subgroup of hypertension patients will benefit by sympathetic modulation. RDN improves brachial and central ambulatory BP, arterial stiffness, and total vascular resistance, indicating an improvement of cardiovascular outcome. 111669 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS PEDRO GABRIEL MELO DE BARROS E SILVA1, Otavio Berwanger2, Renato Nakagawa1, Thiago Macedo3, Lucas Tramujas1, Dalton Precoma4, Oscar Dutra5, John H Alexander6, Christopher B Granger6, Renato D. Lopes3 (1) HCor Research Institute, São Paulo, Brazil; (2) Hospital Israelita Albert Einstein, São Paulo, Brazil; (3) Brazilian Clinical Research Institute, São Paulo, Brazil; (4) Pontificia Universidade Católica do Parana, Curitiba, Brazil; (5) Instituto de Cardiologia Fundação Universitária de Cardiologia do Rio Grande do Sul, Porto Alegre, Brazil; (6) Duke Clinical Research Institute, Duke University Medical Center, Durham, NC, USA Background: In clinical practice, there is large variability in hospital length of stay among cases of acute coronary syndrome (ACS). Even in ACS patients without major complications, the ideal timing for hospital discharge is unknown. Objective: To assess the risk of clinical outcomes within 30 days among patients who did not have a major cardiovascular complication in the first 48 hours after the onset ACS. Methods: SECURE PCI trial and ACCEPT registry enrolled patients admitted with ACS (with or without ST elevation). Patients who had a major cardiovascular event (MACE) within 48 hours were excluded from this analysis since these patients would not have been considered eligible for an early discharge. The rate of MACE between 48 hours and 30 days was described in the overall population and according to the type of ACS and timing of PCI. MACE was defined as myocardial infarction, stroke or death. Multivariable analysis was used to identify factors associated with clinical outcomes. The cases of MACE between 48h and 30 days in the SECURE-PCI trial were further evaluated by source documents and classified according to clinical stability before the occurrence of the events in order to identify patients that indeed would be potentially eligible for early discharge in clinical practice. Results: From the 9,244 patients enrolled in the both studies, we did analyze 8,684 (93.9%) patients who had a confirmed ACS and did not have a MACE within 48 hours. At 30 days, the rate of MACE was 4.1% varying from 2.4 to 4.8% according to type of ACS. In the multivariate analysis, the following variables were associated with the risk of MACE: Age (HR 1.25 per 10 years increment; P < 0.01); Diabetes (HR 1.44; P = 0.02); Type of ACS (STEMI HR 3.56; P < 0.01; NSTEMI HR 1.68; P = 0.049) and PCI within 24 hours (HR 0.36; P < 0.01). The overall rate of death at 30 days was 2.4% but was 0.9% among patients who underwent PCI within 24 hours and were stable in the first 48 hours. Conclusion: Patients with ACS undergoing PCI in the first 24 hours who are stable in the first 48 hours after hospital admission presented low risk of MACE in 30 days (mortality <1%). Advanced age, Diabetes, STEMI and PCI after 24 hours were associated with worse 30-day outcomes among patients without MACE after 48 hours of hospitalization due to ACS. Our findings suggest that early discharge of patients around 48 hours seems safe and reasonable and might save costs and resources utilization. 110086 Modality: E-Poster Researcher – Non-case Report Category: NURSING PAULA VANESSA PECLAT FLORES1, Paula Vanessa Peclat Flores1, Ana Carla Dantas Cavalcanti1, Lyvia da Silva Figueiredo1 (1) Universidade Federal Fluminense – UFF Introduction: Heart Failure is a complex syndrome that demands strict commitment to the therapeutic regimen. In addition to the monitoring strategies that will be used, to achieve better results it is vital to motivate the patient to incorporate new habits into the routine of daily life. Objective: To analyze the effectiveness of the motivational interview in the self-care of patients with chronic heart failure. Method: A randomized, multicenter clinical trial where the intervention group and the control group were followed for 60 days in each of the centers. A total of 130 patients were included, where the intervention group received 3 appointments per motivational interview, with a 30-day interval, while the control group maintained the conventional follow-up in the clinics they attend. The data were evaluated through the Self Care Heart Failure Index 6.2, before and after the intervention, in each of the centers. Among the several statistical calculations performed, the use of mean, median, simple frequency, chi-square, t-test, mann whitney and wilcoxon is highlighted and the effect of the intervention was calculated by Cohen’s d. Results: The global sample (Brazil + Uruguay) has 118 patients, 59 in the control group and 59 in the intervention group. Self-care, evaluated through the sub-scales of maintenance, management and confidence, presented Cohen’s d 0.6723 and p-value (<0.001), indicating medium and significant effect; d of Cohen 0.5086 and p-value (0.187), which means a medium effect but not significant; d of Cohen 0.9877 and p-value (<0.001), showing a high intervention effect with significance. Conclusion: Data from these studies showed that motivational interviewing is effective in self-care of patients with chronic heart failure. Motivational interviewing is a low-cost, effective approach that can be applied by trained professionals who work clinically with patients with heart failure. 110122 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ZAKIR OSSMAN1, Edna Lichucha1, Alcides Munguambe1, Lucio Ribeiro1, Karen Sliwa3, Albertino Damasceno2, Ana Olga Mocumbi1 (1) Instituto Nacional de Saude – INS; (2) Núcleo de Investigação, Departamento de Medicina, Hospital Central de Maputo, Mozambique; (3) Cape Heart Institute, University of Cape Town Introduction: Delivery of regular long-acting intramuscular benzathine penicillin G (BPG) injections is the most effective method for secondary prophylaxis to prevent recurrence of acute rheumatic fever (ARF). Objective: We aimed to assess the adherence rates to BPG regimens for prevention of ARF in a low-income setting. Methods: Between November/2017 and October/2018 we profiled a cohort of patients with RHD on secondary prophylaxis at two hospitals in Mozambique; then we prospectively assessed adherence to secondary prophylaxis using monthly BPG injections. Cultures obtained from throat swabs collected at the 12th month of prophylaxis on 78 patients selected randomly, and were examined by gram stain, catalase test and CAMP test to detect Group A Streptococcus. Results and Discussion: We enrolled 121 patients, mostly adolescents (mean age 20.8 years; SD 5.9) and females (77; 63.6%). Isolated or combined mitral regurgitation was the commonest lesion (107 patients; 88.4%), followed by aortic regurgitation (54; 44.6%) and mitral stenosis (21; 17.4%). The mean follow up was 19 months. The adherence rate was 86.8%, corresponding to 2228 injections applied out of the 2555 expected; only 1708 (66.8%) were administered at the expected date and 59 patients (49%) were fully compliant. Testing for GAS throat colonization in patients randomly selected on month 12 was negative. One fatal event occurred in relation to BPG injection, not fulfilling criteria for anaphylaxis. Conclusions: RHD patients on secondary prophylaxis in urban hospitals in Mozambique had good adherence and acceptable compliance to monthly BPG injections. The incidence of ARF during follow-up was low and no classic anaphylaxis occurred. Further research is warranted to address gaps in delivery of secondary prophylaxis. 110435 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM YAROSLAVSKAYA E.I.1, Migacheva A.V.1, Krinochkin D.V.1, Shirokov N.E.1, Gorbatenko E.A.1, Korovina I.O.1, Osokina N.A.1 (1) Tyumen Cardiology Research Center, Tomsk National Research Medical Center, Russian Academy of Sciences Introduction: Data regarding the influence of arterial stiffness on longitudinal myocardial strain has been scarce. This is especially important for patients who have undergone a complicated course of COVID-19. Objective: To investigate the associations between brachial-ankle pulse wave velocity (baPWV) and left ventricular (LV) longitudinal strain 3 months after COVID-19 pneumonia. Methods: 369 patients (52 ± 11 (from 19 to 84) years; 50.9% women) 3 months ± 3 weeks after discharge after COVID-19 pneumonia were prospectively enrolled into the study. All participants underwent conventional echocardiography, including 2D speckle-tracking echocardiography. Measurements of baPWV were made at the same day as echocardiography in 322 patients. Parameters of LV global longitudinal strain (LV GLS) and segmental longitudinal strain were studied in 296 patients with optimal visualization quality in echocardiography. Both baPWV and LV longitudinal strain were measured in 243 patients. Results: Three months after discharge, obesity was noted in 46.5% of patients, cardiovascular diseases were diagnosed in 73.4%. Arterial hypertension occurred in 71.5% of patients, coronary artery disease – in 22.5%. The median baPWV were 13.3 [11.8; 15.1] cm/s and 13.4 [11.9; 15.1] cm/s for the left and right sides, respectively. Mean LV ejection fraction was 67.8 ± 5.0%, mean LV GLS was –19.6 ± 2.5%. The baPWV showed a weak correlation with longitudinal strain of LV basal level (r = 0.289 for the right side and r = 0.272 for the left side, both p < 0.001) and of LV mid level (r = 0.229, p < 0.001 for the right side and r = 0.218 for the left side, p = 0.001). The baPWV showed a correlation of medium strength only with the longitudinal strain of LV basal anterior segment (r = 0.303 for the right side and r = 0.309 for the left side, both p < 0.001). There were no correlations between baPWV and strain of LV apical level segments. Conclusion: In patients after COVID-19 pneumonia 3 months after discharge baPWV increase associated with deterioration of LV longitudinal strain at basal and mid levels, more pronounced in LV basal anterior segment. 110457 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUIZ GUILHERME PASSAGLIA1, Luiz Guilherme Passaglia1, Carolina Teixeira Cunha Érika1, Erika Nunes de Oliveira Rodrigues1, Flavia Mariana Mendes Diniz1, Darkiane Fernandes Ferreira1, Tiago Almeida de Oliveira2, Maria Augusta Duarte Abreu2, Regina Bicalho Gomes de Faria2, Pedro Henrique Coelho Pinto2, Antonio Luiz Pinho Ribeiro2 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte, MG – Brasil; (2) Faculdade De Medicina da Universidade Federal de Minas Gerais (UFMG) Introduction: In Brazil, low adherence to care guidelines is one of the reasons for the high mortality from acute coronary syndrome (ACS). In view of this, the Brazilian Society of Cardiology (SBC) in partnership with the American Heart Association and with the support of the Ministry of Health developed the Best Practices in Cardiology Program (BPC Program), whose objective is to evaluate the rates of adherence to the SBC guidelines for in institutions of the Unified Health System before and after the implementation of the project. Method: Prospective observational study with data collection from May 2016 to December 2021. The performance measures analyzed were drugs prescribed at hospital discharge. The primary outcome of the study consisted of the evaluation of performance measures with a minimum stipulated target of 85% of global adherence. Results: In this sample, 932 patients were included, with a mean age of 61.6 ± 12.1 years, 69.2% of whom were male, with a low level of education (77.1% with incomplete secondary education or less) and low family income (91.5% with ≤5 minimum wages a month). The main comorbidities were current or previous smoking (67.1%), arterial hypertension (65.7%), diabetes (29.7%), and dyslipidemia (23.0%). In the characterization of ACS, ST-segment elevation infarction corresponded to 68.3%, with Killip classification ≤2 in 75.5% of the sample. Most of the patients (92.2%) underwent coronary angiography, which showed the following pattern of coronary disease: 24.1% trivascular, 16.9% bivascular, 17.5% univascular, with the anterior descending artery being affected in 21.8% of the cases. Coronary angioplasty of the culprit’s vessel was performed in 64.9% of the cases, and only 5.4% of the sample was referred to coronary artery bypass graft surgery. Analysis of performance measures showed aspirin (98.5%), beta-blockers (94.5%), angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers (96.3%) and statins (96.6%) at hospital discharge. There were 27 deaths during hospitalization (2.9%). Discussion: The predominance of males, age ≥ 50 years, and high prevalence of comorbidities show characteristics of ACS patients already known by epidemiological data extensively published in the medical literature. Performance measures indicate good adherence to ACS care guidelines in the hospital. Conclusion: Adherence to quality programs such as the BPC Program is an essential step in improving care for patients with ACS. 110441 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION DIANE XAVIER DE ÁVILA1, Ricardo Vivacqua Cardoso Costa2, Salvador Manoel Serra3, Marcelo Westerlund Montera2, Evandro Tinoco Mesquita1, Alexandre Siciliano Colafranceschi2 (1) Complexo Hospitalar de Niterói; (2) Hospital Pró-Cardíaco; (3) Instituto Estadual de Cardiologia Aloysio de Castro Introduction: Cardiopulmonary exercise testing (CPET) is a well-established clinical tool to predict outcome, stratifying cardiovascular risk and help to select candidates for heart transplantation (HTx) or left ventricular assist devices (LVAD) in patients with chronic heart failure (CHF). Purpose: To evaluate CPET measurements in advanced CHF patients that are being considered for HTx or LVAD and its association to early mortality regardless of the performed procedure. Methods: Maximum intensity CPET was performed on a treadmill and ramp protocol in 73 patients with CHF and reduced ejection fraction, NYHA functional classes III and IV between 2012 and 2021. Measurements derived from CPET were the following: peak V‘O2, V’O2 at the anaerobic threshold (AT), percentage of the V’O2 of the anaerobic threshold in relation to the peak, the slope of oxygen consumption efficiency (OUES), Oxygen uptake efficiency plateau (OUEP), VE/VCO2 slope, maximum heart rate (HR), circulatory power (CP), and the relation (VE/VCO2 slope)/(VO2peak). Results: Seventy-three patients, 75% males. Mean age of 70 ± 9 years. Almost half (47%) had ischemic etiology. There were no complications related to CPET. Ten patients were transplanted, six had an intracorporeal LVAD implanted and the reminder (57 patients) were kept in supervised physical rehabilitation program. There were 12 deaths, 2 in HTx, 2 in LVAD, 8 in the rehabilitation group. Mean follow-up among the survivors was 43 months ± 40,6 and it was 12,1 ± 10,3 months in those who died. CPET derived measurements between survivors and non-survivors, respectively, were as follows: V‘O2 peak (mL.kg–1.min–1): 12,6 ± 4,6 and 8,5 ± 2,8 (p = 0,001); the V’O2 AT (mL.kg–1.min–1): 9,8 ± 3,3 and 6,0 ± 3,0 (p = 0,0004); VE/VCO2 slope: 34,2 ± 12,1 and 67,3 ± 65,6 (p = 0,0002); R peak: 1,1 ± 0,2 and 1,0 ± 0,1 (p = 0,009); T½, in seconds: 136,1 ± 47,8 and 167,9 ± 78,5 (p = 0,03); HR reduction at the first minute 16,7 ± 13 and 7,2 ± 5 (p = 0,009); OUES (L.min–1): 1,2 ± 0,4 and 1,0 ± 0,3 (p = 0,104) and CP [(ml O2.kg–1.min–1).mmHg] 1.530 ± 671,6 and 960,6 ± 363,6 (p = 0.005), OUEP (ML/L): 34,2 ± 8,3 and 25,1 ± 8,3 (p = 0.001) and the relation VE/VCO2 slope and V‘O2 peak were 3,1 ± 2,0 e 11,4 ± 19,5 (p = 0.001). Conclusion: The predisposition to early death could be stratified by V‘O2 peak, V’O2 AT, VE/VCO2 slope, T½, recovery HR, CP, the relation VE/VCO2 slope)/(V‘O2 peak) and notably, OUEP can be more one powerful predictor for early intervention due to its association with severity. 110448 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM PETELINA T.I.1, Musikhina N.A.1, Garanina V.D.1, Avdeeva K.S.1, Sharoyan Y.A.1, Gapon L.I.1, Yaroslavskaya E.I.1 (1) Tyumen Cardiology Research Center, Tomsk National Research Medical Center, Russian Academy of Sciences Introduction: By given pandemic status, the viral infection has shown how SARS-CoV-2 can affect patients involved in other noninfectious epidemics that have been gaining momentum for years. Objectives: To perform a prospective analysis of blood biomarkers in patients with cardiovascular (CV) diseases (CVD) who underwent COVID-19 associated pneumonia with and without type 2 diabetes mellitus (DM2); to assess the nature of their relationship with instrumental parameters and to identify indicators of long-term adverse CV events. Methods: Out of 380 patients with SARS-CoV-2 associated pneumonia participating in the study, we used data of 65 patients. They were divided into 2 groups: group 1 included patients with CVD: arterial hypertension (AH) in combination with coronary artery disease (CAD) without DM2 (n = 45), group 2 – patients with CVD and DM2 (n = 20). Patients were examined at baseline and 3 months after discharge. We evaluated parameters of general blood test; biochemical parameters; instrumental parameters – ABPM, PWV in right and left elastic arteries, echocardiography with the study of left ventricular (LV) apical myocardial strain, computer tomography of lungs. Results: The analyzed leukocyte parameters and their index coefficients – increase in neutrophils/lymphocytes ratio and decrease in lymphocytes/CRP ratio were more significantly changed in DM2 group. Patients in both groups had a significant excess of baseline max CRP concentrations with decrease in parameters after 3 months, but with persistent excess values in group 2 (p < 0.0011). 3 months after discharge patients with DM2 had levels of hs-CRP, IL-1b and TNFa (p < 0.05) that exceeded both the reference values and those in group 1, which reflected the presence of more pronounced vascular inflammatory potential for possible adverse events in this group of patients in post-COVID period. In addition, the exceeding values of NT-proBNP, SBP and DBP 24, PWV-L and apical LV myocardial strain in group 2 compared to group 1 may be indicators of occurrence and progression of adverse CV events in patients with DM2. Conclusions: In groups of patients with AH and CAD, which differ only by the presence of DM2, it is clearly seen that comorbid condition can significantly affect the development of adverse CV complications due to increased inflammatory potential of blood parameters, increased stiffness of the vascular wall, and the presence of myocardial longitudinal strain of LV apical segments. 110901 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ALBEN SIGAMANI1, PALLAV SINGH1, NUMEN CARE TEAM1 (1) NUMEN HEALTH Coronary Artery Disease (CAD) is the number one killer of cardiovascular diseases; yearly, nine-million deaths. CABG was first done in India in 1975; the surgery has grown exponentially, to nearly 100,000 every year. Ischemic cardiomyopathy (IC) and delays in undergoing an evidence-based recommended CABG contribute to significant mortality and morbidity over five years of survival (>35% composite event rate). Cardiac rehabilitation, an evidence-based, effective secondary prevention, is scarce in India; there is just one seat for every 30,000 new IC patients. Numen health is an online digital health platform that organizes virtual cardiac rehabilitation. The program is delivered at the patient’s home via a mobile application. Referrals are received from cardiologists and cardiac surgeons at the time of discharge following a coronary artery event or procedure. On establishing contact and intent to perform cardiac rehabilitation, the patient is subjected to several online tele assessment sessions to help the patient and the care team arrive at a baseline of their health status. The discharge summary of their most recent admission is used for setting Specific Measurable, Achievable, Relevant, and Timely (SMART) Goals. All patients are categorized as per age, gender, known history of pre-existing chronic diseases, medication consumption pattern, physical activity input, potential energy output, detailed analysis of the daily diet, habits, and identification of possible nutritional deficiencies. The program is subscription-based, and the fee is collected either monthly. Between February 2021 and April 2022, 83 patients post CABG have been referred to the program. This represents 10% of the overall number of patients who participated in the program and were referred from 15 cities and 33 doctors across India. They spent a median of 90 days (ranging between 30 to 210 days) on the program and had 600 interactive sessions with the assigned health care manager and other health experts. The age was between 38 and 72 years; 53 (64%) were males. 35(42%) had baseline type 2 diabetes, 8(10%) had significant comorbidity. There were zero deaths, and 35(42%) had achieved their assigned goals. Digital health can bridge the widening gap of providing cardiac rehabilitation to the growing prevalence of IC in India. Virtual cardiac rehabilitation effectively delivers comprehensive secondary prevention to patients post CABG and any coronary event or intervention. 110458 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LUIZ GUILHERME PASSAGLIA1, Luiz Guilherme Passaaglia1, Carolina Teixeira Cunha Érika1, Erika Nunes de Oliveira Rodrigues1, Flavia Mariana Mendes Diniz1, Darkiane Fernandes Ferreira1, Tiago Almeida de Oliveira2, Maria Augusta Duarte Abreu2, Regina Bicalho Gomes de Faria2, Pedro Henrique Coelho Pinto2, Antonio Luiz Pinho Ribeiro2 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte, MG – Brasil; (2) Faculdade De Medicina da Universidade Federal de Minas Gerais (UFMG) Introduction: In Brazil, low adherence to guidelines is one of the reasons for the high mortality from heart failure (HF). In view of this, the Brazilian Society of Cardiology (SBC) in partnership with the American Heart Association, and with the support of the Ministry of Health, implemented the Best Practices in Cardiology Program (BPC Program), whose objective is to evaluate the rates of adherence to the SBC guidelines in institutions of the Unified Health System before and after the implementation of the project. Method: Prospective observational study with data collection from May 2016 to December 2021. The performance measures analyzed were the medications prescribed at hospital discharge and the scheduling of a return appointment. The primary outcome of the study consisted of the evaluation of performance measures with a minimum stipulated target of 85% of global adherence. Results: In this sample, 431 patients were included. The mean age was 59.0 ± 14.6 years, 55.0% were men and 85.4% had a previous diagnosis of HF. The main comorbidities were arterial hypertension (43.4%), atrial fibrillation/flutter (41.1%), diabetes (26.0%), acute myocardial infarction (16.2%), and hypothyroidism (16.0%), with a high rate of current and/or previous smoking (51.0%). The main etiology of HF was Chagas (25.7%), followed by idiopathic (23.0%), ischemic (22.3%), and valvular disease (15.3%). The predominant hemodynamic profile was wet and warm (67.4%), followed by wet and cold (25.0%). At admission, 61 (14.2%) patients underwent heart transplantation. The mean left ventricle ejection fraction was 35.9% (±17.0), and 83 (19.3%) patients died during hospitalization. The analysis of performance measures showed a return appointment schedule (94.6%), beta-blocker (94.8%), angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers (97.3%), and spironolactone (80.9%) at hospital discharge. Discussion: The clinical characteristics of patients with a high prevalence of comorbidities reflect the profile and complexity of patients hospitalized at the hospital and highlight the need for multidisciplinary care. The values of the performance measures indicate good adherence to HF care guidelines, except for the prescription of spironolactone at hospital discharge. Conclusion: Adherence to quality programs such as the BPC Program is an essential step in improving care for patients with HF. 110472 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES EDUARDO MARINHO TASSI1, Emilia Matos do Nascimento1, Marcelo Abramoff Continentino1, Basilio de Bragança Pereira1, Roberto Coury Pedrosa1 (1) Universidade Federal do Rio de Janeiro (UFRJ) In Chronic Chagas Cardiomyopathy (CCC), studies are needed to identify arrhythmogenic risk factors in patients where moderate to severe ventricular dysfunction is not present. The direct relationship between sympathetic neural activity and norepinephrine is known. In patients with heart failure, sympathetic hyperactivity is persistent. However, in CCC, there are indicators that the sympathetic system is heading towards exhaustion as cardiac involvement progresses. Objectives: To verify the dependence between frequent ventricular extrasystoles (FVE), left ventricular ejection fraction (LVEF), extension of fibrosis by cardiac magnetic resonance (CMR) and urinary norepinephrine (NOREPI) measurement in CCC with preserved or minimally compromised LVEF. Methods: The presence of ventricular extrasystoles >720 in 24h was analyzed on Holter. In CMR, the biventricular ejection fraction, presence of segmental alteration and detection and quantification of fibrosis mass using the delayed enhancement technique were evaluated. NOREPI was measured using the Muskiet method. The correlation coefficient matrix was calculated to measure the predictive ability of the variables to predict another, with a significant p-value of p < 0.05. A total of 59 patients were included. Mean age was 57.9 + 10.94 years. FVE was detected in 28 patients. The variable fibrosis showed a negative correlation with the LVEF found (R of –0.61) and with NOREPI (R of –0.68), as well as the variable FVE showed a weak negative correlation with the LVEF (R of –0.33) and to NOREPI (R of –0.27). LVEF, on the other hand, showed a positive correlation with NOREPI (R of 0.83). The variables fibrosis, LVEF and NOREPI showed a high power of association with each other according to Cramér’s V statistics (fibrosis and NOREPI 0.64, LVEF and NOREPI 0.63 and fibrosis and LVEF 0.53). Conclusion: In patients with CCC with preserved or slightly reduced ejection fraction, it is confirmed that the arrhythmogenic substrate is already present in this population, with dependence between NOREPI levels, LVEF and myocardial fibrosis, but not with the presence of FVE (>720 in 24h). 112420 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MIGUEL MALUF1, Miguel Angel Maluf1, Alfredo Eyer Rodrigues1, Maria Eduarda Siqueira1, Ana Fatima Salles1 (1) Universidade Federal de São Paulo; (2) Hospital Alemão Oswaldo Cruz – SP Background: Patients with tetralogy of Fallot, undergoing Right Ventricular Remodeling, in childhood, with or without pulmonary valve reconstruction, evolve, in the late follow-up, with pulmonary insufficiency and Right Ventricular dysfunction, requiring the implantation of a pulmonary prosthesis. The anatomical variations of the pulmonary artery, associated with the presence of calcifications, dilations, or stenosis as a result of surgeries performed, require adequate planning in the surgical approach for Transcatheter Pulmonary Valve Replacement – TPVR. Material: A new Expandable Polyurethane Stent Valve, implanted by catheter, in pulmonary position has been developed and approved in Biocompatibility, Physical, Hydrodynamic, Fatigue, Experimental, and Ultrastructure Study of explanted sheep prostheses after 24 months of follow-up, analysis, following ISO 5840-3, 2015. Method: In a group of 45 adult patients, in the late follow-up of surgical correction of Tetralogy of Fallot, with late follow-up, at São Paulo Federal University, with an indication for TPVR, they were classified into 6 groups according to the anatomical aspects of the pulmonary artery, analyzed by CT Angiography and scheduled minimally invasive procedure intervention, TPVR: Tipe.1: Pulmonary Valve Insufficiency (PVI) Tipe.2: PVI + Pulmonary Trunk (PT) stenosis Tipe.3: PVI + PT aneurysmal dilation Tipe.4: PVI + PT + Right Pulmonary Artery (RPA) + Left PA (LPA) stenosis Tipe.5: Pulmonary prosthesis dysfunction Tipe.6: Valved Conduit (VC) disfunction Through post-processing images by CT Angiography, it was possible to enlarge them to their natural size, followed by 3D printing, inelastic and transparent plastic mass, keeping the interior of the hollow cardiac cavities. Custom prosthesis manufacturing: 3 transverse diameters (TD) are measured: TD1: At the level of the Pulmonary Ring; TD2: In the middle third of the TP and TD3: At the level of the origin of the RPA and LPA. Also, a longitudinal measure (LM), allows for knowing the length of the prosthesis. Conclusion: The strategy for the treatment of residual defects, after correction of the Tetralogy of Fallot, is based on the need to have customized devices to obtain successful and lasting results. 110529 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING FLAVIA VERNIN DE OLIVEIRA1, Gabriel Cordeiro Camargo1, Martha Valério Tavares Pinheiro1, Adriana Soares Xavier de Brito1, Renee Sarmento de Oliveira1, Daniella Braz Parente1, Renata de Mello Perez1, Rosana Souza Rodrigues1, Ana Maria Pitella de Souza Leite1, Gilberto Portela1, Andréa Silvestre de Sousa1, Renata Junqueira Moll Bernardes1 (1) INSTITUTO D’OR DE PESQUISA Introduction: Non-Alcoholic Fatty Liver Disease (NAFLD) is the principal cause of chronic hepatic disease in developed countries, reaching almost 30% of the adult population. It has a large spectrum of histological presentation, from steatosis to cirrhosis. It is a multisystemic disease, affecting a variety of organs, including heart and vascular system, and these are the principal cause of death for these patients, beyond the hepatic disease. Interstitial fibrosis is a factor of worse prognosis for heart and liver, individually, but the correlation between cardiac fibrosis and liver fibrosis has not been studied yet. Purpose: To define the frequency and grade of early myocardial fibrosis in patients with NAFLD and correlate with the intensity of hepatic fibrosis. Methods: Forty-four patients with NAFLD were included; prospectively, the patients performed echocardiogram, myo-intimal carotid measurement by ultrasound, coronary calcium score by computed tomography, cardiac and hepatic magnetic resonance imaging using elastography and T1 mapping sequences; twenty-eight were allocated in the group without fibrosis or with discrete hepatic fibrosis (Group 1) and sixteen patients with significant hepatic fibrosis in Group 2, defined by the magnetic hepatic elastography. Results: The mean age was 57.9 years and 59% were women. The group with significant hepatic fibrosis had also more cardiac interstitial fibrosis, compared with Group 1. The median of myocardial ECV for Group 1 was 22.7% and for Group 2 was 26%, with p = 0.002. The correlation between hepatic and cardiac fibrosis was 0.45, with p = 0.02, even when adjusted for age and body mass index. We also found a negative correlation between hepatic fibrosis and the Global Longitudinal Strain of the left ventricle (r = –0.36 and p 0.016) and with the volume of the left atrium (r = 0.30 and p = 0.04). Conclusion: The study suggests the presence of a correlation between hepatic fibrosis in patients with NAFLD and the presence of diffuse interstitial myocardial fibrosis detected by the cardiac T1 mapping magnetic resonance. 110556 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MARCOS ROBERTO QUEIROZ FRANÇA1, André Assis Lopes do Carmo1, Gustavo de Araújo Silva1, André Dias Nassar Naback1, Reynaldo de Castro Miranda2, Anna Terra França3, Henrique Barroso Moreira2, Vitor Freitas Fontes1, Leandro Garambone de Cerqueira1, Lucas Ruback1, Antonio Luiz Pinho Ribeiro1, Bruno Ramos Nascimento1 (1) Serviço de Cardiologia e Cirurgia Cardiovascular e Centro de Telessaúde do Hospital das Clínicas da UFMG, Belo Horizonte – MG, Brazil; (2) Hospital Felício Rocho, Belo Horizonte – MG, Brazil; (3) Hospital Mater Dei, Belo Horizonte – MG, Brazil Introduction: Catheter ablation is a well-established therapy for atrial fibrillation (AF), with a promising impact on heart failure (HF) outcomes. We aimed to evaluate the impact of AF ablation on echocardiographic and clinical parameters in patients with HF in Brazil, and to assess factors associated with improvement of the left ventricular ejection fraction (LVEF). Methods: Patients diagnosed with HF and LVEF <50%, who underwent radiofrequency AF ablation in 5 high-volume centers, were prospectively enrolled. All patients underwent standard transthoracic echocardiography before the procedure and during clinical follow-up, and the analysis by the examiner was considered. The primary outcome was LVEF normalization (LVEF ≥ 50%) at follow-up. Clinical, echocardiographic, and procedure-related variables associated with the primary outcome were assessed by univariate logistic regression. P-value <0.05 was considered significant. Results: From 2018 to 2022, 77 patients were included, being 52 (68%) males, mean age 66 ± 12 years. Of these, 27 were in NYHA functional class 3/4, 64 (83%) had persistent AF, and only 3 had previous ablations. Pre-procedural LVEF was 38 ± 7%, with 24 (31%) having LVEF < 35%. Complications occurred in only 3 patients (2 in vascular access and 1 endocarditis). In the 13 ± 11 month follow-up, there was a substantial improvement of the LVEF to 54 ± 14% (p < 0.001), and 54 (70%) achieved the primary outcome of LVEF normalization. Fifty-three patients (69%) had LVEF improvement of ≥10%, only 7 remained in NYHA class 3/4, and AF recurred in 13 (17%). Three patients died during follow-up, and none had LVEF improvement. Predictors of LVEF normalization were: pre-ablation LVEF (OR = 1.23, 95%CI 1.11–1.36), baseline left atrial diameter (OR = 0.86, 95%CI 0.79–0.94), ischemic (OR = 0.29, 95%CI 0.08–0.98) and Chagasic (OR = 0.14, 95% CI 0.03–0.78) etiologies and prescription of amiodarone (OR = 0.26, 95%CI 0.09–0.78) and ACEi/ARB (OR = 0.24, 95%CI 0.07–0.81, p = 0.02). In the multivariable model, independent predictors were: baseline LVEF (OR = 1.20, 95% CI 1.06–1.27, p = 0.005), left atrial diameter (OR = 0.88, 95%CI 0.77–0.99, p = 0.03), ischemic (OR = 0.13, 95%CI 0.02–0.85, p = 0.03) and Chagasic (OR = 0.08, 95%CI 0.01–0.76, p = 0.03) etiologies. Conclusion: AF ablation in patients with HF resulted in remarkable echocardiographic and functional improvement, with low complication rates. Less clinical severity and less morpho-functional impairment were associated with LVEF normalization. 110563 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY EVELYN COSTA CRUVINEL1, Aline Teodoro Mendes1, Denise Mayumi Tanaka2, Fernanda Regina Moraes3, Gustavo José Luvizutto1, Marilita Falangola Accioly1, Luciana Duarte Novais Silva1, Douglas Reis Abdalla4, Eduardo Elias Vieira de Carvalho1 (1) Programa de Pós-graduação em Fisioterapia da Universidade Federal do Triângulo Mineiro.; (2) Departamento de Clínica Médica da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo.; (3) Curso de Fisioterapia da Universidade de Uberaba.; (4) Departamento dos Cursos de Saúde do Centro Universitário Talentos Humanos. Introduction: Aging is a physiological process that leads to decreased heart rate variability. Physical training is an established therapy that induces increased heart rate variability, and transcranial Direct Current Stimulation (tDCS) potentiates this beneficial effect. However, a large part of the elderly population does not have access to adequate physical training programs. Instead, meditation is an easily accessible and useful tool to induce small increases in heart rate variability. However, no studies have evaluated the effects of the association of meditation with tDCS on heart rate variability. Purpose: To evaluate the influence of meditation associated with tDCS on heart rate variability in the elderly. Methods: Eleven older adults of both sexes, aged 69.1 ± 7.2 years, were studied. Heart rate variability was collected in the supine position, in a quiet environment, free from the circulation of people and external influences, lasting 80 minutes, with 5 minutes of initial rest to control heart rate, followed by recording the RR intervals at 20 minutes baseline, 15 minutes meditation, 20 minutes tDCS and 20 minutes final/post- tDCS. The meditation technique was mindfulness, applied by a qualified and experienced therapist; tDCS was applied by an anodic current of 2 mA, following the international standards for the system (EEG10–20). Outcomes were analyzed using the ANOVA model with fixed effects. Results: It was observed a significant increase of the heart rate variability indexes with the techniques association: RMSSD from baseline (27.2 ± 35.9 ms) to post-meditation (33.6 ± 43 ms) and post-tDCS (36.7 ± 50.7 ms), p = 0.04; SDNN from baseline (23.8 ± 26.3 ms) to post-medidation (27 ± 28.3 ms) and post-tDCS (31.8 ± 34.5 ms), p = 0.0001; Low-frequency component from baseline (369.6 ± 602.4 ms2) to post-meditation (352.8 ± 599.1 ms2) and post-tDCS (672.2 ± 1034.1 ms2), p = 0.0001; High-frequency component from baseline (207.4 ± 375.5 ms2) to post-meditation (599.7 ± 1068.5 ms2) and post-tDCS (896.8 ± 1958.7 ms2), p = 0.0009; SD1 from baseline (19.2 ± 25.4 ms) to post-meditation (23.8 ± 30.4 ms) and post-tDCS (26 ± 35.9 ms), p = 0.02; e SD2 from baseline (27.4 ± 27.6 ms) to post-meditation (28.9 ± 27.2 ms) and post-tDCS (36 ± 34 ms), p = 0.0001. Conclusion: The results of the present study documented for the first time that the application of anodal tDCS can significantly enhance the beneficial effects of meditation on heart rate variability indexes in the elderly. 110632 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING YAROSLAVSKAYA E.I.1, Krinochkin D.V.1, Shirokov N.E.1, Gorbatenko E.A.1, Osokina N.A.1, Migacheva A.V.1 (1) Tyumen Cardiology Research Center, Tomsk National Research Medical Center, Russian Academy of Sciences Background: COVID-19 can result in myocardial injury in the acute phase. The late cardiac consequences of complicated course of COVID-19 is limited. Purpose: To study the prevalence of disorders and the relationship of left ventricular global longitudinal strain (LV GLS) in patients with proven COVID-19 pneumonia 3 months after discharge from the hospital. Methods: 369 patients underwent a comprehensive clinical examination 3 months ± 3 weeks after discharge after COVID-19 pneumonia. The mean age was 52 ± 11 (from 19 to 84 years), 50.9% women. Standard echocardiography and myocardial strain assessment were performed. LV GLS was studied in 296 (80%) examined patients with optimal visualization quality. LV GLS was considered reduced when its value was above –18%. Results: 3 months after discharge, obesity was noted in 46.5% of patients, cardiovascular diseases were diagnosed in 73.4%. Arterial hypertension (AH) occurred in 71.5% of patients, coronary artery disease – in 22.5%. LV GLS was reduced in 25.1% of the patients. LV GLS showed no relationship with the patient age, NYHA functional class and LV ejection fraction (EF). Reduced LV GLS was independently associated with AH (OR 1.174; 95% CI 1.056–1.305; p = 0.003), obesity (OR 1.295; p < 0.0001), male sex (OR 0.734; 95% CI 0.657 –0.821; p < 0.001). LV GLS showed positive correlations of medium strength with echocardiographic parameters: length of the right (r = 0.308) and left ventricles (r = 0.306), LV mass (r = 0.326), anterior-posterior left atrial (LA) dimension (r = 0.303, all p < 0.0001). LV GLS showed a weak positive correlation with the severity of lung lesions during hospitalization (r = 0.134; p = 0.032), diastolic blood pressure (r = 0.230; p < 0.001), LV posterior wall thickness (r = 0.255, p < 0.001) and interventricular septum (r = 0.188, p = 0.001). Conclusions: Reduced LV GLS 3 months after COVID-19 pneumonia was observed in 25.1% of survivors and is associated with AH, obesity, and male sex. LV GLS was not associated with the patient age, NYHA functional class and LV EF. The correlation between impaired LV GLS and the severity of lung lesions in hospitalization was weak, the correlations with the ventricles length, LV mass, and LA anterior-posterior dimension were of medium strength. 110778 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS ALBERTO FRISOLI JUNIOR1, Elaine Azevedo1, Amanda kimura1, Angela Paes1, Valdir Ambrosio1 (1) Universidade Federal Sao Paulo The diversity of concepts of sarcopenia makes it difficult to use in clinical practice. Low performance on functional tests (functional sarcopenia) has been shown to be associated with severe outcomes, while low muscle mass (anatomic sarcopenia) has controversial results. Functional sarcopenia has phenotypes determined by the accumulation of physical dysfunctions, whose predictive value for mortality is not yet established. Objective: To evaluate if Functional Sarcopenia phenotypes are associated with mortality in elderly people with cardiovascular disease (CVD). Methods: SARCOS is a prospective cohort study on Sarcopenia and Osteoporosis with mortality in elderly with cardiovascular disease. Sample: Elderly outpatients of the Federal University of São Paulo – Brazil. Physical functions were evaluated by: walking speed (WS) in 4.5 mts, the time to sit and stand up from chair test (CST) and hand grip strength (HGS), by dynamometer. Appendicular muscle mass (AMM) was measured by DXA. AMM/height2, HGS, WS and CST were evaluated in quartiles. Functional sarcopenia (FS) phenotype was characterized according to the presence of weakness (HGS < 26 Kgf M, <16 Kgf W), low WS (LWS, if WS < 0.5 m/s) and high time to CST (HCST > 25s): Robust = 0, Pre- Functional sarcopenia = 1, Functional Sarcopenia ≥2. Mortality was assessed by telephone contact at 6 and 12 months. Significant mortality variables and low AMM according to EWGSOP II* and FNIH# were used in the regression. Results: Among the 555 subjects, 57.5% (n = 319) were women, mean age 77.91(7.27). Weakness occurred n = 151 (27.2%), LWS n = 156(28.1%) and HCST n = 129(23.2%). The prevalence of phenotypes: Robust n = 281 (50.6%), Pre FS n = 155 (27.9%), and FS n = 119 (21.4%). Mortality was 4.7% (n = 26), of which, n = 5 (19.2%) were robust, n = 7 (26.9%) Pre FS, and n = 14 (53.8%) had FS, (p < 0.001). The FS phenotype was characterized by being female, higher age, lower HGS and WS, higher Time to CST, higher functional loss (IADL and ADL), lower depression score, lower AMM/h2 and higher percentage of total body fat, compared to other phenotypes. In regression analyses for mortality, Pre FS OR: 1.84 (0.45–7.49, p = 0.390)* and OR: 2.30 (0.56–9.48, p = 0.247)#; and FS OR: 4.98(1.35–18.40; 0.016)* and OR: 7.06(1.71–29.09; p < 0.007)#. Conclusion: In the older adults with cardiovascular disease, Functional Sarcopenia phenotype predicts higher mortality compared to the other phenotypes, regardless of low muscle mass and CVDs. 110761 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT SOLDATOVA A.M.1, Kuznetsov V.A.1, Enina T.N.1, Bogdanova D.S.2, Benzineb F.T.2 (1) Tyumen Cardiology Research Center, Tomsk National Research Medical Center, Russian Academy of Sciences; (2) Tyumen State Medical University Purpose: To access the prevalence of frailty and its impact on long-term prognosis in patients with implanted devices for cardiac resynchronization therapy (CRT). Methods: 77 patients with congestive heart failure (74% men, 26% women; mean age 58.7 ± 10.7 years) with NYHA class II–IV were enrolled. Frailty index (FI) was calculated using 31 parameters (the ability to perform daily activities, clinical status, laboratory markers, comorbidities). Based on the median of FI value patients were identified as not frail (<0.375; n = 41; 53%), and frail (≥0.375; n = 36; 47%). Results: The mean follow-up period was 38.0 [21.0; 66.0] months. The 5-year survival was 87.8% for not frail patients compared to 52.8% for frail patients (Log rank p < 0.001). In univariate analysis the presence of frailty was significantly associated with long-term mortality (HR 6.108; 95 CI 2.207–16.907; p < 0.001). After adjustment for age, gender, NYHA class, left ventricular ejection fraction, presence of left bundle branch block and QRS width frailty remained a significant factor associated with 5-year mortality (HR 5.763; CI 95% 1.837–18.083; p = 0.003). Conclusions: Frailty is widespread in patients with heart failure and implanted devices for CRT and it has an independent association with the risk of 5-year mortality. 110798 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY PRISCILLA MORETTO1, Priscilla Moretto1, Tatiane Boff Centenaro3, Gabriela dos Santos3, Fernando Rosa Inácio1, Ana Inês Gonzáles1 (1) Centro Universitário Estácio de Sá de Santa Catarina; (2) Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí – UNIDAVI; (3) Universidade do Estado de Santa Catarina – UDESC Introduction: In physical exercise programs aimed at Cardiovascular Rehabilitation, it is important to emphasize the adequate monitoring of these patients for care in the event of a cardiovascular event. Given the above, questions arise whether these care and monitoring are performed by physical therapists who work with the Pilates Method in patients with Cardiovascular Diseases (CVD’s) and if it occurs in an adequate and safe way. Objectives: To assess the level of knowledge and safety measures adopted by physiotherapy professionals working in pilates studios regarding the care provided to patients with cardiovascular diseases. Methodology: Cross-sectional research, with a non-probabilistic and quantitative sample, where professionals with active CREFITO, certified in Pilates and active in the method in the states of Brazil were invited. Those who did not provide all the data were excluded from the research. There was the development of a self-response questionnaire containing information related to the professional and the assessment of the level of knowledge regarding the approach and safety measures adopted with the Pilates Method in exercise programs for patients with CVD‘s. Professionals were invited to respond digitally and by publicizing CREFITOS and universities in Brazil. The TCLE was considered electronically signed when the volunteer accepted to participate in the research through an electronic form (Google Forms). The analysis of the study data was carried out using descriptive statistics, based on measures of central tendency (mean and standard deviation), performed in the SPSS statistical program for Windows® version 2.0. Results: 134 physical therapists answered the questionnaire, 98 (73.1%) do not have a training course in the cardiovascular area, 81 (60.4%) have practical knowledge in cardiopulmonary resuscitation, 68 (50.7%) are not up to date regarding rehabilitation guidelines and approximately more than 90% of professionals consider it important to check safety measures regarding vital signs before, during and after each session. In the event of any cardiovascular intercurrence, only 69 (51.5%) of the professionals stated that their team is prepared to assist. Conclusion: Most professionals do not have training to care for this type of patients, and have little knowledge of updating in the area, at the same time they consider it necessary to measure vital signs and perception of effort in the sessions. 110832 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MAKI KOMIYAMA1, Yuka Ozaki1, Yoichi Sunagawa2, Yasufumi Katanasaka2, Masafumi Funamoto2, Kana Shimizu2, Hajime Yamakage1, Noriko Sato-Asahara1, Hiromichi Wada1, Tatsuya Morimoto2, Koji Hasegawa1 (1) Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto, Japan; (2) Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Background: Inflammation of blood vessels is key in the process of coronavirus disease 2019 (COVID-19) aggravation. Besides, risk factors for COVID-19 such as cardiovascular disease is strongly associated with smoking habits. Previous studies have reported that neutrophil to the lymphocyte ratio (NLR), the inflammation marker, is associated with the onset and prognosis of cardiovascular (CV) disease. Smoking is a potent CV risk factor, and smoking cessation significantly reduces CV risk. However, the effects of smoking cessation on the NLR remain unknown. Purpose: The purpose of this study is to demonstrate the effect of smoking cessation on the NLR. Methods: Among smokers visiting our smoking cessation clinics, we examined changes in the NLR and CVD biomarkers before and after smoking cessation. A total of 389 individuals (301 men and 88 women) were enrolled in the study. Results: The median NLR was significantly reduced after successful smoking cessation (before: 1.8 [1.5, 2.5], after: 1.7 [1.3, 2.4]; p < 0.001). In a linear regression model adjusted for sex, percent change in NLR comparing before and after smoking cessation was significantly and positively correlated with percent changes in C-reactive protein (β = 0.260, p = 0.001), α1-antitrypsin-low density lipoprotein (β = 0.151, p < 0.05), and serum amyloid A-low density lipoprotein (β = 0.325, p < 0.001). Conclusion: Our study demonstrated for the first time that smoking cessation significantly reduces the NLR in tandem with markers of inflammation and oxidative stress. These results may suggest that smoking cessation improves the risk of developing cardiovascular diseases and more severe symptoms of COVID-19. Moreover, smoking cessation recovers airway ciliary clearance and immune function as early as one month. Thus, smoking cessation awareness is strongly encouraged as part of the public health measures aiming to limit the global impact of COVID-19. 110803 Modality: E-Poster Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION HENRIQUE COTCHI SIMBO MUELA1, Guilherme Passassi1, Angelina Clara Cassoma Francisco1, António Gerson Bastos Francisco1, Isaura da Conceição Almeida Lopes1, Gade Satuala Vasco Miguel2 (1) Department of Physiology, Faculty of Medicine, Agostinho Neto University, Luanda, Angola; (2) Cardiothoracic Surgery Department, Girassol Clinic, Luanda, Angola Introduction: In 2017, the American College of Cardiology/American Heart Association (ACC/AHA) published a new guideline (JNC 8) on the treatment of hypertension (HTN) that changed the diagnostic standard of high blood pressure (BP) from a BP threshold of 140/90 mmHg, as set by the seventh report of the Joint National Committee (JNC 7) to a BP of 130/80 mmHg. Objective: To carry out a comparative study on the frequency of HTN and the profile of BP levels in a sample of adult inhabitants in Luanda, Angola, based on the most recent American HTN guidelines. Methods: We carried out a cross-sectional study was, which included 1480 individuals who participated in the study on high blood pressure between May 2018 and June 2019, and who were recruited at Shopping Xyami, Angolan Public Television (TPA) and Congolenses’ market. Sociodemographic, anthropometric and clinical data were collected. Each participant had his/her BP taken with three measurements on the arm, after a 5-minute rest, using a semi-automatic Omron® device (Kyoto, Japan), with a 1-minute interval between. HTN was defined as the mean of systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg according to JNC 7 and the mean of SBP ≥130 mmHg and/or DBP ≥80 mmHg according to JNC 8 or use of antihypertensive drugs for both guidelines. Results: The sample was composed mostly by men (69.30%), young people (mean 39.74 ± 11.55 years old, range 18 to 94 years old). Alcohol consumption (74.6%) and overweight/obesity (44.9%) were the most prevalent risk factors. The prevalence of HTN was twice as high using the criteria proposed by the JNC 8 guideline compared to the cutoff points proposed by the JNC 7 (60.20 vs. 34.70; X2 = 519,03; p < 0.001). Both the knowledge and the treatment as well as the control of HTN were worse when using the JNC8 guideline definitions compared to JNC 7 guideline (X2 = 519,03; p < 0,001). Age, weight and alcohol consumption were the main predictors of blood pressure changes. Conclusion: The use of JNC 8 definition resulted in a significant increase in the prevalence of high blood pressure, placing the BP of a significant proportion of adults in the hypertension range. 110804 Modality: E-Poster Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION HENRIQUE COTCHI SIMBO MUELA1, Angelina Clara Cassoma Francisco1, Guilherme Passassi1, António Gerson Bastos Francisco1, Isaura da Conceição Almeida Lopes1, Gade Satuala Vasco Miguel2 (1) Department of Physiology, Faculty of Medicine, Agostinho Neto University, Luanda, Angola; (2) Cardiothoracic Surgery Department, Girassol Clinic, Luanda, Angola Background: Arterial hypertension (HTN) is an important public health challenge worldwide. Data regarding the burden of hypertension is essential for the development of effective strategies for both treatment and control. Studies on the prevalence and awareness of hypertension in Sub-Saharan Africa (SSA) and especially in Angola are still scarce. Objective: To assess the prevalence, awareness, treatment and control of HTN in a sample of dwellers of Luanda. Methodology: We carried out cross-sectional study in a sample of dwellers of Luanda, Angola. Sociodemographic data and cardiovascular (CV) through questionnaires. Blood pressure was measured with semi-automatic sphygmomanometer (Omron®, Model HEM-7131-E), after 5 minutes resting and seated. Three blood pressure (BP) measurements with a 1-minute interval between were used to determine SBP and DBP in each patient. The average of the two last blood pressure readings was used for the analysis. Hypertension was defined as SBP and/or DBP ≥ 140/90 mmHg or current use of antihypertensive drugs. Controlled hypertension was defined as blood pressure <140/90 mmHg under antihypertensive drugs. Results: Between May 2018 and June 2019, we carried out a cross-sectional study including 1480 individuals was carried out. The majority of the sample was young (mean age 39.74 ± 11.55 years old) and men (69.30%). Alcohol consumption (74.6%) and overweight/obesity was the most prevalent risk factors. The HTN prevalence was 34.9% in the sample. Among the hypertensive patients, 245 (47.4%) were aware of their disease and only 190 (36.8%) were under hypertension treatment; among those were taking drugs, only 68 (35.8%) were under control, defined as BP <140/90 mmHg. Conclusion: HTN prevalence was high; the awareness, treatment and control levels were low. 110806 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION HENRIQUE COTCHI SIMBO MUELA1, Fernando Muhongo Sandala1, José António Tito2, Guilherme Mendes Lima Franco1, António Gerson Bastos Francisco1, Isaura da Conceição Almeida Lopes1 (1) Department of Physiology, Faculty of Medicine, Agostinho Neto University, Luanda, Angola; (2) Cardiology Department, Sagrada Esperança Clinic, Luanda, Angola Background: Inflammatory response is one of the main mechanisms in the pathogenesis of atherosclerosis and its progression. The neutrophil-lymphocyte ratio (NLR) has been proposed as an inflammatory biomarker and potential risk and prognosis predictor in cardiovascular disease (CVD). Objective: To assess the association between the neutrophil-lymphocyte ratio and the severity of coronary artery disease. Methods: An observational and retrospective study was carried out, including 56 patients aged ≥18 years (41 men and 15 women) who underwent diagnostic and/or therapeutic coronary angiography from 2014 to 2019 at Sagrada Esperança Clinic, Luanda, Angola. Demographic data, risk factors and comorbidities, biochemical tests and full blood count were collected from the patients’ medical reports. The NLR was calculated as the ratio between the total neutrophil and lymphocyte counts from the patients‘ full blood count. For the statistical analysis purposes, the sample was divided into two groups according to the median of NLR (median: 2.02): patients with NLR ≤ median and patients with NLR> median. The statistical significance was set at 5%. Results: The group of patients with CAD was relatively older than the group without CAD, although without any significant difference (58.49 ± 8.76 vs. 54.74 ± 8.7, p = 0.137). Likewise, the age was similar between groups considering the level of NLR (57.89 ± 8.60 vs. 56.54 ± 9.12, p = 0.571). The risk of having CAD and it being obstructive was twice as high in the group with NLR above the median (NLR > 2.02) compared to the group with a lower NLR (OR: 2.25, CI: 0.722–1.012 and 2.17, CI: 0.717–6.550, respectively). However, in general, our data suggested that there was no association between the neutrophil-lymphocyte ratio and the presence of CAD, nor with its severity (χ2 = 1.991, p = 0.259 and χ2 = 0.760, p = 0.562, respectively). Conclusion: The risk of CAD occurrence, as well as the occurrence of obstructive CAD was twice higher in the group with higher neutrophil-lymphocyte ratio. 110817 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING SASHA KJAEVA ANASTASOVA1, Prof, d-r Srbinovska -Kostovska1, D-r. Paljoskovska-Jordanova1, D-r. Shehu1, D-r.Risteski1, D-r.Debreshlioska2, D-r. Angelovska2, D-r.Nivicka-Kjaeva3, D-r.Kjaev4 (1) University Clinic of Cardiology, Skopje, North Macedonia; (2) University Clinic of Pulmoallergology, Skopje, North Macedonia; (3) University Clinic of Ophthalmology, Skopje, North Macedonia; (4) University clinic of Gynecology, Skopje, North Macedonia Aim: The aim of the study was to evaluate which echocardiographic parameters in assessment of right ventricular function may contribute to risk stratification in patients with severe/very severe chronic obstructive pulmonary diseases (COPD) in correlation with biomarker Nt-proBNP. Material and methods: We have analyzed 38 patients with severe/very severe COPD, stages 3 and 4 according to Gold classification. All patients with heart diseases were excluded from the study. Additionally, all patients were divided into two subgroups: patients with acute exacerbation and chronically stable patients. Echocardiography, analysis included several parameters with emphasis to right chamber echo parameters in correlation to natriuretic peptide. Special emphasis was given to 3 predominant echocardiographic parameters: TAPSE, S’ wave assessed by tissue doppler of the free wall of the right ventricle and FAC%. Results: Of a total number of 38 patients, 81,6% had elevated values of NT-proBNP and 18,4% had values of NT-proBNP in normal ranges. Analysis of the average values of NT-proBNP showed much higher values in the group of patients with acute exacerbation where the average value of NT-proBNP was 786,73 pg/ml in correlation with chronically stable patients, where NT-proBNP was 141,17 pg/ml. All 20 patients with acute exacerbation had elevated NT-proBNP values (above 125 pg/ml). In the group of patients with acute exacerbation, 13 patients (65%) out of 20 had S’ wave from the tissue doppler below 0,095 m/sec and 8 patients (22,6%) had TAPSE below 16 mm. The group of chronically stable patients (who were 18 in number), showed not convincing results. In this group 61% (11 patients) of the patients had elevated NT-proBNP, 4 of them had S’ wave from the tissue doppler below 0,095 m/sec and TAPSE was in normal range(>16 mm). FAC was below 35% in one patient, where NT-proBNP was 345,4 pg/ml. Conclusion: In patients with chronic obstructive pulmonary diseases (COPD), echocardiographic parameters TAPSE and S’ wave assessed from the Tissue Doppler of the free wall of the right ventricle can be significant prognostic markers for disease monitoring. These parameters, especially S’ wave, can also be a marker for acute deterioration of the condition of the patients with COPD. 110833 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES RAFAEL ALEXANDRE MENEGUZ MORENO1, J. Ribamar Costa Jr.1, Nisia Lira Gomes1, Auristela IO Ramos1, Alfredo N. Ferreira-Neto1, Dimytri Siqueira1, Mercedes Maldonado1, Zilda Meneghelo1, Cesar A. Esteves2, Sérgio Braga1, Amanda Sousa1 (1) Instituto Dante Pazzanese de Cardiologia; (2) Universidade Federal de São Paulo (UNIFESP) Background: Percutaneous balloon mitral commissurotomy (PMBC) is an attractive therapeutic approach in patients with mitral stenosis. The aim of this study was to assess the immediate and long-term clinical, echocardiographic and haemodynamic outcomes of PMBC in patients with severe pulmonary hypertension (PH). Methods: Among all procedures (in more than two decades of experience), PMBC was performed from 1987 until 2011 at a single-center in 147 patients who had significant PH defined as baseline pulmonary artery mean pressure (PAMP) (systolic pulmonary pressure >75 mmHg). All-cause mortality, need for mitral valve replacement (MVR) or new PMBC, and valve restenosis were evaluated during follow-up yearly. Results: Mean age was 33.8 ± 12.8 years and 83.6% (123 patients) were women. Primary success was achieved in 89.8% of the patients (132 patients). Mitral valve area (MVA) increased from 0.83 ± 0.17 cm2 to 2.03 ± 0.35 cm2 (p < 0.001), and at 20-years, mitral valve area was 1.46 ± 0.34 cm2 (p = 0.235). Systolic pulmonary artery pressure decreased from 87.0 ± 6.0 mmHg to 60.0 ± 0.9 mmHg (p < 0.0001). The rates of all-cause mortality, need for MVR, new PMV, and valve restenosis were 0.67%, 20.0%, 8.78% and 30.4%, respectively, in long-term follow- up (mean 15.6 ± 4.9 years). Conclusions: PMBC is a safe and effective technique for the treatment of patients with mitral stenosis and PH. A significant decrease in pulmonary pressure was observed after commissurotomy. Although there was a gradual decrease of MVA at long-term follow-up, most patients remained asymptomatic and without major adverse events. 112450 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM FATHIMA AAYSHA AAYSHA CADER1, Ishmum Zia Chowdhury2, Saidur Rahman Khan1, M. Maksumul Haq1, Mashhud Zia Chowdhury1 (1) Ibrahim Cardiac Hospital & Research Institute; (2) BIRDEM General Hospital Introduction: The impact of the COVID-19 pandemic on cardiology services globally has been variable, with a paucity of contemporary data comparing pre- and post-pandemic trends over a long period in a South Asian setting. Objectives: We aimed to report annual trends of cardiovascular services delivery and procedure volumes from 2019 to 2022, to assess the impact of the COVID-19 pandemic, and recovery of services at a tertiary cardiac centre in Bangladesh. Methods: Data on patient visits (outpatient and emergency), admissions, procedures and catheterization volumes were collected from January 2019 to February 2022 via hospital electronic records. Differences for each month of the preceding year were expressed as a percentage (%Δ). Trends (2019 to 2022) were graphically depicted via line diagrams. Results: Following significant reductions of all cardiology services in 2020 (1), particularly ER visits (Δ–59.5%; p = 0.0), cardiology services delivery and procedure volumes had recovered, reaching almost pre-pandemic levels by Q1 of 2021 (Figure 1). A steep and significant decline of admissions and procedures was seen in March–April 2021 as compared with 2020, coinciding with the Delta variant surge (2). By Q4 of 2021, patient visits and procedures, both outpatient and catheterization volumes had once again reached near pre-pandemic levels (3). During the Omicron surge in early 2022, a small decline in outpatient visits (Δ–10.9%) and outpatient procedures (Δ–6.83%) was observed in January–February. However, in-patient admissions (Δ4.39%) and catheterization laboratory procedures (Δ5.7%) showed a rise in February 2022 as compared with the preceding month, with ER visits showing the steepest rise (Δ 41.9%). Notably, although ER visits remained relatively blunted post-pandemic (2020–2021), this trend was not reflected in outpatient visits/procedures, in-patient admissions and catheterization procedures, all of which increased to pre-pandemic levels by the end of 2021 (1–3). This may be explained either by patients presenting at index to the outpatient department, instead of the ER; or, a higher proportion of patients being directly referred to our centre for admission from the peripheries. Conclusion: Two years on from the pandemic, cardiology services and cath lab volumes have reached almost pre-pandemic levels in 2022, except for ER visits which remain low, albeit gradually rising. 110840 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUIZ FERNANDO KUBRUSLY1, Douglas Mesadri Gewehr2, Taiane Belinati Loureiro Kubrusly3, Fernando Bermudez Kubrusly3, Luiz Fernando Kubrulsy1 (1) Mackenzie Evangelical School of Paraná, Curitiba, Paraná, Brazil; (2) Denton Cooley Institute of Research, Science and Technology, Curitiba, Paraná, Brazil; (3) Curitiba Heart Institute, Curitiba, Paraná, Brazil Background: The practice of pretreatment with oral P2Y12 inhibitors in ST-elevation acute coronary syndrome (STEACS) remains common; however, its association with improved cardiovascular outcomes is unclear, since no large RCT has addressed this issue. Objective: We aimed to evaluate the association of oral P2Y12 inhibitor pretreatment in STEACS patients with cardiovascular and bleeding outcomes. Methods: PubMed, MEDLINE, Embase, Cochrane, Scopus, Web of Science were systematically searched for studies that compared pretreatment with P2Y12 versus no pretreatment in STEACS, and reported efficacy and safety outcomes. A meta-analysis using a fixed and random effects model was used to calculated outcomes of interest. Heterogenicity was assessed with I2 statistics. Results: A total of 3 RCTs and 14 observational studies assigning 91,771 patients to either pretreatment (65,598 patients) or no pretreatment (26,171 patients) were included. Follow-up ranged 7 days to 19 months. Medications included clopidogrel, prasugrel and ticagrelor. At 30 days, P2Y12 pretreatment was associate with lower 30-day mortality (risk ratio [RR], 0.71; 95% CI, 0.56–0.91; p = 0.006; I2 = 75%), stent thrombosis (RR, 0.33; 95% CI, 0.12–0.95; p = 0.04; I2 = 83%), 30-day major bleeding (RR, 0.81; 95% CI, 0.74–0.90; p < 0.0001; I2 = 0%). No difference in the incidence of 30-day myocardial infarction (MI), target vessel revascularization (TVR), MACE (death/MI/TVR), stroke, pre-PCI TIMI 0 and post-PCI TIMI 0–2 was observed. Subgroup analysis for 30-day mortality, including only randomized studies, indicated a trend in favor of no pretreatment, but without statistical significance (RR, 1.49; 95% CI, 0.89–2.52; p = 0.13; I2 = 38%). Conclusion: In this study, pretreatment with oral P2Y12 inhibitors among patients with STEACS before cath lab, compared with treatment once coronary anatomy is known, was associated with decreased all-cause mortality and bleeding risk. 110852 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION CHRISTIANE DIAS MAUÉS1 (1) Smart Vascular Technologies EI; (2) Federal Government of Brazil ME/MCTI In recent years, nanotechnology has been a strong ally to medical technology advances, and in this sense, one of the greatest applications concerns drug carrying and releasing systems. The use of nanostructured systems to carry out drug delivery allows the development of new systems for an efficient transport of these drugs, where a controlled release is achieved to the diseased tissues of living systems. These drugs are linked to nanostructures in a controlled way, commonly called functionalization, along with other attached molecules – biomarkers. Smart nanoparticles as liposomes could still be functionalized for a better targeting to the target tissue, preventing the side effects of the drug they carry. As a pioneer project, this privilege holds a non polymeric multidrug eluting coronary stent, which spatial structure is represented by six configurations, associated to a biological matrix (coating) in which nanoparticles, more precisely functionalized liposomes, are stored and grouped into sequential layers, each one differing the type of pharmacological agent to be applied, as well as increased coefficients of biodegrability applied to this support matrix, conferring continuously and selectively control of drug releasing pulse. In this sense, nanofunctionalized liposomes, in addition to being cell membranes models, act as excellent biocompatible systems for formulation, carrying and release of drugs to organic tissues. 110846 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION MICHAŁ LEWANDOWSKI1, Ilona Kowalik1 (1) National Institute of Cardiology, Warsaw, Poland Background: Prediction of sudden cardiac death remains a significant challenge. There is some evidence that ventricular ectopic activity could be regarded as a predictive marker. Objectives: We carried out an analysis to explore whether premature ventricular complexes (PVCs) are a risk factor in implantable cardioverter-defibrillator (ICD) interventions. Materials and methods: The study method was a RR interval series analysis (n = 184) of arrhythmic events and controls from the ICD. Study group consisted of patients with a mean age of 55 ± 27 years; 74% of them were male, 85% were secondary prevention patients, 62% had coronary artery disease (CAD), 15% hypertropic cardiomyopathy (HCM), 15% dilated cardiomyopathy (DCM), and 8% diseases of other etiol¬ogy. The mean follow-up time was 64 months (range: 3–126 months). The study population was divide into patients with at least 1 appropriate intervention ventricular tachycardia/ventricular fibrillation (VT/VF) (group A, n = 101) and controls without interventions (group B, n = 83). The number of PVC/4000 RR cycles, the shortest coupling intervals between a PVC and preceding R as well as the number of PVCs of very short (180–220 ms), short (220–280 ms) and different cycle lengths (CL) as well as the incidence of short-long-short (SLS) sequences were compared. Results: The number of PVCs/4000 RR cycles was significantly higher in group A (263 ± 32 compared to 43 ± 17, p < 0.0001). The mean shortest PVC CL was significantly shorter in group A (320 ± 13 compared to 400 ± 38, p = 0.029). The number of PVCs with a very short CL was 1 ± 0.4 compared to 0.1 ± 0.1 (p = 0.028). The number of PVCs with a short CL was 5 ± 1.2 compared to 0.6 ± 0.4 (p = 0.0007) in groups A and B, respectively. The incidence of SLS sequences was significantly higher in group A than in group B (67 (94% of patients) and 4 (33% of patients) respectively (p < 0.0001)). Conclusions: Significant differences were found in the characteristics of PVCs and SLS sequences between patients with appropriate ICD interventions and controls. A newly developed basic computer program called PCRR was applied for RR interval analysis. This simple method could be a predictor of PVC burden, heart rhythm variability and life-threatening arrhythmias in different populations. 110878 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARIA JULIANA RODRIGUEZ GONZÁLEZ1, Andres Felipe Barragán Amado1, Maria Fernanda Tamayo Tamayo1, John Alexander Ramirez1, Julián Gelves1, Esther Campos1, claudia Patricia Jaimes Castellano1 (1) Lacardio Introduction: In the setting of hypertrophic phenotype, the hypertrophic cardiomyopathy (HCM) is the most prevalent genetic disorder and responds to the 60% of the cases; with variable penetrance and expression. Genotyping is a step in its approach to defining prognosis and establishing phenocopies. Mutations in some genes encoding sarcomere proteins like cardiac myosin-binding protein C and β-myosin heavy chain are the most prevalent affected, representing 30–40% of all pathogenic or likely pathogenic variants. Aim: Describe the genotyping of our first cohort of hypertrophic phenotypes in a reference heart failure clinic. Method: Data was taken for in-Hospital patients irrespective of the cause of hospitalization or out of hospital who required a cardiovascular imagen test; the only requirement was to have a cardiac magnetic resonance or transthoracic echocardiography with a hypertrophic phenotype defined by any wall over 15 mm or 13 in patients with a family history of HCM between 01/01/2019–31/12/2021. Descriptive statistics are presented as median (range) or % of affected patients. Results: Patients with hypertrophic phenotype were 45, the average age was 54 years (ds: 19,73), 52% were women, 37% had a previous diagnosis of hypertension, the MYH7 was detected in 36% of the overall patients, and the total gene mutations were 55 and the unknown significance variants represented the 69%. Nine of these patients had multiple mutations and only three patients had non-sarcomere genes mutation (they had late-onset Fabry disease, amyloidosis, and Noonan). On the imaging aspects, the average diameter was 17,86 mm (ds: 4,16), the most affected segment was anteroseptal basal with 45% and the average left ventricular ejected fraction was 59,3 (ds: 15,01). Conclusion: These results provide important knowledge about genotyping patients with hypertrophic phenotype, to determine the possible diagnosis, the gene mutation, the type of variant, and the significance. This study shows that in our population, the pathogenic variants represent 27% of the total, similar data in comparison with the available publications. This is the first study in Colombia that reveals genotyping in hypertrophic phenotype could let us establish some mutations that carry a worse prognosis and establish family approaches. 111055 Modality: E-Poster Researcher – Non-case Report Category: PSYCHOLOGY MAKI KOMIYAMA1, Hajime Yamakage1, Noriko Satoh-Asahara1, Yuka Ozaki1, Tatsuya Morimoto2, Yuko Takahashi1, Koji Hasegawa1 (1) Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto, Japan; (2) Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Depressive tendency and nicotine dependency are factors related to the failure of smoking cessation. Women generally have a higher depressive tendency and difficulty in smoking cessation than men. However, the impact of sex differences on the relationship between nicotine dependency and depressive tendency remains unclear. We evaluated 727 patients (496 men and 231 women) who visited our outpatient clinic for smoking cessation therapy and compared various parameters measured between sexes during consultation. Age, duration of smoking, and daily cigarette consumption were significantly higher in men during the first visit. Women had significantly higher self-rating depression scale (SDS) scores and took significantly more antidepressant drugs than men. The SDS score significantly correlated with the Fagerström test for the nicotine dependence score and with daily cigarette consumption in women, but not in men. Thus, the present study demonstrates the differential relationship of depressive tendency with tobacco use or nicotine dependency in men and women, which might reflect sex differences in response to psychological stress. We previously reported that depressive tendencies of patients with neurosis improve even in the initial stages of the smoking cessation treatment (i.e., within two weeks after starting the treatment), but further studies are required for the sex difference for the improvement. 110894 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CRISTIANE DA CRUZ LAMAS1, Giovanna Ianini Ferraiuoli Barbosa1, Rafael Quaresma Garrido1, Wilma Félix Golebiovski1, Clara Weksler1, Diego Santos2, Caio Sambo2, Vitor Milczwski2, Milena Paixão3, Flavio Tarasoutchi3, Tânia Mara Varejão Strabelli3, Rinaldo Focaccia Siciliano3 (1) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (2) Department of Infectious Diseases, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil; (3) Heart Institute (InCor), Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil Introduction: Prosthetic valve endocarditis (PVE) is found in 10 to 30% of contemporary series of infective endocarditis (IE) and has a high mortality rate. Our aim was to describe cases of late PVE and analyze mortality-related risk factors. Methods: Two quaternary cardiac surgery institutes were the study scenarios. Data were collected from 2011–2021 in site 1, and 2006–2021 in site 2. Patients were prospectively identified using site-specific procedures to ensure consecutive enrollment. Only adult patients with definite IE according to the modified Duke criteria were included. Studied variables were those present in the International Collaboration in Endocarditis case report forms. Statistical analysis was performed with the SPSS v26 software. Variables with p values less than 0.1 were included in the model. Results: There were 250/559 (44.7%) episodes of LPVE in site 1 and 77/438(17.6%) in site 2 during the study periods. Males accounted for 207/327 (63.3%) of patients, mean age ± SD was 55.5 ± 17.3 years. Rheumatic valve disease was present in 161/326 (49.4%) and congenital heart disease in 29/327(8.9%). Healthcare-associated IE was present in 83 (25.4%) and 132/326(49.4%) patients were in heart failure (NYHA class III/IV). Etiologic agents were defined in 275(84.1%) of cases, and oral streptococci were responsible for 107/327(32.7%). Death was the outcome for 101/327(30.9%). Risk factors for death were assessed by multivariate analysis and those found to be statistically associated were: age over 60 years (OR = 2.4; 95% confidence interval [95%CI]: 1.2–5; p = 0.014), heart failure (NYHA class III or IV) (OR = 4.1; 95%CI = 1.8–8.3; p < 0.0001), the presence of perivalvular abscess/fistula/perforation on admission echocardiography (OR = 2.8; 95%CI = 1.3–6.1; p = 0.009) and being referred from another hospital (OR = 2.6; 95%CI = 1.25–5.4;p = 0.011). Conclusion: Previous series have described that late PVE is found in older patients, but our cohort shows younger patients and a high frequency of rheumatic valve disease. A quarter of patients acquired IE in a healthcare setting, highlighting the relevance of this mode of acquisition and the extra care needed in infection control in patients with prosthetic valves. Similarly to other studies, destructive disease and heart failure were associated with death, and mortality was higher than in native valve IE. 110902 Modality: E-Poster Researcher – Non-case Report Category: DYSLIPIDEMIA KU MING YING1, Shirley Tan Siang Ning1, Crystal Tan Sing Yee1, Tiong Lee Len1, Saiful Shakirin bin Rosli1, Hwang Siaw San2, Ong Tiong Kiam3, Alan Fong Yean Yip3 (1) Sarawak General Hospital; (2) Swinburne University of Technology Sarawak Campus; (3) Sarawak Heart Centre Introduction: Lipoprotein(a) [Lp(a)] is known to have prothrombotic, proinflammatory and proatherogenic properties contributing to Atherosclerotic Cardiovascular Disease (ASCVD) development and progression. Previous studies have shown ethnic differences in plasma Lp(a) levels. Objective: We aim to determine the plasma Lp(a) concentrations among Malaysians with Acute Myocardial Infarction (AMI). Methods: This prospective study involved AMI patients admitted to a tertiary hospital in Sarawak between August 2018 and March 2019. Blood samples were collected during hospital admission and stored at –80°C. Plasma Lp(a) concentrations were determined by quantitative sandwich enzyme immunoassay technique (Elabscience, USA). Results: A total of 240 AMI patients were recruited, with mean age of 56.4(±10.90) years and 88.8% males. Ethnic distributions were 42.9% Malay, 30.4% Chinese and 26.7% Iban/Bidayuh. The mean Lp(a) concentration and baseline LDL-C level were 5.3(±7.49) mg/dL and 3.2(±1.15) mmol/L respectively. Mean Lp(a) concentrations among Malay, Chinese and Iban/Bidayuh patients were similar [5.9(±6.69) vs 5.3(±10.46) vs 4.5(±3.80) mg/dL; p = 0.340]. Patients with or without prior statin therapy demonstrated no significant difference in Lp(a) concentration [5.7(±9.71) vs 5.1(±5.53) mg/dL; p = 0.521]. Only 1.7% of patients were found to have Lp(a) >30 mg/dL. Patients presenting with 1-year MACE (AMI, stroke or cardiovascular death) were found to have significantly higher Lp(a) compared to those who did not [7.9(±7.67) vs 4.96(±7.41) mg/dL; p = 0.042]. Conclusions: Our study showed that the mean Lp(a) concentration among our AMI patients were lower compared to other published studies. However, those who experienced 1-year MACE had significantly higher Lp(a) concentration compared to the rest of the study population. 110904 Modality: E-Poster Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE SASHA KJAEVA ANASTASOVA1, Prof, d-r. Srbinovska-Kostovska1, d-r Pajloskovska-Jordanova1, d-r Shehu1, d-r Risteski1, d-r.Debreshlioska2, d-r Angelevska2, d-r.Nivicka-Kjaeva3 (1) University clinic of cardiology, Skopje, North Macedonia; (2) University clinic of pulmo-allerology, Skopje, North Macedonia; (3) University clinic of ophthalmology, Skopje, North Macedonia Aim: The aim of the study was to evaluate chronic obstructive pulmonary diseases (COPD) in correlation with natriuretic peptide biomarker Nt-proBNP with special emphasys to the subgroup of acute exacerbation patients with COPD. All patients analysed were hospitalized. Material and methods: We have analysed 94 patients with COPD devided in 4 groups according to Gold classification system. Gold class I (18 pat.), Group II (18 pat.), Gold class III and IV (28/30 patients). Additionally, all patients were divided into two subgroups: patients with acute exacerbation and chronically stable patients. From the whole group of 94 patients, 63 were patients with acute exacerbation while 31 was were classified as chronically stable patients. Results: Natriuretic peptide was measured in all 94 patients with the average value above reverence ranges in all pts and it was 236.27 pg/ml. Gold class I/II included patients only with acute exacerbation. In both groups (Gold I/II), values od Nt-proBNP were at a higher level (159, 62 pg/ml). The average value of Nt-proBNP in Gold class III, in the group of acute exacerbation was 492,60 pg/ml versus 147,48 pg/ml in the group of chronic stable patients while in Gold class IV these values were 307,44 versus 151,74 pg/ml. The average values in acute patients are far higher then in the chronic group of stable COPD patients with in appropriate Gold class. Such a difference is almost 3,5 times higher in Gold class 3 and 2 times in Gold class 4. Conclusion: According to the results here is a clear and statistically highly significant difference in the values of Nt-proBNP in relation to acute and chronic COPD patients, especially in Gold class III and IV. In the acute group, this is all due to rich inflammatory process and deterioration of the lung function which is replicated on the right heart cavities resulting in high release of Nt-proBNP. Deterioration of lung function leads to right heart strain and weakness and increased excretion of Nt-proBNP. 110924 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CRISTIANE DA CRUZ LAMAS1, Nícolas de Albuquerque Pereira Feijóo2, Thatyane Veloso de Paula Amaral de Almeida2, Mariana Giorgi Barroso de Carvalho2, Gabriel Santiago Moreira1, Léo Rodrigo Abrahão dos Santos2, Rafael Quaresma Garrido1, Bruno Zappa1, Giovanna Ianini Ferraiuoli Barbosa1, Wilma Félix Golebiovski1, Marcelo Goulart Correia1, Clara Weksler1 (1) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (2) Unigranrio-Afya Introduction: Infective endocarditis is a severe disease, with high mortality. Its features in a developing country such as Brazil differ substantially. Our aim is to describe a series of adult patients with IE cared for in a cardiac surgery referral center, highlighting its specificities. Methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Patients were identified prospectively using site-specific procedures to ensure consecutive enrollment. Statistical analysis was performed using the Jamovi 1.6 and R 4.0.1 softwares. Results: There were 435 episodes of IE in the study period, and mean age ± SD was 47.8 ± 17.3 years; 283(65.1%) were males. Community-acquired IE occurred in 284 (65.4%), hospital-acquired in 112 (25.8%). Early prosthetic valve IE (PVE) accounted for 47/435(10,8%) cases and late PVE for 77(17,7%). Previous 170 (39.3%) had previous cardiac surgery, congestive heart failure in 173 (40.0%), chronic renal failure in 91 (21.0%), coronary artery disease in 60 (14.0%), diabetes mellitus in 55 (12.6%). Main predispositions to IE were rheumatic valve disease (RVD) in 133 (31.5%), valvular prosthesis in 28.5%, congenital heart disease in 61 (14.0%), previous IE in 51 (11.8%) and intravenous drug use in 5 (1.2%). Vegetations were seen in the mitral valve in 214 (49.4%) and aortic valve in 180 (41.6%), in intracardiac devices, in 33 (7.6%). Fever was seen in 91.7%, new regurgitant murmurs in 54.4%, embolism in 48.3%, splenomegaly in 20.4%; Osler’s nodes, Janeway lesions, subconjunctival hemorrages and splinters were seen in less than 5% each. CRP levels were high in 82.3% and ESR in 58.9%. Blood cultures were taken in 98.6% of episodes, but were positive in only 67.1%. The most frequently isolated pathogens were viridans group streptococci, VGS(21%), S.aureus (11%), and enterococci(11%). Main complications were acute heart failure, in 263 (60.7%), acute renal failure (33.9%), myocardial abscess (22.6%). Surgery was indicated for 373 (86.1%) and effectively done for 316 (79.8%). Inhospital mortality was 109/425 (25.6%). Conclusions: In our quaternary center in Rio, left-sided IE predominated, with negative blood cultures or VGS as main findings. RVD was the main predisposition. Surgical indication was frequent, due to referral bias, and mortality was overall similar to the literature. 110942 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION IWONA KORZENIOWSKA-KUBACKA1, Anna Mierzyn’ska PhD2, Ewa Rydzewska PhD1, Edyta Smolis-Bąk PhD1, Rafał Dąbrowski MD, PhD1 (1) National Institute of Cardiology, Warsaw, Poland Department of Coronary Artery Disease and Cardiac Rehabilitation; (2) Department of Rehabilitation. Medical Centre of Postgraduate Education Gruca Orthopaedic and Trauma Teaching Hospital, Otwock, Poland Introduction: Hostility and its behavioral components, anger and aggression are psychosocial risk factors for coronary heart disease. The impact of exercise training on these negative emotions in patients after myocardial infarction (MI) without psychological support, has been poorly studied. Objective: The purpose of the study was to evaluate the effectiveness of physical training on the level of negative emotions, the cognitive aspect of adaptation to disease, and physical capacity in patients after MI who participated in cardiac rehabilitation. Methods: We enrolled 60 post-MI men and women in the study. All of them underwent an 8-week training program(TP) consisting of 24 interval trainings 3 times a week, started on average 40 days after MI. Before and after completion of TP, patients underwent a symptom-limited exercise test with analysis of maximal workload, duration, HR and BP at rest and during effort. The Buss-Perry Aggression Questionnaire assessing the level of negative emotions and the Brief Illness Perception Questionnaire were performed with results analysis in the entire group and subgroups of men, women, patients under 60 years of age (younger) and over 60 years of age (older). Results: After TP a significant reduction in the general level of negative emotions was found only in the group of younger: 67.8 ± 4.6 vs 63.9 ± 3.7 points, P < 0.01. Furthermore, a significant reduction in the sense of the impact of the disease on life was found in younger 6.96 ± 0.5 vs 5.48 ± 0.5points, P < 0.01. Furthermore, there was a significant improvement in overall adaptation to the disease in the subgroup of women from 40.6 ± 2.2 to 35.7 ± 1.9 points, P < 0.05. Physical capacity increased significantly in all studied groups. Conclusions: Participation in rehabilitation based on exercise trainings without psychological intervention not only improved physical capacity, but also beneficially contributed to a decrease in negative emotions and had a positive effect on disease adaptation especially in younger patients under 60 years of age after MI. 110949 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING SASHA KJAEVA ANASTASOVA1, Prof.D-r.Elizabeta Srbinovska-Kostovska1, D-r Elena Grueva-Nastevska1, D-r Savetka Paljovskovska-Jordanova1, D-r Dean Risteski1, D-r Danica Petkoska-Spirova1, D-r Enes Shehu1, D-r Irina Angelovska2, D-r Angela Debreshliovska2 (1) University clinic of cardiology, Skopje, North Macedonia; (2) University clinic of pulmo-allerology, Skopje, North Macedonia Aim: The aim of the study was to evaluate the echocardiographic parameters used to assess right ventricular function and pulmonary hypertension in patients with chronic obstructive pulmonary disease (COPD) according to their specificity and sensitivity and disease progression. Material and methods: We have analysed 94 patients with COPD (Gold class I–IV). The 13 echo-cardiography parameters important for assessment of right ventricular function and pulmonary hypertension due to their sensitivity and specificity and progression of the disease were evaluated: basal dimension of the right ventricle(DV bazal), right atrium(DA), right atrial area(DA area), S‘wave of the right ventricle of TDI, TAPSE, functional area change (FAC%), (SPAP), Vmax of tricuspid regurgitation, acceleration time of pulmonary artery (AT), pulmonary vascular resistance (PVR), myocardial performance index of the right venricle (MPI), global strain of the right ventricle(GL strain), collapsibility of vena.cava inferior >/<50%. Results: Predictors of disease progression with high specifity and sensitivity are the parameters: MPI DV TDI, Global strain of DV and collapsibility of v.cava inferior less then 50%. Predictors of disease progression with high specifity and low sensitivity are: DV bazal, DA, DA area, S TDI, TAPSE, FAC, SPAP, V max TR. Predictors of disease progression with low specifity and high sensitivity are parameters: shortened acceleration time of the pulse Dopler of the pulmonary valve and the development of pulmonary vascular resistance. Conclusion: Echocardiography is a non invasive and useful method for evaluation and follow up the patients with COPD. All this indicates that the values of certain echo parameters can help us detect disease progression with high sensitivity, specifity or both. 110955 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MICHAŁ LEWANDOWSKI1, Rafał Waligóra1, Paweł Syska1, Ilona kowalik1 (1) National Institute of Cardiology, Warsaw, Poland Background: The condition for SICD therapy is passing an electrocardiographic screening test (ECG-s). A patient is considered suitable for implant if at least one surface ECG lead (sense vector) is acceptable for all tested postures. Objective: Identifying the rate of failure, the predicting factors and clinical importance of extended ECG-s with right sided lead position and lying on the left and right side of the body. Methods: ECG-s is based on a manufacturer-specific measurement tool. We performed this test in 50 consecutive pts with indication for SICD using standard protocol(lead at the left sternum border: sitting, standing and lying) and extended for right parasternal lead location and 2 additional position: lying on the left and right side. We analyzed age, gender, BMI, heart rate, rhythm(sinus/atrial fibrillation), QRS duration, PR interval, corrected QT interval(QTc), echocardiography parameters, etiology. Results: ECG-s failure occurred in 4/50(8%) of patients. Screening results: 1 vector failure rate with the standard device and electrode positions: primary(P) 12%,seconadary(S) 17%, alternative(A) 40%, P vs. A and S vs. A p < 0,001. Testing with an electrode positioned on the right sternum border- 1 vector failure: (P) 10%,seconadary(S) 17%, alternative(A) 48%, P vs. A and S vs. A p < 0,001. In case of failure on left side 50,14 and 6% of pts passed the test with the right sided lead position for P, S and A vector respectively, p < 0.001. Identified factors predicting ESG-s passing vs. failure included: QTc interval 445,32 ± 35,4 vs. 480,6 ± 37,6 ms for any vector(p = 0,034), BMI:26,7 ± 4,7 vs. 29,9 ± 4,7 for vector A(p = 0,008), EF: 43,3 ± 20,1 vs. 34,0 ± 16,2 for vector A(p = 0,027). Sense vector failure rate with the left parasternal lead location lying on the left and right side of the body was concordant with lying on the back, p = 0,65. For the right parasternal lead location the rate failure was lower lying on the right side for all sense vectors: P, S and A(p = 0,045; 0,083 and 0,020) respectively. Conclusions: ECG screening failure rate for SICD is comparable with previous reports. The right parasternal lead location screening enables important clinical findings before implantation and in case of lead reposition during procedure, thus we recommend to screen routinely on both sternum borders. Some additional body position testing: lying on the left and right side of the body, mimicking sleeping/rest could be an important element to determine eligibility for SICD. 110961 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MICHAŁ LEWANDOWSKI1, Paweł Syska1 (1) National Institute of Cardiology, Warsaw, Poland Background: Young implantable cardioverter-defibrillator (ICD) recipients present high rate of inappropriate shocks(IS) and complications. Some of them seem to be underestimated. The subcutaneous ICD system was developed to eliminate lead-related complications and was proved to be effective. S-ICD appears to be a good therapy option and an alternative to transvenous implantable cardioverter-defibrillator (TV-ICD) in pediatric and young patients population. Aims To report on our clinical experience with TV-ICD and S-ICD therapy in patients under thirty years of age. Methods: We reviewed database of ICD recipients in our institution between 1996 and 2021(25 years) and have chosen 115 pts consecutively implanted up to and including the age of 30 years. We retrospectively analyzed the rate of appropriate and inappropriate interventions, lead complications rate, infection rate, mortality and treatment options. Results: The study group: 84 TV-ICD(age: 6–30, BMI: 16,3–22,2) and 31 S-ICD (age: 15–30, BMI; 15,6–31,1) patients. The mean follow-up in analyzed groups was 159 ± 48 and 36 ± 2 months respectively. Abnormal ventricular function: EF < 30 occurred in compared groups: 12/84(14%) and 5/31(16%) respectively, p = 0,3. 24/84pts(28%) received ≥1 appropriate therapy(AT) for VT/VF(ATP or shock) in TV-ICD and 3/31pts(10%) in S-ICD groups. 25/84 pts(30%) had one or multiple (IS) and 0% in compared groups respectively, p = 0,02. There were 18/84 (21.5%) ventricular lead dysfunctions(reimplantation of a new system) in TV-ICD and 0% in S-ICD groups, p = 0,025. An infection rate (endocarditis or device pocket) was 6/84 (7%) in TV-ICD group with complete system removal and 1 pts (3%) wound infection in S-ICD group successfully treated with antibiotics. Mortality rate was 6/84 pts (7%), caused by ventricular lead dysfunction, end stage heart failure or heat transplantation fatal result in TV-ICD group. There was no death in S-ICD group. Conclusions: Endocardial ICD implantation in children and young adults is a feasible and effective procedure in a 25-year follow-up but the rate of complications is high in this population. S-ICD recipients did not experienced lead failures, systematic infections or inappropriate shocks. S-ICD appears to be a good therapy option in life-threatening arrhythmias, preventing from some serious T-ICD complications in young patients. The method and follow-up period effect is present between the TV-ICD and S-ICD in analyzed groups. 110968 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH PRANAS SERPYTIS1, Egle Majauskiene1, Pranas Serpytis1 (1) Vilnius University, Faculty of Medicine, Lithuania; (2) Vilnius University, Clinic of Cardiac and Vascular Diseases, Centre of Cardiology and Angiology, Lithuania Introduction: Physical activity is associated with improved health outcomes for adolescents. Many of the benefits are observed with an average of at least 60 min of moderate-to-vigorous intensity physical activity daily (1). Unfortunately, more than 80% of school-going adolescents did not meet current recommendations for daily physical activity (2). Objective: We aim to analyze physical activity trends and their association with stress, weight, screen time, and other factors in Lithuania adolescents. Methods: A prospective observational cross-sectional study enrolled students from High Schools in Lithuania’s capital city. A survey was administered to a cohort of students aged 13–18 years. Data from a questionnaire about behavioral risk factors, academic performance, stress level at school, and physical activity were collected. Stress levels were measured by 10 points system. Data were analyzed using SPSS v28 statistical package. Results: The study population consisted of 589 adolescents (61.4% female and 38.5% male participants). The mean age was 15.79 ± 2.81 years. Adolescents engage in physical activity for 3.30 ± 1.91 days a week. Only 18.34% of adolescents’ (12.98% female vs 26.87% male; p < 0.001) physical activity lasts more than 420 min a week (60 min daily). Adolescents who exercise more, experience less stress at school (p < 0.001). Whereas screen time lasts 6.80 ± 3.23 hours daily and is associated with higher stress levels at school (p < 0.001). Smoking and alcohol consumption were significantly more common among girls (smoking: 22,37% vs 22,70%; p = 0,632; alcohol: 27.73% vs 21,15%; p < 0.001). Conclusions: More than 80% of adolescents in Lithuania did not meet recommendations for daily physical activity. Girls are less active than boys and tend to have more addictions. To avoid health consequences, it is necessary to strengthen education about cardiovascular risk factors and a healthy lifestyle benefits. 110978 Modality: E-Poster Researcher – Non-case Report Category: CARDIO-ONCOLOGY LOURENÇO SAMPAIO DE MARA1, Mariana Kleis Pinto da Luz Lodi3, Suellen Cristina Roussenq3, Magnus Benetti2 (1) Curso de Pós Graduação em Cardio-Oncologia SBC/INC/INCA, Rio de Janeiro, RJ, Brasil; (2) Universidade do Estado de Santa Catarina-UDESC Centro de Ciências da Saúde e do Esporte – CEFID, Núcleo de Cardioncologia e Medicina do Exercício Santa Catarina, SC, Brasil,; (3) Centro de Pesquisas Oncológicas – CEPON, Santa Catarina, SC, Brasil Introduction: Bone marrow transplantation (BMT) remains the best clinical alternative for the treatment of leukemias and chemotherapy-refractory lymphoid cancers. There is an increase in the survival of these patients, but with cardiovascular sequelae, which can be minimized by the effects of exercise. Objective: To analyze the effect of exercise on the gain of cardiovascular capacity in patients undergoing allogeneic BMT. Methods: Pilot study of a randomized controlled clinical trial with the Brazilian Registry of Clinical Trials, registry RBR-887gqvs. Fifteen patients with leukemia and lymphoma who were eligible for BMT participated, nine were randomized to the exercise intervention group (EIG), and six to the control group (CG). The EIG performed aerobic and resistance exercise during hospitalization, followed by a cycle ergometer protocol at Day Hospital, three times a week until the 100th day after BMT, with assessments of cardiovascular capacity by the six-minute walk test, which occurred before BMT or time 1 (T1), at hospital discharge or time 2 (T2) and 100 days post BMT or time 3 (T3). Data were evaluated using the SSPS software version 20.0. Results: The mean age of patients in the EIG was 38.7 (±8.7) years with 78% men and for the CG, 41.7 (±15.4) years with 50% men, with no difference between the groups (p = 0.860). In the comparison between EIG and CG regarding cardiorespiratory functional capacity, there was no difference in T1 (727.5 ± 160 × 640 ± 209; p 0.232) and in T3 (885 ± 379.1 × 790 ± 162.8; p 0.794) and an increase in functional capacity in favor of the EIG in T2(813.7 ± 192 × 530 ± 209.3; p 0.009). Comparing the distances covered with the predicted value within the groups, it was observed that the EIG covered distances significantly greater than the predicted in the three different times: T1 (598.9 ± 39.7 × 727.5 ± 164.9; p = 0.038); T2 (598.9 ± 39.7 × 813.7 ± 192.7; p = 0.011); T3(598.9 ± 39.7 × 885 ± 379.1; p = 0.039). The CG covered a greater distance than predicted only in T3 (578.5 ± 21.9 × 790 ± 162.8; p = 0.046). There was no significant difference between the average distance covered predicted for the EIG and the CG (598.9 ± 39.7 × 578.5 ± 21.9; p = 0.131). Conclusion: Patients who perform an exercise program during and after hospitalization for allogeneic BMT benefit from a gain in cardiorespiratory capacity. 110982 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARCELO WESTERLUND MONTERA1, Louise Freire1, Fabiola Traverso1, Beatriz Robert1, Isaura Rocha1, Daniele Cordeiro1, Ana Maria Medeiros1, Ana Amaral Dutra1 (1) Hospital Procardiaco Introduction and/or Fundamentals: The treatment of acute decompensated heart failure (ADHF) in clinical practice is usually carried out by a cardiologist not specialized in heart failure (HF). The implementation of specialized HF protocols (HFP) in the treatment of ADHF has shown benefits in improving the outcomes and quality of care. Objectives: To assess the benefits of HFP versus usual care(UC) in patients with ADHF in terms of clinical outcomes in in-hospital mortality(IHM), length of stay(LS), and readmission at 30 days(R30d), and the quality of care through the analysis of the medication prescription rate at hospital admission and discharge. Materials and methods: Retrospective non-randomized study of a cohort of 834 pts with ADHF and HFrEF(LVEF < 50%) admitted to a private hospital in Rio de Janeiro, between 01/2015 and 12/2021 381 pts were submitted to HFP and 453 pts to the UC defined by the attending physician. All pts were evaluated for their clinical characteristics, LVEF by echocardiogram, natriuretic peptide levels. The outcomes evaluated were:LS, IHM and R30d. The rate of prescription of beta-blockers (BB) and converting enzyme inhibitors or angiotensin II receptor blockers (ACEI/ARB) and spironolactone(spiron.) were also evaluated at hospital admission and discharge. Results: There was no significant difference between the groups in terms of age, sex, ADHF etiology, systolic blood pressure at admission, BNP and NT-proBNP levels and LVEF. HFP pts had a lower rate of IHM(11% vs 20%;p < 0.0001), and LS(7.4 ± 6.9 vs 10.5 ± 12.8 days;p < 0.001), and with no significant difference regarding R30d (4.3% vs 7.2%; p = 0.2). At admission the HFP pts showed a higher rate of ACEI/ARB prescription (45% vs 36%; p < 0.006) and higher dosage of intravenous furosemide (120 ± 60 mg vs 60 ± 30 mg, p < 0.01) and no difference in BB prescription rate(75% vs 70%; p < 0, 15). At discharge, the HFP pts had a higher rate of prescription of ACEI/ARB (89% vs 43%; p < 0.0001) and spiron. (50% vs 27%; p < 0.0001) and furosemide(72% vs 62% p = 0.05) and no difference in BB prescription rate(87% vs 84%; p = 0.2). Conclusions: The treatment of pts with ADHF with HFP compared to UC, had an impact on improving in-hospital clinical outcomes with lower IHM and LS and they also had a better quality of care with a higher rate of prescription of ACEI/ARB and higher dosage of intravenous furosemide at admission, and a higher rate of prescription of ACEI/ARB, spiron. and furosemide at hospital discharge. 110986 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARCELO WESTERLUND MONTERA1, Louise Freire1, Arnaldo Rabischoffsky1, Amarino de Oliveira1, Luiz Antonio Carvalho1, Ana Amaral Dutra1 (1) Hospital Procardiaco Objectives: Identify the clinical characteristics and complementary tests that allow the differentiation of Takotsubo from Acute Myocardial Infarction (AMI) with ventricular dysfunction, at hospital admission. Materials and methods: Retrospective study of a cohort of patients admitted to a private hospital in Rio de Janeiro, from 2010 to 2020, with a diagnosis of Takotsubo and AMI with ventricular dysfunction (LVEF < 40%). The variables evaluated were: 1) clinical characteristics: age, gender, clinical presentation, presence of decompensating factor (DF), systolic blood pressure (SBP) and heart rate (HR). 2) ECG changes: T wave inversion, ST segment changes, pathological Q wave; 3) changes in the echocardiogram (ECO) regarding the location and type of segmental contraction 4) Levels of TpI, BNP, NT-proBNP, leukocytes and CRP. All patients initially underwent coronary angiography. The t test for independent samples, chi-square and logistic regression were used. Results: 65 pts were evaluated with Takotsubo and 75 pts with AMI. There were no differences between the groups regarding age, clinical presentation, SBP and HR. There were also no differences regarding the levels of BNP and NT-proBNP, TpI, leukocytes and CRP at admission. Pts with Takotsubo had a higher prevalence of females (84.6% vs 13.8%, P < 0.0001) and identification of a DF in 78% of Takotsubo pts. The ECG in the pts with Takotsubo showed a higher prevalence of T wave inversion (38% vs 12.5%; p = 0.0005) with no differences in the other ECG alterations compared to the pts with AMI; The ECHO of the pts with Takotsubo showed a higher prevalence of akinesia in Apical (69% vs 32%; P < 0.001), anterior (23% vs 5.5%; P < 0.003) and Lateral (32% vs 2.7%, P < 0.0001)walls. In the logistic regression, the variables that showed high significance and predict the diagnosis of Takotsubo were:female (OR:9.8;CI:3.1–30.8); T wave inversion on ECG(OR: 5.8; CI: 1.45 to 23.71); Apical (OR:36.12;CI:4.8–271.7) and Anterior akinesia (OR:17.3;CI:2.8–109.8) on ECHO. Conclusões: In the differential diagnosis of Takotsubo from AMI on hospital admission, the presence of female Pts, with DF, T-wave inversion on ECG, anterior or apical akinesia on ECHO, strongly suggest the diagnosis of Takotsubo in the face of AMI with ventricular dysfunction. 110998 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ANDREA VIRGINIA FERREIRA CHAVES1, Débora Nascimento da Nóbrega3, Marcelo Santos Veloso4, Eveline Barros Calado5, Manuel Markman2, Anabel Lima Vieira2, Milca Dantas da Silva1, Brivaldo Markman Filho5, Luydson Richardson Silva Vasconcelos3, Dinaldo Cavalcanti de Oliveira5 (1) Rarus – Serviço de Doenças Raras de Recife; (2) Hospital Agamenon Magalhães, Recife, PE; (3) Instituto Aggeu Magalhães; (4) Hospital Alfa, Recife; (5) Universidade Federal de Pernambuco – UFPE Introduction: Molecular investigation of hypertrophic cardiomyopathy (HCM) through genetic panels makes it possible to identify phenocopies with left ventricular hypertrophy (LVH), as Fabry disease (FD). New biomarkers are being studied, including microRNAs (miRNAs). Objective: To investigate miRNAs as pathogenicity markers in the cardiac variant of FD. Methods: A total of 131 patients (pts) with HCM who underwent genetic investigation for FD were included, of which 76 were through GLA sequencing gene and 55 by the genetic panel for HCM. Results: In 7 pts (5.34%) mutations were found in the GLA gene and the following variants were identified: 1 c.967C>A(p.Pro323Thr), 4 c.352C>T(p.Arg118Cys) and 2 c.937G>T(p.Asp313Tyr). Pts with mutations in the GLA gene were submitted to the panel for HCM: 3 pts (p.Arg118Cys) had mutations in other genes and in 4, there were no other detecting variants. The family screening revealed 17 individuals with mutations in the GLA gene, and 2 siblings were identified with LVH with the c.937G>T(p.Asp313Tyr) variant. The pts and the carriers of variants in the GLA gene were submitted to the determination of lyso-Gb3 and the analysis of the following miRNAs: miR-1291, miR-214, and miR-505. In the comparison between individuals with and without LVH, there was no significant difference for miRNAs, however, there was a tendency for overexpression in individuals with LVH, especially for miR-1291. The expression of miR-214 in subjects with LVH was significantly lower than in subjects without LVH (p = 0.0416). There was a significantly higher expression for miR-505, both in carriers of variants in the GLA gene without LVH and in those with LVH (p = 0.0195). The miR-505 and miR-214 showed a significant and directly proportional correlation with lyso-Gb3 in pooled pts (with and without LVH). All subjects had normal lyso-Gb3 levels. Conclusion: In this study, provides evidence that increased miR-505 and miR-1291 expression and miR-214 under-expression in those with LVH may be pathogenic markers in FD. Further investigations of these microRNAs with a larger number of individuals are necessary. 111032 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY INGRID LOUREIRO DE QUEIROZ LIMA1, RICARDO ZAUTIH SILVA1, FABIO AUGUSTO PINTON2, JOSE MARIANI JUNIOR3 (1) HOSPITAL IRMANDADE SANTA CASA DE SAO PAULO; (2) HOSPITAL SIRIO LIBANES; (3) HOSPITAL ISRAELITA ALBERT EINSTEIN Objective: To evaluate whether puncture guided by fluoroscopy was more effective for accessing the common femoral artery (CFA) than puncture guided by anatomical parameters alone. Background: Although radial vascular access is becoming the preferred route of access for most procedures in interventional cardiology, CFA remains the major vascular access used in practice. Properly accessing the femoral artery reduces the incidence of vascular complications; however, in some cases, the anatomical parameters may not adequately guide the appropriate route of access to the CFA. Methods: Single-center, prospective, controlled, randomized study, performed between June and December of 2015, evaluated 158 patients subjected to elective coronary angiography guided by fluoroscopy or angiography guided by classical anatomical parameters alone. In all patients, angiography was performed at the site of vascular access and was considered appropriate if the access route to the CFA was above the bifurcation of the CFA and below the inferior epigastric artery. Results: Seventy-nine patients underwent puncture guided by anatomical parameters alone, and 79 underwent puncture guided by fluoroscopy. Puncture guided by fluoroscopy achieved greater success rates than did the use of anatomical parameters alone (92.4% vs. 64.6%; p < 0.001). In all groups analyzed, the one guided by fluoroscopy had more appropriate punctures than did the group using anatomical parameters alone. The independent predictors of inadequate puncture were age > 65 years and puncture guided by anatomical parameters alone. Conclusions: Puncture guided by fluoroscopy increased the likelihood of adequate access to the CFA compared with puncture guided by anatomical parameters alone. 111048 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PALJOSKOVSKA JORDANOVA SAVETKA1, D-r Danica Petkoska Spirova1, D-r. Sasha Kjaeva Anastasova1, D-r Dejan Risteski1, D-r Emilija Lazarova Trajkovska1, D-r Enes Shehu1, D-r Aleksandra Eftimova1, Prof. D-r Marijan Bosevski1 (1) University Clinic of Cardiology Introduction: Peripheral arterial disease (PAD) is a condition in which the arteries are narrowed and they can’t carry as much blood to the outer parts of the body. Carotid artery disease (CAD) causes a narrowing of the major blood vessels that supply the brain. Both are known to be specific manifestations of atherosclerosis. We aimed to evaluate patients with documented CAD who also have PAD and investigate the risk factors that should be considered when talking about these vascular diseases. Material and methods: This study included a population of 1031 patients with documented carotid artery disease is defined as a presence of plaque or stenosis confirmed with B-mode ultrasonography. 66,6% were men and 33,5% were women. The average age was 64.44 years. We investigate how many patents with CAD have PAD and evaluate the potential risk factors. Results: This analysis reveals that 1030 out of 1031 patient (99,9%) have CAD and 1023 out of 1031 patient (99.2%) also has PAD. The most common risk factor is HTA in 1026 patients or 99.5% followed by diabetes mellitus in 1025 patients or 99.4%, tobacco smoking in 958 patients or 92.9%, HLP in 958 patients or 86.8% and also obesity found in 351 patients or 34%. Conclusion: PAD is strongly associated with carotid artery disease. According to our study, these diseases seem to occur more commonly in men that women. We can conclude that the most prevalent factors that can influence the risk of developing an outcome are hypertension and diabetes mellitus. In accordance with this, these patients need more intensive medical management to prevent complications. 111244 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CONRADO ROBERTO HOFFMANN FILHO1, Conrado Roberto Hoffmann Filho1, Benjamin Massao Harada1, João Paulo Souza Brighenti1, Michele Mendonça1, Beatriz Roca1, Gilmar Sidnei Erzinger2, Esther Botelho1, Gabriel Erzinger2, Jaqueline Barp1, João P.P. Ribeiro2 (1) Hospital Regional Hans Dieter Schmidt; (2) Univille Universidade da Região de Joinville Introduction: Acute myocardial infarction remains the leading cause of death in Brazil and worldwide with high mortality rates, especially when adequate treatment is not performed. During the period of the COVID-19 pandemic, the demand for care services caused by acute coronary syndromes (ACS) was reduced by several factors. Objetives: The present study was carried out to evaluate the management of our institution in the treatment of ACS during the COVID-19 pandemic. Methods: Retrospective cohort study of patients treated at a tertiary center from 01/01/21 to 01/31/21, through the analysis of hospital’s electronic medical records. 582 patients were selected. SPPS software was used. Student’s T test to compare numerical variables. Categorical variables were expressed as frequencies and percentages and compared using the chi-square test. Pearson’s correlation used as needed. Quantitative variables were expressed as mean and standard deviation. Results: Mean age did not differ between men and women 62.1% and 62.8% respectively. The ACS were divided in unstable angina (UA) in 30.6% and 32.1%, Non ST Elevation Myocardial Infarction (NSTEMI) 36.5% and 31.5% and ST elevation myocardial infarction (STEMI) 32.3% and 36.2%. Among coronary lesions >70%, Left anterior descending 61.4% and 64.1%, right coronary 45.6% and 49.3%, circumflex 28.9% and 35.8%, left main >50% 1.1% and 6.3%. The presence of left main lesion was higher in men. Significant positive correlation was observed between age and left descending lesion p = 0.04. In-hospital death was greater among older (67.7 and 61.9 years respectively) p = 0.003. Despite the number of deaths being significantly higher (p = 0.001) in STEMI (4.6%) compared to NSTEMI (2.4%) and UA (0.5%), the mean age was significantly lower in the first p = 0.03. Angioplasty was the most used treatment with 68.2% of cases and myocardial revascularization in 5%, p = 0.0005. Regarding treatment, about 87% used ASA, 80% antiplatelet drugs, statins 88%, ACEI/ARB 88.9% beta-blockers 85.6%, spironolactone 16.5% and insulin 8.6%. Comparing our data with literature, we found that the in-hospital mortality rate is similar (7.6%), the mean age is lower (62% and 68% respectively). The number of coronary angioplasty is higher (68.2% and 38.5%). And the number of revascularizations is lower (5% and 7.8% respectively). Conclusions: The pandemic reduced the number of patients who went to the hospital, however the standard treatment of ACS was maintained. 111065 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RICARDO MOURILHE-ROCHA1, Daniel Xavier de Britto Setta1, Marcelo Luiz da Silva Bandeira1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Eric Costa de Almeida1, Julia Paulo Mourilhe Rocha1, Bruno Reznik Wajsbrot1, Roberta Siuffo Schneider1, Ricardo Mendes Carneiro1, Vitor Hugo Mussi Campos1, Fernando Oswaldo Dias Rangel1 (1) HOSPITAL PRÓ-CARDÍACO Background: Acute myocardial infarction is a prevalent disease with high morbidity and mortality. The recognition of mortality predictors allows the identification of patients at higher risk of death. Objective: To identify the risk predictors of in-hospital mortality. Materials and Methods: Observational, retrospective, cohort study of 398 patients admitted with a confirmed diagnosis of AMI, with and without ST-segment elevation (STEMI and NSTEMI) between January 2018 and January 2022. Data were subsequently analyzed by SPSS software. Results: Among the comorbidities analyzed, mortality in the presence of previous diseases was heart failure (HF) occurred in 20.6% and 6.3% in those without HF (p = 0.03); previous stroke (stroke) in 21.4% and without stroke of 6.5% (p = 0.04); with atrial fibrillation (AF) 22% and without AF in 5.9% (p < 0.01). The mortality found in patients with different characteristics was: Killip I was 2.5% and in Killip >= 2 was 31.7% (p < 0.001); EF > 50% was 1.2% and EF <= 50% was 14% (p < 0.001); STEMI was 13.7% and NSTEMI was 4.7% (p = 0.02); without angioplasty was 13.3% and with angioplasty was 6.1% (p = 0.027). Binary logistic regressions were performed to check if such factors are independent predictors of risk, being statistically significant: ejection fraction <50%, OR 6.2 95% CI (1.6–23.6), p = 0.007; and Killip classification >= 2, 95% CI (3.0–25.7), p < 0.001. Conclusions: The predictors of mortality were the presence of EF <= 50% and Killip >= 2. Such factors are related to the extent of myocardial injury caused by AMI and the presence of previous heart disease. Early recognition of these circumstances may allow the development of care aimed at reducing this outcome. 111071 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY ANA INÊS GONZÁLES1, Ana Inês Gonzáles1, Amanda da Silva1, Gabriella Lavarda do Nascimento1, Mario Bernardo-Filho2, Danúbia da Cunha de Sá-Caputo2, Anelise Sonza1 (1) Universidade do Estado de Santa Catarina (UDESC) – Centro de Ciências da Saúde e do Esporte (CEFID); (2) Universidade do Estado do Rio de Janeiro Introduction: In the management of cardiovascular diseases (CVD), the adequate exercise prescription is an essential condition. In this scenario, the whole body vibration exercises (WBV) appear as a promising therapeutic resource. Objective: To investigate the effects of WBV alone or associated with other types of exercises in the management of CVD. Methods: This study is a systematic review following the PRISMA guidelines and registered PROSPERO under number CRD42021230663. The databases used were PubMed, Cochrane, PEDro, Lilacs, and Science Direct, from the beginning of the databases until January 2021. The descriptors related to WBV and CVD used were: [(Hypertension OR Heart Diseases OR Heart Failure OR Coronary Artery Disease OR Myocardial Infarction) AND (Whole Body Vibration OR Whole-Body Vibration)]. The following inclusion criteria were determined:studies such as controlled and randomized clinical trials, quasi-randomized controlled trials, comparative studies with or without simultaneous controls, case studies, case series with ten or more consecutive cases, cross-sectional studies, and pilot studies. The population was composed by adults (≥18 years), of both sexes, with a clinical diagnosis of CVD, submitted to intervention with the exercise of WBV. Studies in Portuguese, English, and Spanish were included. The selected studies were assessed for quality, risk of bias, and level of evidence. Results: Three studies were included, with CVD submitted to WBV as an isolated intervention (35 females/12 males). There was variation in the parameters applied in the WBV. There were no abnormal cardiovascular responses in the three studies, and no adverse events were reported. The reactive hyperemia index (RHI) showed a significant increase after WBV in several subgroups and the muscle strength measure showed a significant increase in muscle strength when compared to the control group in the two studies. There were improvements in the parameters of systolic and diastolic blood pressure, pulse pressure, augmented pressure, augmentation index, AIx adjusted to 75 beats per minute, first systolic and second systolic peak and mean arterial pressure. Conclusion: The use of different IVC protocols, alone, to improve hemodynamic, cardiovascular, vascular/arterial and muscular parameters in individuals with CVD is plausible and can be considered a safe and effective training resource. 111077 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ANA INÊS GONZÁLES1, Ana Inês Gonzáles1, Yolanda Gonçalves da Silva Fontes1, Daiane Pereira de Lima1, Tatiane Boff Centenaro1, Tales de Carvalho1 (1) Universidade do Estado de Santa Catarina (UDESC) – Centro de Ciências da Saúde e do Esporte (CEFID) Introduction: Cardiac Rehabilitation is considered an effective strategy in the treatment of individuals with Cardiovascular Diseases and has substantive evidence base on its benefits. However, in Brazil there is a predominance of programs based on rehabilitation centers that provide access to a minority of patients, providing low adherence to rehabilitation. Given this situation, alternative models are plausible to be tested, with home-based CR being a possibility to expand access and adherence of individuals. Objective: To compare adherence, quality of life (QoL), functional capacity and perception of barriers in patients undergoing center-based (CB) and home-based (HB) CR. Methods: Individuals with Coronary Artery Disease, randomized into two groups (CB and HB), were submmited to a 12 week of aerobic aerobic training with walking and patient education. Exercise prescription was individualized, based on heart rate (HR) determined by the ventilatory thresholds of the cardiopulmonary exercise test. In all, 36 physical exercise sessions were proposed. The sessions in the CB group were all supervised and monitored in person and in the HB only 3 supervised (one per month) and 33 at home (streets, squares, parks), with remote monitoring and monitoring (asynchronous). Adherence to sessions was evaluated by the average percentage of sessions performed. QOL and functional capacity were measured before and after 3 months using Short Form 36 and peak oxygen consumption, respectively. Barriers were verified after 3 months by the Scale of Barriers for CR (EBRC). Results: 21 (32.3%) individuals participated in the study, distributed in CB (N = 11) and HB (N = 10). HB showed higher adherence in the number of prescribed sessions (p < 0.001) and extra sessions (p = 0.001). Regarding QoL, only a significant intergroup difference was observed. In the HB group, in the domains of functional capacity (p = 0.04), limitations due to physical aspects (p = 0.04) and emotional aspects (p = 0.04). In the CB group, regarding the functional capacity domains (p = 0.01) and limitations due to physical aspects (p = 0.01). There was no difference between groups and between groups for peak VO2 values. Regarding the perception of barriers, the CB group had the highest total score (p < 0.01). Conclusions: HB adherence was higher, with better results in the emotional aspect of QoL and lower perception of barriers. 111076 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS DANIEL XAVIER DE BRITTO SETTA1, Marcelo Luiz da Silva Bandeira1, Bruno Reznik Wajsbrot1, Julia Paulo Mourilhe Rocha1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Claudia Lanzillotti Weksler1, Roberta Siuffo Schneider1, Ricardo Mendes Carneiro1, Vitor Hugo Mussi Campos1, Fernando Oswaldo Dias Rangel1, Ricardo Mourilhe-Rocha1 (1) HOSPITAL PRÓ-CARDÍACO Background: Acute myocardial infarction (AMI) is a prevalent disease with high morbidity and mortality. The measurement of quality indicators in health care enables the recognition of weaknesses and the construction of action plans to improve care. One of the main indicators is the utilization rate of survival modifying medications at the time of hospital discharge, whose internationally recommended goal is at least a 90% utilization rate. Objectives: To evaluate the rate of use of survival modifying drugs in AMI care [ASA, P2Y12 inhibitors, beta-blockers, statins and iECA or ARB (the latter in the presence of ventricular dysfunction) at hospital discharge]. Methods: This is an observational, retrospective, cohort study of 398 patients admitted with a confirmed diagnosis of AMI with and without ST-segment elevation between January/2018 and January/2022. Data were collected by a trained healthcare professional team and subsequently analyzed by SPSS software. Results: The diagnosis of STEMI (68.8%) was predominant. Drug utilization rates at discharge in the general population and subgroups were: AAS = 96.9% (97.6% in NSTEMI and 95.2% in STEMI; p = 0.23), P2Y12 inhibitors = 93.5% (93.9% in NSTEMI and 92.5% in STEMI; p = 0.59), beta-blockers = 86.9% (81.3% in NSTEMI and 83.8% in STEMI; p = 0.4), iECA or ARB = 82.9% (71.0% in NSTEMI and 60.0% in STEMI; p = 0.23), statins = 93.8% (93.5% in NSTEMI and 94.3% in STEMI; p = 0.79). Conclusions: The ASA, P2Y12 inhibitor, and statin utilization rates were within the recommended target. However, the usage rates of beta-blockers and ACEI or ARB were lower. The recognition of these indicators allows the initiation of strategies to expand the use of these drugs and improve outcomes in the medium and long term. 111102 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE ADELLE CRISTINE LIMA CARDOZO1, José Icaro Nunes Cruz1, Giulia Vieira Santos1, Jade Soares Dória1, Camille Marques Aquino1, Juliana Maria Chianca Lira1, Philipi Santos Soares1, Diego Maldini Borba de Lima1, Mariano César de Souza Reis1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital; (3) Primavera Hospital Introduction: Several studies analyze the influence of Spirituality/Religiosity (S/R) on cardiovascular diseases, considered the leading causes of global morbidity and mortality. The brazilian Updated Cardiovascular Prevention Guideline (2019) shows the importance of S/R in healthcare and recommends its approach in clinical practice. Objectives: To describe the clinical profile and S/R in cardiology patients and to evaluate the acceptance of the S/R approach during the medical consultation. Methods: Cross-sectional, descriptive study. The sample included patients from cardiology outpatient clinics of three hospitals in Sergipe (Brazil). A questionnaire of self elaboration and two scales were applied: Duke Religiosity Index (DUREL) and Brief Multidimensional Measure of Religiosity/Spirituality (BMMRS). The values obtained in the constructs and domains of the scales were entered into the database so that they scored directly proportional to the level of S/R. Results: 130 patients were included in the study (mean age 60.6 ± 11.2 years). 62.3% were female. The prevalences of hypertension, dyslipidemia, coronary artery disease, and diabetes mellitus were 73.8%, 59.2%, 40.8%, and 35.4%, respectively. 97.6% of the patients believe in God and 79.8% believe that S/R helps to cope with the diseases. 96.9% would like to have S/R addressed in a doctor’s appointment, however 78.1% stated that this topic was never addressed by their doctors. 96.9% said they were Christian (76.2% Catholic, 19.8% Evangelical, and 2.4% Spiritualist), 3.8% said they had no religion, and 0.8% were Umbanda believers. The domains with the highest mean scores on the BMMRS were “Values and Beliefs” (7.1 ± 1.2; scale maximum: 8.0) and “Religious and Spiritual Overcoming” (25.0 ± 2.9; scale maximum: 28.0). The means of the constructs organizational religiosity, non-organizational religiosity, and intrinsic religiosity (DUREL) were 4.1 ± 1.5, 4.6 ± 0.8, 13.8 ± 2.1, respectively, all of them considered high. Conclusions: Cardiology patients had a high level of religiousness in this study. Almost the totality of the sample believed that S/R should be addressed during medical consultations. Despite this, most stated that their physicians never addressed these topics during consultations, denoting a mismatch between clinical practice and the recommendation of the Guideline. 111090 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY RICARDO MOURILHE-ROCHA1, Bruno Reznik Wajsbrot1, Ricardo Mendes Carneiro1, Eric Costa de Almeida1, Julia Paulo Mourilhe Rocha1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Claudia Lanzillotti Weksler1, Ana Amaral Ferreira Dutra1, Tiago Azevedo Costa Mattos1, Fernando Oswaldo Dias Range1, Daniel Xavier de Britto Setta1 (1) HOSPITAL PRÓ-CARDÍACO Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice. Epidemiological data have shown that AF is a huge public health problem due to its association with a high risk of death and functional loss. Characterizing mortality predictors allows identifying patients at higher risk for evolution to death. Objective: To identify risk predictors for in-hospital mortality in patients with atrial fibrillation. Methods: Retrospective and consecutive cohort of patients hospitalized for acute atrial fibrillation between January 2018 and December 2021 at a tertiary hospital in Rio de Janeiro. Demographic, clinical, laboratory and mortality data were analyzed, obtained from a database collected by reviewing electronic medical records and subsequently performing univariate and multivariate statistical analysis using SPSS 20.0. Results: There were 819 patients, with a male predominance (57.6%), mean age of 71 +– 14.4, 72.6% aged >65 years, 66.4% hypertensive, 21.7% diabetic, 12% with HF, 8.2% with stroke or previous TIA, 21.6% with previous coronary artery disease and 8.6% with CABG surgery. The median of CHA2DS2-VASc was 3 and the median of HASBLED was 2. There was reversion to sinus rhythm in 69.9% of the patients. The general mortality of the population was 0.9%. Mortality in patients with CHA2DS2-VASc >= 3 was 1.2% vs 0 in those with CHA2DS2-VASc <3 (p = 0.02). In patients with CHA2DS2-VASc >= 2 or +, 0.9% died vs no death in patients with CHA2DS2-VASc <2 (p = 0.092). Analyzing the VASc variables (from CHA2DS2-VASc), if variables are present it increases mortality by more than 3 times (0.5% vs 1.8%; p = 0.071). Another important parameter was age >= 75 years, which almost triples mortality (0.5% aged < 75 years vs 1.3% aged >= 75 years (p = 0.193). All other variables showed no relationship to the mortality. Conclusion: The only predictor of mortality in this population was CHA2DS2-VASc >= 3. Early recognition of higher-risk patients can provide a more targeted care plan aimed at early discharge without increasing negative outcomes. 111113 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE ADELLE CRISTINE LIMA CARDOZO1, José Icaro Nunes Cruz1, Jade Soares Dória1, Camille Marques Aquino1, Gabriela de Oliveira Salazar1, Bruna Souza Matos de Oliveira1, Diego Maldini Borba de Lima1, Giulia Vieira Santos1, Juliana Maria Chianca Lira1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital; (3) Primavera Hospital Introduction: Despite advances in treatment, chronic coronary syndrome (CCS) persists as an important cause of global morbidity and mortality worldwide. Spirituality/Religiosity (S/R) has been pointed out as an important dimension in health care, including cardiovascular diseases. Objectives: To evaluate S/R in patients with CCS and its association with Resilience. Methods: This is an observational, cross-sectional, analytical study. The sample included patients from cardiology outpatient clinics of three hospitals in Sergipe. The patients included were divided into two groups: I) CCS; II) Control. Three questionnaires were applied: Duke Religiosity Index (DUREL); Brief Multidimensional Measure of Religiosity/Spirituality (BMMRS), and Connor-Davidson Resilience Scale for Brazil-10. The scales were quantified ordinally according to the progression of the degree of S/R, so that the higher the average on the scale, the higher the level of S/R. Mean scores on the scales were compared using Student’s t-test. Demographic characteristics and comorbidity profile were described in terms of means and frequencies and compared between groups using the Chi-square or Fisher’s exact test. Results: Were included 130 patients, of whom 53 (40.8%) belonged to the CCS group and 77 (59.2%) to the Control group. Ages were not different between the CCS and Control groups (62.2 vs. 59.4 years; p > 0.05). There was also no difference between the groups for the following variables: marital status, work status, diabetes mellitus, hypertension, sedentary lifestyle, and obesity (p > 0.05), however, dyslipidemia was more prevalent in the CCS group (80.0% vs. 50.7%; p < 0.001). According to the BMMRS, patients in the CCS group believe that “God punishes them” more than control patients (3.1 vs. 3.5; p < 0.05). In addition, the CCS group believed that “people in their religious communities would offer them less comfort in difficult times” than patients in the Control group (2.8 vs. 3.2; p < 0.05). There were no differences between the groups for the DUREL or the Resilience Scale. Conclusions: The results suggest that patients with CCS are more likely to believe that “God punishes them” and that “people from their religious communities would offer them less comfort in difficult times”. There was no correlation between S/R and Resilience. Studies with larger sample sizes are needed to better understand this last association. 111123 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARTA AMORIM1, Diana Ferrão1, Marta Carreira1, Fernando Nogueira1, Pedro Ribeirinho Soares1, Sergio Madureira1, Rita Gouveia1, Catarina Elias1, Ana Neves1, Joana Pereira2, Patrícia Lourenço3 (1) Heart failure clinic of the Internal Medicine Department of Centro Hospiralar e Universitário São João; Faculdade de Medicina da Universidade do Porto; (2) Heart failure clinic of the Internal Medicine Department of Centro Hospiralar e Universitário São João; (3) Heart failure clinic of the Internal Medicine Department of Centro Hospiralar e Universitário São João; Cardiovascular Research and Development Unit of Faculty of Medicine of Porto University Introduction: Low Chloride (Cl) has been associated with worse prognosis in heart failure (HF). Still, results are controverse and mainly based in old Randomized controlled trials. Objectives: To assess determinants of Cl levels and to study the prognostic role of Cl in a contemporary real-world chronic HF population. Methods: Retrospective analysis of chronic ambulatory HF patients followed in a HF clinic. Adult patients with history of HF with left ventricular systolic dysfunction (LVSD) attending the clinic between January 2012 and May 2018 were included. Patients with no Cl measurement were excluded. Follow-up: until February 2022. Primary endpoint: all-cause mortality. Multivariate linear regression analysis to assess independent determinants of Cl level. The prognostic impact of Cl was evaluated by Cox-regression analysis. Cl was assessed both as a categorical (cut-off 101 mmol/L – percentile 33.3) and as a continuous variable. Multivariate adjustment was performed accounting for: age, gender, LVSD, NYHA class, arterial hypertension, diabetes, atrial fibrillation, ischaemic aetiology, BNP, renal function, serum sodium, loop and thiazide diuretics and evidence-based therapy. Results: We studied 859 patients. Mean age 70 years, 66.3% male, 44.9% had ischaemic HF and 46.7% had severe LVSD; 84.3% were on renin-angiotensin system inhibitors (RASi), 92.8% on beta blockers and 29.1% on mineralocorticoid receptor antagonists. Cl ranged from 85 to 116 mmol/L. Independent determinants of decreased Cl levels were Diabetes, severe LVSD, higher NYHA class and lower natremia. RASi medication associated with higher Cl and loop and thiazide diuretics predicted lower serum Cl. During a median follow-up of 56 (29–90) months 464 (53.4%) patients died: 45.9% in those with Cl > 101 mmol/L and 66.6% in the remaining, p < 0.001. Patients with Cl ≤ 101 mmol/L) had a multivariate adjusted HR of death of 1.27 (95% CI 1.02–1.57), p = 0.03 compared to those with higher Cl. Per each 2 mmol/L increase in Cl levels the all-cause death HR was 0.94 (95% CI: 0.89–1.00), p = 0.048. Conclusions: Low Cl is an independent predictor of all-cause mortality in HF with LVSD. Patients with Cl ≤ 101 mmol/L have a 27% increased risk of death and per each 2 mmol/L Cl increase there is a 6% decrease in the death risk. Main determinants of lower Cl are diuretic use, concomitant diabetes, worse LVSD and higher NYHA class; use of RASi and higher sodium levels are independently associated with higher Cl levels. 111134 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARCUS VINICIUS SIMÕES1, Káryta Suely Macedo Martins1, Antonio Carlos Barros-Filho1, Minna Moreira Dias Romano1, Denise Mayumi Tanaka1, Paulo Louzada-Junior1, Edecio Cunha-Neto2, José Antonio Marin-Neto1 (1) Faculdade de Medicina de Ribeirão Preto – USP, Ribeirão Preto, SP, Brasil; (2) Incor – Instituto do Coração – FMUSP Background: The essential histopathologic lesion in chronic Chagas’ cardiomyopathy (CCC) is a relentless low grade myocarditis with increased production of cytokines. Recent experimental studies have suggested that pentoxifylline, an immunomodulator that reduces the production of TNF-alfa, may be associated to a better evolution of CCC. Purpose: We tested the effect of pentoxifylline in cytokines profile and cardiac function in patients with CCC. Methods: This is a prospective double-blind clinical study, enrolling 38 CCC patients (age: 60 +/– 13 y.o., 66% male, LVEF = 48 +/– 7%), randomized to pentoxifylline (PTX, n = 19), 400 mg 3x/day for 6 months or placebo (PLC, n = 19). At baseline and post-treatment, patients underwent assessment of serum cytokines (IL6, IL10, TNF-alfa) and 2D-Echo with measurement of LVEF, indexed LV end diastolic volume (LVEDVi), LV wall motion score index (WMSi), tricuspid annulus systolic excursion (TAPSE). Mixed effects ANOVA for repeated measures was used to test the treatment effect between groups. Results: The table summarizes the results. We observed a strong trend toward reduction of TNF-alfa and increase of IL10 values in the PTX group, but no significant effect in cardiac function variables. Conclusion: Despite its immunomodulator effect, pentoxifylline was not associated to significant improvement in cardiac function in patients with CCC. 111145 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY JULIANA RIBEIRO GOUVEIA REIS1, Laissa de Cássia Alves2, Karine Siqueira Cabral Rocha2, Alessandro Reis3, Juliana Ribeiro Gouveia Reis1 (1) Instituto Pró-Vida; (2) Centro Universitário de Patos de Minas; (3) Hospital Santa Casa de Misericórdia de Patos de Minas Metabolic syndrome is characterized by a set of cardiovascular risk factors such as arterial hypertension, insulin resistance, hyperinsulinemia, glucose intolerance/type 2 diabetes, central obesity and dyslipidemia. People with this condition have low functional capacity. The cardiopulmonary stress test is considered the gold standard in the assessment of physical capacity, however, it requires specific equipment and a specialized team. Therefore, the Shuttle walking test becomes a more practical option to estimate VO2 max in this population. The purpose of this study was evaluate the measurement of indirect VO2 in people with metabolic syndrome and compare it with predicted values for normality. This is a cross-sectional study with a quantitative approach. The inclusion criteria for the study were patients with metabolic syndrome who were being treated at the State Center for Specialized Care in Minas Gerais/Brazil, aged between 18 and 59 years. Patients with SatO2 < 88%, severe arterial hypertension, aortic coarctation, and decompensated heart failure were excluded. Patients underwent anthropometric assessment and then the Body Mass Index was calculated. The test was started after analyzing the contraindications and the VO2 obtained and the maximum expected VO2 were recorded. The results were analyzed using descriptive statistics, mean and standard deviation using the Wilcoxon test. The significance level was set at 0.05. The project received approval from the Research Ethics Committee under opinion number 3,153,984. Thirty patients participated in this research, 15 men and 15 women. The mean age of the male group was 55 years (±3.6) and the mean age of the female group was 49 years (±3.6). The mean BMI of the male group was 32.45 (±3.44). The female group had a mean BMI of 33.77 (±4.31). Regarding the VO2 of male patients, an average VO2 value of 12.84 (±2.20) was observed, while the average VO2 maximum was 72.90 (±2.12), with p value <0.05. In female patients, there was a lower value compared to males, with an average VO2 obtained of 12.52 (±2.20), and VO2 max with an average of 36.31 (±4.49), with a value of p < 0.05, also with mean values below the male participants. It is concluded that individuals with metabolic syndrome have low VO2 max values when compared to the expected values for this population and women have lower rates. 111149 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY VIVIANE PAIVA DE CAMPOS1, Ana Carolina Krauspenhar Gluszczuk2, Nathalia Jacques Pereira2, Francisca Moura Strebel3, Fernanda Lucchese-Lobato4 (1) Instituto de Cardiologia/Fundação Universitária de Cardiologia Programa de Pós-graduação em Ciências da Saúde: Cardiologia; (2) Universidade Federal do Rio Grande do Sul; (3) Universidade Federal de Ciências da Saúde de Porto Alegre; (4) Irmandade Santa Casa de Misericórdia de Porto Alegre Background: About 320,000 preterm (PT) infants are born in Brazil annually. Of these, it is estimated that 10–15% have congenital heart disease (CHD). Infants with CHD are born with normal body weight and lose it substantially after birth. The stunted growth and low weight characteristic of CHD infants are associated with poor developmental and surgical outcomes. Due to the challenges that infants with CHD face they are less likely to breastfeed, which leaves them more vulnerable to a diminished immune system, incidence and severity of diseases, mortality, prolonged hospital stay, and developmental delays. Objective: To verify the prevalence of breastfeeding in PT infants with CHD admitted to a reference children’s hospital in Southern Brazil between 2019–2022. Methods: This is a descriptive cross-sectional retrospective study. Data from medical records of PT infants (<37 weeks gestational age) with CHD, ages 0–12 months submitted to surgical procedures between 01/19 to 03/22, were analyzed. Infants with genetic syndromes were excluded from the analyses. Demographic, anthropometric, and clinical data were collected from electronic charts and entered into the REDCap data platform. Statistical analyses were performed using the SPSS v. 25 software. Results: The 62 infants included in the study were mainly late PT (34–36 weeks and 6 days; 68%), female (53%), white (73%), acyanotic CHD (58%), and SUS users (77%). Seventy percent of all infants had appropriate weight for gestational age at birth. Most infants were formula fed (73%), while only 27% received breast milk, either exclusively (3%), complemented with solids (5%), or mixed breast milk and formula (19%). Fifty-two percent used tube feeding exclusively. Only 13% of tube-fed infants received breast milk. At the time of surgery, 65% were severely stunted, 56% severely underweight and 50% extremely thin/thin in BMI. Conclusions: The findings suggest that premature infants with CHD in Southern Brazil are more likely to have an adequate nutritional profile at birth with increasing deficits and significant weight loss in the pre-surgical period. Due to the low prevalence of breastfeeding and high use of exclusive tube feeding, we conclude that it is of utmost importance to encourage breastfeeding and nutritional programs to promote weight gain and better immunity before surgery. Future multidisciplinary programs should focus on milk bank collection and inclusive breastfeeding pre-surgical protocols. 111197 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RAMAKRISHNAN SIVASUBRAMANIAN1, Chandini Suvarna1, Praveen Aggarwal1, Sandeep Seth1, Neeraj Parekh1, Meenakshi Sharma2, Sheikh Vamik1, Ambuj Roy1, Ganesan Karthikeyan1, Sandeep Singh1, Rajiv Narang1, Balram Bhargava2 (1) All India Institute of Medical Sciences (AIIMS), New Delhi, India; (2) Indian Council of Medical Research (ICMR), New Delhi, India Background: Outcomes of ST elevation myocardial infarction (STEMI) are suboptimal in India. Pre-hospital thrombolysis is an established practice in developed countries for timely reperfusion in STEMI. Mission DELHI (Delhi Emergency Life Heart Attack Initiative) is a pilot project for pre-hospital thrombolysis using motorbike ambulance service by trained paramedics at the patient’s doorstep in a selected geographical area of New Delhi. To the best of our knowledge, this is the first study of prehospital thrombolysis in India. Objective: In this pilot phase, we report the safety and feasibility of prehospital delivery of care in STEMI. Methods We covered a geographical area of 5-km around our institution. A command center was set up and the patients were required to call a dedicated helpline number. Upon initial screening, a motorcycle ambulance was dispatched to the caller’s location. A brief history, physical examination and an ECG were obtained. ECG was electronically transmitted to the command center and thrombolysis with Tenecteplase was given at patient’s doorstep after evaluation of the ECG by a cardiologist. Results: A total of 26 STEMI patients (mean age 56.2 years; 88.5% male) were treated either at home (46.2%), public places (23.1%), place of work (15.4%) or at small clinics (15.4%). Time taken to reach the patient location was 15.1 ± 6.1 min, and call to ECG time was 25.0 ± 6.8 min. Out of the 26 patients with STEMI, 15 patients were thrombolysed at a pre-hospital location with a door to needle time of 31 ± 11.1 min. Out of these, 13 patients had successful thrombolysis and had undergone further MI management. There was one death in a patient with cardiogenic shock. We also managed a total of 60 patients diagnosed with NSTEMI. Conclusions: In this pilot study, we have demonstrated the feasibility and safety of a telemedicine supported pre-hospital thrombolysis by paramedics at the patient’s location. Such an approach is likely to significantly improve thrombolytic therapy usage and reduce ischemic times, and thereby could result in significant improvements in STEMI outcomes in a country like India. 111208 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING JOSÉ LUIZ BARROS PENA1, Isabel Cristina Gomes Moura1, Marcos Victor Prosdocimi Diniz1, Fabiano Maia Linhares1, Pedro Manuel Marques Cristóvão1, Eduardo Back Sternick1, Amanda Marcos de Oliveira2, Gabriela de Campos Viveiros2, Rafaela Santos Garcia2, Eduardo Felipe Souza de Deus2, Tatiane Pires da Silva2, Tarcila Paoli Marques Moreira2 (1) Faculdade Ciências Médicas de Minas Gerais; (2) Hospital Felício Rocho Introduction: Due to its various clinical manifestations, Fabry disease (FD) is a commonly misdiagnosed rare condition. Treatment depends ideally on early disease detection and timely enzyme replacement therapy to improve prognosis. Echocardiography may be an efficient noninvasive technique to detect structural and functional heart consequences resulting from the disease. This study aimed to determine whether new echocardiographic modalities may contribute to more improved diagnoses. Material and methods: Prospective and transversal study of 15 patients (Pts) with FD. During one examination, conventional transthoracic, bi-, and tridimensional echocardiography as well as strain/strain rate speckle tracking were measured. Specific echocardiographic data were obtained and exported to a workstation for subsequent evaluation. Statistical analyses were performed with 5% significance using the R program, version 3.1.3. Results: Nine Pts (60%) were male, and the mean age was 39 ± 11.9 years. Pts presented increased left atrium volume index, mean left ventricle (LV) septum, and posterior wall thickness. LV hypertrophy was found in varying degrees in 8 (53%) FD Pts. Mean myocardial mass value measured by the 4D technique was 110.9 ± 8.1 grams. A binary appearance of LV endocardial border (“binary sign”) and hypertrophy of the anterolateral papillary muscle were detected in 9 (60%) of Pts. Mitral regurgitation was present in 4 (26.7%) Pts, mitral valve prolapse in 2 (13.3%), and aortic regurgitation in 1 (6.7%). Four (26.7%) Pts presented RV hypertrophy. Global mean longitudinal 2D strain measured –19.5 ± 3.4%, and 4D circumferential strain measured –17.7 ± 3.5%. We identified peak systolic strain reduction in basal and mid inferolateral segments in 10 (66.6%) Pts. By analyzing the bull‘s-eye plot. Conclusion: Echocardiography is an accurate, robust tool in Fabry disease evaluation. Compared to conventional measurement techniques, advances in 3D and speckle tracking echocardiography have augmented and improved anatomical and functional data. 111251 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION FERNANDA SANCHES AGUERA GROCHOCKI1, LAÍS SANCHES AGUERA1, CRISTINA PELLEGRINO BAENA1, JOSE ROCHA FARIA NETO1 (1) PONTIFÍCIA UNIVERSIDADE CATÓLICA DO PARANÁ – PUCPR Introduction: Cardiovascular diseases are the leading cause of mortality in the world. The identification and correction of modifiable risk factors are the cornerstone of cardiovascular prevention. However, this purpose in performing periodic health assessment (check-up) in asymptomatic individuals still has its effectiveness questioned. Objective: To analyze whether the periodic health assessment can be a tool for the control and treatment of cardiometabolic risk factors and whether the impact of this assessment is different according to the presence of these factors. Methods: A retrospective cohort was carried out, including data from patients seen consecutively in 2015 and 2016, at a check-up service in Curitiba, Paraná. The cardiometabolic profile of the patients was evaluated and a comparison was made between variables of interest obtained in 2015 and 2016. It was also evaluated whether the variation from one year to the other was different between patients who presented cardiometabolic alterations in the first year compared to those who presented variables without alterations. Microsoft Excel and SPSS Statistics programs were used for calculations, using Student’s T test for paired samples or Wilcoxon test, according to normality tests. Results: A total of 478 patients were included, 80.3% of whom were male and with a mean age of 46.29 years (SD ± 8.06). The following altered variables showed improvement: triglycerides (p < 0.05), HDL (p < 0.001), LDL (p < 0.001), non-HDL (p < 0.001), fasting glucose (p < 0.001) and glycated hemoglobin (p < 0.001). Evaluating altered anthropometric data, there was an improvement in Body Mass Index(BMI) (p < 0.01), Systolic Blood Pressure (p < 0.001), Diastolic Blood Pressure (p < 0.001) and male waist circumference (p < 0.05). Analyzing the altered variables, there was a change to values within the normal range in 21% of cases of total cholesterol, 29% triglycerides, 26% HDL, 27% LDL, 32% non-HDL, 7% BMI, 87% systolic blood pressure, 84% Diastolic blood pressure, 10% waist circumference, 54% blood glucose, 43% glycated hemoglobin. (p < 0.001). Conclusion: Periodic health assessment seems to have a positive impact on the control and treatment of cardiovascular risk factors. This effect was mainly observed in patients who already had altered laboratory and anthropometric variables in the first evaluation. 111254 Modality: E-Poster Researcher – Non-case Report Category: CARDIO-ONCOLOGY DANIANE RAFAEL1, Juliana Bueno Refundini1, Adriana A. da Silva1, Ana Cristina Camarozano1, Claudio Leinig Pereira da Cunha1, Sérgio Lunardon Padilha1, Admar Moraes de Souza1, Ana Karyn Ehrenfried de Freitas1 (1) Universidade Federal do Paraná – UFPR Chemotherapeutics brought greater survival to breast cancer patients, however, they can generate cardiotoxicity with important dysfunction. The echocardiogram (Echo), has enabled early detection of myocardial dysfunction. Our purposes was to obtain, with Echo, the incidence of left ventricle dysfunction after chemotherapy, analyzing the Ejection Fraction (EF) in 2 dimensions-Simpson Method (2D) and in 3 dimensions (3D), and to identify early contractile changes using the Longitudinal Strain – Speckle-Tracking (ST). A prospective study studied 37 women, mean age 48 ± 11 years with breast cancer in the beginning of chemotherapy between 2013 and 2015. Echo was performed at baseline, after 3 and 6 months, evaluating 2D EF and 3D EF in addition to the contractile evaluation using the ST and peak systolic velocity at the base of the lateral wall (lateral S’) on tissue Doppler. The patients were also evaluated for signs, symptoms, possible risk and protective factors contributing to myocardial dysfunction overall. Dyspnea was the only symptom detected (23%). We observed that the altered ST in the 1st exam proved to be a predictor of 3D EF reduction in the 3rd exam (p = 0.049). The lateral S‘ reduced at 6 months, corroborating the change in systolic function. Both risk and protective factors did not affect the parameters evaluated. There was a progressive decrease in EF after exposure to cardiotoxic chemotherapeutic agents, which became more evident by 3D analysis. The evaluation of the ST showed significant changes associated with the period of administration of anthracycline, and an ST altered in the beginning of the treatment was a predictor of EF reduction at 6 months of treatment. These findings support the importance of these new Echo techniques in the follow up of these patients. 111263 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES AMBUJ ROY1, Ambuj Roy1, Nitish Naik1, Sharma Sanjiv1, Sandeep Singh1, Gautam Sharma1, Ramakrishnan Lakshmy1, Rohit Bhatia1 (1) All India Institute of Medical Sciences (AIIMS) Background: Rheumatic mitral stenosis (MS) is associated with significant morbidity, especially ischemic stroke(IS) and continues to be very prevalent in developing countries. Clinical predictors of IS are poor and is common even among those not in atrial fibrillation (AF). Silent brain infarct (SBI) is an important predictor and surrogate of future ischemic stroke. In this study, we assessed the prevalence of SBI in patients with MS in normal sinus rhythm (NSR) and AF. Methods: Consecutive patients with more than moderate mitral stenosis underwent non-contrast MRI. Standard MRI sequences and definitions for infarct were used. Patients with prior intervention and surgery for MS were excluded as were those with prior clinical IS or transient ischemic attack (TIA), left atrial clot, and severe concomitant other valval lesions. Results: Of 211 patients who underwent MRI, 140 were in NSR and 71 in AF. 59% were females, 88% had severe MS, 23%, 24% and 53% were in NYHA Class I, II and III respectively. A total of 44 (21%) patients had SBI. The baseline variables in those with normal MRI scans and with SBI were as in Table 1. Only NYHA functional class and not AF was significantly different in patients with SBI. Conclusions: SBI is common among patients with MS including those in NSR. Only NYHA class was different in the two groups. Better predictors of IS beyond rhythm are needed in patients with MS to decide on IS preventive strategies. 111280 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY ABISAI DOS SANTOS SANTANA1, Giovana Côrte Real Ruffier2, Mauro Felippe Felix Mediano1, Daniel Arthur Barata Kasal1 (1) Instituto Nacional de Cardiologia; (2) Universidade do Estado do Rio de Janeiro Introduction: The assessment of physical performance in the preoperative evaluation of open-chest heart surgery (OHS) can help to establish the most appropriate moment for the intervention. There are noninvasive and simple methods that can be used at the bedside, in order to evaluate physical performance in these patients. Objective: To evaluate physical performance in the preoperatory period of elective OHS and establish associations with surgical recovery outcomes. Methods: Adult patients subjected to elective OHS, either coronary artery bypass grafting (CABG) or valvular replacement (VR) surgery, at a quaternary hospital were recruited for the study. Physical performance was measured with 30-s chair-stand-test (30sCST), timed up and go test (TUGT) and hand grip, performed during surgical admission, before the intervention. Surgical risk was estimated with the Euroscore II risk score. Clinical data were obtained using medical records. The outcomes evaluated were mechanical ventilation time (MVT), admission time (AT), and in-hospital death (IHD), and their associations with the physical tests were evaluated with either logistic (IHD) or linear (MVT, AT) regressions (p < 0.05). Results: One hundred sixty-six individuals were evaluated, most patients were male (65%), aged 58.3 ± 11.3 years, and with body mass index of 27 ± 4.4 kg/m2. Left ventricular ejection fraction (Teicholz) was 59.4 ± 15.5%, and New York Heart Association functional classes I, II, III and IV were 8, 70, 53, and 3%, respectively. Euroscore II was 3.1 ± 2.1, 98% of surgeries employed extracorporeal circulation, and IHD was 7.2%. In the unadjusted analysis, the hand grip was associated with IHD (OR 0.90; p = 0.007), but the association was not significant after adjustments for age and sex, or Euroscore II (OR 1.00 p = 0.93 and OR 0.92 p = 0.06, respectively). None of the physical tests were associated with either AT or VMT. Conclusions: The present results suggest a lack of association between the 30sCST and TUGT physical tests and the recovery variables after OHS studied. Preoperatory hand grip offered an association with mortality after surgery, but significance was borderline after adjustment for Euroscore II. The contribution of physical fitness before OHS to surgery outcomes may be evaluated with additional methods and in particular groups such as the elderly in future studies, in order to explore this important association. 111769 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY LUCAS CARVALHO DIAS1, Luiz Eduardo Montengro Camanho2, Eduardo Benchimol Saad2, Charles Slater1, Luiz Antonio Oliveira Inacio Junior2, Gustavo Vignoli Santos2, Ricardo Mourilhe Rocha1 (1) Hospital Universitario Pedro Ernesto; (2) Hospital Pró-Cardíaco Introduction: The hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease, strongly related to the occurrence of malignant ventricular arrhythmias and sudden death. Objective: Describe the clinical data and the occurrence of appropriate and inappropriate therapy in a subgroup of HCM patients with implantable cardioverter-defibrillators. Methods and results: In a cohort of 720 consecutive and retrospective patients, monitored from March 2006 to December 2021, with 57 of this total being diagnosed with HCM. The male sex was found in 42 (74%), and the average age was 54.6 years. ICD implantation by primary prophylaxis occurred in 49 (86%) patients and in 8 (14%) by secondary prophylaxis. The report of unexplained syncope was observed in 50 (88%) patients. At the time of ICD implantation, the records showed that 51 (89%) patients were in NYHA functional class 1 and the average LVEF was 59,4%. The electrocardiographic analysis showed that the mean QRS was 118 ms and that 25 (44%) had an LBBB pattern. During the ICD monitoring period, 11 patients (19%) were observed with a record of VF/VT and appropriate therapy. Inappropriate therapy was observed in 7 patients (12%), all in the AF group, which occurred in 25 (44%) patients throughout the record. Conclusion: Despite the not insignificant number of inappropriate therapies, the ICD has been shown to be an effective therapy in preventing sudden cardiac death and in the clinical management. 111563 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION CAMILA MACIEL DE OLIVEIRA1, Roberto Luis Zagury3, Luiza Borcony Bolognese4, Davi Casale Aragon5, Livia Mandina da Graça Couto2, Marcela Carvalho2, Thaíza dos Anjos2, Mercedes Balcells6, Clemente Nobrega2, Chunyu Liu7 (1) Universidade Federal do Parana (UFPR); (2) Klivo LLC; (3) Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione (IEDE); (4) Pontificia Universidade Catolica de Minas Gerais (PUC-Poços); (5) Universidade de São Paulo (USP-RP); (6) Massachusetts Institute of Technology; (7) Boston University Digital therapeutics, an emerging type of medical approach, is defined as evidence-based interventions through qualified software programs that help prevent, manage, or treat chronic diseases. We aim to describe the clinical impact of a digital therapeutic product of a Brazilian startup company that focuses on individual needs as an essential shift for the ongoing management of patients with chronic conditions such as diabetes, hypertension, and dyslipidemia. This program is a model of behavior change based on an intensive lifestyle intervention method and seeks to manage cardiovascular risk factors in adults aged ≥ 18 years. It was sponsored by health plans and healthcare provider organizations and was free for patients. During the management process, patients were remotely supervised by nurses on a weekly basis via phone calls intended at health education and assessment of laboratorial outcomes. An app was developed to improve the communication between patients and nurses, but e-mails and text messages were also used. To improve control of glycemic events in patients with diabetes, a glucometer was sent to the patient’s home and was connected to the app. When abnormal glucose parameters were detected, the patient was contacted according to an established protocol. Outcomes of interest were evaluated at baseline and after 3 months. For a total of 2544 patients (53.35 ± 13.45 years; 50% male) distributed in 21 Brazilian states (40% in São Paulo), it was possible to verify significantly improved glycemic and lipid profile (total cholesterol and LDL-cholesterol) after follow-up for 3 months. A1c decreased (9.13 ± 2.10 versus 7.14 ± 1.69; p < 0.01), and so did fasting glucose (127.19 ± 51.96 versus 122.51 ± 42.40; p = 0.03), total cholesterol (176.66 ± 50.11 versus 161.36 ± 45.72; p = 0.01), and LDL-cholesterol (100.42 ± 40.11 versus 89.87 ± 33.82; p = 0.03). No difference was observed for HDL-cholesterol or triglycerides in this sample. This program has shown promising findings related to glycemic and lipid profiles, providing insights into the management of cardiovascular risk factors, which will allow us to reshape this unique therapeutic approach for the Brazilian population continuously. 111319 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT PEDRO GABRIEL MELO DE BARROS E SILVA1, Denilson Campos Albuquerque3, Marcus Vinícius Simões5, Renato Delascio Lopes6, Conrado Hoffmann7, Paulo Roberto Nogueira8, Helder Reis9, Fabio Akio Nishijuka10, Lidia Zyntynski Moura11, Fernando Bacal3, Evandro Tinoco Mesquita3, Múcio Tavares de Oliveira Junior3 (1) IP-Hcor; (2) Hospital Samaritano Paulista; (3) Sociedade Brasileira de Cardiologia; (4) Hospital Copa D’Or; (5) Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo; (6) Brazilian Clinical Research Institute; (7) Hospital Regional Hans Dieter Schmidt; (8) Fundação Faculdade Regional de Medicina de São José do Rio Preto; (9) Hospital de Clínicas Gaspar Viana; (10) Hospital Naval Marcílio Dias; (11) Irmandade Santa Casa de Misericórdia de Curitiba; (12) Instituto do Coração (INCOR HC FMUSP) Background: Heart failure with preserved ejection fraction (HFpEF) accounts for almost half of the HF cases. However, little is known about the characteristics, medical therapies, and long-term prognosis of patients hospitalized with HFpEF in Latin America. Methods: BREATHE was a nation-wide prospective registry that included patients hospitalized due to acute heart failure (AHF) in Brazil. In-hospital management as well as 12-month clinical outcomes were assessed. In the current analysis, patients were classified according to left ventricular ejection fraction (LVEF) in 3 groups: <40% (HFrEF), 40 to 49% (HFmrEF) and ≥50% (HFpEF). Results: A total of 3,013 patients were included with a median follow-up of 346 days. In 1,204 patients, the LVEF was assessed during the first 24 hours of hospitalization with 28.1% of patients classified as HFpEF. The patients with HFpEF were older (70.5 ± 16.4 vs 63 ± 15.9; P < 0.01) had more female patients than HFrEF ones (57.5% vs 30.4%; P < 0.01). Among comorbidities, hypertension (81.4% vs 70.5%) and atrial fibrillation (40.5% vs 27.2%) were more common in patients with HFpEF (both P < 0.01), while history of myocardial infarction was more common in HFrEF (18.9% vs 29.4%; P < 0.01). The creatinine was 1.5 ± 1.0 mg/dL and similar among the 3 groups but the level of BNP/NT-ProBNP was >2 times lower in the HFpEF population. The wet-cold profile occurred in 5.9% of the patients with HFpEF and in 12.6% of patients admitted with HFrEF (P = 0.04). The main cause of decompensation was poor adherence in the HFrEF patients, and infection in HFpEF. At hospital admission, almost 54% of HFpEF patients were using renin-angiotensin-aldosterone inhibitors or beta-blockers, and 19.5% were using spironolactone. There was no enhancement in the use of these medications during the 12 months follow-up. Intra-hospital mortality was similar among the 3 groups of LVEF (average 10.5%). After discharge, the mortality and hospital readmission rates at 365 days were not statistically different in all groups (23.3 deaths for 100 patient years with 51% readmission rate at 12 months in the HFpEF group). Conclusions: In this large multicentre nationwide Brazilian prospective registry of AHF, patients with HFpEF presented different clinical characteristics but similar prognosis compared to patients with lower ejection fraction during one year of follow-up. Improvement in the medical care is necessary to minimize complications in this high-risk population. 111345 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO RAMOS NASCIMENTO1, Washington Luis S. Ramos2, Gisele Lobo Pappa2, Edson Roteia Araújo Júnior2, João Francisco Barreto da Silva Martins2, Wagner Meira Júnior2, Sander Luis Gomes Pimentel1, Juliane Franco1, Kaciane Krauss Bruno Oliveira1, Maria Carmo Pereira Nunes1, Antonio Luiz Pinho Ribeiro1, Erickson Rangel do Nascimento2 (1) Serviço de Cardiologia e Cirurgia Cardiovascular e Centro de Telessaúde do Hospital das Clínicas da UFMG, Belo Horizonte – MG, Brazil; (2) Departamento de Ciência da Computação da Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brazil Introduction: Cardiac involvement seems to impact prognosis of COVID-19. Bedside echocardiography (echo) holds promise for early prediction of unfavorable outcomes, and artificial intelligence may be an additional tool to overcome personnel limitations. We propose a spatial-temporal deep learning-based approach for automatic prediction of mortality of inpatients with COVID-19 with echo images. Methods: Patients admitted in 2 reference hospitals in Brazil in 90 days with confirmed moderate and severe COVID-19, based on the Berlin criteria, underwent clinical and laboratory evaluation, and focused bedside echo (GE Vivid IQ), following admission, with remote interpretation by telemedicine in Brazil and the US. Independent echo predictors of all-cause mortality were assessed, after adjustment for clinical variables. Our image dataset consists of 737 videos (Mpeg) collected from included patients in 3 different probe positions: apical 4-chamber and parasternal long and short axis. It was used to predict the patients’ outcome (discharge or death) in a 10-fold cross-validation procedure manner. We used a 2-stream deep neural network composed of two identical ResNet-18 Convolutional Neural Networks (CNN) – originally developed for Rheumatic Heart Disease diagnosis – followed by attention units to extract relevant spatial and temporal features from RGB and Optical Flow frames. A final softmax layer is used for classification, and accuracy measures are presented with 95% confidence intervals (CI). Results: Total 163 patients were enrolled, mean age was 64 ± 16 years, 107 (66%) were admitted to intensive care and in-hospital mortality was 34% (N = 56). Independent predictors of mortality, after adjustment for clinical and demographic variables, were age ≥ 63 years (OR = 5.53, 95%CI 1.52–20.17), LVEF < 64% (OR = 7.37, 95%CI 2.10–25.94) and TAPSE < 18.5 mm (OR = 9.43, 95% CI 2.57–35.03), C-statistic = 0.83. During training, we rescaled videos to 224 × 224 pixels and used temporal jittering to select a final clip with 32 frames per video in random batches of size 6. Our proposed method achieved an exam-wise accuracy of 61.21% (95%CI 50.87–71,55), sensitivity of 59.31% (95%CI 36.12–82.5) and specificity of 65.18% (95%CI 45.08–85.28) for mortality. Conclusion: Automatic detection of high-risk echo findings in COVID-19 inpatients at bedside seems feasible and, with more research, can improve mortality prediction at the point-of-care, especially in low-income settings. 111379 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES EZEQUIEL J ZAIDEL1, Lara Gheco1, Luis Guillermo García Chamorro1, Erna Florencia Segovia1, Diego Carvallo Claros1, Elena Vargas Parra1, Agustín Monzón1, Brian Perotti1, María Inés Sosa Liprandi1, Álvaro Sosa Liprandi1 (1) Sanatorio Güemes, Buenos Aires, Argentina Introduction: Migratory movements have led to Chagas disease (CD) being diagnosed in non-endemic areas. There are few epidemiological studies on the prevalence and behavior of the disease in urban areas. Aim: To describe the clinical profile of patients with CD evaluated in an urban area and to identify the frequency of high risk cases. Methods: Retrospective cohort study of patients who were hospitalized or evaluated in outpatient clinics, in a third level center from Buenos Aires. Patients over 18 years of age with a diagnosis confirmed by serology were selected. The patients were evaluated by clinical examination and complementary studies in accordance with current standards. A descriptive analysis was performed and the risk scores of Viotti, Rassi, Pinho Ribeiro, and Sousa were analyzed. Results: Data from 46 patients were analyzed, with a mean age of 61 ± 11 years, and 41% female. Main origin of patiets was nothern provincies from Argentina, as well as Bolivia and Paraguay. The diagnosis was made mainly by medical check-up due to risk factors for ECH (30%) or by finding in blood banks (10.8%). The median time of evolution from the date of diagnosis of the disease to the cutoff made for this analysis was 6 years (IQR 1–12). Only one case in this cohort had received trypanocidal treatment. The prevalence of cardiomegaly was 51% by radiography, but the diastolic diameter by echocardiogram was 51 mm ± 7 mm with a mean ejection fraction of 58%. 21% presented right bundle branch block, 4% left anterior hemiblock, 18% atrial fibrillation, and 6% low voltage and ventricular extrasystoles. Fifty percent had symptomatic heart failure, 23% had implanted cardiac devices, and 17% had cerebrovascular accident (CVA). No cases of digestive or neurological pathology were found. By Viotti score, 41% had a high risk of progression. Using Rassi score, 10% had a high risk of mortality, but by the Pinho Ribeiro score, only 1 patient had a high risk. When analyzing the risk of stroke, 16% of the cohort presented high risk by Sousa score, however, anticoagulation in this cohort was prescribed only to subjects with atrial fibrillation or those who had already had a stroke. Conclusions: The evaluation of patients with urban CD is a growing challenge for cardiologists from urban settings: Using current scores, the proportion of high risk cases was variabe, highlighting the need of better stratification tools. 111386 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY IEDA PRATA COSTA1, Eduardo Arrais Rocha1, Almino Cavalcante Rocha Neto1, Roberto Lima Farias1, Daniele Melo Leopoldino1, Cristiane Liberato1, Ronaldo Vacsoncelos Távora2 (1) Hospital Universitário Walter Cantidio – universidade Federal Ceara; (2) Hospital de Messejana _ SESA/CE Introduction: Cardiovascular death is the main cause of death in chronic Chagas‘ heart disease (CCC). Syncope in CCC can be caused by bradyarrthymias (sick sinus syndrome-SSS and conduction disorders) or ventricular arrhythmias. Objectives: To evaluate the association of syncope and pre-syncope with important unfavorable outcomes (SSS, severe conduction system disorders – HV >70 ms and VT/VF) in the electrophysiological study (EPS) in patients with CCC. Methods: This is a prospective cohort study, including 52 patients with CCC from the postgraduate’s research project, 48 of whom underwent an EPS with or without previous use of antiarrhythmic drugs; with a mean age of 57 + 10.2 years; 62.5% male. PTs were classified into two groups: I- With syncope/pre-syncope and II- Without syncope. The chi-square test and Fisher’s exact test were used for statistical analysis. Results: The clinical characteristics were the mean: Rassi score was 8.43 + 4.8 points (39.5% low risk); 43.7% used antiarrhythmics; 8.3% had Functional Class III/IV and 79.1% (38pt) had syncope/pre-syncope. In group I, we observed that 31.2% (10pt) were at low risk of Rassi and 71.8% (23pt) had altered EPS (1 by SSS, 1 by prolonged HV and 21 by VT). In group II, we found 56.2% (9pt) low risk and 25% (4pt) had altered EPS. Unfavorable outcomes in the EEF were greater in group I than in group II (p = 0.002). Comparing the risk of Rassi and the presence of altered EPS, in the groups we have: Group I- Low risk- 60%, Intermediate- 71.4% and high- 80% altered EPS (p = 0.83). Group II – Low risk – 11%, Intermediate 42.8% altered EPS(p = 0.57). Conclusions: Syncope/pre-syncope was a predictor of unfavorable outcomes in the EPS. The presence of altered EPS was similar in the different Rassi risk scores in both groups. 111401 Modality: E-Poster Researcher – Non-case Report Category: NURSING ÉRICA SOBRAL GONDIM1, Antonia Elizangela Alves Moreira1, Ana Camila Gonçalves Leonel1, Amanda da Costa Sousa1, Emiliana Bezerra Gomes1 (1) Universidade Regional do Cariri – URCA Introduction: The electrocardiogram (ECG) subsidizes an assertive nursing care plan aimed at cardiac conduction abnormalities. But there are difficulties in performing and interpreting the test, which demonstrates the need for teaching strategies for its understanding and skills development. Objective: To report a teaching strategy on ECG shared between a professor, a student in teaching internship and an undergraduate teaching assistant. Method: Experience report of theoretical and practical teaching about ECG, carried out in October 2021 with 36 undergraduate nursing students, in a public university in Northeast, completed in two phases: theoretical, (skills training) and practical (simulation and case studies). Results: The implementation of the strategy used theoretical-practical association in a transversal way, which stimulated the development of knowledge, skills and attitudes, emphasizing the creativity and improvisation demonstrated due to the scarcity of materials at the public university. Finally, the promoters of the teaching strategy shared the potentialities and difficulties perceived in the process. The main potentiality was the motivation demonstrated by the students as a result of the interactivity and dynamics of the class, which stimulated their interest and active participation in both moments of the strategy. The biggest obstacle was the scarcity of resources, making it necessary to use low-fidelity simulation to replace the equipment and electrodes themselves. The heterogeneity of the learning process was also mentioned as a difficulty, since there are different ways to build knowledge aimed at different audiences, but it was evident from the students‘ reports that the strategy guided the necessary aspects of the teaching theme. Conclusion: Reporting the experience of teaching with simulation allowed the promoters to improve their strategies based on the students‘ feedback and the development of the activity. We suggest studies that enable the development of skills with the use of dynamic and participatory strategies. 111415 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY SEBASTIÁN DARÍO PERALTA1, Marcelo Omar Bettinotti1, Carlos Maximiliano Giuliani1, Guillermo Jubany1, Luis Murillo1, Juan Grieve Bruno1, Ezequiel José Zaidel1, Luis Carlos Sztejfman2, Matías Sztejfman2 (1) Sanatorio Güemes, Buenos Aires, Argentina; (2) Sanatorio Finochietto, Buenos Aires, Argentina Introduction: Current transcatheter aortic valve replacement (TAVR) procedures may be performed with an ultra-minimalistic approach which improves hospital resources. Aims The aim was to describe in hospital length of stay (LOS) and vascular events of TAVR procedures using ultra minimalisitc approach prior and during the COVID-19 pandemic. Methods: Consecutive TAVR cases from two centres between Jan 2019 and June 2021 were analyzed. Ultra-minimalistic approach included: all in one (AIO) femoral single puncture, conscious sedation, self-expanding valves, and femoral closure devices. Intra-pandemic cases were considered since March 2020 according to country’s COVID 19 uptake. Conventional descriptive and comparative statisitics were performed with Epi Info V7. Results: 37 cases were included, 21 (56%) before the pandemic and 16 intra-pandemic. Mean age was 82 (6) years and 59% were female. 73% had no prior conduction abnormalities, 35% had concomitant peripheral artery disease, 8% porcelain aorta. Median EuroScore was 5.8 (IQR 4.2–8), STS 5.4 (IQR 3.6–7.2), mean ejection fraction 56% (SD 11%) and mean gradient at baseline 45 mmHg (SD 12). A self-expandable valve was used in all cases. A predilatation was used for all cases. There were no deaths, no bleeding events, no strokes nor infections, 1 case of vascular complication (iliac dissection, resolved during the same procedure) and 1 case of permanent pacemaker implantation (2.7%). The rate of mild paravalvular leak was 43% and moderate leak 5.4%. Median length of stay was 2 days (IQR 2–3). 16 cases (43%) were performed intra-pandemic, without differences in procedural duration (58 minutes vs 56 pre-pandemic, p = 0.56), nor in LOS (median = 2 days in both groups, p = 0.24). 16% of the cases were discharged at day 1. Conclusion: Ultra-minimalisitc TAVR was safe and associated with short LOS prior and during COVID-19 pandemic. Physicians and patients must consider these findings in the context of the pandemic with risk of in-hospital infections and low healthcare capacity. These results must be considered also for TAVR cost-effectiveness analysis. 112184 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING SERGIO RODRIGO BERALDO1, Sergio Rodrigo Beraldo1, Tiago Augusto Magalhaes1, Augusto Hiroshi Uchida2, Rafael Willain Lopes3, Douglas Carli Silva4, Erico Luiz Camacho5, Gabriel Nunes Rodrigues5, Paulo Roberto Maia6, Ana Clara Beraldo Muniz6, Carlos Eduardo Rochitte1 (1) Computed Tomography and Cardiovascular Magnetic Resonance Sector from Heart Institute (InCor), University of Sao Paulo Medical School.; (2) Hospital Israelita Albert Einstein; (3) Nuclear Medicine Sector of Hospital do Coração -HCOR; (4) Siemens Healthineers; (5) Corpus Computed Tomography and Magsul MND Nuclear Medicine; (6) Sapucai Valley University School of Medicine Background: Patients with low to intermediate probability of CAD presenting exercise treadmill test (ETT) consistent with myocardial ischemia is a common scenario and may lead to other complementary tests, frequently myocardial scintigraphy (SPECT). Purpose: We sought to evaluate the diagnostic performance of myocardial perfusion (CTP), and Fractional Flow Reserve Derived from Computed Tomography – (cFFR) compared to SPECT in a population of patients with inducible ischemia on exercise treadmill tests (ET), using coronary computed tomography angiography (CTA) as the reference method. Methods: Sixty patients (58.4 ± 9.2, 37 men (61.7%), underwent clinical evaluation and two non-invasive imaging exams: SPECT and CTA with CTP during stress with dipyridamole. Results: SPECT showed significantly lower diagnostic accuracy than CTP (AUC 0.62 vs. 0.72, p < 0.001). The cFFR and minimal luminal area showed higher diagnostic accuracy than perfusional methods (AUC = 0.86 and 0.90). Coronary calcium score (CAC) showed AUC of 0.72 for the detection of obstructive CAD by CTA. In a per-patient analysis, CAC score and CTA were the strongest predictor of revascularization (AUC 0.87 and 0.94 p < 0,001). Conclusions: CTP alone presented higher accuracy than SPECT in patients with positive ETT with low or intermediate probability of CAD. Adding the cFFR and CAC data increases the diagnostic accuracy by computed tomography. These results suggest the use of CTP, cFFR or CAC in clinical practice might be adequate alternatives to SPECT in this scenario. 111437 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY GABRIEL ASSIS LOPES DO CARMO1, Bárbara Carolina Silva Almeida1, Gabriela Zamunaro Lopes Ruiz1, Renato Braulio1, Ana Cristina Carioca1, Fábio Morato Castilho1, Cláudio Leo Gelape1, Bruno Rodrigues Pereira1, Luiza Moreira Gomes1, Ana Carolina Sudário Leite1 (1) Universidade Federal de Minas Gerais (UFMG) Introduction: Vasoplegia after cardiac surgery is a well described complication, closely related to increased mortality. It is associated with use of angiotensin converting enzyme inhibitor or angiotensin receptor blocker. Therefore, we routinely discontinue these medications before elective surgeries. However, we still face several cases of vasoplegia. Objective: Describe clinical characteristics associated with vasoplegic syndrome after elective cardiac surgery. Methodology: Prospective cohort analysis of cardiac surgery patients in a public hospital in Brazil between 2016 and 2021, excluding heart transplant. Results: We enrolled 406 patients, median age 57 (46;66) and 214 (52,7%) females. 287 (70,7%) had valve surgery, 99 (24,4%) coronary artery bypass graft, 29 (7,1%) congenital heart correction, 17 (4,2) aorta procedures and 33 (8,1%) other cardiac surgeries. Univariate analysis showed that age, 64 (57;73) vs 55 (44;65), p < 0,001, chronic obstructive pulmonary disease, 47,1% vs 16,5%, OR = 4,51 (1,68–12,15), p0,004, renal replacement therapy (RRT), 57,1% vs 17%, OR = 6,49 (1,42–29,66), p = 0,021, and aortic surgery, 41,2% vs 16,7%, OR = 3,49 (1,28–9,50), p = 0,018, were associated with vasoplegia. After multivariate analysis, age, OR = 1,068 (1,041–1,095), p = 0,005, RRT, OR = 5,77 (1,12–29,69), p < 0,001, and aorta procedures, OR = 3,45 (1,17–10,17), p = 0,025, remained statistically significant. Conclusion: Our study shows that older age, RRT and aortic procedures are associated with higher incidence of vasoplegia in elective cardiac surgeries. All these factors are non-modifiable, but could add some information in the decision to perform a surgery in selected patients, as well as planning best intra and post operative monitorization strategies. Therefore, the diagnosis of vasoplegia could be made as soon as possible and appropriate therapy could be started earlier. 111439 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY BRUNA AFFONSO MADALOSO1, Nelson Samesima1, Nancy Maria Martins de Oliveira Tobias1, Caio de Assis Moura Tavares1, Horacio Gomes Pereira Filho1, Mirella Espanhoto Facin1, Carlos Alberto Pastore1 (1) Clinical Unit of Electrocardiography – Instituto do Coração (INCOR), Hospital das Clínicas FMUSP Background: The electrocardiogram (ECG) is a powerful tool for differential diagnosis among a group of pathologies called J-wave syndrome. The vectorcardiogram (VCG) can be used as a complementary method to the ECG in several dubious alterations. Purpose: We carried out a VCG analysis; after conceiving a novel parameter (JT-distance), that quantified the visual VCG change observed, in most Brugada type-1 patients and that allows diagnosis of the Brugada ECG pattern. Methods: We selected ninety-six ECGs (test cohort) with J-point elevation in V1/V2, ECG superior leads and VCGs, all performed on the same day. The VCG measurement by Frank method (JT-distance) was designed in transverse and right sagittal planes by three lines drawn 1) at the final third of the QRS loop, comprehending J-point; 2) at the initial portion of the T loop; 3) a parallel of the J-point line at the beginning of T loop. JT measure was determined by the distance between parallels. A validation cohort of thirty-five patients was also established. Results: JT-distance ≥1.5 mm (transverse plane) and JT-distance >1.25 mm (right sagittal plane), differentiated Brugada type-1 from type-2, early repolarization and others, with 95% sensitivity,68% specificity (p < 0.05). JT-distance <1.5 mm (transverse plane) and JT >1.25 mm (right sagittal plane) had 100% sensitivity, 85% specificity (p < 0.05) for Brugada type-1 diagnosis (Figure 1). Validation cohort showed JT distance ≥1.5 mm (transverse plane) could differentiate both Brugada types from others with sensitivity of 61%, specificity of 94%, (p = 0.0009). When JT distance (transverse plane) was <1.5 mm and in right sagittal plane was >1.25 mm, we found sensitivity of 83%,specificity of 94%, for Brugada type-1 diagnosis. (p = 0.001). Both cohorts showed very similar Cohen’s kappa levels (0.65 vs 0.77, test and validation cohorts, respectively). Conclusions: The novel vectorcardiogram measurement (JT-distance) presented a new diagnostic criterion to identify Brugada pattern with a practical and possible reproducible method. Nevertheless, prospective studies should also be performed to confirm these findings. 111440 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ARCHNA SINGH1, Sakshi Shukla1, Sandeep Aggarwal1 (1) All India Institute of Medical Sciences, New Delhi Aim: Adiponectin is a cytokine produced by adipocytes that act on specific receptors of several tissues through autocrine, paracrine, and endocrine signaling mechanisms. Adiponectin circulates in different oligomeric isoforms, with different biological effects. Circulating levels of adiponectin decline under conditions of metabolic stress, including obesity and metabolic syndrome, and are associated with decreased adiponectin signaling. Adiponectin has insulin-sensitizing effects and antiatherogenic properties. Higher adiponectin has been associated with decreased CVD risk. Methods: We divided female participants into 3 categories based on their BMI: normal weight control (C) = 24.28, overweight (OW) = 26.28, and non-diabetic obese (NDO) = 39.40. We estimated adiponectin mRNA expression (n = C: 31, OW: 21, and NDO: 67), serum levels (n = C: 44, OW: 29, and NDO:75) and fasting lipid profile. The Kruskal-Wallis and one-way ANOVA tests were applied for between-group analysis. Results: Overall group analysis for adiponectin mRNA levels and serum levels showed no significant differences; however, a significant difference was noted between C and NDO groups at both mRNA (0.0000) and serum (p-value: 0.0032) levels. A significant difference was seen between groups for all lipid profile parameters i.e., HDL (mean: C: 54.67, OW: 47.79, NDO: 42.28), LDL (mean: C: 96.54, OW: 97.73, NDO: 113.17), TC (mean: C: 165.82, OW: 154.92, NDO: 184.84), TG (mean: C: 114.56, OW: 107.40, NDO: 151.78) and VLDL (mean: C: 22.92, OW: 21.47, NDO: 32.73). Adiponectin gene expression correlated negatively with serum adiponectin levels and with HDL and ApoA1 levels. Conclusion: Lower adiponectin levels could contribute to an increased CVD risk in obesity, an effect that could be mediated via changes in HDL. 111474 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CARLOS HENRIQUE DEL CARLO1, Antonio Carlos Pereira Barretto1, Mucio Tavares de Oliveira Junior1, Alfredo José Mansur1, Antonio de Padua Mansur1, Sergio Jallad1, Juliano Novaes Cardoso1, André Barbosa de Abreu1, José Antonio Ramos Neto1, Roberto Kalil Filho1 (1) Heart Institute (InCor) University of Sao Paulo Medical School Background: Heart failure (HF) is a clinical syndrome with high morbidity and mortality. Optimal treatment can lead to improvement in ventricular remodeling in HF with reduced ejection fraction (HFrEF). Objective: To analyze the characteristics and prognosis of patients (pts) with HFrEF who evolved with HF with recovery ejection fraction (HFrecEF): left ventricular ejection fraction (LVEF) >40%. We also analyzed the outcomes of pts with any degree of LVEF improvement. Methods: We analyzed the electronic medical records of outpatients treated at a tertiary medical center in São Paulo-SP (Brazil) diagnosed with heart failure (ICD-10: I50) in 2017 and followed up until December 31st, 2020. There were included 4068 pts with an initial echocardiogram (ECHO1) presenting LVEF ≤40% (HFrEF). Data from a second evolutionary echocardiogram (ECHO2) were analyzed and pts were reclassified into HFrEF, HFrecEF, or any LVEF improvement (LVEF in ECHO2 > ECHO1). The clinical characteristics and prognosis of pts with respect to HFrecEF and those with any degree of LVEF improvement were analyzed. Statistical methods: Mann-Whitney U test, chi-square test or Fisher’s exact test, univariate and multivariate logistic regression analysis, and survival using the Kaplan-Meier method (p = Log-Rank). Results: Of the 4068 pts studied, age 61.3 ± 13.4 years, 65% male, with regard to etiology: idiopathic cardiomyopathy (46.6%), ischemic (27.5%), hypertensive (14.2%), Chagas disease (7.6%), valvular (4.2%); the mean initial LVEF: 30.0 ± 6.7%. Comparing LVEF in ECHO1 and ECHO2, 2594 pts (63.8%) showed any degree of LVEF improvement and 1250 (30.7%) evolved with total or partial LVEF recovery. Pts with HFrecEF had a higher initial mean LVEF: 32.2 ± 6.6% vs 29.0 ± 7.5% (p < 0.001). The ECHO2 showed an important improvement in the LVEF among pts with HFrecEF (50.9 ± 7.5% vs 29.4 ± 6.4%, p < 0.001). Female gender, hypertensive and valvular etiologies were associated with HFrecEF, while male gender, chagasic etiology, and history of stroke were independently associated with non-recovery of LVEF. Pts with HFrecEF had lower mortality (16.5% vs 30.1%, p < 0.001). The reduction in mortality was also observed in pts with any LVEF improvement (21.9% vs 33.0%, p < 0.001). Conclusion: HFrecEF was observed in 30.7% of pts with HFrEF and was accompanied by an improvement in prognosis. A reduction in mortality was also observed in pts with any degree of LVEF improvement. 111477 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA CAROLINE DARIVA CHULA1, Ana Caroline Dariva Chula1, Márcia Olandoski1, Elias Teixeira Krainski2, Lucas Henrique Olandoski Erbano1, Bruna Olandoski Erbano1, Raisa Natalia Dotto1, Lucas Baena Carstens1, Nicolle Amboni Schio1, Amanda Zanlorenzi1, Rafaela Lima Camargo1, José Rocha Faria-Neto1 (1) Pontificia Universidade Catolica do Parana – PUC PR; (2) Universidade Federal do Parana – UFPR Background: Many countries have reported an increase in the number of out-of-hospital deaths from cardiovascular diseases during the COVID-19 pandemic, which may be due to factors intrinsic to the disease or social conditions arising from the pandemic. Objective: To analyze possible differences in the temporal trend of out-of-hospital cardiovascular deaths during the COVID-19 pandemic in the year 2020 in Brazilian capitals. Method: This is a time series, whose data were collected on the websites https://transparencia.registrocivil.org.br/especial-covid, based on the Civil Registry Information Center, and https://opendatasus.saude.gov.br/dataset/bd-srag-2020, from the Influenza Epidemiological Surveillance Information System. On the first website, the numbers of deaths from COVID-19 and deaths from out-of-hospital cardiovascular causes were collected, week by week, from the 2nd to the 35th epidemiological week of 2019 and 2020, in Brazilian capitals. On the second website, data on hospitalizations for COVID-19 in the capitals were collected daily between March and October 2020, by date of first symptoms and by date of hospitalization itself, and tabulated weekly for combination by epidemiological week. The 2020 time series of total out-of-hospital cardiovascular deaths (TMCV-EH) was considered as the outcome. The time series effect of COVID-19 data was tested to explain the variation across the studied weeks of 2020. COVID-19 hospitalization data were tested as potential predictors of MCV-EH, as were death data from COVID-19. The TMCV-EH in the corresponding period of 2019 was used for comparison. Results: There was an increase in the TMCV-EH in 2020 compared to the non-pandemic year of 2019 and a correlation in the variation of these deaths with the COVID-19 data, with a peak in late April and early May when the relative risk, in the sum of the data, reached twice. There was also heterogeneity in the distribution of MCV-EH when analyzing each capital, with a predominance in the north and northeast regions. The highest relative risks stand out, compared to 2019, in peak periods in some capitals such as Salvador with 3.8, Recife with 3.6, São Luis with 5.6, Belém with 7.5 and Manaus with 9. 5 times. Rio de Janeiro presented a relative risk in the peak period of 2.2 times and São Paulo of 1.6 times. Conclusion: There was an increase in the number of out-of-hospital cardiovascular deaths during the COVID-19 pandemic, in Brazilian capitals. 111486 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MARIANE VENTUROLI FERREIRA BENAVENTE1, Márcia Olandoski1, José Rocha Faria Neto1 (1) Pontifícia Universidade Católica do Paraná – Escola de Medicina Introduction: During COVID-19 pandemic, several countries reported reduction in hospital admissions for cardiovascular diseases (CVD), including acute coronary syndromes (ACS), in addition to excess cardiovascular mortality due to direct and indirect effects of the pandemic. In Brazil, there are studies demonstrating reduction in hospitalizations, increase in in-hospital lethality and excess deaths from CVD. There are no studies evaluating the impact of the pandemic on hospitalizations and mortality from CVD in the state of São Paulo, the country’s most populous state, which concentrates the largest number of COVID-19 cases and deaths. Understanding this impact is essential for the implementation of public health strategies to reduce morbidity and mortality from these conditions. Purpose: The aim of this study was to assess the impact of the pandemic on ACS admissions and mortality from CVD in the state of São Paulo. Methods: We performed a retrospective observational study by analyzing the number of ACS hospital admissions – angina pectoris (AP) and acute myocardial infarction (MI) and the number of deaths from CVD – MI and unspecific cardiovascular causes (UCVC), in-hospital and at home, between January 1st, 2020, and December 31st, 2021. Data from 2018 and 2019 were used for comparison. Results: In 2020, there was a 10% decrease in hospitalizations for ACS (18% for AP and 4% for MI) compared to 2019. In 2021 reduction was of 12% (27% for AP and 2% for MI). In 2020, there was a 10% decrease in deaths from MI (11% in-hospitals and 7% at home) compared to 2019, while in 2021 there was a 12% increase in the number of MI deaths compared to 2020, 11% in-hospitals and 15% at home, reaching pre-pandemic levels. In 2020, there was a 50% increase in deaths from UCVC (33% in-hospitals and 127% at home) and in 2021 there was an increase of 11% in the number of these deaths, 11% in-hospitals and 10% at home. The decrease in ACS hospitalizations was followed by increase in deaths from MI and UCVC causes in both 2020 and 2021. Conclusion: In the state of São Paulo the pandemic had impact reducing ACS hospitalizations and increasing deaths from CVD, specially UCVC and at home, which remained above baseline levels throughout the analyzed period. 111495 Modality: E-Poster Researcher – Non-case Report Category: PSYCHOLOGY MARCIA MOURA SCHMIDT1, Filipa Waihrich de Oliveira1, Camila de Matos Ávila1, Brenda Pereira Nunes2, Fernanda Lucchese-Lobato3 (1) Instituto de Cardiologia/Fundação Universitária de Cardiologia; (2) Hospital Geral de Caxias do Sul; (3) Hospital da Criança Santo Antônio, Irmandade Santa Casa de Misericórdia Background: Heart failure (HF) is a systemic disease characterized by deterioration of the heart. There are high rates of morbidity, mortality, and re-hospitalization. Cardiology Societies recommend the inclusion of self-care in the treatment. Objective: This study aimed to verify whether a psychoeducation intervention can reduce hospital readmissions, improve quality of life, and promote post-traumatic growth in patients with HF at 1-yr follow-up. Methods: Parallel randomized clinical trial study with HF patients from a regional hospital in Southern Brazil. Patients were invited to join the study at their first outpatient appointment after hospital discharge. All participants completed the WHO quality of life (WHOQOL-BREF) and the post-traumatic growth inventory (PGI) questionnaires at both pre- (T1) and 6 months post-(T2) intervention assessments. Randomization envelopes were opened after the first interview. The patients in the control group (CG) continued to carry out their regular outpatient consultations per medical instructions. Patients in the intervention group (IG), in addition to the regular visits, had two additional individual follow-ups, of 1 hour each, with an interval of 7 days in between, to promote health and psychological education. About one year later, hospital readmission was assessed through medical records and phone call patient report. Statistical analyses were performed using SPSS software v. 24.0. Results: A sample of 142 patients was recruited at T1 (72 in the CG and 70 in the IG). While at T2, 19 dropped out, and 123 patients (63 in the CG and 60 in the IG) were reassessed after 315 ± 198 days. The participants were 65% male, with 64 ± 11 years old, 58% had low income and 67% had less than high school. The risk of readmission was reduced by 54% (p = 0.050). There was an improvement in the total quality of life (p = <0.05) and positive psychological growth (p < 0.001) in the IG at T2. Conclusions: The intervention proved to be protective for patients with HF in a regional hospital in Southern Brazil. A 2-session psychoeducational intervention was effective in reducing readmission rates by half compared to the CG, as well as improving quality of life and promoting positive psychological growth. Future brief culturally sensitive psychoeducation programs should be implemented in Brazilian hospitals to improve knowledge about HF and patient self-care, reducing the burden that HF creates to our health system. NCT 04870918. 111496 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS DR. CHANDINI SUVARNA1, Praveen Aggarwal1, Ramakrishnan Sivasubramanian1, Sandeep Seth1, Neeraj Parekh1, Sheikh Vamik1, Ambuj Roy1, Ganesan Karthikeyan1, Sandeep Singh1, Rajiv Narang1, Balram Bhargava2, Meenakshi Sharma2 (1) Chandini Suvarna; (2) Praveen Aggarwal; (3) Ramakrishnan Sivasubramanian; (4) Sandeep Seth; (5) Neeraj Parekh; (6) Sheikh Vamik; (7) Ambuj Roy; (8) Ganesan Karthikeyan; (9) Sandeep Singh; (10) Rajiv Narang; (11) Balram Bhargava; (12) Meenakshi Sharma Background: Non–ST-segment elevation myocardial infarction (NSTEMI) are common manifestations of coronary artery disease (CAD) which is the leading cause of death in India. Despite advances in the treatment of ACS, pharmacologic therapy remains underused and is often delayed. Mission DELHI (Delhi Emergency Life Heart Attack Initiative) is a pilot project for pre-hospital thrombolysis using motorbike ambulance service by trained paramedics at the patient’s doorstep in a selected geographical area of New Delhi. Objective: We sought to determine the feasibility and safety of prehospital care and risk stratification of NSTEMI. Method: We covered a geographical area of 10-km around our institution. A command center was set up and the patients were required to call a dedicated helpline number. Upon initial screening, a motorcycle ambulance was dispatched to the caller’s location. A 12 lead ECG was obtained as soon as possible after first medical contact by paramedic and electronically transmitted to a dedicated command center. Evaluation of the ECG was done by cardiologist. Patients with suspected NSTEACS, administration of platelet aggregation inhibitors, anti-cholesterol drugs and transferred to the emergency department for further diagnostic assessment and therapeutic decision. On site troponin testing was done when needed. Patients with persistent symptoms despite initial therapy may be transferred directly to a catheterization laboratory. Result: A total of 60 NSTACS patients (mean age 57.7 years; 68.3% male) were treated either at home (66.7%), public places (15%), place of work (11.7%) or at small clinics (6.7%). Time taken to reach the patient location and take an ECG was 27.9 ± 8.2 min. Nearly 56.7% of patients received a loading dose of aspirin, clopidogrel and atorvastatin pre-hospital. Nearly two third of patients (46.7%) received medication for HTN, followed by 66.7% of patients given cardiac medications and 60% patients given other medications including antacid/antiemetic. Conclusions: In this study, we have demonstrated the prehospital acquisition and transmission of ECG by paramedics was feasible. An early diagnosis of the majority of cases with NSTEMI and initiation of basic pharmacotherapy was possible prehospitally. Such an early diagnosis and risk stratification could improve outcome, which needs to be assessed in larger studies. 111914 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ILIANA REGINA RIBEIRO MENEZES1, Giovanni Possamai Dutra1, Letícia de Sousa Peres1, Nathalia Duarte Camisão1, Mariana Moreno Canário da Silva1, Renata Mexias Abdala Felix1, Thiago Moreira Bastos da silva2, Anna Butter1, Henrique Custódio Goudar1, Bruno Ferraz de Oliveira Gomes1, João Luiz Fernandes Petriz1, Gláucia Maria Moraes de Oliveira2 (1) HOSPITAL BARRA D’OR; (2) UNIVERSIDADE FEDERAL DO RIO DE JANEIRO Introduction: There is evidence that the clinical outcomes of COVID-19 vary according to its variants. Given the high risk of infection-related mortality, recognizing the outcomes in each variant may contribute to understanding the severity of the disease. Objective: To evaluate the association of mortality, myocardial injury, lung parenchyma involvement and use of mechanical ventilation, and length of hospital stay with the most prevalent variants of COVID 19 in Brazil. Methods: Patients admitted to an intensive care unit during the COVID-19 pandemic, with an RT-PCR confirmed diagnosis of COVID-19 were included. The following outcomes and clinical features were analyzed in each period: mortality, length of hospital stay, myocardial injury, lung parenchyma involvement, and use of mechanical ventilation (MV). We divided our sample according to periods where some variants were more prevalent: March to October/2020 (beta); November/2020 to July/2021 (P.1); August to December/2021 (delta) and January to April/2022 (omicron). The variables were analyzed using the chi-square test (categorical) and ANOVA (continuous). Results: 1454 patients were included, mean age = 59.8 ± 17.0 years, 62.6% men. Occurred 269 deaths (18.5%) during the study period (mean follow-up = 338 ± 209 days). We observed a greater association with mortality in the beta variant: beta (23%), P.1 (16.6%), delta (16.6%), and omicron (12.1%) with p = 0.015. Myocardial injury was more prevalent with the omicron variant: beta (32.7%), P.1 (49.2%), delta (53.5%), and omicron (60.6%) with p < 0.001. Lung involvement >50% was more common in omicron: beta (13.0%), P.1 (14.3%), delta (13.6%) and omicron (27.6%) with p < 0.001. No difference was observed in MV need. The average length of hospital stay was higher in beta: beta (20.6 days), P.1 (14.3 days), delta (13.5 days), and omicron (9.6 days), p < 0.001. Finally, the Average time of MV use was higher in beta and P.1: beta (21.4 days), P.1 (21.1 days), delta (14 days), and omicron (9 days). Conclusion: The beta variant was associated with greater mortality and length of hospital stay, while the omicron variant was associated with myocardial injury and shorter mechanical ventilation time in patients hospitalized in intensive care for COVID 19. 111523 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING DIEGO LOPEZ OTERO1, Alberto Otero Cacho2, Brais Diaz Fernandez1, Maria Bastos Fernandez1, Xoan Sanmartin Pena1, Vicente Perez Munuzuri2, Alberto Perez Munuzuri2, Jose Ramon Gonzalez Juantey1 (1) Universitary Clinical Hospital of Santiago de Compostela, Cardiology. CIBERCV, Santiago De Compostela, Spain; (2) FlowReserve Labs S.L. Santiago de Compostela Purpouse: To validate a new model of Fractional Flow Reserve obtained from a coronary computed tomography angiography (CCTA) (FFRct) (figure 1A), as well as obtaining new emerging parameters, less studied, such as ΔFFRct (Figure 1B), wall share stress (WWS) (figure 1C) and stenosis resistance (SR). Methods: We included patients referred by the chest pain unit for ischemic heart disease screening, selecting for invasive study those with at least one coronary lesion >50% in the CCTA. Patients with unadequate CCTA interpretation were excluded. To eliminate possible biases, the results of the invasive FFR were unknown at the time of applying this new model to obtain the FFRct value. Results: 26 patients (32 lesions) were included. 86.6% were males and a mean age 57.4 ± SD 11.7 years. In most cases, as these were low-risk patients, and critical disease was excluded, a FFR value >0.80 was obtained. To study the correlation between invasive FFR values and those obtained by the computational model, Spearman’s ρ coefficient and R2 were calculated. There were no false negative or positive cases. The correlation was high in all the parameters studied (>0.6), being positive for the FFRct and negative for ΔFFRct, WWS and SR. The plot in Figure 1D compares the FFRct under steady hyperaemic conditions as a function of the invasive FFR, note the good agreement between the numerical and invasive methods. Conclusions: Our new model was shown to have a good correlation with the invasively measured FFR in this serie of low-medium risk patients. The clinical usefulness of the new parameters in isolation is yet to be determined, but they could be helpful in cases where the FFRct value is in the gray area (0.75 ≤ FFRct ≤ 0.8), specially the ΔFFRct. 111547 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES EZEQUIEL J ZAIDEL1, Álvaro Sosa Liprandi1, Joaquin Perea1, Matías Ariel Oliva1, Pablo Perel2, Kavita Singh2, Dorairaj Prabhakaran2, Lana Raspail2, Karen Sliwa Hahnle2 (1) Sanatorio Güemes, Buenos Aires, Argentina; (2) World Heart Federation Introduction and aims: Vulnerable patients with chronic Chagas Disease (CD) may have worse outcomes if hospitalized due to COVID-19. As CD is a neglected tropical disease, it is under-represented in international COVID-19 studies. Current data is scarse and with diverse results. The aim of the present analysis was to describe the clinical profile of patients with known chronic CD during COVID-19 hoapitalization and to analyze if CD patients had different clinical outcomes. Methods: WHF Covid-19 and cardiovascular (CV) Disease registry is a unique COVID-19 prospective registry, as it included cases from different continents and economic strata. CD was a specific subgroup of analysis, and different centres mainly from latin america included CD cases in the registry. A conventional descriptive and comparative analysis was performed. Results: A total of 5313 Covid-19 patients were enrolled in 40 hospitals from 23 countries. Among them, 36 were reported as chronic CD cases. The mean age of CD was similar to non-CD patients (56.4 ± 15 vs 57 ± 16 years, p = NS) but the proportion of women was higher (61.1% vs 40.4%, p = 0.011), and had higher rate of baseline heart failure (16.6% vs 5.4%, p < 0.01), prior pacemaker (3.5% vs 1.1%, p < 0.01) and use of CV drugs (75% vs 46.1%, p < 0.01). During hospitalization, the rate of invasive ventilation was higher in CD (8.3% vs 7.5%, p < 0.01), as well as inotropes requirement (8.3% vs 6.8%, p < 0.01), and ventricular arrhythmias (11.4% vs 1.09%, p < 0.01) or heart blocks (11.4% vs 1.4%, p < 0.01). Regarding outcomes, in-hospital death was 5.7% in CD patients and 13.2% in non CD patients (P = 0.38), and no additional deaths occurred at 30 days (0% vs 2.6% in non CD cases, p = 0.054). Conclusions: Altough a limited number of Chagas disease patients were included, they seem to have more CV disease at baseline than non-CD patients, more CV events during COVID-19 hospitalization but similar short term outcomes. 111530 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING ANDREA ROCHA DE LORENZO1, Ivanete Costa2, Augusto Santos Tavares3, Gabriel Grossman2, Rafael Lopes3, Ronaldo SL Lima1 (1) Clinica de Diagnóstico por Imagem; (2) Hospital Moinhos de Vento; (3) Hospital do Coração-HCOR Background: The COVID-19 pandemic- and more specifically, the lockdown period- caused unquestionable effects on non-COVID healthcare, with reductions in the access to exams and treatments. As myocardial perfusion imaging (MPI) has a well-established role in the evaluation of coronary artery disease (CAD), the diagnosis of CAD might have been affected, with possible long-term adverse consequences. Understanding of the temporal trend of MPI utilization is central in this context. Objective: This study aimed to evaluate the impact of the COVID-19 pandemic on MPI performance and results in 3 Brazilian, Nuclear Cardiology laboratories. Methods: Consecutive patients, with or without known CAD (prior myocardial infarction or revascularization), who underwent stress/rest MPI at 3 Brazilian private Nuclear Cardiology laboratories were studied. The number of tests, indication (diagnostic vs known CAD), frequency of abnormal tests, and frequency of ischemic MPI (those with reversible perfusion defects) were registered in three 30-day time intervals: immediately pre-pandemic, lockdown, and post-lockdown. Variables were compared using Fisher’s exact test; p < 0.05 was considered statistically significant. Results: From a total of 1027 MPI studies, 443 were pre-pandemic, 127 were during lockdown (a 71% reduction), and 457 were post-lockdown. Diagnostic tests were 70% of the total before, 61% during lockdown, and 67% after (p = 0.05). The frequency of abnormal MPI tests increased in patients without known CAD in the lockdown period, returning to prior levels after lockdown (11%, 19%, and 11%, p = 0.05), although ischemic tests remained stable (7%, 9%, and 8%). Conclusions: A large reduction of MPI performance occurred during lockdown, with less diagnostic tests. There was no significant difference in the frequency of myocardial ischemia among the 3 intervals, although abnormal MPI tests increased during lockdown in patients undergoing MPI for diagnostic reasons. The altered pattern of MPI utilization caused by the pandemic might have future effects on CAD diagnoses, and therefore should be further and continuously assessed. 111536 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SORAYA ARAÚJO1, Patrícia Vieira2, Andrea Bento3, Carolina Cohen3, Regina Próspero4, Amira Awada4, Sônia de Castilho5, Vanessa Pirolo Vivancos6 (1) Cardiovascular Advocacy Group; (2) Brazilian Association of Family Hypercholesterolemia; (3) Collaborate with the Future; (4) Rare Lives Institute; (5) ADJ Diabetes Brazil; (6) Botucatuense Association of Diabetic Care The Cardiovascular Disease Advocacy Group (GAC) promoted a campaign of Cardiovascular risk awareness named “Faça de Coração – Teste seu Risco Cardiovascular”, as a part of the campaign for World Heart Day. Our group applied a poll to stakeholders at the Legislative Assembly of the State of São Paulo- Brazil to mobilize one of the critical stakeholders to this day. This type of approach provides quick evidence of knowledge from the people who are responding and, at the same time, provokes reflections on the subject. The aim of this study was to verify the perception of parliamentarians about cardiovascular diseases (CVD) and their impact on public health in Brazil. Method: A poll conducted by the GAC in the Legislative Assembly of São Paulo, from 06/09 to 30/08 of 2021 was applied. The poll consisted of eleven multiple-choice questions (five questions about their health status; two questions about public policies; four questions about specific knowledge of CVD) available on the Googles Forms platform. The questions were presented to the 94 parliamentarians who compose the Legislative Assembly of São Paulo (LASP) in virtual visits, witnessed and sent by WhatsApp and e-mail. A descriptive analysis was performed. Results and conclusions: Of the 94 members within a current mandate in the Legislative Assembly, 70 responded to the poll. 84,3% were male; 62,9% aged 36 to 59y; 18,6%, 18 to 35y; and 18,6% aged >60y. In the self-report health status, all are non-smokers, 74% practice physical activity; 52,9% made health check-up; 80% know about their cardiovascular risk; 14,3% has hypertension; 4,3% High Cholesterol, 2,9% Diabetes and 1,4% Obesity. 42% be a part of any collegiate of parliamentarians at LASP to find the best strategies to promote health. Most respondents demonstrate a good knowledge of cardiovascular disease. It is very worrying to note that 91,4% do not believe that the current public policies are effective in the management and control of CVD. However, this is the first step to changing this scenario. 111548 Modality: E-Poster Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MARIANO DUARTE1, Mariano Duarte2, Analia Aquieri1, Javier Coyle1, Claudio Yaryour3, Carlos Reyes Toso2 (1) Laboratorio Hipertensión Arterial, Hospital de Clínicas Facultad de Medicina UBA; (2) Laboratorio de Fisiopatología Cardiovascular UA 2 Facultad de Medicina UBA; (3) División Urgencias Hospital de Clínicas Facultad de Medicina UBA Introduction: The rapid regulation of blood pressure allows the continuous adaptation of the cardiovascular system. This function is carried out by baroreflex (BR). Despite the hypertensive (HT) damage to the arterial wall and BR structures are located within it, the impact of hypertension (HYP) on the BR function has not been fully studied. We hypothesized that HYP has decreased BR function caused by increased arterial stiffness (AS). Materials and methods: In prospective study, 101 patients over 65 years of age were included; G1: HT G2: Normotensive (NT). BR function: A continuous rhythm strip in lead DII of electrocardiogram and the Valsalva maneuver was performed. Chronotropic response at the end of the test was established by the lowest R-R recorded up to 5 beats post-Valsalva. According with AHA, BR function was established by the greatest variation in heart rate (smallest R-R) within 5 immediate beats over Valsalva. Results 2 populations: A-BR Nomofunction: increase 10 and 30 beats. B-BR hypofunction, increase <10 beats. Assessment of AS by means of pulse wave velocity (PWV): It is performed by Complior System device, the “gold standard” for the non-invasive measurement. Results: 101 patients (43 men) were studied, 76 of them were HT (147.2 ± 15.7 mmHg) and the rest NT (130.2 ± 10.8 mmHg) p < 0.05. BR hypofunction was observed in 60 of the 76 HT and only in 3 of the 25 NT. PWV was higher in the HT vs. NT (10.54 vs 8.58 m/s) p < 0.001. Conclusions: As hypothesized, 8/10 in G1 had decreased BR function and only 1/10 in G2. Best predictors of BR hypofunction were sought by multivariate analysis were HYP and AS. So, AS could be one of the causal links between HYP and BR hypofunction and would explain the HT baroreceptor desensitization. 111558 Modality: E-Poster Researcher – Non-case Report Category: ANTICOAGULATION CHRISTIAN HENGSTENBERG1, Nicolas M. Van Mieghem2, Rosa Wang3, Xiaomei Ye4, Ling Shi4, Shien Guo4, Cathy Chen3, James Jin3, Xin Ye3, Martin Unverdorben3, George Dangas5 (1) Department of Internal Medicine II, Division of Cardiology, Vienna General Hospital, Medical University, Vienna, Austria; (2) Department of Cardiology, Erasmus University Medical Centre, Thoraxcenter, Rotterdam, the Netherlands; (3) Daiichi Sankyo, Inc., Basking Ridge, NJ, USA; (4) Evidera PPD, LLC, Bethesda, MD, USA; (5) Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai Hospital, New York, NY, USA Introduction: ENVISAGE-TAVI AF (NCT02943785; N = 1426) was a randomized, open-label trial that compared edoxaban vs VKAs in patients with atrial fibrillation (AF) after transcatheter aortic valve replacement (TAVR). Noninferiority was shown with edoxaban vs VKAs for the primary endpoint, net adverse clinical events. The effect of edoxaban or VKA-based therapy on patient-reported treatment satisfaction remains unknown. Objective: To assess patient-reported treatment satisfaction and convenience with edoxaban vs VKAs in patients with AF after TAVR. Methods: The Perception of Anticoagulation Treatment Questionnaire 2 (PACT-Q2) is a validated patient-reported outcome instrument that was used to assess patients’ satisfaction and convenience with their anticoagulant treatment. A mixed-effect model for repeated measures assessed the least squares mean difference (LSMD) in PACT-Q2 scores between edoxaban- and VKA-treated patients. Cohen’s effect size (ES) evaluated clinical meaningfulness of the differences, defined as ES ≥ 0.2. Results: A total of 1107/1426 (77.6%) patients (edoxaban, n = 585; VKA, n = 522) had evaluable PACT-Q2 data and were included in this analysis. Edoxaban- vs VKA-treated patients had similar baseline characteristics. Edoxaban-treated patients reported significantly higher overall PACT-Q2 scores for treatment satisfaction than VKA-treated patients (LSMD [95% confidence interval], 6.1 [4.5–7.7], P < 0.05), with a clinically meaningful difference (ES, 0.4 [0.3–0.5]). Results were similar in the convenience dimension (Figure). Conclusions: ENVISAGE-TAVI AF patients found edoxaban to be more convenient and had greater treatment satisfaction than those who received VKAs. For patients with AF after TAVR, edoxaban may elicit an improved patient experience compared with VKAs. 111569 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM FATHIMA AAYSHA AAYSHA CADER1, Ishmum Zia Chowdhury2, Syeda Rifat Mahmud1, Isha Abdullah Ali1, Saidur Rahman Khan1 (1) Ibrahim Cardiac Hospital & Research Institute; (2) BIRDEM General Hospital Introduction: There is little data from South Asia on the impact of the COVID-19 pandemic on acute myocardial infarctions (AMI) admissions and outcomes, particularly in the two years following the onset of the pandemic. Objective: We aimed to compare AMI admissions and outcomes over two time periods since the onset of the pandemic at a tertiary cardiac care centre with catheterization laboratory facilities and referrals from across the country. Methods: In this cohort study conducted at our tertiary cardiac centre, we collected and compared data of all patients diagnosed and admitted with AMI over two time periods: between March–August 2020 and March–August 2021. Patient demographics, diagnostic and therapeutic strategies and in-hospital outcomes were statistically analysed and compared by the student’s t test and chi-square tests, as appropriate. Results: A total of 380 AMI admissions were included, 82 (21.6%) in 2020 and 298 (78.4%) in 2021 over the same time periods. Significantly more men presented in both years (69.5% and 83.2% in 2020 and 2021 respectively; p = 0.005). No significant differences in cardiovascular risk factors or type of AMI were seen across both time periods (49.7% vs 52.4% STEMI in 2020 vs 2021). Late presentation of STEMI was 30.5% vs 23.2% in 2020 and 2021 respectively (p = 0.13). 35.9% were vaccinated against COVID19 in 2021 (p < 0.001). Significantly more patients developed cardiogenic shock (40.2% vs 23.8% (Odds Ratio [OR] & 95% confidence interval [CI] 2.15 (1.29–3.61); p = 0.003); heart failure (76.8% vs 57.4%; OR 2.46 (1.40–4.32); p = 0.001), cardiac arrest (19.5% vs 9.7%; OR 2.25 (1.15–4.38); p = 0.015) and required ventilation (19.5% vs 9.1%; OR 2.43 (1.24–4.78); p = 0.008) in 2020, as compared with 2021. In-hospital mortality was numerically higher in 2020 (17.1% vs 9.7%; OR 1.91 (0.96–3.81); p = 0.063). AMI patients were significantly less likely to undergo coronary angiography (54.9% vs 71.1% for 2020 vs 2021; p = 0.005) and percutaneous coronary intervention (35.4% vs 44%; p = 0.001) during index admission in 2020. 45% of STEMI patients were given fibrinolysis overall, with fewer in 2020 as compared with 2021 (20.9% vs 47.2%). Conclusion: In the immediate aftermath of the pandemic, fewer patients presented with AMI in 2020, as compared with similar time periods the next year, in 2021. Significantly fewer patients underwent angiography and PCI in 2020. AMI patients had improved outcomes in 2021, as compared to 2020. 111588 Modality: E-Poster Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION TATIANE DE AZEVEDO RUBIO1, Bruno Rodrigues1, Silvia Elaine Ferreira-Melo1, Lucia Helena Bonalume Tacito2, José Fernando Vilela-Martin2, Moacir Fernandes de Godoy2, Heitor Moreno-Junior1, Juan Carlos Yugar-Toledo2 (1) University of Campinas – UNICAMP; (2) Faculty of Medicine of Sao Jose do Rio Preto -FAMERP Among the blood pressure (BP) regulation mechanisms in RHTN, the expression of autonomic modulation plays an important role. Linear and non-linear analyzes of heart rate variability (HRV) assess the autonomic modulation of the cardiovascular system. Resistant hypertensive patients may present with autonomic dysfunction with different degrees of impairment. The objective of the present study was to evaluate the impairment of the autonomic function of controlled and uncontrolled resistant hypertensive patients, as well as those using beta-blockers. Assessments of autonomic function were performed in 49 resistant hypertensive patients aged between 53 and 82 years. Results: Uncontrolled RHTN patients (U-RHT) present a reduction in HRV and greater expression of sympathetic activity in relation to parasympathetic activity, demonstrated through a statistically significant reduction of the variables SDNN, RMSSD, SD1 and SD2 when compared to the group (C-RHT). In addition, despite the use of beta-blockers, both in RHTN + beta-blockers (RHT+BB) and U-RHT patients, the autonomic balance shows negative changes, that is, lower HRV, compared to the controlled RH group. Conclusion: RHTN patients present a reduction in HRV due to the occurrence of a reduction in parasympathetic activity and a relative increase in the sympathetic component. These results corroborate the importance of interventions on the autonomic nervous system. 112313 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM NATHALIA DUARTE CAMISÃO1, BRUNO FERRAZ DE OLIVEIRA GOMES1, JOÃO LUIZ FERNANDES PETRIZ1, GLAUCIA MARIA MORAES DE OLIVEIRA2, ANNA BUTTER1, HENRIQUE CUSTÓDIO GOUDAR1, RENATA MEXIAS ABDALA FELIX1, THIAGO MOREIRA BASTOS DA SILVA2, MARIANA MORENO CANÁRIO DA SILVA1, LETÍCIA DE SOUSA PERES1, ILIANA REGINA RIBEIRO MENEZES1, GIOVANNI POSSAMAI DUTRA1 (1) Hospital Barra D’or; (2) UNIVERSIDADE FEDERAL DO RIO DE JANEIRO (UFRJ) Introduction: The pandemic caused by the SARS-Cov-2 virus brought the need for and importance of identifying biomarkers that may indicate a worse prognosis and an increase in the long-term mortality of these patients. Objective: Identify biomarkers associated with long-term death in patients hospitalized by COVID-19. Methods: Retrospective study with hospitalized patients with a confirmed diagnosis of COVID-19. The following biomarkers, collected during hospitalization, were analyzed: d-dimer (admission, peak, and discharge), ultrasensitive troponin (admission and peak), C-reactive protein (admission and peak), and platelet count (admission and peak). We used the ROC curve and the area under the curve (AUC) analysis to determine the biomarkers with the best association with all-cause mortality. Results: A total of 1454 patients were included, mean age of 59.8 ± 17.0, 62.6% were men. There were 269 deaths (18.5%) during the study period (mean follow-up = 338 ± 209 days), and 44.7% of patients had myocardial injury. We observed that the highest AUC was obtained with the discharge d-dimer (AUC = 0.900) and the peak d-dimer (AUC = 0.870). The cutoff point proposed by the Youden index was, respectively: 1945 ng/dL (sensitivity 89%/specificity 73%) and 1987 ng/dL (sensitivity 81%/specificity 88%). Conclusions: We observed a high association of mortality (in and out of hospital) with the peak d-dimer obtained before hospital discharge. This data highlights the need for attention to patients who present high levels of this biomarker at the hospital discharge and may indicate the need for preventive therapies for thromboembolic events. 111595 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARLUS KARSTEN1, Gustavo dos Santos Ribeiro2, Luís Fernando Deresz3, Elisabetta Salvioni4, Dominique Hansen5, Piergiuseppe Agostoni6 (1) Universidade do Estado de Santa Catarina (UDESC); (2) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (3) Universidade Federal de Juiz de Fora (UFJF); (4) Centro Cardiologico Monzino (CCM); (5) Hasselt University (UHASSELT); (6) University of Milan (UNIMI) Background: Exercise oscillatory ventilation (EOV) is an abnormal phenomenon observed in chronic heart failure (HF) patients and is associated with a greater risk of adverse cardiovascular events. Purpose: To compare the EOV prevalence of 3 EOV definitions and to predict 2-year all-cause mortality in HF patients. Methods: Cardiopulmonary exercise test data from 233 HF patients were analyzed. Two blinded reviewers identified EOV according to the definitions of Ben-Dov, Corrà, and Leite. Prevalence data were compared by the Cochran’s Q test and McNemar post hoc test. The relative risk of death and Kaplan-Meier survival analysis were applied in a 2-year follow-up. Sensitivity and specificity to predict 2-year mortality were determined by the receiver-operating characteristic curve. Results: EOV prevalence was 16.7, 17.5, and 8.4% by Ben-Dov, Corrà, and Leite’s definitions. There was no difference in EOV prevalence between Ben-Dov and Corrà. Both were different from Leite (p < 0.01). The 2-year death risk was 1.9 (1.0–3.7), 2.8 (1.6–5.2), and 2.0 (0.9–4.3) applying Ben-Dov, Corrà, and Leite’s definitions. Table 1 shows the sensitivity and specificity to predict 2-year mortality. Conclusion: EOV definition’s choice directly affects the number of EOV cases. Ben-Dov and Corrà definitions seem to have similar predictive power. Corrà definition shows better sensitivity to predict 2-year mortality. 111589 Modality: E-Poster Researcher – Non-case Report Category: PSYCHOLOGY SIRLEI PEREIRA NUNES1, Sirlei Pereira Nunes1, Bellkiss Wilma Romano1, Flávio Tarasoutchi1, Danielle Misumi Watanabe1 (1) InCor – Instituto do Coração do Hospital das Clínicas da FMUSP Introduction: The high mortality from cardiovascular diseases compromises significantly the Quality of Life (QoL) of patients (pacs) in all aspects, especially physical, psychosocial and mental health, making it necessary to rethink care in this population, implementing care, carried out and shared by a team of multidisciplinary. Objective: Verify the surgical influence the valve on the improvement of QoL at perspective of pacs. Method: Retrospective randomized observational longitudinal study of pacs surgical valve disease from a public health institution in the State of São Paulo, by a multidisciplinary team: clinical and surgical cardiologists, nurses, psychologists and others. Psychology assessment occurred in three moments: preoperative(preop), postoperative (posop) in the nursery ward and postoperatively (posop B), six months after the procedure from hospital discharge. 995 pacs were evaluated in preop, 517 posop in nurse and 144 pacs in posop B, March/2018 to February/2020, using semistructured interview technique and the SF 36 instrument that measures QoL from the individual’s perception of their general health status- score from 0 to 100. Results: The results showed an improvement in QoL the SF 36, emphasizing physical, social, emotional and mental health aspects, see table 1(one). Conclusion: Value-based care through the surgical valve disease line of care was effective in providing pacs with na improvement in their QoL. 111644 Modality: E-Poster Researcher – Non-case Report Category: NURSING FERNANDA CARNEIRO MUSSI1, Flávia Silva Ferreira1, Maria Cecilia Gallani4, Jules Ramon Brito Teixeira2, Thiago Ferreira Sousa3, Francisco José Gondim Pitanga5, Fernanda Michelle Santos Silva1, Brenda Silva Cunha1 (1) Escola de enfermagem, Universidade Federal da Bahia, Salvador, Bahia, Brazil.; (2) Escola de Enfermagem, Universidade Estadual de Feira de Santana, Feira de Santana, Bahia, Brazil.; (3) Centro de Formação de Professores, Universidade Federal do Recôncavo Baiano, Amargosa, Bahia, Brazil; (4) Faculté des sciences infirmières, Université Laval, Québec, Québec, Canada.; (5) Escola de Educação Física, Universidade Federal da Bahia, Salvador, Bahia, Brazil. Introduction: Sedentary behavior (SB) has been associated with chronic disease and all-cause mortality even in sufficiently active people. College students are referred to as a group exposed to SB, but little is known about this behavior in nursing students. Objective: To estimate the amount of time nursing students, spend in sedentary behavior in their daily lives and to examine the association between time in sedentary behavior and sociodemographic, academic, and behavioral variables. Method: Cross-sectional study, with 286 university students. Pearson’s Chi-square or Fisher’s Exact test and Multiple Logistic Regression were used. Sedentary behavior ≥8 hours per day (h/day) was common and risks were corrected by the Delta estimation method, obtaining the prevalence ratio and 95% confidence interval. A statistical significance of 5% was adopted. Results: Sedentary behavior ≥8h/day was identified for 53.5%. Older college students were 33.0% less in sedentary behavior ≥8h/day compared to younger students; with ≥3 hours of the out-of-class study showed 1.23 times more sedentary behavior ≥8h/day compared to those with <3 hours; with ≥4 subjects showed 1.58 times more sedentary behavior ≥8h/day compared to those with ≤3; insufficiently active showed 1.25 times more sedentary behavior ≥8hr/day compared to those who met the recommendation and who used sleep medications showed 1.46 times more sedentary behavior ≥8h/day compared to those who did not. Conclusion: It is necessary to combat sedentary behavior especially in younger college students, with accumulation of subjects, sleeping drugs, and insufficiently active. 111660 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JOSÉ RAMÓN LANZ-LUCES1, Fernando Augusto Alves da Costa11, Luís Fernando Escobar Guzman2 (1) Instituto Paulista de Doenças Cardiovasculares (IDPC); (2) Hospital Beneficência Portuguesa de São Paulo (BP) Introduction: Dilated aortic root (DAo) is a risk for cardiovascular events besides left ventricular hypertrophy (LVH). There is a lack of a useful index pondering both scenarios. Objective: To evaluate an electrocardiographic index in DAo and LVH comparing echocardiographic consensus values and those derived using the wall chest thickness (CT). Methodology: 631 patients, 236 hypertensive (HT) and 395 non-hypertensives (nHT), the diameter of the aortic root was based on the golden number (Phi) and the derived formula CT × 1.33 = Ao, using a cut-off >15% for enlargement. The index stemmed from the R + S amplitude sum in leads D1 + D2 + D3 and for values <23 mm. The index was also compared with Cornell and Sokolow-Lyon-Rappaport indexes and stablished criteria for Dao and LVH using T-test and CH2 test as appropriate. Results: The index prevalence was similar between groups (HT:42.7 vs. nHT: 37%, p = 0.15). There were differences in echocardiographic measurements (aorta, septum, posterior wall, and ventricular mass, p < 0.05). The index failed to be related to LVH, on the contrary it was associated to DAo derived from CT in the general population (OR: 2.189 CI95%: 1.546–3.100, p < 0.001) and in NTH (OR:3.010 CI95%: 1,869–4.847). Similar results for actual Dao criteria in the NTH (OR:3.078 CI95%:1.224–7.738, p = 0.012). Cornell index showed similar odds however, the test-sensibility was far less than the novel index, 8.4% vs 71.2%. Conclusion: This novel index was advantageous separating those with a DAo based CT measure along with literature consensus criteria for non-hypertensive patients. 111670 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY LUIZ SÉRGIO FERNANDES DE CARVALHO1, Alice Pacheco Santos4, Ana Carolina Machado Rodrigues da Cunha4, Guilherme Albuquerque Gruber4, Ana Claudia Cavalcante Nogueira2, Alexandre Anderson de Souza Munhoz Soares2, Gustavo Alexim3 (1) Clarity Healthcare Intelligence; (2) Instituto Aramari Apo; (3) HRS-DF – Hospital Regional de Sobradinho do Distrito Federal; (4) UCB – Universidade Católica de Brasília Introduction: The epidemiology of acute coronary syndromes (ACS) has gone through dramatic changes in recent decades, with an increase in its incidence in young individuals (under 55 years, ACSy) and a relative decrease in older individuals (ACSo). The management of ACS in young patients with moderate to high atherosclerotic load after the primary event still needs elucidation, since these individuals retain a long life expectancy. Objective: To compare clinical outcomes and cost of care in individuals with premature CAD and moderate to high atherosclerotic load undergoing Coronary Artery Bypass Graft (CABG) or percutaneous coronary intervention (PCI). Method: The study population consisted of the retrospective B-CaRe:QCO assortment, which includes all patients admitted with ACS in public hospitals in Federal District between 2013 and 2015 and who underwent cardiac catheterization with SYNTAX score >28. Outcomes were adjudicated with death certificates and medical record data. The primary clinical outcome was composite by CV death and recurrent ACS (MACE), followed by secondary endpoints: all-cause death and AMI. To assess indirect and direct costs, we evaluated, in international dollars (Int$) per year, the cost of lost productivity due to illness and death, ambulatory costs, and costs of new hospitalizations. Multivariate and propensity score paired (PEP) analyses were performed. Results: Among 1088 subjects (111 treated by CABG and 977 with PCI) aged <55 years accompanied for 6.2 (IQR 1.1) years, 304 primary events were observed. MACE was observed in 20.7% of the CABG group and 28.8% of the PCI group (p = 0.037). All-cause deaths were seen in 8.1% of the CABG group and 6.6% (p = 0.450) of the PCI group. AMI was seen in 6.3% of the CABG group and 15% of the PCI group (p = 0.014). On multivariate analysis, PCI was associated with higher hazard ratio (HR) for MACE, 1.227(95% CI 1.004–1.499; p = 0.04577), and in PEP HR = 1.268 (95% CI 1.048–1.548; p = 0.0271) compared to the CABG group. Although we observed no statistical differences in direct costs, the cost of lost productivity was higher in the PCI group (Int$4,511 (IQR 18062)/year vs Int$3,578 (IQR 13198)/year; p = 0.049] compared to CABG. Conclusion: In younger, high-atherostatic load ACS patients, the surgical approach is associated to lower occurrence of major long-term adverse cardiovascular effects, besides being associated to lower indirect costs. 111703 Modality: E-Poster Researcher – Non-case Report Category: PERIOPERATIVE EVALUATION BRUNO FERRAZ DE OLIVEIRA GOMES1, Thiago Moreira Bastos da Silva2, Leticia de Sousa Peres1, Iliana Regina Ribeiro Menezes1, Nathalia Duarte Camisão1, Mariana Moreno Canário da Silva1, Renata Mexias Abdala Felix1, Giovanni Possamai Dutra1, Anna Butter1, Henrique Custódio Goudar1, João Luiz Fernandes Petriz1, Gláucia Maria Moraes de Oliveira2 (1) Hospital Barra D’Or; (2) Universidade Federal do Rio de Janeiro Background: Myocardial injury in non-cardiac surgery (MINS) occurs in 10–25% and increases mortality in 30 days. Prediction tools can help in the creation of preventive strategies. Methods: Retrospective cohort study including all non-cardiac surgery patients admitted to a postoperative care unit who stayed at least one night in this unit and had at least one measurement of high-sensitive cardiac troponin. Clinical characteristics and the occurrence of MINS were assessed. All variables were included in the classification tree statistical model, a machine learning method, where variables with p < 0.05 were included in the analysis. Results: 2230 patients were included, mean age = 63.7 ± 16.2 years, and 55.6% women. The prevalence of MINS was 9.4%. The classification tree is available in Figure 1. The variables selected by the model were: age, RCRI score, high-risk surgery, previous myocardial infarction, and creatinine. This model has an accuracy of 90.6%. Conclusion: Age, RCRI score, high-risk surgery, previous myocardial infarction, and creatinine were predictors of MINS and were included in the classification tree. This model exhibited high accuracy and may be useful in the early identification of these patients. 111681 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARIA TERESA CABRERA CASTILLO1, MILENA DAVI NARCHI1, FLAVIA CUNACIA D‘EVA1, ROBERT TANAKA1, ADRIANA FUCCI1, DEBORA CLOTILDE CAROLLA1, LUISA MURAKAMI1, FARID SAMAAN1, RUI FERNADO RAMOS1, GUSTAVO BERNARDES DE FIGUEIRDO OLIVEIRA1, ARI TIMERMAN1 (1) INSTITUTO DANTE PAZZANESE DE CARDIOLOGIA Introduction: Cardiovascular disease (CVD) is the leading cause of death in Western countries, with heart failure being the final pathway of heart disease. Despite this, few patients are referred for Palliative Approach (PA). There is no consensus about the indication or the right time to refer patients to PA, whose main objective is to improve the patient’s quality of life. Objective: Characterize the epidemiological profile of patients with heart diseases with indication for PA in a hospital specialized in cardiology. Methods: This is a cross-sectional study with analysis of 554 patients included for PA from Feb 2018 to Feb 2022. Frequency, median, percentage, mean, maximum and minimum value were used for statistical analysis. Results: The sample comprised of 554 patients (P), for PA 270(57%) were male, mean age 71 ± 14 years, 179(37%) Catholic, 91(20%) Evangelical, With most frequent medical history of arrhythmia 242(51%), ventricular dysfunction 297(62%), Coronary artery bypass graft surgery 109 (23%) and stroke 175(15%). The most frequent clinical complains were: Dyspnea 329(59.30%), Lower limb edema 96(17.39%), diagnosis at the time of PA was renal failure 351 P (74.4%) and of these 148 (31%) needed hemodialysis, Heart failure 177(31.9%) (HF), Sepsis/Septic shock 146(26,3), stroke 113(20%). The mean ejection fraction was 40%. As for performance in activities of daily living we used the Palliative performance Scale (PPS) where 282 (71,1%) needed life support. We evaluated the prognosis with the PPI scale, 345(56.8%) with high short-term mortality. Regarding treatment, 277(58,7%) of patients with PA had CVC, 277(58.7%) received broad spectrum antibiotics, 196(41.5%) dobutamine, 138(30%) noradrenaline, 164 (34.7%) on mecanichl ventilation 130(27.5%) on hemodialysis 116(24.6%) sedated, 81 (8%) with IAP. Among these patients 295(52.9%) died during hospitalization and 126 (22%) were able to be discharged. Only 26(4.7%) did not accept the PA protocol, the most frequent site of the PA was 245(51.1%) in the ICU, followed by the ward with 227 (48%) P. Conclusion: In our sample we identified that patients with heart disease in PA still receive futile interventions and therapies, and the most frequent site of palliation is the ICU. This shows a later indication for PC, in patients with advanced HF and at the end of life. Strategies and institutional protocols are needed for an earlier indication, in order to promote integral care and better qualy of life. 111684 Modality: E-Poster Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PAULO ZIELINSKY1, Paulo Zielinsky1, Júlia Foresti1, Débora Raupp1, Daniela Babinski Guimarães1, Kelly Zucatti1, Vitória Gomez1, Vitória Aragon1, Izabele Vian1, Eduarda Bonamigo1 (1) Institute of Cardiology of Rio Grande do Sul (IC/FUC) Omega-3 (DHA) has been reccomended to adequate fetal development during gestation. Since it is a substance with intense anti-inflammatory action, such as dietary polyphenols, its effects upon the fetal heart and circulation are unknown and safety of its administration during gestation is not yet established. This study was designed with the purpose to investigate if maternal supplementation of omega-3 is followed by alterations of flow dynamics on fetal ductus arteriosus, at the final trimester of pregnancy, through a double blind, placebo-controlled, randomized clinical trial by groups. Pregnant women between gestational 27 and 28 weeks without cardiac alterations at fetal echocardiography were included, with exclusion of those taking nonsteroidal anti-inflammatory drugs or other substances with this effect, such as polyphenol-rich foods. The intervention group received supplementation of 450 mg of DHA/day in gastroresistent capsules and was compared to the placebo group, after 8 weeks. The participants in both groups were submitted to fetal Doppler echocardiography, assessment of dietary polyphenol and omega-3 consumption, as well as serum prostaglandin levels. Intergroup and intragroup data were evaluated. The study ended up with 24 participants in each group. After 8 weeks, Doppler echocardiographic parameters of ductal flow and serum prostaglandin levels in both groups did not show significant intergroup differences (systolic velocity: p = 0.59; diastolic velocity: p = 0.53; pulsatility index: p = 0.29; serum prostaglandin levels: p = 0.40) The expected intragroup differences as a result in increase in gestational ages were present. The results of this study suggest that omega-3 supplementation is safe at the final third of pregnancy, not causing alterations in the dynamics of the fetal ductus arteriosus flow, at the doses used, despite its potential anti-inflammatory action. 111685 Modality: E-Poster Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE TIAGO FERNANDES1, Fernanda Roberta Roque1, Vander José Neves2, João Lucas Penteado Gomes1, Andre Casanova Silveira1, Graziela Amaro Vicente Ferreira Saraiva4, Suliana Mesquita Paula3, Camila Paixão Jordão5, Rodrigo Souza4, Luciana Venturini Rossoni3, Maria Urbana Pinto Brandão Rondon4, Edilamar Menezes Oliveira1 (1) Laboratory of Biochemistry and Molecular Biology of Exercise, School of Physical Education and Sport, University of Sao Paulo; (2) President Tancredo de Almeida Neves University Center; (3) Department of Physiology and Biophysics, Institute of Biomedical Science, University of Sao Paulo; (4) School of Physical Education and Sport, University of Sao Paulo, Sao Paulo; (5) Heart Institute Arterial hypertension (AH) is a multifactorial syndrome characterized by high levels of blood pressure. Aerobic exercise training (ET) has been used as an important non-pharmacological treatment of AH, since it mitigates cardiovascular remodelling and blood pressure; however, the mechanisms involved are poorly understood. Our analysis of the miRNA’s expression profile by microarray, we selected the miRNA-205 for being one of the most differentially expressed miRNAs in AH, being neutralized by ET, and for targeting many genes involved in the vascular process. Therefore, we aim to evaluate the therapeutic potential of ET and gene therapy with AAV9-miRNA-205 treatment in spontaneously hypertensive rats (SHR), and the circulating miRNA-205 expression in AH patients. SHR aged 6 months and the respective controls Wistar Kyoto (WKY) were divided into four experimental groups (n = 7/group): SHR, trained SHR (SHR -T), WKY and trained WKY (WKY-T). The swimming ET consisted of 10-week, 1 × day/5 × a week/10 weeks. ET promoted reduction in blood pressure in SHR and resting bradycardia in trained animals. We observed a reduction in VO2max accompanied by cardiac hypertrophy and skeletal muscle atrophy. Moreover, these animals showed an increase in wall:lumen ratio of the femoral artery and muscle arteriole. ET corrected these changes in the peripheral vessels. AH increased 300% miRNA-205 levels by real-time PCR paralleled with a decrease of target genes VEGF, Akt, Bcl-2, eNOS, Itga5, TGFα and p70S6K levels by western blot compared to WKY group in skeletal muscle. In contrast, ET counteracts miRNA-205 and target genes levels toward control. Also, we used an adeno-associated virus 9 (AAV9) delivery methods to inhibit miRNA-205 in cardiovascular and muscle systems. Interestingly, AAV9-mediated miRNA inhibition (single systemic injection of AAV: 2 × 1012 viral genome particles) reduced AH-induced high levels of blood pressure compared to AAV-9 control around 25 mmHg in SHR. Indeed, miRNA-205 inhibition attenuated cardiac remodelling in SHR. In AH patients, the circulating levels of miRNA-205 were higher when compared to the normotensive group. Together, these results support the hypothesis that the morphofunctional changes arising of AH may be regulated by miRNAs and target genes; and ET participates in restoring the cardiovascular system. Thus, AAV9-mediated miRNA-205 inhibition appears as perspectives for the potential therapeutic use of miRNAs in the treatment of AH. 111930 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RAFAEL MODESTO FERNANDES3, Carolina Aslan Ribeiro Brito5, Carolina Costa da Silva Souza5, Vitor Queiroz de Castro Souza5, David Le Bihan4, Andrea A. Vilela1, Rodrigo B. M. Barretto2, Elizabete S. Santos1, Amanda G. M. R. Sousa1, Ari Timerman1 (1) Dante Pazzanese Institute of Cardiology, São Paulo, Brazil; (2) Instituto do Coração (INCOR), University of São Paulo, Brazil; (3) D’Or Institute for Research and Education (IDOR), Hospital Aliança, Salvador, Brazil; (4) Grupo Fleury- São Paulo; (5) Bahiana School of Medicine and Public Health, Salvador, Brazil. Background: Cardiovascular disease is the leading cause of death in Brazil and worldwide. In the context of diastolic dysfunction, echocardiography is the best non-invasive diagnostic method. However, the comparison of the two main guidelines for evaluate left ventricular diastolic function in acute coronary syndrome (ACS) is scarce. Objective: To compare the prognostic value of both guidelines of American Society of Echocardiography, from 2009 and 2016, in patients with ACS. Methods: This is a sub-analysis of a prospective cohort observational study with 109 patients admitted to the emergency with ACS. The follow up was performed within 1 year and combined outcome was cardiovascular death or new heart failure. We used the non-parametric Spearman method to assess the correlation between the categories of diastolic function according to the guideline used. The Cox model and the Log rank test with Kaplan-Meier curves were used to compare the prognostic value of categorizing patients according to the guideline used. The results were expressed as hazard ratio with a confidence interval of 95%. Results: The mean age was 63 years ±11, most male patients (73.4%) and the predominant color of patients was white (60.4%). Among the main risk factors, the most frequent for coronary artery disease was dyslipidemia (78%), followed by systemic arterial hypertension (77.1%) and sedentary lifestyle (65.9%). The study identified a mean borderline ejection fraction, a high E/E’ ratio, and LV diastolic and systolic volumes presented means above normal. The most common electrocardiographic change on admission was T-wave inversion (45%), and NSTEMI was the main clinical diagnosis (74%). The categories in which there was a greater disagreement between the guidelines were grade II diastolic dysfunction and normal function. The correlation between the diagnosis of diastolic dysfunction when compared to the use of the 2009 and 2016 guidelines was weak (R = 0.56 by Spearman’s method; p < 0.001). By the Kaplan-Meier curves, dividing the groups into with or without LA pressure elevation, the distinction between the evolution of the two groups is significant when performed by the 2016 guideline (Log Rank = 8.17; p = 0.04). Conclusion: The current guideline (2016) for the assessment of left ventricular diastolic dysfunction showed a higher prognostic value of combined outcome of cardiovascular death or new heart failure within one year, when compared to the guideline of 2009 in patients. 111722 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ALEXANDRA CORRÊA GERVAZONI BALBUENA DE LIMA1, Caroline Barreto Cavalcanti1, Paula Fernandes Freitas Lima1, Barbara Cunha Barreto2, Bruno Ramos Carneiro2, Gabriela de Oliveira Silva2, Maria Alice Ramalho Bragatto2, Ilan Sousa Figueirêdo3, Wenisten Jose Dantas da Silva3, Erick Giovani Sperandio Nascimento3, Luís Moreira da Silva de Azevedo Meireles4, Sergio Henrique Rodolpho Ramalho5 (1) North Wing Regional Hospital – Brasilia – Brazil; (2) School of Health Sciences – Brasília – Brazil; (3) Manufacturing and Technology Integrated – Campus SENAI CIMATEC, Salvador, Bahia, Brazil; (4) MDI Industrial, Salvador, Bahia – Brazil; (5) Coordinator of the Clinical Research Center of the Brasilia of the Dasa Hospitals Network – Brasilia – Brazil Background: Dyspnea is challenge in hospitalized individuals. The reasons for dyspnea are often multifactorial and the evaluation may use different and expensive methods (ex.: arterial blood gas, natriuretic peptide measurement, echocardiogram, computed tomography). This objective of this study was to assess the accuracy of an artificial intelligence (AI) capnography waverform and the presence of heart or pulmonary disease using the AI technology. Methods: In this prospective observational study, of patients admitted to a public hospital in Brasilia, Brazil, from September 2021 to February 2022, deep learning algorithms were developed and validated using capnography waveforms acquired from patient monitoring. The classification model was a binary classifier of individuals with healthy patients and sick patients (heart disease or pulmonary disease or heart and pulmonary disease) by analyzing biosignal waveforms. We assessed the model performance using area under the receiver operating characteristic curve (AUC-ROC), Accuracy, Sensitivity, Recall, F1-score, and Specificity. Results: Among them, 286 cases were taken as the experimental group, 61 (21%) heart disease, 69 (24%) pulmonary disease, 85 (30%) heart and pulmonary disease and 71 (25%) without heart or pulmonary disease (healthy patients). The AI model revealed a true positive (sick patients) of 88% and a true negative (healthy patients) of 82%. Therefore, the AI capnography waveform algorithm showed an average performance of 86% of Accuracy, 85% of Specificity, 85% of Recall and 83% of Precision, 84% of F1-score, and 85% of AUC-ROC. Conclusions: Capnography is a non-invasive, low-cost, and easy-to-use equipment. The capnography waveform could predict the presence or absence of heart or pulmonary disease based on biosignals acquired using noninvasive patient monitoring. The research showed that the design of AI information processing can assistant the clinical diagnostic and evaluation of patients. 111727 Modality: E-Poster Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES RAFAEL ALEXANDRE MENEGUZ MORENO1, Luiz Eugenio B. Prota-Filho1, Caio C.V. Queiroz1, Fabricio C. Wohnrath1, Felipe A.C. Carboni1, Gisele R.C. Silva1, Joselyn I.P. Castro1, Wandemberg S. Silva1, Auristela I.O. Ramos1, Nisia L. Gomes1, Sergio L.N. Braga1, J. Ribamar Costa Jr1 (1) Instituto Dante Pazzanese de Cardiologia Background: Percutaneous mitral balloon commissurotomy (PMBC) remains the preferred treatment for patients with severe symptomatic rheumatic mitral stenosis (MS) and suitable anatomy. The objective of this study was to propose a new score for the prediction of immediate and late success. Methods: This is a retrospective, single-center, single-arm registry encompassing all 1915 consecutive patients with rheumatic mitral stenosis recruited and referred to PMBC between August 3rd 1987 and July 19th 2010. All data were previously collected and recorded in a dataset. Clinical status was determined according to the New York Heart Association (NYHA) classification. Long-term outcome was a composite of incidence of major adverse cardiac events (cardiovascular death, new PMBC or mitral valve repair surgery) up to 24 years of clinical follow-up (from 1988 until December 3rd, 2011), including cardiovascular death, need for new PMBC, or mitral valve replacement surgery. Results: Mean patient age was 36.8 ± 12.9 years, most (86.4%) were female, and Wilkins score was between 9–11 in 49.1% of patients. In the multivariate analysis, the predictors of immediate success were age (odds ratio [OR], 0.98; 95% confidence interval [CI], 0.96–0.99; p = 0.01), left atrium size (OR, 0.96; 95% CI, 0.93–0.99; p = 0.01), mean pre-procedure mitral gradient (OR, 0.93; 95% CI, 0.89–0.96; p < 0.001), intermediate Wilkins score 9–11 (OR, 0.62; 95% CI, 0.40–0.94; p = 0.02), and high Wilkins score ≥12 (OR, 0.35; 95% CI, 0.16–0.76; p < 0.01). For prediction of late events, age (hazard ratio [HR], 0.98; 95% CI, 0.97–0.98; p < 0.001), New York Heart Association class III–IV (HR, 1.50; 95% CI, 1.18–1.92; p < 0.001), left atrium size (HR, 1.02; 95% CI, 1.02–0.04; p < 0.01), and high Wilkins score ≥12 (HR, 2.02; 95% CI, 1.30–3.15; p < 0.01) were significant. Two nomograms were developed using significant predictors from the model (one for immediate results and another for long-term results). Conclusions: In this large population, not only the Wilkins score, but also clinical and hemodynamic features, seem to be relevant in predicting immediate and late success for patients with rheumatic MS who underwent PMBC. 111724 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DOMINGOS SAVIO BARBOSA DE MELO2, Domingos Savio Barbosa de Melo2, Antonio Carlos Pereira Barreto1, Aristea Izabel de Oliveira2, Rodrigo Savio de Oliveira Melo2, Breno Domingos de Gusmao Melo2 (1) Heart Institute- InCor-FMUSP; (2) Ergocardio Medicina Diagnosis and Research Background: Rapid up-titration of beta-blockers (BB) in the treatment of heart failure (HF) during hospitalization has yet to be tested. Objectives: The purpose of this research was to evaluate the correlation between the rapid up-titration of beta-blockers and functional capacity in patients hospitalized for HF using the 6-minute walk test (6MWT) as a parameter. Methods and Results: 92 patients with advanced HF (New York Heart Association [NYHA] IV) and left ventricle ejection fraction (LVEF) <45% were hospitalized for clinical compensation and followed-up for one year after discharge. The patients were distributed into two groups: 46 in the treatment group (TG) and 46 in the control group (CG). During hospitalization, they were divided randomly for BB rapid up-titration with an increase of dosage every two days (TG), or by the usual treatment regime (CG). Statistical analysis used: Student’s t-test, Mann-Whitney, Chi-square and Fisher’s exact test, Kaplan-Meier for survival, using the Log-rank test for comparison and the hazard ratio (HR) calculated with the Cox model. P < 0.05 was considered significant. The exercise tolerance analysis was performed at the entrance, three months and one-year intervals and the walking distance was stratified into four levels (NV): NV1 <300 meters (m), NV2 = 300–375 m, NV3 = 375–450 m and NV4 > 450 m. At the entrance moments, three months and one year the distance traveled for the TG/CG were, respectively, 206.62 ± 33.54/185.09 ± 32.32 meters, 384.55 ± 77.54/336.55 ± 65.23 meters, and 422.17 ± 98.57/387.41 ± 76.66 meters. It was verified that there was an increase in walking distance for the two groups studied (p = 0.002). However, the distance walked in the TG was significantly higher than in the CG (p < 0.001). Conclusions: The rapid up-titration of beta blockers dosage during hospitalization of end-stage HF patients compared to the usual treatment regimen in advanced HF is safe, effective, and promotes increased functional capacity through 6MWT analysis. 111743 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DOMINGOS SAVIO BARBOSA DE MELO2, Antonio Carlos Pereira Barretto1, Aristea Izabel de Oliveira2, Rodrigo Savio de Oliveira Melo2, Breno Domingos de Gusmao Melo2, Domingos Savio Barbosa de Melo2 (1) Heart Institute- InCor-FMUSP; (2) Ergocardio Medicina Diagnosis and Research Background: Rapid up-titration of beta-blockers (BB) in the treatment of heart failure (HF) during hospitalization has yet to be tested. Objectives: Our purpose for this research is a new model for administering beta-blockers in patients with end-stage HF in order to, after clinical compensation of the patient, promote more rapid dosage increments than the conventional method or usual care procedures. We also sought to ascertain whether or not this model for administering beta-blockers could modify the impact of HF in relation to the enhancement of the quality of life (QOL) as measured by the Minnesota Living with Heart Failure questionnaire. Methods and Results: 92 patients with advanced HF (New York Heart Association [NYHA] IV) and left ventricle ejection fraction (LVEF) <45% were hospitalized for clinical compensation and followed-up for one year after discharge. The patients were distributed into two groups: 46 in the treatment group (TG) and 46 in the control group (CG). During hospitalization, they were divided randomly for BB rapid up-titration with an increase of dosage every two days (TG), or by the usual treatment regime (CG). Statistical analysis used: Student’s t-test, Mann-Whitney, Chi-square and Fisher’s exact test, Kaplan-Meier for survival, using the Log-rank test for comparison and the hazard ratio (HR) calculated with the Cox model. P < 0.05 was considered significant. The pcts responded to the questions of the MLHFQ. This battery of 21 items uses a scale for answers from (0–5). Total compilation of scores varied from 0 to 105. A lower score reflects a better QOL. Among the total group of 92 patients studied, initially, LVEF average was 27.02%, end-diastolic diameter average was 66.15 mm, and end-systolic diameter average was 57.04 mm. For the TG, the average dosage of Carvedilol reached 34.37 mg/per day; and with the CG, 13.99 mg/per day. In the initial evaluation, the pcts showed an average score of 82 points in the two separate groups evaluated. With treatment, the TG moved to a score of 63, while the CG scored 73. There was a reduction of 23% in the MLHFQ score for the TG in relation to the CG group (p < 0,001). Conclusions: Beta blockers rapid optimization dosages during hospitalization of end-stage HF patients is safe and promotes an enhanced patterns of QOL as measured by MLHFQ, indicating that more elevated dosages of BB is probably responsible for this positive outcome than that observed with a usual treatment schedule. 111747 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES ANDRE SCHMIDT1, Maria Fernanda Braggion-Santos1, Marcel Koenigham Santos1, Gustavo Jardim Volpe1, Henrique Turin Moreira1, José Antonio Marin-Neto1 (1) Faculdade de Medicina de Ribeirão Preto – USP Introduction: Myocardial fibrosis (MF) identified in Cardiac Magnetic Resonance (CMR) is a recognized prognostic marker in Chronic Chagas Cardiomyopathy (CCC). Since CMR is not widely available, it seems relevant to identify markers of MF in other methods that may indicate the need for a CMR in this neglected disease. Objective: We sought to investigate the relationships between the presence of ventricular premature beats (VPB) in an Electrocardiogram (ECG), Non-sustained ventricular tachycardia (NSVT) on a Holter exam, and the presence of MF at CMR. Methods: Consecutive CCC patients with an ECG, Holter, and CMR within one year were selected from our institution database, and the presence of VPB on ECG, NSVT on Holter, and MF on CMR were collected, as well as demographic and clinical data. Descriptive statistics, Fisher exact tests, and logistic regression analysis were performed. A 5% level of significance was established. Results: Inclusion criteria were fulfilled by 121 patients, 53% females, 56 ± 14 years, and most (93%) were in NYHA class I or II. The mean left ventricular ejection fraction was 48 ± 14%. Amiodarone was used by 22 (18%) patients at the physician’s discretion. Twenty-three (19%) patients resting ECG presented VPBs, and 38 (31%) patients had NSVT on Holter. MF was identified in 94 (78%) CCC patients. Concomitant occurrence of VPBs on ECG and NSVT on Holter in the same patient was low (12%) but MF was present in all of those with both VPB and NSVT. VPBs in ECG indicated an odds ratio (OR) of 7.9 (CI: 1.02–61.9; P = 0.04) of having MF, as 22 (96%) out of 23 with VPB had it. NSVT on Holter presented an OR of 16.9 (CI: 2.2–129.8; p = 0.007) as 37 (97%) out of 38 patients with NSVT had MF on CMR. Amiodarone use did not significantly (p > 0.05) influence the results of each exam to predict MF. Conclusions: Electrocardiogram and Holter may present findings indicating MF occurrence in CCC. Although rare in resting ECGs, VPB significantly increases the odds of finding MF. The presence of NSVT has a much higher chance of finding MF in a CMR exam. These findings may indicate a subgroup of CCC patients that should be followed closely due to the possible presence of myocardial fibrosis. 111933 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY ALTAIR ARGENTINO PEREIRA JUNIOR1, Ana Inês Gonzales2, Darlene Aparecida Pena1, Tales de Carvalho3, Alexandro Andrade3, Sabrina Weiss Sties1 (1) Centro Universitário Avantis – UNIAVAN; (2) Centro Universitário para o desenvolvimento do Alto Vale do Itajaí – UNIDAVI; (3) Universidade do estado de Santa Catarina – UDESC Introduction: In the context of cardiac rehabilitaion, the practice of regular physical exercises is considered a mandatory therapeutic strategy, since it significantly reduces the morbidity and mortality of patients with heart disease. However, especially in individuals with low exercise tolerance, the overtraining syndrome could occur. Objective: Evaluate the presence of signs of overtraining syndrome in patients with coronary artery disease undergoing cardiac rehabilitation. Methods: Seventy-seven adults participated in the cardiac rehabilitation program were analized. For early detection of overtraining syndrome was used the Brunel Mood Scale. Data were evaluated by descriptive statistics. Results: In this study, the prevalence of overtraining syndrome among patients with coronary artery disease was 11.68%. The tension domain score was 4.44 (±3.25), depression 4.22 (±3.07), anger 0.78 (±1.09), fatigue 4.89 (±3.52) and mental confusion 3.33 (±2.87) were high, while the vigor domain (9.67 ± 3.24) was low, indicating signs of overtraining syndrome. Conclusion: Some patients with coronary artery disease showed signs of overtraining syndrome. Taken together, these results indicate this target population requires attention to the need for adopt strategies aiming at preventive measures. 111935 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING RAFAEL MODESTO FERNANDES3, Rafael Modesto Fernandes3, Andre Moises de Oliveira Nunes5, Hayala Machado Cavalcante Conceição5, Loren Lacerda Rodrigues5, David Le Bihan4, Andrea A. Vilela1, Rodrigo B. M. Barretto2, Elizabete S. Santos1, Amanda G. M. R. Sousa1, Ari Timerman1 (1) Dante Pazzanese Institute of Cardiology, São Paulo, Brazil; (2) Instituto do Coração (INCOR), University of São Paulo, Brazil; (3) D’Or Institute for Research and Education (IDOR), Hospital Aliança, Salvador, Brazil; (4) Grupo Fleury – São Paulo; (5) Bahiana School of Medicine and Public Health, Salvador, Brazil. Background: The analysis of left atrial (LA) function can add important information to the understanding of cardiovascular diseases. Therefore, to assess its functioning the use of volumetric measurements is adopted. The maximum LA volume is the most echocardiography parameter used, however, some studies demonstrated that minimum LA volume is better to correlate with atrial dysfunction. Purpose: The purpose of this study was compare the accuracy of the phasic volumes of the left atrium in determining LA dysfunction identified by the two-dimensional strain. Methods: This observational, cross-sectional study admitted 109 participants with diagnosis of acute coronary syndrome non-ST-segment elevation. The exams were performed within 72 hours of admission. LA volume was defined by the mean of measurements performed in the four and two-chamber apical windows using Simpson’s method. The phasic volumes of the left atrium were measured using an electrocardiogram synchronized with the device. Reservoir LA strain less than 21% was the cut off to LA dysfunction. Results: The phasic LA volume variables analyzed were maximal (LAVMAX), minimal (LAVMIN) and before atrial contraction (LAVBAC) volume, and each variable indexed for body surface (LAVIMAX, LAVIMIN and LAVIBAC). Therefore, a Roc curve was generated for each volume variable to assess which would be more accurate in predicting left atrial dysfunction. The Youden index was used to determine the cutoff point for each one of them. The areas under the Roc curves were: 0.83 (LAVIMIN), 0.81 (LAVMIN), 0.78 (LAVBAC), 0.76 (LAVIMAX), 0.74 (LAVIBAC) and 0.71 (LAVMAX). Conclusion: This study concluded that phasic volumes were good determinants of left atrial dysfunction identified by 2D strain. The minimum left atrial volume was the better correlated with left atrial dysfunction. 111782 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY INGRID LOUREIRO DE QUEIROZ LIMA1, THIAGO CAVALCANTE1, LUIZ CARLOS DE LIMA1, FERNANDO LUIZ WESTPHAL1 (1) UNIVERSIDADE FEDERAL DO AMAZONAS; (2) HOSPITAL DO CORAÇÃO FRANCISCA MENDES Background: Troponin T (TnT) and troponin I (TnI) are myocardium necrosis biomarkers very sensible and specific used to evaluate a myocardial injury, and their rise after coronary angioplasty correlate with worst prognoses. Complications associated with this procedure are a well-recognized cause of elevated myocardial necrosis biomarkers. In its absence, the exact causes of elevated troponin are merely speculative. Objective: To evaluate the relationship between troponin elevation and clinical and anatomical variables after coronary angioplasty in the absence of complications. Method: It is an epidemiological study evaluating 124 patients submitted to coronary angioplasty from December 2018 to December 2019 in the catheter laboratory of University Hospital Francisca Mendes, using clinical variables and troponin doses before, 6 hours and 12 hours after the procedure. Results: Of all 124 analyzed patients, 63,3% were male and mean age of 63 years old. The mean implanted stents per patients was 1,66. Hypertension was the most prevalent comorbidity. Eighty patients (64,5%) presented high troponin dosage (compared to 99th percentile) and 32 (25,8%) showed troponin dosage above five times the 99th percentile. When analyzing all patients presenting troponin dosage above 99th percentile, none of the clinical characteristics were significantly related to troponin alteration. While analyzing procedure-related characteristics, we observed that troponin elevation above 99th percentile is significantly correlate with the number of implanted stents, the number of treated arteries (p < 0,001 and p = 0,046 respectively), procedure duration (p = 0,032), and contrast media volume (p = 0,008). The number of implanted stents and number of treated arteries were still associated with troponin elevation above five times the 99th percentile. Conclusion: Clinical characteristics were not associated with troponin elevation above the 99th percentile. However, the procedure duration, contrast media volume, number of treated arteries, and number of stents implanted are significantly associated with troponin elevation when studying the procedure characteristics. Amongst the subgroup of patients with troponin levels above five times the percentile, clinical history of smoking, the number of implanted stents and stent length were significantly associated with the elevation of this biomarker above five times the 99th percentile. 111805 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY GABRIEL ASSIS LOPES DO CARMO1, Bárbara Carolina Silva Almeida1, Gabriela Zamunaro Lopes Ruiz1, Renato Braulio1, Ana Cristina Carioca1, Fábio Morato Castilho1, Cláudio Leo Gelape1, Bruno Rodrigues Pereira1, Luiza Moreira Gomes1, Ana Carolina Sudário Leite1 (1) Universidade Federal de Minas Gerais (UFMG) Introduction: Rheumatic valve disease (RVD) has a high prevalence in Brazil, but remain poorly studied. Different from developed countries, valvar cardiac surgeries are the most performed procedure in several public hospitals in Brazil. However, impact of RVD on cardiac surgery is not well established. Objective: Describe baseline characteristics and prognosis of cardiac valve surgery patients in a public hospital in Brazil. Methodology: Prospective cohort analysis of cardiac surgery patients between 2016 and 2021, excluding heart transplant. Results: We enrolled 416 patients in the study, median age 57 (47;67) and 377 (49,8%) females. Overall, 287 (69,2%) procedures were elective, while 115 (27,7%) were urgent and 13 (3,1%) emergencies. Most procedures, 330 (79,3%), were isolated valve surgery, followed by combined valve and coronary artery bypass surgery (CABG), 36 (8,6%), valve and aorta, 19 (4,6%), valve and congenital, 8 (1,9%), valve, CABG and aorta, 2 (0,5%), and valve plus other cardiac surgery in 21 (5,1%). 147 (23,3%) had previous cardiac surgery. Diabetes was present in 69 (16,6%), 47 (11,3%) non-insulin dependent and 22 (5,3) insulin dependent. 25 (6%) had a history of previous myocardial infarction, 40 (9,6%) had stroke, 21 (5%) chronic pulmonary disease, 141 (43,9%) atrial fibrillation, 143 (44%) had rheumatic valve disease and 8 (1,9%) were on renal replacement therapy. Heart failure was diagnosed in 303 (73,5%) and 50 (12,1%) had a NYHA IV classification. Median ejection fraction was 63% (55;68), PSAP was 42 (31;55) and creatinine 0,98 (0,8;1,15). Observed in-hospital mortality was 13,0%. Univariate analysis showed that age, 67 (56;74) vs 56 (46;66), p = 0,002, endocarditis, 14 (30,4%) vs 40 (10,8%), OR = 3,60 (1,77–7,31), p < 0,001, mitral replacement plus tricuspid repair, 18 (23,4%) vs 26 (11,6%), OR = 2,34 (1,18–4,63), p = 0,013, and non-elective procedures, 27 (21,1%) vs 27 (9,4%), OR = 2,57 (1,44–4,60), p = 0,001 were related to increased mortality. Rheumatic etiology was a protective factor, 17 (9,3%) vs 37 (15,9%), OR = 0,54 (0,29–0,99), p = 0,045. In multivariate analysis, only age, OR = 1,058 (1,025–1,091), p = 0,001, endocarditis, OR = 4,51 (1,717–11,855), p = 0,002, and mitral replacement plus tricuspid repair, OR = 2,903 (1,325–6,362), p = 0,008, remained statistically relevant. Conclusion: Our study shows that older age, endocarditis and mitral replacement combined with tricuspid repair are associated with increased mortality after cardiac valve surgery. 111816 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES JOSÉ EDIVALDO DOS SANTOS1, José Victor Furtado Jacó de Oliveira2, Maria Letícia de França Oliveira2, Rafael Alexandre Meneguz-Moreno2 (1) Hospital de Urgências de Sergipe; (2) Universidade Federal de Sergipe – Campus Lagarto Introduction: The implementation of Telecardiology (TCL) in the state of Sergipe, Brazil, is a viable, promising and innovative strategy. Regarding specifically STEMI (ST-elevation myocardial infarction), it is known that mortality is directly proportional to the first medical contact to balloon time. The objective of the study is to evaluate data regarding patients with STEMI regulated by TCL service in Sergipe and referred to a tertiary hospital. Methods: This is a cross-sectional, retrospective, descriptive study, reporting data from June 2021 to April 2022 involving TCL service. Data were collected from electronic medical records, through the e-SUS program. Results: The resulting analysis population included 444 patients presented with STEMI during 11 months, being 288 (65%) male, mean age 61 years old, ranging from 27 to 102 years. Patient had previous hypertension (55%), diabetes mellitus type 2 (32%), dyslipidemia (12,8%), smoking (12,8%), previous coronary artery disease (5,6%), obesity (4,0%) and 68 (15,3%) reported no comorbidities. From all patients presented with STEMI during the analyzed period, 294 (66,2%) patients were referred to immediate PCI (percutaneous coronary intervention), 51 (11,4%) had been submitted to thrombolytic (TB) therapy, and 99 (22,2%) were not treated with reperfusion therapy within 12 hours of symptoms onset, yet 47 (47,4%) from these 99 patients were referred to urgent coronary angiography after TCL cardiologists evaluation. Among TB group, 32 (62,7%) patients were referred to a CICU (Coronary Intensive Care Unit) immediately after reperfusion, 2 (4%) were referred to rescue PCI and 17 (33,3%) have not been transported. The reasons for choosing TB therapy were, as follows, long distance (21), no transport available (6), no CICU vacancy (6), choice of attending physician (5) and not informed (13). There were 22 (5%) deaths among all patients, 4 from PCI group, 4 from TB group and 14 from the other group. Conclusion: At the beginning of TCL service in Sergipe, there were some patients that still have no access to PCI. However, with the consolidation of TCL, this flowchart was reorganized and since October 2021, there was no lack of vacancy. Therefore, TCL has improved the quality of STEMI care in the state of Sergipe (northeast Brazil). Strategies must still be implemented to increase PCI rates, such as improvement on medical education, in order to recognize symptom and trigger the TCL service more quickly. 111830 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY GABRIEL ASSIS LOPES DO CARMO1, Bárbara Carolina Silva Almeida1, Gabriela Zamunaro Lopes Ruiz1, Renato Braulio1, Ana Cristina Carioca1, Fábio Morato Castilho1, Cláudio Leo Gelape1, Bruno Rodrigues Pereira1, Luiza Moreira Gomes1, Ana Carolina Sudário Leite1 (1) Universidade Federal de Minas Gerais (UFMG) Introduction: Knowing baseline clinical information is important to compare results between different institutions. Brazil has a particular socioeconomic scenario and disease profile that make users of public health system (SUS) unique. Objective: Describe baseline characteristics and prognosis of cardiac surgery patients in a public hospital in Brazil. Methodology: Prospective cohort analysis of cardiac surgery patients between 2016 and 2021, excluding heart transplant. Results: We enrolled 633 patients, median age 58 (48;67) and 377 (49,8%) females. Most procedures, 406 (64,1%), were elective, followed by urgent, 109 (31,4%), and emergent, 28 (4,4%). Most surgeries were valvar, 416 (65,5%) followed by coronary artery bypass surgery (CABG), 188 (29,6%), aorta, 43 (6,8%), congenital heart disease, 33 (5,2%), and other surgeries, 50 (7,9%). 147 (23,3%) had previous cardiac surgery. Diabetes was present in 133 (20,9%), 88 (13,9%) non-insulin and 45 (7,1) insulin dependent, myocardial infarction in 76 (12%), stroke in 59 (9,3), chronic pulmonary disease in 29 (4,6%), atrial fibrillation in 150 (23,6%), rheumatic valve disease in 134 (21,1%) and 13 (2%) were on renal replacement therapy. Heart failure was diagnosed in 370 (58,9%) and 56 (8,8%) had a NYHA IV classification. Median ejection fraction was 62% (54;68), PSAP was 38 (29;51) and creatinine, 0,97 (0,8;1,18). Mean and median EUROSCORE II were 3,978 (±6,591) and 1,8 (0,990;4,020), respectively. Observed in-hospital mortality was 13,3%. Conclusion: Our results show a much higher than predicted mortality according to EUROSCORE II. However, our population has some important differences from that evaluated in EUROSCORE II study, especially a higher prevalence of rheumatic valve disease. Also, more commonly patients were operated on an urgent basis. Our population had more similarities with the InsCor Risk Assessment study performed in Brazil, which found a general mortality of 8,9%, but included only CABG and valve surgeries. Due to these specific baseline characteristics, local developed score would be more appropriate while evaluating cardiac surgery candidates in Brazil. 111860 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION MARCELO CARVALHO VIEIRA1, Fernanda de Souza Nogueira Sardinha Mendes1, Paula Simplício da Silva1, Gilberto Marcelo Sperandio da Silva1, Flavia Mazzoli-Rocha1, Andrea Silvestre de Sousa1, Roberto Magalhães Saraiva1, Vitor Barreto Paravidino2, Luiz Fernando Rodrigues Junior3, Alejandro Marcel Hasslocher-Moreno1, Pedro Emmanuel Alvarenga Americano do Brasil1, Mauro Felippe Felix Mediano1 (1) Evandro Chagas National Institute of Infectious Disease, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.; (2) Institute of Social Medicine, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil; (3) Department of Research and Education, National Institute of Cardiology, Rio de Janeiro, RJ, Brazil Introduction: The association between functional capacity and quality of life (QoL) in individuals with chronic Chagas cardiomyopathy (CCC) is still poorly investigated, with the few studies including indirect measures of functional capacity, limiting the validity and the interpretation of the results. Objective: The present study aimed to evaluate the association between functional capacity (quantified by cardiopulmonary exercise test [CPET]) and QoL in individuals with CCC. Methods: A cross-sectional analysis using baseline data from PEACH study, a randomized clinical trial that evaluated the effects of exercise training in patients with CCC, was performed. QoL was assessed using the SF-36 questionnaire. Sociodemographic, anthropometric, clinical, cardiac function and maximal progressive CPET variables were retrieved from PEACH study dataset. Generalized linear models adjusted for age, sex, and left ventricular ejection fraction were performed to evaluate the association between CPET variables and QoL. Results: After adjustments for potential confounders, VO2peak and VO2AT were both positively associated with physical functioning and physical component summary (PCS). Double product was positively associated with physical functioning, general health perceptions, and PCS, whilst heart rate recovery <12bpm at the first minute (HRR) was negatively associated with physical functioning, role limitations due to physical problems, bodily pain, and PCS. VE/VCO2 slope presented a negative association with all mental scales of SF-36: vitality, social functioning, role limitations due to emotional problems, mental health, and mental component summary. HRR <12bpm was negatively associated with vitality and mental health. Double product was positively associated with vitality. The CPET variables that most explained the QoL variation in the adjusted models were VO2AT (50% for physical functioning and 36% for PCS), VO2peak (31% for physical functioning and 21% for PCS), VE/VCO2 slope (45% for mental health and 31% for mental component summary), and HRR < 12 bpm (20% for vitality). Conclusions: The association between CPET variables and QoL reinforces the importance of CPET inclusion for a more comprehensive evaluation of individuals with CCC. Intervention strategies aiming to improve functional capacity may also promote additional benefits on QoL and should be incorporated as a treatment strategy for patients with CCC. 111901 Modality: E-Poster Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION MARCELO CARVALHO VIEIRA1, Fernanda de Souza Nogueira Sardinha Mendes1, Paula Simplício da Silva1, Gilberto Marcelo Sperandio da Silva1, Flavia Mazzoli-Rocha1, Andrea Silvestre de Sousa1, Roberto Magalhães Saraiva1, Fernanda Martins Carneiro1, Luiz Fernando Rodrigues Junior2, Alejandro Marcel Hasslocher-Moreno1, Pedro Emmanuel Alvarenga Americano do Brasil1, Mauro Felippe Felix Mediano1 (1) Evandro Chagas National Institute of Infectious Disease, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.; (2) Department of Research and Education, National Institute of Cardiology, Rio de Janeiro, RJ, Brazil Introduction: Physical exercise has been described as an efficient and safe strategy to improve quality of life (QoL) in heart failure (HF). However, there is a lack of information about its influence on the QoL of patients with chronic Chagas cardiomyopathy (CCC). Objective: The present study aimed to investigate the effect of physical exercise training on the QoL of patients with CCC. Methods: PEACH study was a single center, superiority randomized parallel-group clinical trial of exercise training versus a control group with no exercise training. The sample comprised Chagas disease patients with CCC, left ventricular ejection fraction <45% or HF symptoms (CCC stages B2 or C). QoL was assessed at baseline, after three months, and at the end of follow-up (six months) using SF-36 questionnaire. Patients randomized for the intervention group performed physical exercise (aerobic exercise, strength training and stretching exercises) for 60 minutes, three times a week, for a period of six months. Patients in the control group were not provided with a formal exercise prescription. During the study, patients from both groups underwent monthly appointments with the same cardiologist, and with other specialists, if necessary. In addition, patients in both groups received identical nutritional and pharmaceutical counseling during the study. Longitudinal analysis of the effects of physical training on QoL, considering the interaction term (group × time) to estimate the rate of changes between groups in the outcomes (represented as beta coefficient), was performed using mixed linear models. Analyses were adjusted to the baseline QoL values. Results: There were significant improvements in physical functioning (β = +10.7; p = 0.02), role limitations due to physical problems (β = +25.0; p = 0.01) and social functioning (β = +19.2; p < 0.01) scales during the first three months in the exercise group compared to control group. No significant differences were observed between groups after six months of follow-up. Conclusion: Exercise training was associated with short-term improvements in physical and social aspects of QoL and should be incorporated as a treatment strategy for patients with CCC. 111898 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BRÁULIO CRUZ MELO1, Larissa Rebeca da Silva Tavares1, Irlaneide da Silva Tavares3, Antônio Guilherme Cunha de Almeida1, Antônio Carlos Sobral Sousa2, João Vitor Andrade Pimentel2, Emerson de Santana Santos2, Enaldo Vieira de Melo2, Giovanna Medeiros Resende1, Evelen Rouse de Souza Santos1, Diego Henrique da Silva Tavares1, Joselina Luzia Menezes Oliveira2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe; (3) Hospital Primavera Introduction: Hypertrophic Cardiomyopathy (CMH) is the most common genetic disease in the world, and can lead their carriers to death, therefore making it’s correct diagnosis fundamental, along with the follow-up and genetic counseling of patients and their family. Methods: This is a cross-sectional, prospective and analytical study, with patients diagnosed with CMH in Sergipe between January 2021 and April 2022, being also subjected to genetic study. Objective: To describe the patient population under CMH investigation. Results: 80 patients were studied, 49 (61%) being of male gender, with the average age of 49. MRI was performed in 50 patients and positive for CMH in 45 (90%); 71 patients have resting echocardiogram, 60 of which cover CMH criteria. The most relevant echocardiographic variant for CMH is septal thickness, with an average of 15,5 mm on echocardiogram and 16,5 on MRI. It’s obstructive form was present at the MRI in 20% of the cases (10 patients), ischemia in 18 (39% in the septal wall), and fibrosis in 40 patients (80%). The doppler echocardiogram under physiological stress was performed in 21 patients, 11 on the treadmill, 2 on the bicycle and 6 on both methods which made possible the identification of 7 individuals with labile obstructive form. Finally, in the genetic study, the most found genes were MYBPC3 and MYH7 (28 and 12%, respectively), in agreement with whats already described in literature, in addition to 30% of cases being identified as phenocopies. Conclusion: The CMH can appear more frequently than expected and present variable expressiveness and penetration. Ocasionally, phenocopies and dislead it’s diagnosis. The echocardiographic evaluation under physiological stress can identify higher risk individuals (labile obstructive CMH). 111910 Modality: E-Poster Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES ANDRE SCHMIDT1, Maria Fernanda Braggion-Santos1, Marcel Koenigkam Santos1, Gustavo Jardim Volpe1, Henrique Turin Moreira1, José Antonio Marin-Neto1 (1) Faculdade de Medicina de Ribeirão Preto – USP Background: Chronic Chagas’ cardiomyopathy (CCC) is a consequence of several insults resulting in myocardial fibrosis (MF). Detection of MF is possible noninvasively through cardiac magnetic resonance (CMR). However, it is an expensive and not widely available method. Objective: This study aims to correlate ECG Selvester score (or QRS score) with quantification of MF and left ventricular ejection fraction (LVEF) through CMR analysis, in patients with CCC. Methods: Retrospective analysis of ECG and CMR exams, performed within one year of 194 patients with CCC. Rassi score was evaluated in 171 patients. Continuous variables were presented as median and interquartile range (IQR)and categorical data were summarized as percentages. Spearman’s rank correlation coefficient was used to evaluate the correlation between estimated MF by QRS score and quantified MF by CMR, and LVEF. Bland-Altman plot was applied to compare percentage of MF estimated by QRS score and measured by CMR. Patients were divided into tertiles based on QRS score values and Kruskal-Wallis test was applied to compare MF and LVEF among the groups. Receiver Operating Characteristic curves (ROCcurve) were generated to define cut-off values with the best accuracy to identify MF >= 10% of left ventricular mass, MF >= 12.3 g, LVEF < 50%, or LVEF < 35%. According to Rassi score classification, patients were separated in 3 risk groups, and QRS scores were evaluated by the Kruskal-Wallis test. P < 0.05 was considered statistically significant. Results: A total of 98 participants were women. Median age was 56 years(IQR: 44–67). Correlation between QRS score and MF by CMR was moderate (r = 0.45,p < 0.001), as well as the negative correlation between QRS score and LVEF (r = –0.42, p < 0.001). Bland-Altman plot showed worse agreement between methods for higher values of QRS score, and a trend to overestimate percentage of MF. QRS score >= 5 was correlated to MF >= 12.3 g, MF >= 10% of left ventricular mass, and LVEF < 50%. QRS score >= 6 was associated to LVEF < 35% with good specificity. Patients classified as high risk by Rassi score had a median QRS score of 5 points (IQR: 3–9). Conclusion: Higher QRS score in CCC had a moderate correlation with higher burden of MF and worse LVEF. QRS score values tend to overestimate percentage of MF, whereas lower values of QRS seem to have a better correlation with CMR. Selvester scores above 5 or 6 points are associated with more LV impairment, and potentially related to worse outcomes. 111923 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIEL ASSIS LOPES DO CARMO1, Bárbara Carolina Silva Almeida1, Gabriela Zamunaro Lopes Ruiz1, Renato Braulio1, Ana Cristina Carioca1, Fábio Morato Castilho1, Cláudio Leo Gelape1, Bruno Rodrigues Pereira1, Luiza Moreira Gomes1, Ana Carolina Sudário Leite1 (1) Universidade Federal de Minas Gerais (UFMG) Introduction: Neurological complications after heart surgeries are relatively frequent. Status epilepticus (SE) is particularly relevant due to its association with increased mortality. However, little is known about its pathogenesis and risk factors. Objectives: To evaluate the incidence of SE and identify its predictors after heart transplant. Methods: Prospect cohort of patients referred to heart transplantation in a university health facility in Brazil. Results: Between April of 2014 and November of 2021, 211 patients were referred to heart transplantation, median age 51 (40;58) and 73 (34,4%) females. In the early postoperative period, 28 (13.27%) patients had SE. Univariate analysis showed that INTERMACS 1 or 2 profile, OR = 4,88 (2,03–11,71), p < 0.001, and cardiorenal syndrome on renal replacement therapy (RRT), OR = 3,87 (1,59–9,39), p = 0.009, were associated with SE. Multivaltivariate analysis showed that only the INTERMACS 1 or 2 classification remained associated with SE, OR 3.88 (CI = 1.43–10.51), p = 0.008. The Hosmer-Lemeshow test had a value of p = 1.000. The occurrence of SE was not associated with increased mortality, OR 1.65 (CI = 0.61–4.45), p = 0.393. Conclusion: The occurrence of SE was only correlated with the INTERMACS 1 and 2 profile, a well know critical hemodynamic state with low cardiac output (CO). SE may be related to sudden increase in CO after cardiac transplantation, leading to an imbalance in cerebral blood flow. In the present analysis, SE was not correlated with higher mortality, possibly due to the small sample size. 111961 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING MARIA CATARINA DE MELO DIAS GUERRA1, Augusto Hiroshi Uchida1, José Antônio Franchini Ramires1, Eduardo Cavalcanti Lapa dos Santos1, Renata Ávila Cintra1, Jürgen Beuther1, Nevelton Heringer Filho1, Tiago Augusto Magalhães1, William Azem Chalela1, Guilherme Garcia1, Leonardo Filipe Benedeti Marinucci1, Carlos Eduardo Rochitte1 (1) Instituto do Coração (InCor – FMUSP) Introduction: Myocardial infarction (MI) size is a key predictor of prognosis in post-MI patients. Cardiovascular magnetic resonance (CMR) is the gold standard test for MI quantification but is still limited due to restricted availability in daily routine. Previous studies have documented the capability of different electrocardiogram (ECG) markers for prognosis assessment soon after ST elevation myocardial infarction. Selvester QRS scoring detects scar, although the reported performance varies. A simplified version of this score showed comparable predictive value for infarct size in a prospective study including 201 patients at a median of 2 days after a revascularized MI (area under the curve = 0.64 vs. 0.67). Methods: Retrospective, observational, single-center study. The sample was obtained through analysis of medical records with a record of clinical diagnosis of MI who underwent CMR to assess myocardial viability. Those who did not present delayed enhancement with an ischemic pattern on CMR and those whose CMR revealed a diagnosis other than MI or did not have satisfactory image quality were excluded. Patients reporting a new acute coronary syndrome in the interval between the available ECG and the CMR were also excluded. The ECG performed 7 days after the infarction and with a maximum interval of 1 year from the CMR were analysed by an evaluator with extensive experience in electrocardiography. Results: The mean age of patients was 61.3 years and 75% were men. Conventional infarction identification through the presence of a pathological Q wave occurred in 172 of the 245 patients included (70.2%). In the subgroup of 192 patients in which CMR was already analyzed, the median modified Selvester score was 5 and the fibrosis mass was 23.2% ± 11.3 of the left ventricular mass. There was no statistically significant correlation between the modified Selvester score and fibrosis mass on CMR. Conclusions: After seven days of acute coronary syndrome, the modified Selvester score was not a predictor of fibrosis mass. This correlation had been previously described in smaller studies that evaluated this score at admission of acute MI. Intraventricular conduction disorders and ventricular hypertrophy are factors that may impact the correlation between the Selvester score and fibrosis mass. 111962 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIEL ASSIS LOPES DO CARMO1, Bárbara Carolina Silva Almeida1, Gabriela Zamunaro Lopes Ruiz1, Renato Braulio1, Ana Cristina Carioca1, Fábio Morato Castilho1, Cláudio Leo Gelape1, Bruno Rodrigues Pereira1, Luiza Moreira Gomes1, Ana Carolina Sudário Leite1 (1) Universidade Federal de Minas Gerais (UFMG) Introduction: The Society for Cardiac Angiography and Interventions (SCAI) shock classification was published in 2019, but not yet validated in patients referred to heart transplant. Objectives: To evaluate the association between different groups of the SCAI shock classification and the occurrence of death among patients undergoing heart transplant. Methods: Prospect cohort of patients referred to heart transplant in a university health facility in Brazil. Results: Between April 2014 and November 2021, 211 patients underwent heart transplantation, median age 51 (40;58) and 73 (34.4%) females. Baseline population characteristics were similar among the 5 different groups of the SCAI classification, with the exception of insulin-requiring diabetes mellitus (15.1%, 21.4%, 1.5%, 8.3% and 5.6%, p = 0.038), use of intra-aortic balloon prior to surgery (0.0, 4.6%, 20.0%, 55.6%, p < 0.001), days of hospitalization before surgery [0 (0;0); 1.5 (0;22); 30(15;59); 30(15;60); 39 (27;44)], platelet count [191000 (159250;246500); 214500 (172500;287500);221000 (176000;285500); 191000 (146000;250000); 184500 (139500;252500)] and RNI [1.51 (1.22; 2.39); 3.07 (1.65; 4.51); 1.46 (1.23; 2.37); 1.30 (1.12; 1.55) and 1.34 (1.18; 1.65)] respectively for groups A, B, C, D and E. Death occurred in 4 (7,4%) patients in group A, 2 (14,3) in group B, 4 (6,2) in group C, 15 (25,0%) in group D and 7 (38,9%). Compared to group A, patients in classification B had OR 2.083 (0.341–12.733), p = 0.595, those in group C OR 0.820 (0.195–3.444), p = 1.000, group D OR 4.167 (1.288–13.481), p = 0.012 and group E 7.955 (1.979–31.972), p = 0.004, for death. The ROC curve for the SCAI rating was 0.695 (0.590–0.799). Patients classified as D or E had an OR 4.832 (2.146–10.879), p < 0.001. Multivariate logistic regression analysis showed that group D or E classification was the only variable associated with mortality, OR 4.028 (1.397–11.610), p = 0.010. Conclusion: SCAI shock classification D or E was associated with increased risk of death after heart transplantation. The model had a limited capacity of discrimination, probably due to similar risk of death in groups A, B and C. 111972 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANA CAROLINA MICHELETTI GOMIDE NOGUEIRA DE SÁ1, Elton Junio Sady Prates2, Deborah Carvalho Malta1 (1) Nursing Post Graduate Program. Nursing school, Federal University of Minas Gerais (UFMG). Belo Horizonte, MG, Brazil.; (2) School of Nursing at the Federal University of Minas Gerais (UFMG). Belo Horizonte, MG, Brazil. Introduction: Cardiovascular diseases (CVD) are the main cause of morbidity and mortality in the world and in Brazil and are caused by unhealthy lifestyles, genetic factors, social and health inequalities. These diseases cause disability and loss of quality of life. Consequently, this situation exacerbates the social and economic burden for society, governments and health services. Evidence has identified that physical inactivity is associated with increased coronary risk. However, there are still scientific gaps about the association of leisure-time physical activity (LPA) and cardiovascular risk in Brazil, even in view of the magnitude of CVD. The National Health Survey (PNS) performed laboratory tests and blood pressure (BP) measurements, thus, it was possible to explore, in an unprecedented way, the effect of AFL practice on cardiovascular risk in a representative sample of Brazilian adults. Objective: To analyze the association between AFL practice and cardiovascular risk in Brazilian adults. Methods: Cross-sectional study, with data from the PNS, between 2014–2015, in 8,952 adults. Prevalence and 95% confidence intervals were estimated for the practice of LPA stratified by sex, according to the Framingham risk score and its components. It was defined as sufficient AFL, the practice ≥150 minutes per week of light or moderate intensity or 75 minutes per week of vigorous intensity. The risk score was classified as low, medium and high. Differences were evaluated using the chi-square test or analysis of variance (p ≤ 0.05). Results: The highest prevalence of sufficient LPA practice was associated with low cardiovascular risk in men (17.9%; 16.0–20.0) and women (12.0%; 10.9–13.4) (p ≤ 0.05). For risk score components, sufficient AFL prevalence was higher: in men aged 18 to 29 years (10.43%; 8.9–12.1), with high-density lipoprotein (HDL) between 35–44 mg/dL (9.8%; 8.5–11.3), total cholesterol (TC) between 160–190 mg/dL (11.2%; 9.8–12.8), untreated BP < 120 mmHg (11.4%; 9.9–13.1), who do not have diabetes (25.5%; 23.6–27.6) and are non-smokers (23.3; 21.4–25.3); and in women with HDL between 50–59 mg/dL (4.43%; 3.8–5.2), untreated BP < 120 mmHg (11.50%; 10.24–12.89) and not are smokers (16.2%; 14.9–17.5) (p ≤ 0.05). Conclusion: The practice of AFL is associated with a decrease in cardiovascular risk in Brazilian adults. Thus, it is important to encourage the practice of AFL in the country. These results can support CVD prevention actions and policies. 111973 Modality: E-Poster Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY SEBASTIÁN DARÍO PERALTA1, Marcelo Omar Bettinotti1, Guillermo Jubany1, Ezequiel José Zaidel1, Luis Murillo1, Juan Grieve Bruno1, Luis Carlos Sztejfman1, Matías Sztejfman1, Carlos Maximiliano Giuliani1, Ramón Gomes Marquez1, Pedro Piccaro de Olivera1, Alexandre Schaan de Quadros1 (1) LATAM CTO registry Background: The newest techniques for chronic total occlusions (CTO) percutaneous coronary interventions (PCI) may improve technical success. Purpose: To describe safety and efficacy of ADR (antegrade dissection and reentry) technique as initial revascularization strategy. Methods: Multicentric registry from Latin American countries. We analyzed baseline characteristics and outcomes of cases using ADR as primary strategy or bailout of antegrade wire escalation (AWE). Retrograde approach cases were excluded. Results: From 1875 patients analyzed, 50 were planned primary ADR (pADR) and 1825 planned primary AWE (pAWE). pADR was preferred in older patients, with a history of revascularization (CABG: pADR 33.3% and pAWE 13.4%, p < 0,001; PCI: 66.6% and 48.8% respectively, p = 0.012). Longer CTOs (30 mm [22–41] and 21 mm [15–30], p < 0.001), and moderate or severe calcification (62% and 42.6%, p = 0.008) were associated with the selection of pADR instead of pAWE. There was a significant correlation between increasing J-CTO score (Chi2 = 37, df = 5, p < 0.001), and use of pADR. pAWE had a success rate of 88.45%, and pADR of 76.67%. For pAWE and bailout ADR, the use of a dedicated device was related to the highest rates of success (92.31% and 82.69%, respectively). Short term outcomes were similar between groups. Conclusions: In Latin America, ADR was safe and effective, both as primary or bailout strategy, even when used for higher complexity lesions. The use of dedicated devices was related to a higher success rate. 111999 Modality: E-Poster Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS POLLIANNA DE SOUZA RORIZ1, José Victor de Sá Santos3, Murilo Jorge da Silva3, Victor Luis Peixoto Pereira Botelho3, Marcelo Vincenzo Sarno Filho3, Bianca Aparecida Colognese3, Márcio Andrade Barreto Filho4, Rhanniel Theodorus Helhyas Oliveira Shilva Gomes Villar1 (1) Protocolo IAM – SAMU Salvador; (2) Hospital Ana Nery; (3) Universidade Federal da Bahia; (4) Escola Bahiana de Medicina e Saúde Pública Introduction: An integrated healthcare system plays an important role in ST Elevation Myocardial Infarction (STEMI) assistance. Published data emphasize that the number of hospitalizations, diagnostic and therapeutic procedures performed in STEMI assistance in the pandemic context have decreased. Objective: Evaluate if there was a change in STEMI care assistance by the Acute Myocardial Infarction Protocol (AMIP) team, as reperfusion strategies and mortality in Salvador’s health public system during the pandemic of Covid 19. Methods: Ambispective cohort of STEMI patients attended by the AMIP in Salvador. Epidemiologic data, assistance times, reperfusion strategies and intra-hospitalar mortality were compared between pre-pandemic period (jan/2019–feb/2020) and pandemic (march/2020–dec/2021) – groups I and II, respectively. Statistical significance was considered for p < 0.05. Results: Group I was composed of 542 patients and group II of 932. The mean age and male sex frequency were similar in both groups. There was a higher frequency of hypertension (73.2% × 67.8%, respectively; p = 0.03) and typical A/B pain (91.2% × 85%, respectively; p = 0.01) in group I. Other comorbidities, clinical presentation, Killip and GRACE showed no differences between groups. Controversially, there was no difference in the median symptom-first medical contact time [I–120 (50–270)min × II–123 (60–268.5)min, p = 0.32]. Door-to-ECG time was even shorter in group II [29 (14–70)min] compared to I [35 (16–92.7)min], p = 0.01. There was a shorter door-to-needle time in group II compared to I [111.5 (76–174.8)min × 140 (91.8–202.3)min, respectively; p = 0.002] as well as door-to-balloon time [219 (169.3–317.8)min × 253 (175.8–356.5)min; p = 0.03]. Regarding reperfusion strategies, more patients underwent Percutaneous Coronary Intervention (PCI) in II × I (78.3% vs. 73.5%, p = 0.04), due to a higher frequency of pharmaco-invasive strategy in the second group (II–17.7% × I–3.2%, p < 0.001). There was no difference for primary PCI between groups (57.3% I × 58.8% II; p = 0.64). In-hospital mortality did not differ between the groups (I–17.3%) and (II–16.1%), p = 0.56. Conclusion: During the pandemic, there was a switch in the reperfusion strategy supported by AMIP in order to offer more room to fibrinolysis. The troubled scenario caused by the COVID-19 pandemic did not have a major impact on mortality of patients with STEMI in Salvador. 112000 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ARN MIGOWSKI ROCHA DOS SANTOS1, Arn Migowski1, Gustavo Tavares Lameiro da Costa1, Helena Cramer Veiga Rey1 (1) Instituto Nacional de Cardiologia (INC) Introduction: In Brazil, early neonatal mortality is responsible for more than 50% of deaths in the 1st year of life and congenital heart diseases accounts for 40% of malformations. Newborn pulse oximetry screening(POS) test for critical congenital heart defects between 24–48 hours of life is non-invasive, easy-to-perform and highly specific. Objectives: To estimate the prevalence of POS in Brazil, as well as identify the factors associated with it and the prevalence of positive screening results. Methods: Prevalence was estimated based on results of the most recent National Health Survey, a nationwide population-based cross-sectional Brazilian study. The adjusted marginal prevalence ratios were estimated from a logistic regression model based on Wilcosky & Chambless approach(R prLogistic package). Results: The prevalence of POS was 66.3% (95% CI: 65.5–67.1; N = 3,140,023). The screening prevalence in children born in private funded hospitals (PFHs) was higher than in the National Unified Health System (SUS): 78.1% (76.7–79.5) vs 61.1% (60.2–62.1), respectively. Major regions comparison shows important differences. In the North the prevalence in PFHs (64.9%; 59.7–70.1) was lower than in the South (82.5%; 79.4–85.6) and the Southeast (81.5%; 79.3–83.6) and it is even lower when compared with SUS in the North (44.0%;42.4–45.6). An ordinance was published in Aug 2018, allowing SUS to finance after-screening diagnosis to investigate congenital heart diseases, increased the prevalence of screening in SUS: 57.6% (56.2–59.1) before vs 64.6% (63.3–65.9) after the ordinance. Prevalence of positive screening tests was 9.2%(8.9–9.5) in SUS and 7.8% (7.3–8.3) in the PFHs. The proportion of these newborns that underwent complementary exams after screening was lower in SUS than in the PFHs (40.8%; 40.5–41.1 vs 57.2%; 56.7–57.7). In the multivariate model, the main independent predictors of POS were the prevalence of other newborn screening tests, specially the red eye reflex (PR = 2.03; 1.90–2.16) and newborn hearing test (PR = 1.75; 1.64–1.87). Conclusions: Inequalities were found in the prevalence of screening between major regions, as well as in SUS and PFHs. Government financial incentive has reduced this inequality between public and private, although the percentage of complementary exams after positive screening was also unequal. The main independent predictors of screening prevalence were those related to the organization of health services. 112004 Modality: E-Poster Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANTONIO GABRIELE LAURINAVICIUS1, Antonio Gabriele Laurinavicius1, Alessandra Cristina Vieira1, Julia Luzo Elias Thame1, Rafael Gonçalves Zimmer1, Henrique Marino de Medeiros1, Jorge Tadeu Campos Paixão1, Fernanda Consolim Colombo1, Marcio Gonçalves Sousa1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Out-of-Office Measurement of Blood Pressure (BP) is recommended in addition to office BP for the diagnosis and follow-up of hypertensive patients. Ambulatory Blood Pressure Monitoring (ABPM); Home Blood Pressure Monitoring (HBPM); and Self-Monitoring of BP (SMBP) are the currently available options and their indication may vary according to the context. The aim of the present study was to assess how Out-of-Office Measurement of BP takes place in clinical practice and its impact on BP control in a public tertiary outpatient clinic. Methods: We evaluated 225 consecutive patients seen at a high-complexity public outpatient facility (mean age: 66.7 ± 11.9 years; female: 62.7%). All patients were routinely requested to perform SMBP according to a prespecified institutional protocol. ABPM and HBPM were indicated for selected cases at the discretion of the attending physician. Patient Adherence to Out-of-Office Measurement of BP was labeled into 5 possible categories: a) No Measurement; b) ABPM; c) HBPM; d) Adequate SMBP e) Inadequate SMBP. Patient Adherence was also stratified according to sex, age, number of antihypertensive drugs, schooling, length of follow-up at the facility, comorbidities and availability of BP monitor at home. Rates of BP control were related with Patient Adherence, as well as with the aforementioned variables. Results: 87.5% of the study population reported having a BP monitor at home. However, adding up the 5 possible categories, adequate Out-of-Office Measurement of BP was available in only 46.7% of the sample (40.9% of the patients did not bring any measurement; 13.8% underwent ABPM; 32.9% adequate SMBP; 12.4% inadequate SMBP; 0% HBPM). Availability of a BP monitor at home (p < 0.001) and the number of antihypertensive drugs in use (p = 0.019) were strongly associated with adherence to SMBP. Prevalence of smoking was 2 folds higher (7.5% vs 3.4%) in those who returned without SMBP. Rate of BP control based on office BP was 42.6% (79.5% of the sample was under ≥3 classes of antihypertensive drugs). Out-of-office BP measurements were not associated with higher rates of BP control (p = 0.377), but allowed to identify a White Coat Effect (WCE) in 1 out of 3 patients with uncontrolled BP according to office BP (WCE prevalence: 29% among uncontrolled patients vs 3.9% among controlled ones. Conclusions: Out-of-Office Measurement of BP is still an unmet need in the treatment of hypertension. 112035 Modality: E-Poster Researcher – Non-case Report Category: CARDIO-ONCOLOGY MONICA DE MORAES CHAVES BECKER1, Gustavo Freitas Alves de Arruda1, Diego Rafael Freitas Berenguer1, Roberto de Oliveira Buril1, Felipe Alves Mourato1, Paulo José de Almeida Filho2, Brivaldo Markman Filho1, Simone Cristina Soares Brandão1 (1) Universidade Federal de Pernambuco; (2) Real Hospital da Beneficência Portuguesa Introduction: Studies have suggested that the assessment of cardiac 18F-FDG uptake may be an early metabolic marker of cardiotoxicity. Echocardiography is the method of choice for the evaluation and follow-up of patients undergoing chemotherapy (CT) with anthracyclines, especially through the Global Longitudinal Strain (GLS) technique, which would change earlier than the drop of the left ventricular ejection fraction (LVEF) in patients with reduced myocardial performance. Objective: The objective of this study was to show the cardiac metabolic and functional behavior pre and post-CT with anthracyclines, in patients with lymphoma and to evaluate the relationship between metabolic alteration and cardiac performance. Methods: 18F-FDG PET-CT and strain echocardiography were prospectively performed before and after CT. On PET-CT, the maximum standardized uptake (SUV) value of 18F-FDG was measured in the interventricular septum (IVS) and aorta – blood pool. An increase above 30% in the uptake of 18F-FDG in the IVS was considered significant. On echocardiography, LVEF and GLS were measured. After CT, patients were divided into two groups according to the drop in GLS ≥15% from baseline. Data were compared pre- and post-CT and between groups. Results: Twenty-four consecutive patients were selected (mean age 44,5 ± 18 years, 58,3% female and 66,7% with non-Hodgkin’s lymphoma). The mean LVEF did not show significant difference before (63,5% ± 4,6%) and post CT (64,9% ± 4.4%). Subclinical left ventricular dysfunction (GLS drop ≥15%) occurred in seven (29,2%) patients, during or after CT. Significant increase in IVS FDG uptake occurred in 13 (54,2%) patients. The pre-CT median SUV maximun SIV/SUV maximun aorta ratio increased from 0.84 (IQ 0.73–1.01) to 1.06 (IQ 0.84–1.93) after CT, p = 0.02. However, when we compared this relationship between the groups, there was no significant difference. Conclusions: In this casuistic, we did not observe a significant reduction in LVEF after chemotherapy. However, about 29% of patients presented a decrease in cardiac performance assessed by the strain echocardiography and more than half had a metabolic change in IVS. The relationship between metabolic alteration and subclinical cardiac performance needs to be better clarified. 112042 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY ADRIANO MÁRCIO DE MELO MILANEZ1, Marina Coelho Feitosa2, Yury Pifano Varela2, Josué Viana de Castro Neto3, Flávio Duarte Camurça1 (1) Cardiovascular surgeon, Cardiovascular Surgery Department, Hospital Antônio Prudente, Fortaleza, Ceará, Brazil.; (2) Medical school student, The University of Fortaleza – UNIFOR, Fortaleza, Ceará, Brazil; (3) Cardiovascular surgeon, Professor of Surgery, The University of Fortaleza – UNIFOR, Fortaleza, Ceará, Brazil. Introduction: The Right Inferior Periareolar Access (RIPA), also known as the Brazilian Technique, has been progressively applied in minimally invasive cardiac surgery (MICS) for atrioventricular valve and septal defect procedures. It provides good functional and aesthetic results, while using a similar fourth intercostal access when compared to the usual inframammary minithoracotomy. We aim to describe a Right Superior Periareolar Access (RSPA), performed one intercostal space above the RIPA, with advantages. Methods: The RSPA consists of a videoassisted minithoracotomy in the 3rd intercostal space through a superior periareolar incision of 3 cm enlarged by a wound protector (Image 1). The cardiopulmonary bypass was established through a femoral platform and cannulation of the right internal jugular vein was added when necessary. A 5 mm port was inserted through the 2nd intercostal space for non dominant hand instrumentation initially and for clamp placement afterwards. The camera port was inserted through the 5th intercostal space. This technique was performed in 39 patients: 30 with atrial septal defects, 5 submitted to mitral valve reconstruction and 4 undergoing mitral valve replacements. Twenty four were females. The mean age was 40. Results: The RSPA provided adequate surgical fields for all procedures. The cardioplegia infusion in the ascending aorta was easily done. Conversion to full sternotomy was necessary in one patient due to bleeding. The median lenght of intensive care unit stay was 1 day and the median postoperative hospital stay was 3 days. There were no postoperative wound infections and no postoperative mortality. The RSPA provided easy access to cardiovascular structures, contributing to effective, functional and safe procedures. The resulting surgical scars were very subtle, and did not compromise the areola. Conclusion: The MICS procedures applying the RSPA technique were effective and safe. Other remarkable advantages were adequate access to the aorta and atriums, low lenght of stay in the intensive care unit and in the hospital and good aesthetic results. 112055 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING MARIA CATARINA DE MELO DIAS GUERRA1, Augusto Hiroshi Uchida1, José Antônio Franchini Ramires1, Eduardo Cavalcanti Lapa dos Santos1, Renata Ávila Cintra1, Jürgen Beuther1, Nevelton Heringer Filho1, Tiago Augusto Magalhães1, William Azem Chalela1, Guilherme Garcia1, Leonardo Filipe Benedeti Marinucci1, Carlos Eduardo Rochitte1 (1) Instituto do Coração (InCor – FMUSP) Introduction: Fragmented QRS (fQRS) is an easily evaluated electrocardiographic parameter and has been associated with alternation of myocardial activation due to myocardial scar. Methods: Retrospective, observational, single-center study. The sample was obtained through analysis of medical records with a record of clinical diagnosis of myocardial infarction (MI) who underwent cardiac magnetic resonance (CMR) to assess myocardial viability. Those who did not present delayed enhancement with an ischemic pattern on CMR and those whose CMR revealed a diagnosis other than MI or did not have satisfactory image quality were excluded. Patients reporting a new acute coronary syndrome in the interval between the available electrocardiogram (ECG) and the CMR were also excluded. The ECG performed 7 days after the infarction and with a maximum interval of 1 year from the CMR were analysed by an evaluator with extensive experience in electrocardiography. Results: The mean age of patients was 61.3 years and 75% were men. Conventional infarction identification through the presence of a pathological Q wave occurred in 172 of the 245 patients included (70.2%). Fragmentation of the QRS complex was identified in 102 of the 245 patients included (41.6%). The prevalence of fQRS was significant even in smaller MI and no correlation was found between the presence of fQRS and the mass of fibrosis on CMR. A higher prevalence of fQRS was observed in infarcts predominantly in the lateral and anterior walls (figure 1). Conclusions: This study revealed a significant prevalence of fQRS in patients after the acute phase of infarction, even in those with a lower mass of fibrosis. Although the analyzed sample of lateral infarctions is still limited, the high prevalence of fQRS may be a relevant finding, considering the low sensitivity of the classic criteria for identifying lateral infarction on the ECG. Figure 1 – Prevalence of QRS fragmentation according to the wall most affected by the infarction. 112063 Modality: E-Poster Researcher – Non-case Report Category: CARDIOGERIATRICS ALEXANDRA CORRÊA GERVAZONI BALBUENA DE LIMA1, Luana de Oliveira Alves1, Laila Morais Nahass Franco1, Pedro Pinto Machado2, Pedro Henrique Parcianello Teixeira2, Luciana Bartolomei Orru D’Avila1 (1) North Wing Regional Hospital – Brasília – Brazil; (2) School of Health Sciences– Brasília – Brazil. Background: Heart failure (HF) increases significantly in relation to advancing age. With aging, not only do age-related, morphological, and physiological cardiovascular changes predispose to HF, there is also increased prevalence of comorbid conditions that compound cardiac limitations (e.g., renal insufficiency) and others that tend to overwhelm limited cardiovascular reserves (e.g., infections and ischemia). The HF among very old adults, the prevalence, the complications in acute HF demand more studies. Objective: Evaluate the clinical features and prognostic evolution of individuals 80 years and older with acute HF. Methods: Retrospective and observational study of patients 80 years or older, hospitalized with acute HF at North Wing Regional Hospital, Brasília, Brazil, January 2017 to May 2020. Patients who did not meet the clinical criteria of HF, with missing or incomplete information in the medical record or were not accessible by telephone were excluded. The minimal follow-up were 12 months and final date were 05.21.2021. Clinical profiles, clinical history, blood test, and mortality were analyzed. The patients were divided into groups: 80 to 84 years and ≥85 years. Results: A total of 119 individuals (66 and 53) were included, 45% and 47% male, 92% and 83% hypertensive. The mean age of the groups were 81.7 ± 1.3 years (80–84) and 88.6 ± 3.5 years (85–97), hemoglobin 11.9 ± 2.4 mg/dL and 10.8 ± 2.0 mg/dL, creatinine 1.44 ± 1.9 mg/dL and 1.78 ± 1.4 mg/dL, left ventricular ejection fraction 51.8 ± 15.7% and 58.6 ± 11.8%, and follow-up 29.1 ± 25.9 days and 28.4 ± 24.6 days. The presence of pulmonary congestion 40% and 45%, peripheral edema 45% and 43%, and atrial fibrillation (AF) 34% and 35%. During the hospitalization, hemodialysis 15% of both groups, vasoactive drugs 28% and 18%, intensive care unit 45% and 39%, and mortality rate 21% and 18%. In multivariate analysis deaths were related to hypertension (HR = 0.54, 95%CI = 0.29–0.98, p = 0.046), pulmonary congestion (HR = 3.38, 95%CI = 1.97–5.79, p = 0.000), peripheral edema (HR = 4.12, 95%CI = 2.30–7.38, p = 0.000), AF (HR = 0.58, 95%CI = 0.34–0.98, p = 0.043), and hemoglobin (HR = 0.84, 95%CI = 0.75–0.95, p = 0.007). Conclusion: In our study simple clinical features as pulmonary congestion, peripheral edema and hemoglobin showed association with mortality. Consideration of disease prognosis according to factors that predict mortality can help to better define the care plan and promote palliative and supportive care. 112078 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH PRISCILA VALVERDE DE OLIVEIRA VITORINO1, Alice PInheiro Ribeiro1, Edison Nunes Pereira2, Gustavo Carvalho Marcelino1, Ana Carolina Arantes2, Ademir Schmidt1, Weimar Kunz Sebba Barroso2 (1) Pontifícia Universidade Católica de Goiás; (2) Universidade Federal de Goiás Introduction: Sedentary behavior (SC) is a habit present in worldwide, with increasing percentages in the last decade, especially among adolescents. With the COVID-19 pandemic, CS increased, especially due to social isolation and school closures. Objetives: Carachterize adolescens regarding sociodemographic and economic conditions, aspects related to school and work and general health; classify adolescents in terms of SC and compare CS according to gender. Method: Analytical cross-sectional study, conducted with adolescents (10 to 19 years), enrolled in 19 schools of Goiás. Electronic data collection took place from November 2020 to August 2021. The form contained questions about personal, sociodemographic, lifestyle and CS. Two cutoff points were considered for SC: ≥6 and ≥ to 3 hours/day. The project was approved by Ethics Committee. Descriptive analysis and comparisons were performed using pearson’s Student and Chi-square T tests. Significance level p < 0.05 was considered. Results: We evaluated 167 students with a mean age of 15.3 DP 2.1 (Table 1). The frequencies of SC 89.1% and 52.7% at the cutoff points of 6 and 3 hours/day, respectively. Conclusions: The sample was characterized by adolescents who did not work, were mainly from social classes B and C and had a low frequency of bad health habits. There was no difference in the frequency of SC in relation to the variables evaluated. 112101 Modality: E-Poster Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE CÉLIDA JULIANA DE OLIVEIRA1, Héryka Laura Calú Alves1, Gabriela de Sousa Lima1, Érica Sobral Gondim1, Emiliana Bezerra Gomes1 (1) Universidade Regional do Cariri – URCA Introduction: Hypertension evolves slowly and is asymptomatic, so it does not usually receive the proper care, resulting in poor adherence to treatment and an increase in cardiovascular events. It is a disease that requires changes in lifestyle for adherence to treatment, which is influenced by several factors, such as religiosity, which includes people’s beliefs, attitudes and values. Therefore, the pilgrimage of Padre Cícero stands out, a religious manifestation that attracts thousands of faithful to the municipality of Juazeiro do Norte-CE, in which the attendance to pilgrims because of the elevation of blood pressure is a constant, making it necessary to study the relationship between religiosity and adherence. Objective: To investigate the influence of religiosity in the treatment adherence of hypertension by pilgrims. Method: Cross-sectional, qualitative, carried out in Juazeiro do Norte, between September 9 and 15, 2018. The question was: “Does religion influence you to comply with treatment for high blood pressure?”. The data collected were organized into thematic categories. The study was approved by the Ethics Committee under opinion number 2,753,051. Results: 338 pilgrims self-reported with hypertension were investigated, male (62.43%), retired (71.06%) and mean age of 64.5 years. When questioned about the influence of religion on treatment, three categories emerged: Category 1 “Faith”: Faith was answered as the influence of religion on compliance with treatment, either alone or in a complementary way, also as a cure for hypertension. Category 2 “I pray for improvement”: Prayers are the most cited as influencing treatment, for comforting, improving thoughts and guiding behaviors, such as waking up to pray and taking medication. Category 3 “Religion is above all”: Church and masses were cited as contributors. Pilgrims report feeling good and associating religious practices with treatment, combining medication intake times with church participation. The people who are in this space represent a support network, especially the priest, by encouraging self-care related to the treatment. Final Considerations: It was noticeable that the pilgrimage and its elements influence the perception of health, illness and treatment. Thus, they can be used as beneficial means of promoting health and adherence to therapy through strategies that enhance the spiritual dimension. 112117 Modality: E-Poster Researcher – Non-case Report Category: PHYSICAL EDUCATION FÁBIO SILVA MARTINS DA COSTA1, Henrique Silveira Costa2, Lucas Frois Fernandes Oliveira2, Matheus Ribeiro Ávila2, Pedro Henrique Scheidt Figueiredo2, Mauro Felippe Felix Mediano3, Luciano Fonseca Lemos de Oliveira1, Manoel Otávio Costa Rocha1 (1) Universidade Federal de Minas Gerais – UFMG; (2) Universidade Federal dos Vales do Jequitinhonha e Mucuri – UFVJM; (3) Fundação Oswaldo Cruz – FIOCRUZ Introduction: Patients with Chagas cardiomyopathy (ChC), in addition to having a worse prognosis compared to other cardiomyopathies, can have important consequences, including reduced functional capacity and quality of life. The exercise training (ET) has emerged as an adjuvant option for treatment of ChC, relieving dyspnea and fatigue caused by heart failure. This study aimed to discuss the main findings of effect of ET in patients with ChC, focusing on the functional capacity, hemodynamic and autonomic function, musculoskeletal system, myocardial function and morphology, and health-related quality of life (HQoL). Methods: This is a systematic review using the descriptors: (“Chagas disease” OR “chronic Chagas cardiomyopathy”) AND (“exercise training” OR “cardiac rehabilitation”). A literature search of the MEDLINE, LILACS, Web of Science, CINAHL, Scopus, PEDro and EMBASE was performed with no data or language restrictions. The study was registered in PROSPERO database (CRD CRD42017064912) and edited following in guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Results: A total of 5 papers (screened from 1.169 studies) reached the inclusion criteria and were included in the present review. The patients with ChC submitted to ET presented improvement of functional capacity (increase in the VO2peak and the walked distance in the 6-minute walk test, but did not reduce the ventilatory efficiency given by VE/VCO2slope), hemodynamic and autonomic function (reduce the HR at rest but do not change the blood pressure; slight increase in the HRV in patients with reduced LVEF and better improvements in the autonomic balance given by increase in the parasympathetic activity and reduction in the sympathetic activity over the heart in patients with preserved LVEF and autonomic dysfunction), musculoskeletal system (increase in the cross-sectional and number of type I fiber, reduce the genes expression involved in the muscle atrophy process, increase skeletal muscle perfusion) and the HQoL (improve domains of vitality and emotional aspects). However, no changes in the myocardial function and morphology were observed. Conclusion: In conclusion, the ET as a low-cost non-drug therapy option seems to provide important benefits in association with the classic treatment of patients with ChC, however, more studies are warranted to confirm these results. 112137 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY CHARLES SLATER1, Luiz Eduardo Camanho1, Eduardo Benchimol Saad2, Luiz Antonio Oliveira Inacio Jr2, Lucas Carvalho Dias1, Gustavo Vignoli Santos2, Ricardo Mourilhe-Rocha1 (1) State University of Rio de Janeiro (UERJ), Rio De Janeiro, Brazil; (2) Hospital Pro Cardíaco, Rio De Janeiro, Brazil Background: Conduction system pacing (CSP) (His bundle pacing and left bundle pacing) is a group of techniques intended to achieve cardiac pacing with a narrow QRS complex. The safety and effectiveness of this technique are not yet entirely understood. Purpose: To describe the implant findings and safety profile of CSP as a first option after 4 years in a single center. Methods: In a period of 42 months, 214 patients were submitted to CSP as a first strategy to restore AV synchrony (pacemakers for AV block or sinus node dysfunction) or as a resynchronization (CRT) strategy (for patients with heart failure and bundle branch block). CSP was implanted in lieu of a conventional right ventricular lead in pacemaker cases, and in addition or in lieu of a coronary sinus lead, in CRT cases, depending on the technical and anatomical findings. Results: The mean age was 76.7 ± 16.4 years, 65% males. 162 patients implanted a CSP lead for a dual-chamber pacemaker, 3 patients for a single chamber pacemaker, 32 patients for CRT-D (CSP lead replacing the coronary sinus lead with a defibrillator), and 13 patients for an optimized CRT (CSP lead plus coronary sinus lead). In 16 patients (7.4%) the technique of choice was His bundle pacing. One patient (0.4%) had subacute lead dislodgement, being submitted to repositioning. 4 patients – intended for CRT (1.8%) didn‘t meet the criteria for His Bundle pacing or left bundle pacing, being submitted to conventional coronary sinus lead placement. There were 10 cases (4.6%) of confirmed lead perforation during the lead septum insertion, with prompt repositioning, all uneventful. No pericardium effusion related to lead perforation was observed. One patient (0.4%) had a pneumothorax, requiring chest tube drainage. Conclusion: Conduction system pacing as a first strategy is a feasible, effective and safe technique, both for pacing and for resynchronization purposes, with complication rates comparable to conventional implantation. 112166 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY DANIELLA CUNHA BRANDAO1, Bruna T.S. Araújo1, Kamyla Maria Alcantara Silva Alves1, Armèle Dornelas de Andrade1, Daniella Cunha Brandão1 (1) Universidade Federal de Pernambuco Introduction: Studies relating respiratory muscle strength and diaphragmatic mobility in heart transplant patients are scarce, especially when correlated with submaximal exercise tests, such as the Glittre ADL-Test, which is still poorly investigated in this population. Purpose: To investigate whether the time required to perform the Glittre ADL-Test correlates with respiratory muscle strength and diaphragmatic mobility. Methods: This was a cross-sectional study with 38 adult individuals aged 21 to 65 years, diagnosed with HF of all etiologies and with reduced ejection fraction. The performance of the Glittre ADL-Test followed the standardization of the literature, using the Total Time to complete the five laps of the test. The assessment of respiratory muscle strength was made by a digital manovacuometer and diaphragmatic mobility was measured by ultrasound in the M. mode. Results: The mean Glittre ADL-Test time was 286.5 seconds and showed significant correlations with maximal inspiratory pressure (MIP) (r = –0.445 – p < 0.01) and maximal expiratory pressure (MEP) (r = –0.531 – p < 0.01) and with diaphragmatic mobility (r = –0.361 – p < 0.05). Conclusions: Our findings suggest an inverse relationship between respiratory muscle strength and diaphragmatic mobility with Glittre ADL-Test performance. If respiratory muscle strength and diaphragm muscle movement are lower, the time taken to complete the test will be longer, suggesting that increased respiratory work may potentiate peripheral muscle fatigue. The impairment of respiratory muscles seems to have important implications on the performance of HF individuals in their daily activities, confirming the need for early identification of patients with respiratory muscle weakness, enabling a targeted intervention in this population. Support: Edital PROPG-UFPE 230760186722020-32; CNPq 421656/2021-7 and FACEPE 0801-4.08/21. 112169 Modality: E-Poster Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CHARLES SLATER1, Luiz Eduardo Camanho1, Eduardo Benchimol Saad2, Luiz Antonio Oliveira Inacio Jr2, Lucas Carvalho Dias1, Gustavo Vignoli Santos2, Ricardo Mourilhe-Rocha1 (1) State University of Rio de Janeiro (UERJ), Rio de Janeiro, Brazil; (2) Hospital Pro Cardíaco, Rio de Janeiro, Brazil Background: Conduction system pacing through direct His bundle of left bundle stimulation is a new alternative to achieve adequate CRT in patients with heart failure with reduced ejection fraction (HFrEF). Purpose: To describe the short and medium term outcomes of CRT through direct conduction system pacing in a single center. Methods: 53 consecutive patients who underwent CRT through direct stimulation of the His bundle or left bundle branch were retrospectively evaluated. All presented in functional class III/IV (NYHA). The CRT response criteria were: improvement of functional class and reverse remodeling criteria (increase in ejection fraction (EF) >10% and/or decrease in left ventricular end-systolic diameter (LVESD) >15%). The patients were evaluated at 1 and 3 months and every six months after the procedure. Results: The mean age was 76,4 ± 14,8 years, 65% males, 42% Ischemic cardiomyopathy. Left bundle branch block (LBBB) was observed in 98% and mean QRS duration was 169 ms; average EF: 28%; mean LVESD and left ventricular end-diastolic diameter (LVEDD) was 53 and 68 mm, respectively. Direct His bundle pacing was the technique of choice in 5 patients, while left bundle branch pacing was used in 48 patients. There were five patients where His bundle pacing of left bundle branch pacing criteria was not met, being submitted to conventional CRT through coronary sinus branches. The mean follow up time was 21 months. Mean QRS duration was significantly shorter after the procedure (128 ms). Improvement of functional class was found in 85,7% of patients. Reverse remodeling criteria was found in 70,5% of patients. 4 patients progressed to end-stage HF and subsequent death. Conclusion: CRT through conduction system pacing is a valid alternative to conventional CRT, with promising results, allowing cardiac resynchronization in patients with HFrEF. 112201 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO FERRAZ DE OLIVEIRA GOMES1, Thiago Moreira Bastos da Silva2, Leticia de Sousa Peres1, Iliana Regina Ribeiro Menezes1, Nathalia Duarte Camisão1, Mariana Moreno Canário da Silva1, Renata Mexias Abdala Felix1, Giovanni Possamai Dutra1, Anna Butter1, Henrique Custódio Goudar1, João Luiz Fernandes Petriz1, Gláucia Maria Moraes de Oliveira2 (1) Hospital Barra D’Or; (2) Universidade Federal do Rio de Janeiro Introduction: Several studies have already scored the predictors of in-hospital mortality in patients hospitalized for COVID-19. 2 years after the beginning of the pandemic, few studies have evaluated predictors of long-term mortality. Objectives: To evaluate clinical and laboratory characteristics predictors of mortality using machine learning techniques. Methods: Retrospective cohort study with patients who were hospitalized with a confirmed diagnosis of COVID-19. Comorbidities, laboratory tests, vaccination status, and clinical characteristics were evaluated. The primary outcome of this study is all cause death occurring in the hospital or after hospital discharge. We used the classification tree model for the survival outcome, available in RStudio 2021.09.0. Results: 1454 patients were included, mean age of 59.8 ± 17.0, 62.6% men. There were 269 deaths (18.5%) during the study period (mean follow-up = 338 ± 209 days). 44.7% of patients had myocardial injury. The following predictive variables were selected by the method: use of mechanical ventilation, age, platelet count at discharge, CRP at discharge, myocardial injury, SAPS3 score, d-dimer peak of hospitalization, dementia, chronic renal failure, COVID-19 vaccination, and use of anticoagulation therapy. The model is available in the figure. Conclusion: In patients hospitalized for COVID-19, patients who required mechanical ventilation, with high CRP at discharge and higher age had the worst long-term prognosis. Other markers have shown promise in predicting more severe patients, such as platelet count at discharge and the occurrence of myocardial injury. 112202 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY CHARLES SLATER1, Luiz Eduardo Camanho1, Eduardo Benchimol Saad2, Luiz Antonio Oliveira Inacio Jr2, Lucas Carvalho Dias1, Gustavo Vignoli Santos2, Ricardo Mourilhe-Rocha1 (1) State University of Rio de Janeiro (UERJ), Rio de Janeiro, Brazil; (2) Hospital Pro Cardíaco, Rio de Janeiro, Brazil Background: Vascular access is the first step to perform cardiac device (CD) implantation. However, vascular access-related complications still remain a major cause of complications in CD implantation, increasing risk, costs, and hospitalization time. Ultrasound has become an important tool in helping vascular access, and its use has increased recently for CD implantation. Purpose: To describe the results after 4 years of US-guided vascular access for implantation of CD. Methods: Over a period of 50 months, 627 patients (average age 77,1 ± 14,6 years – interquartile range: 13 years) were submitted to CD implantation (271 dual-chamber pacemakers, 9 single-chamber pacemakers, 24 CRT-P’s, 167 conduction system pacemakers (His bundle pacing or left bundle pacing), 63 CRT-D’s, 46 dual-chamber ICD’s, 2 single-chamber ICD’s, 4 lead implantations and 41 upgrades for CRT), in a total of 1282 US-guided punctures (longitudinal “out of plane” axillary vein approach) using a “Point of Care” US machine with a 12MHz probe protected with a sterile cover. All punctures were performed on intact skin, followed by an incision. Chest radiography and pulmonary ultrasound were performed in the search for complications immediately after the procedure and the next morning, before discharge. Results: Vascular access was successful in all cases. In 11 cases (1,75%) there was limiting vascular obstruction, requiring contralateral vascular access to complete the procedure. US image also allowed alternative approaches, such as access to a supraclavicular vein (3 cases/0,47%) in patients with complete obstruction of the axillary vein; and cephalic vein puncture (4 cases/0,63%) when the axillary vein was too deep. Pneumothorax was found in one patient (0,16%) through the pulmonary US the next morning and submitted to chest drainage with no further complications. No haemothorax was found in this sample. Conclusion: US-guided vascular access technique allows safe implantation, with complication rates considerably lower than that related in literature. 112215 Modality: E-Poster Researcher – Non-case Report Category: PHYSIOTHERAPY DANIELLA CUNHA BRANDAO1, Jéssica Costa Leite2, Bruna T.S. Araújo1, Armèle Dornelas de Andrade1, Daniella Cunha Brandão1 (1) Universidade Federal de Pernambuco; (2) UNIFACISA Introduction: The Glittre ADL-Test appears as one more test option to assess submaximal exercise tolerance in heart transplant patients. Purpose: To verify the association between the time spent in the Glittre ADL-Test with the variables of the Cardiopulmonary Exercise Test (CPET). Methods: A cross-sectional study evaluating 53 male and female subjects with Heart Failure (HF), NYHA (New York Heart Association) II–III and left ventricular ejection fraction <45%. The individuals performed the CPET on a treadmill with ramp protocol to evaluate the maximum functional capacity and on another day the Glittre ADL-Test, considering the total time to complete the five rounds of the test. A minimum interval of 24 hours and a maximum of 7 days between the tests was respected. Results: Glittre ADL-Test time was negatively and moderately associated with peak oxygen uptake (VO2peak) (r = –0.48; p = <0.001) and oxygen uptake at the first ventilatory threshold (r = –0.475; p = <0.001), besides a low negative association with the time to reach VO2peak (r = –0.345; p = 0.014). Conclusions: We can suggest from the results obtained that the Glittre ADL-Test is an accessible method for assessing individuals with HF and correlates with the oxygen uptake measured by CPET. As clinical implications we have: The Glittre ADL-Test can be a fast and economical alternative for estimating the functional capacity of individuals with HF in the absence of a maximal exercise test. Support: PROPG-UFPE n.23076018672/2020-32; CNPq 421756/2021-7; FACEPE 0801-4.08/21 112236 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MONICA AMORIM DE OLIVEIRA1, Helena Cramer Veiga Rey1 (1) Instituto Nacional de Cardiologia HTN is the most common, identifiable and modifiable risk factor for cardiovascular disease. The prevalence of SAH is about three times higher in obese patients. Weight loss has an impact on the treatment of HTN and this should be encouraged. The aim of the study was to evaluate the prevalence of obesity in a group of 1000 patients over 18 years of age treated in primary care in the city of Rio de Janeiro and its relationship with HTN. Of the 1014 patients included, 737 (72.68%) were hypertensive. A sedentary lifestyle was present in 65.5% of the population studied and there was no difference between the group of patients with and without SAH. The average BMI of the general population was 29.6 ± 6.2 kg/m2. Being 30.1 ± 6 Kg/m2 and 27.8 ± 6.5 Kg/m2 for hypertensive and non-hypertensive patients, respectively, with statistical significance. Obesity was found in 41.2% of the population studied. The distribution according to the WHO BMI classification can be verified in the table and showing the association between HTN and obesity. The treatment of hypertension in the obese requires a targeted approach to obesity. Adopting a healthy lifestyle facilitates weight loss, increases responsiveness to antihypertensive drug therapy, and produces independent beneficial effects on factors. did not present SAH, as a measure to prevent its development. 112240 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY CRISTIANO FARIA PISANI1, Rodrigo M. Kulchetscki1, Bernardo Salvajoli2, Marina P. Mayrink1, Ligia Arteaga2, Jason Cook3, William Stevenson3, João Salvajoli2, Mauricio Scanavacca1 (1) InCor – Heart Institute – University of São Paulo Medical School; (2) ICESP – Instituto do Cancer do Estado de São Paulo; (3) Vanderbilt Heart Background: Stereotactic radiotherapy (SBRT) is a new therapeutic option in cases of recurrent ventricular tachycardia (VT) in patients with structural heart disease where there is a contraindication or a therapeutic failure of the catheter ablation (CA). Objectives: To describe the initial experience of SBRT for recurrent VT in patients with Chagas Cardiomyopathy (CCM) in whom catheter ablation is not an option. Methods: The target sites and SBRT doses were planned based on imaging exams and on bipolar voltage maps from previous CA procedures and new electrophysiologic study was also performed aiming to evaluate the exit site of induced VT. Results: Five patients with CCM and recurrent VT underwent SBRT from July 2021 to April 2022. Most patients were male (60%), mean age 61,4 ± 5, years and EF 22% (Q1: 20 Q3:45). One patient (20%) had two prior catheter ablation and the others only one. The median number of VT episodes in the six and two months prior to SBRT was 13 (Q15.5–Q348.5) and 11 (Q1:2.5;Q3:26.5), respectively. The mean PTV (planning target volume) was 82 ± 17 mL and the ITV (internal target volume) was 29 ± 5 ml, with safe constraints regarding the esophagus and stomach. In a median FU of 194 days, 2 (40%) patients presented VT recurrence. No patients died. The median number of VT episodes reduced from 24 (4.75;52.25) to 2 (0;8.5) (P = 0.068). Conclusion: SBRT is safe and seems to be effective in Chagas Disease patients 112483 Modality: E-Poster Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ANDREY GARANIN1, Andrey Garanin1, Dmitriy Duplyakov1, Irina Mullova1, Oksana Shkaeva1, Polina Duplyakova1 (1) Samara State Medical University Introduction: The pandemic of a new coronavirus infection has shown the need for the development of telemedicine technologies, especially remote medical monitoring using telemonitoring of vital body functions. In some regions, this approach is also justified by the factor of distance and the shortage of certain categories of medical workers. Objective: Decrease of repeated hospitalizations and mortality in patients with chronic heart failure by means of remote monitoring system including arterial pressure monitoring. Methods: 401 patients, who were randomized into two equal groups comparable by sex, age, left ventricular ejection fraction, functional class of heart failure, six-minute walking test results, body mass index, baseline systolic and diastolic blood pressure, and heart rate of the underlying disease and hospitalized for myocardial infarction or decompensation of chronic heart failure, were examined at two research centers. In group 1, remote blood pressure monitoring using oscillometric method was performed using certified tonometers with the ability to transmit the measurement results via cellular communication channel to the research center. Based on the results of the data obtained, the physician was able to contact the patient and adjust the previously prescribed treatment. Group 2 patients performed self-monitoring of blood pressure. All the patients were observed for 3 months. Treatment in both groups was prescribed according to the latest recommendations of the European Society of Cardiology. Results: Group 1 showed a tendency to blood pressure lowering from 130 mmHg to 125 mmHg. In contrast, Group 2 showed an increase in blood pressure from 125 mmHg to 130 mmHg. Group 1 showed 4 hospitalizations related to acute cardiac pathology with a total duration of 30 days; group 2 showed 13 hospitalizations with a total duration of 133 days (p = 0.027; OR; 95% CI 3.4; 1.1–10.8). Total mortality was 6 in group 1 and 11 in group 2 (p = 0.226; OR; 95% CI 1.9; 0.7–5.3). Cardiovascular mortality was 3 in group 1 and 10 in group 2 (p = 0.052; OR; 95% CI 3.5; 0.9–12.9). Conclusions: Remote management of patients with chronic heart failure, including blood pressure monitoring, over 3 months demonstrates a significant reduction in hospitalizations and a trend toward lower total and cardiovascular mortality. 112516 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY OLGA PECHANOVA1, Andrej Barta1, Martina Cebova1 (1) Centre of Experimental Medicine, Slovak Academy of Sciences Background: Aliskiren, a renin inhibitor, has been shown to exert cardio-protective, reno-protective, and anti-atherosclerotic effects independent of its blood pressure lowering activity. However, relatively high dose and frequency of the treatment, which is needed for beneficial effects of the drug, may incur several side effects such as high blood potassium levels, particularly when used with ACE inhibitors in diabetic patients. We hypothesized that gradually released aliskiren from polymer-based nanoparticles could at least partially solve the problem of bioavailability. Therefore, we aimed to determine the effects of aliskiren-loaded polymeric nanoparticles on blood pressure, nitric oxide synthase (NOS) activity and structural alterations of the heart and aorta developed due to spontaneous hypertension in rats. Methods: Twelve-week-old male spontaneously hypertensive rats were divided into the untreated group, group treated with powdered aliskiren or aliskiren-loaded nanoparticles (25 mg/kg/day) and group treated with nanoparticles only for 3 weeks by gavage. Blood pressure was measured by tail-cuff plethysmography. NOS activity, eNOS and nNOS protein expressions, and collagen content were determined in both the heart and aorta. Vasoactivity of the mesenteric artery and wall thickness, inner diameter, and cross-sectional area (CSA) of the aorta were analyzed. Results: After 3 weeks, blood pressure was lower in both powdered aliskiren and aliskiren-loaded nanoparticle groups with a more pronounced effect in the latter case. Only aliskiren-loaded nanoparticles increased the expression of nNOS along with increased NOS activity in the heart (by 30%). Moreover, aliskiren-loaded nanoparticles decreased vasoconstriction of the mesenteric artery and collagen content (by 11%), and CSA (by 25%) in the aorta compared to the powdered aliskiren group. In conclusion, aliskiren-loaded nanoparticles represent a promising drug with antihypertensive and cardioprotective effects. 112557 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY NGO YOUMBA EP NTEP GWETH1, Marie Ndjie Ndzana1, Liliane Mfeukeu Kuate1, Chris Nadège Nganou Gnindjio1, Valerie Ndobo1, Sylvestre Efonle Ngoh1, Samuel Kingue2 (1) HOPITAL CENTRAL DE YAOUNDE (HCY); (2) HOPITAL GENERAL DE YAOUNDE Randomized control trial comparing efficiency between losartan and amlodipine to hypertensive Cameroonian in Yaounde SUMMARY. Introduction: High blood pressure is the first chronic disease in the world and which also increases the cardiovascular risk. The reduction of this particularly high risk in patients from sub-Saharan Africa should be the main goal of care in our setting. Numerous studies on the choice of a better antihypertensive treatment of arterial hypertension have been conducted but the inherent specificities of our populations, in particular a lower renin activity, a tendency towards sodium-water retention and vascular hyperreactivity, impose a customization of the pharmacological treatment in Africans. Although there is a difference in response to antihypertensive classes in African subjects who respond better to amlodipine than to losartan, few study in our context has yet demonstrated this to the best our knowledge. Objective: The main objective of this study was to compare the efficacy of a amlodipine blocker and losartan receptor blocker in Cameroonian hypertensives at the Yaounde Central Hospital. Methods: This was a randomized, controlled clinical trial phase IV. It involved patients with grade 1 or 2 clinical hypertension, naive to antihypertensive therapy. The intervention consisted in starting the recommended average doses in monotherapy for 12 weeks (84 days) of losartan 50 mg in single dose per day in group 1 and amlodipine 5 mg in single dose per day. Group 2. The follow-up were scheduled 2 weeks. The increase in monotherapy doses occurred from the fourth week (S4) when the blood pressure goal was not reached with losartan 100 mg once a day and amlodipine 10 mg once daily for group 1 and 2, respectively. The patients for whom BP was still not controled despite the increase in monotherapy doses adding a low dose of hydrochlorothiazide at 12.5 mg at eight week (S8) to achieve the blood pressure goal. Compliance assessment was done through Morisky’s questionnaire and telephone calls. Results: In all 46 patients initially included, 40 were selected including 21 women and 19 men. The mean age was 54.0 ± 5.5 years in the losartan group 1 and 58.05 ± 7.1 years in the amlodipine group2 (P = 0,350). The two groups were substantially comparable at inclusion for the clinical measurement of blood pressure, the biological parameters tested namely lipid profile, blood ionogram, blood glucose and serum uricemia. After intervention, the mean SBP. 112572 Modality: E-Poster Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY PEDRO GABRIEL MELO DE BARROS E SILVA1, Renato Delascio Lopes1, CR. Hoffmann Filho4, MA. Cavalvante5, C. Miranda6, RB. Esper7, G. Lima8, L. Zimerman9, O. De Souza11, A. Fagundes12, E. Saad10, R. Teixeira12 (1) Brazilian Clinical Research Institute; (2) IP-HCor; (3) Duke Clinical Research Institute, Durham, United States of America; (4) Hospital Regional Hans Dieter Schmidt, Joinville, Brazil; (5) University of the West of Sao Paulo, Presidente Prudente, Brazil; (6) Hospital Madre Teresa, Belo Horizonte, Brazil; (7) Prevent Senior, Sao Paulo, Brazil; (8) Institute of Cardiology of Rio Grande do Sul, Porto Alegre, Brazil; (9) Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; (10) Cardiologia Americas; (11) D’Or Institute for Research and Education, Rio De Janeiro, Brazil; (12) Sociedade Brasileira de Arritmias Cardiacas, Sao Paulo, Brazil There is limited prospective real-world evidence of patients with atrial fibrillation (AF) in Latin America. Methods: RECALL was the first nationwide prospective study of patients in Brazil with known atrial fibrillation. A total of 4,585 patients were included among 89 sites from April 2012 to August 2019. The follow-up was one year by the protocol. Patient characteristics, medications under use and clinical outcomes during the follow-up were collected. Results: From the total of patients enrolled, 41 were excluded from the analysis since they did not have a confirmed diagnosis of AF. The median age was 70 (61–78) years, 46% were women and the majority of the cases were permanent AF (53,8%). The mean CHA2DS2VASc was 3.2 ± 1.6 and the median HASBLED was 2 [2–3]. The most common risk factor was arterial hypertension (77.9%). The median heart rate was 74 [65–85] and the mean ejection fraction was 52.2 ± 2.6 (%). Only 4.4% of patients had history of previous ablation and 30.4% were using anti-arrhythmic. At baseline, 22.0% did not use anticoagulants and 9.1% did not use any antithrombotic therapy. The most common anticoagulant was vitamin K antagonist (VKA) (62.6%) while the remaining 37.4% were direct oral anticoagulants. The main reasons for not using an oral anticoagulant were physician judgment’s (low risk of stroke – 24.6%) and difficult to control (14.7%) or perform INR (9.9%) while patient preference and adverse event represented respectively 5.3% and 4.1%, respectively. Only 42.5% of the INRs at baseline were between 2 and 3. During follow-up, the use of anticoagulants and INR in the therapeutic range increased to 87.1% and 59.1%, respectively. The use of antithrombotic therapy changed not only during follow-up but it was different also according to the CHADSVASC of the patients. Regarding clinical outcomes in one year, 17.8% of patients were hospitalized due to AF and the rates of death, stroke, systemic embolism and major bleeding were 5.76 [5.12–6.47], 2.77 [2.32–3.32] 1.01 [0.75–1.36], 2.21 [1.81–2.70], respectively. Among VKA users, the rate of mortality and bleeding was higher in the group with time in therapeutic range below 60%. Conclusion: RECALL represents the largest prospective registry of patients with atrial fibrillation in Latin America. Our findings highlight important gaps in the treatment of patients which can inform clinical practice and help to guide future interventions to improve the care of these patients. 112591 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY OMAR ASRUBAL VILCA MEJIA1, Bianca Meneghini1, Ligia Cristina Fonseca Hoeflinger1, Fabiane Leticia de Freitas1, Bruno Mahler Mioto1, Luis Augusto Ferreira Lisboa1, Luis Alberto de Oliveira Dallan1, Alexandre Chiapina Hueb2, Mauricio Landulfo Jorge Guerrieri2, Rodrigo Coelho Segalote3, Felipe Cosentino3, Fabio Biscegli Jatene1 (1) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; (2) Hospital das Clinicas Samuel Libanio; (3) Instituto Nacional de Cardiologia do Rio de Janeiro Background: The world is getting older. This change in demographics increases the overall prevalence of coronary artery disease. The growing number of elderlies is not only related to an increase in the number of coronary heart diseases but also frailty, a syndrome that affects at least 10% of the elderly population and is an exceedingly effective parameter for correlating with the adverse effects of aging. Regarding CABG surgery, randomized controlled clinical trials have mainly focused on low-risk, elevated-risk, or high-risk patients, but not on frail patients. Purpose: The aim of this study was to evaluate the impact of coronary artery bypass graft surgery on the frailty index of patients in the 6-month follow-up. Methods: This was a subanalysis of the national, multicenter, randomized, controlled trial called the FRAGILE clinical trial. We analyzed 62 patients aged 60 years or older undergoing coronary artery bypass graft surgery with and without cardiopulmonary bypass in 8 main cardiac surgery centers in Brazil. Fried’s Frailty Criteria were used to classify patients into frail, pre-frail, and non-frail. The parameters evaluated were unintentional weight loss, self-reported fatigue, physical activity level, grip strength, and gait speed. We divided the patients into two groups: the off-pump CABG group (n = 30) and the on-pump CABG group (n = 32). We compared each group for frailty variables before and after surgery using the Mann-Whitney test and Stuart-Maxwell marginal homogeneity using the R software. The study was approved by the local Ethics Committee and all patients signed the informed consent. Results: We observed an improvement in the frailty of patients undergoing CABG. Overall, the number of pre-frail patients doubled in 6 months after surgery (from 11 to 22 patients), and the number of frail patients decreased from 19 to1 patient. Nine patients included in the study no longer had any degree of frailty after 6 months of surgery. No patient was classified as non-frail preoperatively in both groups because it was one of the exclusion criteria of the study. There is no difference between the groups. Regarding the frailty criteria, all tests showed differences in the pre and post-surgical comparison, except the unintentional weight loss variable. Conclusion: CABG played a role in improving frailty at the 6-months follow-up. Both techniques, on-pump and off-pump CABG, showed similar results when comparing frailty features before and after CABG surge. 113890 Modality: E-Poster Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RENATO DELASCIO LOPES1, Pedro Gabriel Melo de Barros e Silva2, Ariane Vieira Scarlatelli Macedo2, Alexandre B. Cavalcanti4, Regis G. Rosa6, Otávio Berwanger7, Viviane C. Veiga8, Luciano C.P. Azevedo9, Murillo O. Antunes10, Otávio Celso Eluf Gebara11, Eduardo Ramacciotti1, Alvaro Avezum12 (1) Brazilian Clinical Research Institute, Sao Paulo, Brazil; (2) Duke University Medical Center, Durham, NC, USA; (3) Hospital Samaritano Paulista, São Paulo, Brazil; (4) HCOR Research Institute, São Paulo, Brazil; (5) Brazilian Research in Intensive Care Network; (6) Hospital Moinhos de Vento, Porto Alegre; (7) Academic Resaearch Organization (ARO) – Hospital Israelita Albert Einstein; (8) BP–A Beneficência Portuguesa de São Paulo; (9) Hospital Sírio Libanês Research and Education Institute; (10) Hospital Universitário São Francisco de Assis na Providência de Deus and Irmandade do Senhor Bom Jesus dos Passos da Santa Casa de Misericórida de Bragança Paulista, Bragança Paulista; (11) Dasa-Hospital Santa Paula, São Paulo, Brazil; (12) International Research Center, Hospital Alemão Oswaldo Cruz The efficacy and safety of anticoagulation in the early phase of COVID-19 is unknown. Methods: APOLLO is an academic-led, multicenter, randomized, double-blinded clinical trial that planned to enroll 1000 patients from 30 sites participating in the Coalition COVID-19 Brazil initiative. Eligible patients with a confirmed diagnosis of COVID-19 with onset symptoms up to 10 days at increased risk for thrombotic complications were randomized to apixaban (2.5 mg twice daily) or placebo for 30 days. The primary outcome was the number of days alive and out of the hospital at 30 days. Secondary outcomes included hospitalization for bleeding (key safety outcome), hospitalization for cardiovascular/pulmonary causes (key secondary efficacy outcome). Results: The trial was stopped prematurely due to slower than expected recruitment and low overall event rates. Among 411 (41% of the initial sample size) patients randomized (207 in apixaban and 204 in the placebo group), the mean (+/– SD) age was 44.0 years (±14.0), 58.4% were women, three patients from the apixaban group withdrew the informed consent, and all the remaining 408 patients completed the 30-day follow-up. The mean time (+/– SD) from symptom onset to randomization was 6.2 (2.4) days. There was no significant difference in the number of days alive and out of the hospital in patients assigned to apixaban and placebo (29.6 ± 1.6 and 29.5 ± 2.4 days, respectively; mean difference 0.18, 95% confidence interval [CI] –0.22 to 0.58; P = 0.38). Among patients assigned to apixaban versus placebo, there was also no significant difference in secondary endpoints including all-cause hospitalization (4.9% vs. 6.9%; odds ratio [OR] 0.70, 95% CI 0.30–1.61), and hospitalization for cardiovascular/pulmonary causes (4.4% vs. 6.9%; odds ratio [OR] 0.63, 95% CI 0.26–1.48). There was no hospitalization due to bleeding and no arterial and venous thrombosis in either study groups. One patient died (in the placebo group) due to a non-cardiovascular cause. Conclusions: In symptomatic, but clinically stable outpatients with COVID-19 and additional risk factors for thrombotic complication, treatment with apixaban 2.5 mg twice daily compared with placebo did not improve the number of days alive and out of the hospital. No major bleeding was observed in the trial population. While the power is limited due to not reaching the intended sample size, our results do not support the routine use of low dose apixaban for outpatients COVID-19. 113909 Modality: E-Poster Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY GWO-PING JONG1 (1) Chung Shan Medical University Hospital; (2) Chung Shan Medical University; (3) Taoyuan Armed Forces General Hospital; (4) National Defense Medical Center; (5) Central Taiwan University of Science and Technology; (6) Taichung Armed Forces General Hospital Background: Epidemiological evidence suggests the association of diabetes with an increased risk of stroke. Clinical studies have investigated the effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors on new-onset stroke (NOS), but the results are inconsistent. Objectives: To determine the association between the use of SGLT2 inhibitors and NOS in patients with type 2 diabetes mellitus (DM). Methods: We conducted a nationwide retrospective cohort study based on the Taiwan Health Insurance Review and Assessment Service database (2016–2019). The primary outcome of the assessment was the risk of incident stroke by estimating hazard ratios (HRs) and 95% confidence intervals (CIs). Multiple Cox regression was applied to estimate the adjusted HR of NOS. Subgroup analysis was also conducted. Results: Among the 232,101 eligible patients with type 2 DM aged ≥20 years, SGLT2-inhibitor users were compared with non-SGLT2-inhibitor users based on age, sex, and the duration of type 2 DM matching at a ratio of 1:2. The event rate per 10 000 person-months was 9.20 (95% CI 8.95 to 9.45) for SGLT2-inhibitor users and 10.5(10.3–10.6) for non-SGLT2-inhibitor users. There was a decreased risk of NOS for SGLT2-inhibitor users (adjusted HR 0.85, 95% CI 0.82–0.88) compared with non-SGLT2-inhibitor users. Results for the propensity score-matched analyses showed similar results (adjusted HR 0.87, 95% CI 0.84–0.91 for both SGLT2-inhibitor users and non-SGLT2-inhibitor users). Conclusion: The risk of developing NOS was lower in patients with SGLT2-inhibitor users than in non-SGLT2-inhibitor users. The decreased risk of NOS in patients with type 2 DM was greater among patients with concurrent use of statins, biguanides, thiazolidinediones, and glucagon-like peptide-1 receptor agonists. We, therefore, suggest that the long-term use of SGLT2 inhibitors may help reduce the incidence of NOS in patients with type 2 DM. 107880 Modality: E-Poster Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANTONIO DE PADUA MANSUR1, Carlos Henrique Del Carlo1, José Antonio Ramos Neto1, André Barbosa de Abreu1, Airton Roberto Scipioni1, Antonio Carlos Pereira Barreto1 (1) Insituto do Coração – HC FMUSP Background: Chronic Chagas cardiomyopathy (CCC) is one of the leading causes of congestive heart failure (CHF) in Latin America and carries a high morbidity and mortality burden. Previously, it was believed that there was no epidemiological and clinical evidence of a gender-associated risk of death in patients with CCC. Purpose: To analyze the mortality of congestive heart failure due to CCC in women and men. Methods: From February 2017 to September 2020, we followed a cohort of patients with CHF (Framingham criteria) due to CCC in a single-center outpatient clinic. Appropriate serologic tests defined Chagas disease. Baseline data included clinical characteristics and echocardiographic findings. Statistical analyses were performed with the Kaplan-Meier (K-M) method to analyze time-to-event data and the Cox proportional hazards methods to search for predictors of death. Results: We studied 733 patients, mean of 61.4 ± 12.3 years, 381 (52%) males. Females were older (63.0 ± 11.9 vs. 60 ± 12.4 years; p = 0.01), had a higher baseline mean left ventricular ejection fraction (LVEF) (44.5 ± 14.6% vs. 37.3 ± 14.8%; p < 0.001), and a lower left ventricular diastolic diameter (LVDD) (56.7 ± 8.9 vs. 62.4 ± 9.4 mm; p < 0.001). Over a 3-years follow-up period, 168 (44%) men and 126 (36%) women died (K-M: log-rank p = 0.002; Figure). Women had more implantable pacemakers (PM) (26.1% vs. 16.5%; p = 0.002) and men more implantable cardioverter-defibrillators (ICDs)(20.7% vs. 12.5%; p = 0.003). Heart transplant occurred in 10.8% of men and 7.4% in women (p = NS). Cox regression for death adjusted for age, previous myocardial infarction, diabetes, previous stroke, chronic kidney disease (CKD), atrial fibrillation, PM, ICD, heart transplant and LVEF, showed, in descending order, previous stroke (HR = 2.4; 95%CL:1.5–3.6), diabetes (HR = 2.0; 95%CL: 1.3–3.1), and CKD (HR = 1.8; 95%CL:1.3–2.6) as the main predictors of death in men, and in women diabetes (HR = 2.2; 95%CL:1.4–3.4), previous stroke (HR = 1.8; 95%CL:1.1–2.9), and CKD (HR = 1.7; 95%CL:1.1–2.7). Conclusions: Women had a better prognosis than men but similar predictors of death. Control of diabetes and prevention of stroke and CKD could significantly reduce the death rate in CHF due to CCC. 107731 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FREDERICO LOPES DE OLIVEIRA1, Giulliano Gardenghi2, Adriano Gonçalves de Araújo1, Maurício Lopes Prudente2, Flávio Passos Barbosa1 (1) Hospital de Urgências Governador Otávio Lage; (2) Hospital Encore; (3) Hospital Samaritano de Goiânia Introduction: Coronary perforation is a potentially fatal complication during percutaneous coronary intervention. Although rare (0.2 to 0.6% of procedures), it is life-threatening, which justifies its immediate diagnosis and correction. Objective: We report two cases of coronary perforation and alternative management of current practice. Case reports: CTO 78 years old, admitted on 04/16/2020 and WlSO, 61 years old on 01/12/22, men with previous AMI of more than 12 hours of evolution (CTO with 36 hours and WISO with 18 hours), still in typica pain refractory to intravenous nitrate, dual product control and potent analgesia, thus opting for invasive stratification in both cases. After anterior descending recanalization, a type III rupture with the presence of pericardial contrast was observed. Heparin action was immediately reversed, the balloon was inflated with low pressure and the echocardiography and cardiac surgery teams were contacted. Manufactured coated stents were made (CTO with balloon catheter segment and WISO with a sterile adhesive skin dressing) successfully correcting its perforations. Discussion: In coronary perforation, reversal of anticoagulation and occlusion of the bleeding orifice are essential to prevent tamponade until definitive treatment (lately only direct suture of the vessel by open thoracotomy). However, with the evolution of materials and techniques, it has been proven that ballooning at low pressures and protamine infusion have high success rates in punctual occlusions. Circulatory assist devices and decompressive pericardiocentesis (Marfan puncture) are still needed, increasing costs, morbidity and mortality. In case of failure, polymerized mesh stents (Graft stent) are able to “seal” the leak to pericardial sack, however, their value is high and their numbering is restricted. Thus, alternatives manufactured stents with venous grafts, parts of coronary balloon catheters, sterile skin patches (such as Tegaderm®) or even intentional infusion/embolization of autologous fat are good examples of low cost. Other techniques that are also costly, such as the use of microcatheters for infusion of “coils” and the implantation of successive smaller-caliber stents (decreasing the lateral holes), are also used. Conclusion: In the cases reported here, the procedures presented were able to contain the bleeding. We understand that disseminating this knowledge, we can contribute with other colleagues in solving this complication. 107875 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY NATHALIE JEANNE MAGIOLI BRAVO-VALENZUELA1, Eliane Lucas4, Juliane R Sousa2, Fernanda C Lemos3, Patrícia S Correia2 (1) Curso de Pós-Graduação em Cardio- Oncologia da Sociedade Brasileira de Cardiologia, Instituto Nacional de Cardiologia e Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brasil; (2) Hospital Federal de Bonsucesso (HFB), Rio de Janeiro-RJ, Brazil; (3) Federal University of Rio de Janeiro, Rio de Janeiro-RJ, Brazil; (4) Faculdade de Medicina do Centro Educacional Serra dos Órgãos (UNIFESO), Teresópolis-RJ, Brazil Introduction: Cardiac myxomas are the most common primary cardiac tumors in adults but rare in children. However, in Carney syndrome, myxomas develop at an earlier age and tend to recur more frequently. SC is considered a familial multiple endocrine neoplasia (such as primary adrenal nodular disease, cardiac and cutaneous myxoma, testicular neoplasia) that results from PRKAR1A gene mutations. Case report: We present the case of a 14-year-old male patient who was admitted to our hospital for exercise-induced fatigue, acute chest pain, and mental confusion. His mother reported a history of high blood pressure since the age of 5 that worsened in the last 2 months. His maternal family history was positive for atrial myxoma. A physical examination revealed skin hyperpigmentation, obesity, and hirsutism. Chest radiographic and electrocardiographic findings were normal. Fundoscopy revealed grade II hypertensive retinopathy. Elevated serum ACTH levels were consistent with primary hypercortisolism. The transthoracic echocardiography showed the image of a single, hyperechoic, and irregular mass located in the left atrium (Figure 1). The mass was voluminous, pedunculated, and adhered to the anterior leaflet of the mitral valve, causing obstruction of the left ventricular inlet blood flow. Surgical resection of the tumor was performed, and the histopathological features confirmed the diagnosis of myxoma. Screening for Carney complex was performed. Testicular ultrasonography and abdominal tomography revealed testicular microcalcifications and adrenal tumors, respectively. The patient underwent an uneventful adrenalectomy and was discharged from the hospital on corticosteroid therapy. PRKAR1A gene sequencing showed heterozygosity for the nonsense variant R228X in the patient and his mother. Conclusion: Atrial myxoma justifies surgical resection due to the risk of cardiovascular embolization, complications, and sudden death. Pediatric cardiac myxomas should raise the suspicion of a Carney complex. The early diagnosis of this syndrome contributes to the optimization of the treatment of these patients and can have a positive impact on their management and prognosis. 107905 Modality: E-Poster Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO TANAJURA GOMES NETO1, Bruna Aparecida Souza Machado1 (1) Clínica Médica Santa Helena – CMSH Introduction: The clinical presentation of myocarditis is variable and often mimics myocardial infarction. The diagnosis of acute myocarditis is frequently empirical and is made based on clinical presentation, electrocardiographic alterations, elevated cardiac enzymes, and theabsence of epicardial coronary artery disease. We report the case of a 46-year-old patient with a history of IgA nephropathy (NIgA) admitted for atypical acute precordial pain associated with ST-segment elevation in the inferiorwall lead. Description: A 46-year-old man with a history of NIgA, SAH, PLD, obesity (BMI 34) and sedentary, using anlodipino/losartan (2.5 mg/50 mg daily), succinato de metoprolol (50 mg daily) and simvastatin (20 mg daily). He was a non smoking and had no family history of coronary heart disease. The patient complained of chest pain into his right hemithorax over the 2 days, stabbing, intermittent, of moderate intensity that traveled into his sternum and right shoulder. The patient looked anxious and high blood pressure (200 × 100 mmHg). His vital signs were stable. Performed ECG and measurement of myocardial necrosis markers (MNM). He was classified as MNM and after improvement he was discharged in a reasonably good condition. ECG: showed ST-segment elevation in the inferior wall. Lab: Hb/Ht: 16/54 U: 35 Cr: 1.39 CKMB: 12 U/L (VR <5 U/L) Troponin: 13 ng/ml (VR: Between 0 and 0.5 ng/ml) Proteinuria 4,488.96 mg/24h Urine volume 1,120 ml Negative thrombophilia investigations. Echo TT: Preserved global and segmental LV systolic function, LV concentric remodeling, and type I diastolic dysfunction. Cate: Myocardial bridge with mild systolic constriction. Cardiovascular magnetic resonance (CMR): Presence of late mesocardial enhancement (non-ischemic pattern) in the inferior basal and inferolateral segments of the left ventricle. ID: Myocarditis. Conclusion: When we came across such a case, some questions were raised, suchas the attempt to correlate myocarditis with the nephrotic picture. The reported case and publications raised bring to light the discussionon the importance of CMR in the diagnosis of acute myocarditis. 108022 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM VICTOR RODRIGUES RIBEIRO FERREIRA1, Amanda Pupim Assunção Toledo1, Maria Fernanda Aguilar de Azevedo2, Maria Christiane Valéria Braga Braile Sternieri1 (1) Braile Cardio; (2) Beneficência Portuguesa de São José do Rio Preto/SP Introduction: The global race for a safe and effective vaccine against SARS-CoV-2 (COVID-19) has challenged scientists around the world. The mRNA-based vaccine was quickly linked to cases of myocarditis. Thus, doubts arose about the evolution of the cases and the therapeutic approach. A descriptive study recently published highlighted 1,626 reports myocarditis among the 192,405,448 individuals vaccinated with mRNA based vaccine and registered in the Vaccine Adverse Event Reporting System. Case Description: SKP, 35 years old, healthy, immunized with a booster dose of the BNT162b2 SARS-CoV2 mRNA vaccine and, after 5 days of administration, developed dyspnea on exertion. Laboratory tests rule out acute COVID-19 infection and elevated NTproBNP (2161 pg/ml) indicated acute heart failure. Echocardiography revealed left ventricular (LV) dilatation, reduction in LE ejection fraction (LVEF = 32%), moderate mitral regurgitation and pulmonary hypertension. The Speckle Tracking study revealed reduced strain rate (–9.6%). Cardiac magnetic resonance imaging confirmed myocarditis, revealing mesocardial late enhancement in several segments, with reduced systolic function and mild pericardial effusion. The standardized treatment was optimized doses of Bisoprolol (10 mg/day), Sacubitril/Valsartan (400 mg/day) and Spironolactone (25 mg/day). There was complete remission of symptoms after 5 weeks of treatment. After 14 weeks, a new echocardiographic study revealed normalization of all previously detected alterations. Conclusions: Writing the history of a disease as it unfolds before our eyes is a challenge for contemporary medicine. Findings are built on everyday experiences and case reports have proved to be valuable in the last two years. Vaccine-induced myocarditis still lacks studies regarding its epidemiology and pathophysiology. But we must stick to reality: vaccination remains the most effective means of combating SARS-CoV2. The data are categorical in affirming the benefits of vaccination for public health and the damage related to this pharmaceutical agent is much lower than the damage caused by viral infection in non-immunized peop. 108531 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY ELIAS ANTONIO YUNES2, Andre Luiz Marins Vera Cruz Porto1, Erika Ferreira de Moura Porto1, Carlos Eduardo Cordeiro Soares1, Wolney de Andrade Martins2 (1) Dr. Beda General Hospital, IMNE group, Campos dos Goytacazes, Rio de Janeiro State, Brazil; (2) Postgraduate Course in Cardiology SBC/INC/INCA, Rio de Janeiro, Rio de Janeiro State, Brazil Introduction: Among neoplastic tumors, secondary implants are 132 times more common than primary cardiac tumors (PCT), which have an incidence of 1.38 cases/100,000. More than 90% of TCPs are benign and only 5% to 6% are malignant. Case Description: Female patient, 24 years old, previously healthy, started progressive dyspnea on exertion 30 days ago. She presented dyspnea with mild exertion, paroxysmal nocturnal dyspnea and lower extremity edema, in addition to intermittent fever spikes. Echocardiogram showed left ventricle with normal dimensions and function, ejection fraction = 69%, large tumor mass occupying almost the entire right atrium (RA). Following the investigation, MRI was requested, which showed an extensive mass with invasion of the RA roof, fixed, heterogeneous, irregular, filling almost the entire cavity, extending to the hepatic and superior vena cava, measuring 106 × 100 × 76 mm. A CT-guided biopsy was performed and the histopathological report indicated partially necrotic tissue fragments with proliferation of pleomorphic epithelioid cells forming slits and vascular channels, which associated with positive immunohistochemistry for CD31 and CD34, led to the diagnosis of angiosarcoma. The tumor was considered unresectable and the proposed conduct was chemotherapy with paclitaxel alone for 21/21 days. Staging with chest, abdomen and pelvis CT also showed bone metastasis in the thoracolumbar spine and hip, in addition to mediastinal invasion. After 2 months, she presented a spontaneous femur fracture. She underwent 7 cycles of chemotherapy and died 10 months after the onset of symptoms due to pulmonary sepsis. Conclusion: This report presents a rare case of malignant PCT in a patient with unusual sex and age group, initial clinical presentation with heart failure, extremely aggressive and with a poor prognosis. The increase in PCT diagnoses is attributed to the advances in imaging technology and the use of multimodality. In malignant PCT, sarcomas represent 64.8%, of which 2/3 are angiosarcomas. It most often affects male patients in the fourth decade of life, and in 75% the RA. They can have important hemodynamic and arrhythmic consequences, depending on their size and location. Despite advances in diagnostic methods and cancer treatment, in case of these tumors there are serious limitations. The median survival at 12 months is 45%, but in cases without resection, it drops to 3.8 months. 108362 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FERNANDA CRISTINA CASTANHO1, Claudia Catelan Carlomagno2, Glaucia de Farias Correia2 (1) Hospital Regional de Sorocaba “Dr. Adib Domingos Jatene”; (2) Curso Intensivo de Cardiologia Clínica Introduction: Kounis syndrome is characterized by the coexistence of cardiac signs and symptoms with allergic clinical manifestations. The pathophysiological mechanism involved is believed to include mast cell activation with release of inflammatory mediators that induce coronary vasospasm and/or rupture of the atheromatous plaque. Among the molecules involved, several cytokines and chemokines, histamine, arachidonic acid products, platelet activating factor, neutral proteases, tryptase and cathepsin-D can be identified. Cardiac clinical manifestations differ depending on the syndrome subtype: type 1 results from coronary spasm in normal coronary arteries; type 2, by spasm or rupture of plaque, in coronary arteries with previous atherosclerosis; type 3, the hypersensitivity reaction leads to previously implanted drug-eluting stent thrombosis. Case report: LFS, 67 years old, male, hypertensive, after contrast injection during coronary angiography, evolved with dyspnea, chest pain, hypertension, skin rash and diffuse coronary spasm. Intracoronary injection of nitroglycerin and intravenous injection of hydrocortisone and diphenhydramine were performed with clinical improvement and resolution of pain. Control coronary angiography demonstrating resolution of the spasm without fixed strictures. After clinical stabilization and initiation of drug therapy with diltiazem, he was discharged asymptomatic and without skin changes. Discussion/Conclusion: Among several proposed mechanisms of immediate hypersensitivity to iodinated contrast, the following stand out: direct effect on the membrane related to osmolarity or chemical structure of the iodinated contrast medium molecule; complement system activation; direct bradykinin formation and IgE-mediated mechanism. In the face of sudden chest pain associated with symptoms of allergy or anaphylaxis, the possibility of Kounis syndrome should always be considered. The approach to the patient must be directed to the coronary event and the allergic reaction that induces it. Vasospasm is the primary mechanism, nitrates and calcium channel blockers should be considered first-line therapy. Corticosteroids play an important role in the treatment of allergic reactions and adrenaline is the mainstay of anaphylaxis treatment, however, it can worsen ischemia. Kounis Syndrome is not uncommon, but it is underdiagnosed and there are no specific clinical guidelines for treatment. 108516 Modality: E-Poster Researcher – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY THIAGO ALEXANDRE FERREIRA PIRES1, LUDMILLA DA ROCHA FREITAS VIEITAS1, HUGO ANDRADE SANTOS1, BRUNO SANTANA BANDEIRA1 (1) HOSPITAL CAXIAS D’OR Introduction: Supraventricular tachyarrhythmias are frequent pathologies in the emergency department and their approach must be systematized and prepared for possible complications. Degeneration of ventricular rhythms is uncommon but potentially serious. Case description: Young patient, without comorbidities, comes to the emergency room with tachycardic palpitations for 30 minutes. Physical examination showed no changes, BP 120 × 80 mmHg. ECG showing regular, narrow QRS tachycardia with a heart rate of approximately 250 bpm. Corrected normal QT interval. A modified Valsalva maneuver was performed, evolving with a brief period of wide QRS polymorphic tachycardia, with subsequent reversion to sinus rhythm with normal QT interval. Normal laboratory tests. Conclusions: Polymorphic ventricular tachycardia (PVT) is rare, with various causes classified according to the QT interval. In the case of prolonged QT, we have the Torsade of bridges. In cases where the QT is normal, a ventricular extrasystole (VES) with a short coupling interval, which is not pause-dependent, causes PVT. In the case in question, the patient had a narrow QRS tachyarrhythmia, suggestive of tachycardia due to nodal reentry, with hemodynamic stability, and the vagal maneuver was chosen as the initial approach. Evolved with PVT with spontaneous reversion to sinus rhythm. PVT started from a ventricular extrasystole that appeared at the end of the T wave, giving rise to the R over T phenomenon. This is an event that can occur in the VES, when it appears in the T wave of the anterior QRS, generating a conduction with a high probability of developing ventricular tachycardia/fibrillation belonging to grade 5 of the Lown classification. This is an unusual event that can occur during an attempt to reverse arrhythmias. 108522 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR SURGERY LARISSA NETO ESPINDOLA1, Christian Martins Macedo2, Beatriz Sant‘ana Isaías Rodrigues1, Elise Sant‘ana Isaías1, Paula Oliveira de Andrade Lopes1 (1) Curso Intensivo de Revisão em Cardiologia Clínica; (2) Hospital Santa Izabel Introduction: Coronary fistulas are rare conditions (0.002% of the population), comprising 48.7% of all coronary anomalies. The pathophysiology and clinical presentation depend on the magnitude of the flow through the fistula and its location. They can cause angina (3–7%), dyspnea (60%), endocarditis (20%, usually associated with Streptococcus viridans), syncope, palpitations, congestive heart failure (HF –19%) and cardiac arrhythmias. They can lead to aneurysmal dilatation of the involved coronary artery. Case report: A 46-year-old male patient suffered trauma to the elbow in May/2018, with local phlogosis and recurrent episodes of fever. In September/2018, he had atypical chest pain and increased fever frequency. He sought medical attention and, after auscultation of a heart murmur, a transthoracic echocardiogram (ECHO) was performed, which showed left ventricular dysfunction and severe aortic insufficiency. Therapeutic treatment for HF was initiated, it evolved with significant decompensation, requiring hospitalization. He was discharged on a surgical schedule for valve replacement. He was referred for preoperative cardiac catheterization, which revealed a circumflex artery (CX) aneurysm with a fistula to the right atrium (RA). Transesophageal ECHO showed significant aortic insufficiency secondary to endocarditis and CX fistula to the superior vena cava (SVC). Coronary computed tomography angiography confirmed atrial branch of the CX with fistula to RAand SVC. Negative blood cultures. Patient used Gentamicin + Ceftriaxone. He underwent aortic valve replacement (biological) and ligation of the fistula drainage ostium in the SVC and the proximal ostium next to the CX. Blood transfusion and vasoactive amines was performed in the immediate postoperative period. Conclusion: Coronary angiography is the main diagnostic method. In some cases the origin and relationship of the fistula to adjacent structures can be difficult to determine and non-invasive techniques can be used. There is general agreement that symptomatic patients should be treated. Closure of fistulas in the proximal segment of the coronary vessel is highly recommended due to the greater propensity for aneurysm and rupture. The prognosis of treated patients is excellent, although it depends on the severity of the shunt and on complications such as HF, pulmonary hypertension and bacterial endocarditis. Recurrence rates after surgical treatment are around 25%. Long-term follow-up is essential due to the possibilit. 108530 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES LARISSA NETO ESPINDOLA1, Christian Martins Macedo2, Elise Sant‘ana Isaías1, Beatriz Sant‘ana Isaías Rodrigues1 (1) Curso Intensivo de Revisão em Cardiologia Clínica; (2) Hospital Santa Izabel Introduction: The global impact of the disease due to infective endocarditis (IE) is practically unknown. IE is a heterogeneous syndrome whose incidence is influenced by multiple factors of the patient himself that determine the risk of infection, including anatomical conditions, injection drug use, in addition to social factors. Case Description: Male patient, 17 years old, using an orthodontic appliance for 1 year, sought the Emergency Unit due to asthenia with 9 months of evolution. Bradycardial, electrocardiogram showing total atrioventricular block (TAVB) and transthoracic echocardiogram (TTE) with signs of right coronary sinus rupture and interventricular septum dissection (IVS). Transesophageal Echo also showed bidirectional flow in the neocavity that formed within the IVS with the presence of perfusion (use of echocardiographic contrast). Vegetation was not seen. Physical examination without peripheral IE stigmata. Antibiotic therapy was started for IE, although blood cultures were negative (patient with dental abscess and visit to a “practical” dentist for braces implantation). Surgical treatment was chosen to close the IVS and excision of possible infected material. During the procedure, an aspect suggestive of a chronic process was observed, with a cavity within the IVS already endothelialized. Opted to occlude entry orifice and reapproximate IVS walls. Definitive pacemaker was implanted (patient on TAVB and with extensive septal lesion). Patient kept under antibiotic therapy for 28 days, when he was discharged in good condition and asymptomatic, after a new TTE confirmed the absence of flow in the topography of the neocavity observed in the preoperative examination. Conclusion: The clinical presentation of IE encompasses a wide spectrum of symptoms, being influenced by multiple factors (pathogen virulence, persistence of bacteremia, extent of tissue destruction (valvular and perivalvular), hemodynamic sequelae, septic embolization, consequences of circulating immune complexes. Due to the close proximity between the atrioventricular node and the proximal intraventricular conduction system and the aortic valve and root, perivalvular extension of infection in this valve is the most common cause of new atrioventricular block of any degree. driving is also a multivariate risk predictor of death associated with IE. 109857 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR IMAGING JULYANA GALVAO TABOSA DO EGITO1, 2. Rafael de Almeida Torres1, 3. Manoela Falsoni1, 4. Remulo José Rauen Jr1, 5. Guilherme Barreto Gameiro Silva1 (1) Hospital Santa Cruz – Curitiba –PR- Brazil Primary cardiac tumors are rare, and about 75% are benign. Myxoma is the most frequent primary cardiac tumor and its most common location is in the left atrium (LA) (75%). Approximately 70% of patients with LA myxoma have cardiac symptoms. The most frequent symptom is dyspnea (70%). The size, location and mobility of the myxoma will determine the symptomatology and severity of valve obstruction, 12% of myxomas are completely asymptomatic. Although cardiac myxomas are histologically benign, they may be lethal because of their strategic position. Therefore surgical removal should be performed as soon as possible. Case report: Female patient, 53 years old, previously dyslipidemic and with a positive family history of coronary artery disease. She was asymptomatic when she underwent a chest tomography due to the diagnosis of covid-19. The exam resulted in increase in the dimensions of the LA and a voluminous hypodense formation. Physical examination showed telediastolic murmur +/4+. A transesophageal echocardiogram described: Echogenic mass occupying the entire atrial cavity measuring 62 × 46.2, suggestive of myxoma. Preoperative coronary artery CT, showed the size and irrigation of the Myxoma (figure 1). The patient underwent removal of the myxoma without intercurrences. The presence of a murmur in a young and previously healthy patient should not be neglected and it is mandatory to complement it with imaging tests for diagnostic elucidation. 108647 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY JOAO LUIZ DE ALENCAR ARARIPE FALCAO1, Lara Lins Afio Ponte2, Breno de Alencar Araripe Falcao3, Sandra Nivea dos Reis Saraiva Falcao1, Ana Carolina Pereira Nogueira de Alencar Araripe Falcao4 (1) Universidade Federal do Ceará; (2) Universidade de Fortaleza; (3) Hospital de Messejana Dr. Carlos Alberto Studart Gomes; (4) Faculdade Christus Background: Transcatheter aortic valve replacement (TAVR) is indicated for treating symptomatic severe aortic valve stenosis (AS) with intermediate-to-high surgical risks. Few reports are available on managing leaflet thrombosis after TAVR with worsening heart failure. Case Summary: A previously fully vaccinated for SARS-CoV-2 infection 86-year-old man with severe AS received a successful TAVR with Edwards Sapien 3 valve. It was prescribed aspirin 100 mg at hospital discharge. Three weeks later, patient developed flu-like symptoms and fatigue. An RT-PCR test diagnosed SARS-CoV-2 infection. D-dimer test was elevated. Serum creatinine increased to 1.9 mg/dl. A new transthoracic echocardiogram showed a subacute aortic prothesis disfunction with mean transaortic pressure gradient of 50 mmHg. Rivaroxaban 20 mg substituted single antiplatelet therapy to treat presumed subacute leaflet thrombosis. After 45 days of anticoagulation a follow-up echocardiogram showed a decrease in mean transaortic valve pressure gradient to 8 mmHg. Symptoms of heart failure has improved gradually. It was planned to mantain anticoagulation for 3 months. Conclusion: A worsening heart failure or an increase in transaortic valve pressure gradient, especially in the early months after TAVR, suggest subacute leaflet thrombosis. SARS-CoV-2 infection may have triggered this thrombotic event. Subacute thrombosis after TAVR can be resolved after using NOAC as in our present case. 108847 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR IMAGING POLYANA EVANGELISTA LIMA1, José Verissimo dos Santos Neto1, Fabio Antonio Amando Granja1, Matheus Pereira Barreira1, Alane Mota dos Santos – Santos, AM1 (1) Universidade Federal do Vale do São Francisco Introduction: Infective endocarditis (IE) is a serious pathology with a high mortality rate. Early diagnosis is essential, since delayed or inadequate treatment can lead to serious complications. Case Report: A 47-year-old patient with mitral valve prolapse evolving to severe mitral valve insufficiency underwent valve replacement surgery with a metallic prosthesis. Five months later, he presented with progressive dyspnea NYHA III/IV for 1 month associated with fever peak. Transesophageal echocardiogram was performed, showing mobile mass in the ventricular surface, measuring 15 × 18 mm, and dysfunction of the metallic prosthesis with stenosis. He underwent cardiac surgery that revealed maintenance of the posterior cuspid of the mitral valve, inadvertently preserved along with the subvalvar apparatus that obstructed the prosthesis, locking the posterior hemi-disc. Usually, the vegetation of IE is positioned upstream of the valve; in this case, it would be positioned on the atrial side of the mitral prosthesis. Following the new guidelines, other imaging modalities, such as positron emission tomography with 18F-fluorodeoxyglucose and single photon emission computed tomography have gained importance in defining IE. Conclusion: We report one of the presentation forms of early dysfunction of a metallic prosthesis in mitral position. This is a case that would possibly have benefitted from the use of new imaging modalities for diagnostic elucidation. 108883 Modality: E-Poster Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARÍLIA PEREIRA2, Lívia Camarinha Franco de Menezes2, Flávia Gurgel3, Anna Esther Araujo e Silva2, Ana Flávia Malheiros Torbey1 (1) Universidade Federal Fluminense; (2) Hospital Universitário Antônio Pedro; (3) Hospital Getúlio Vargas Filho Introduction: Isolated unilateral absence of the pulmonary artery (UAPA) is a rare congenital malformation, prevalence range from 1 in 200,000 to 1 in 300,000. Clinical presentation is variable, making its diagnosis a challenge. Some patients have no symptoms and others may have heart failure and pulmonary hypertension (PHT). Case description: 1-year-old boy with a history of pneumonia at 2 months of life, presented to the pediatric emergency room with fever, cough and dyspnea. The chest radiograph showed unilateral whiteout of the left lung with hyperinflation of the right lung, and deviation of the mediastinum to the left. The diagnosis of comunitary pneumonia was made and penicillin initiated. Despite the important clinical improvement, the radiographic image remained and the patient was transferred for investigation of sustained pulmonary hypotransparency. At admission the vital signs were HR of 84 bpm, RR of 24 rmp, SatO2: 97%; breath sounds were decreased on the left side. The bronchoscopy was performed and showed reduction in the caliber of the division of the left main bronchus, making it difficult to visualize the subsegments, and a pulsation in the inferior right third of the trachea without any obstruction. The Doppler echocardiogram revealed a structurally normal heart with a right aortic arch and no visualization of the left pulmonary artery. CT angiography confirmed the absence of the left pulmonary artery. Conclusion: Isolated UAPA is a very rare congenital malformation and the absence of the left pulmonary artery is twice less frequent than that of the right pulmonary artery. Later age at presentation may result in higher risk of morbidity and mortality. The early diagnosis can prevent the development of PHT, recurrent pulmonary infections and hemoptysis. 108902 Modality: E-Poster Researcher – Case Report Category: PHYSIOTHERAPY RENATA CRUZEIRO RIBAS1, Sabrina Costa Lima de Andrade1, Jessica Blanco Loures1, Estêvão Lanna Figueiredo2 (1) RC Fisioterapia – Reabilitação Cardiovascular; (2) Instituto Orizonti Introduction: Dyspnea and poor exercise tolerance are frequent in Heart Failure with preserved ejection fraction (HFpEF). Respiratory muscle (RM) weakness is an independent predictor of low ventilator efficiency during exercise and contributes to poor prognosis in this population. Inspiratory Muscle Training (IMT) enhances the benefits to aerobic and peripheral muscle training by improving RM fatigue, exercise tolerance and functional status, increasing quality of life (QoL) and reducing mortality. IMT is recommended as part of a cardiac rehabilitation (CR) programme in HFpEF patients. Case report: An 86 year old hypertensive man with reduced functional status on the setting of Covid-19 pandemic lockdown developed HFpEF, with increasing dyspnea (NYHA’s functional class III), peripheral and pulmonary congestion. In sinus rhythm. Ejection fraction 65% and signs of diastolic dysfunction on echocardiogram, elevated NTproBNP. Patient was enrolled in a home-based CR programme for 12 weeks, with 4 sessions/week – 2 supervised and monitored sessions and 2 non-supervised. Medical therapy was optimized (diuretics, espironolactone, empagliflozin, anlodipine, telmisartan). Supervised and monitored sessions included IMT with POWERbreathe (PB)®, set on 30%–40% of S-index, weekly adjusted, and aerobic associated with muscular resistance exercises. The non-supervised sessions included IMT and muscular resistance exercises. Patient was submitted to functional assessment before starting CR, and after 12 weeks, that included dynamic inspiratory strength (S-index), peak inspiratory flow (PIF) and inspiratory volume measured with K5 PB®, Incremental Shuttle Walk Test (ISWT) and 30-second sit-to-stand test (STS). Dyspnea and self-perceived exertion were assessed with the modified Borg Scale and QoL by Minnesota Living with HF Questionnaire (MLwHFQ). Results: Compared to baseline, there was an increase in the % of the predicted S-index (Δ55%), in PIF (Δ2.39%), in inspiratory volume (Δ0.28%), in ISWT (Δ200 m) and in STS (Δ7 repetitions), with reduction in self perceived exertion (very hard to quite hard). There was an important decrease in MLwHFQ (Δ36 points) related to a better perception of QoL. NYHA’s functional class improved and NTproBNP decreased significantly. Conclusion: IMT in CR context, associated with optimized medical therapy, contributed to functional improvements, reducing dyspnea, and amelliorating QoL. This case illustrates how CR can change patient’s prognosis. 108931 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM FIRMINO HAAG FERREIRA JUNIOR1, Camila Louro Mota Branco1 (1) Hospital Geral de São Mateus Objective: To describe a case of Multisystem Inflammatory Syndrome (MIS-C) associated with SARS COV-2 and Chicungunya associated with left ventricular dysfunction in a child admitted to a children’s intensive care unit in a public hospital in the city of São Paulo. Case report: A.S.P.S., female, mixed race, 9 years old, was admitted to the pediatric intensive care unit with fever, dysuria, recent-onset arthralgia, with confluent and hyperchromic micropapular lesions, accompanied by choluria and abdominal pain. On physical examination, she was hypoactive, tachypneic, acyanotic, contacting and oriented. Complete blood count and urine analysis without significant changes. Antibiotic therapy was introduced with Ceftriaxone 2 g/day, Hydrocortisone and clinical support. Serology was requested for SARS COV-2, in addition to serology for Dengue, Chicungunha, Zika and leptospirosis, which showed detectable results for SARS COV-2 and Chicungunha. Abdominal US showing free fluid in the cavity without involvement of target organs. A transthoracic echocardiogram was performed, which showed moderate myocardial dysfunction with associated pericardial effusion without signs of restriction. Opted for the association of pulse therapy with methylprednisolone 10 mg/kg/weight for 3 days, associated with immunoglobulin 1.0 g/kg/weight for 10 days. The control echocardiogram showed preserved ejection fraction, normal cardiac dimensions and mild laminar pericardial effusion. He evolved with progressive improvement of the condition, being discharged asymptomatic and in satisfactory clinical conditions. Conclusion: Multisystem inflammatory syndrome is a rare but serious disease in which children with COVID-19 develop a systemic inflammatory process. Cross-association with other types of viruses is rare, but evident in the literature. Recovery of left ventricular function is very common, and occurs within a few days of starting treatment. The clinical evolution of patients treated early has been favorable, as in the case described above. 108979 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARCELO SABEDOTTI1, Bibiana Guimarães Maggi1, Leandro Gazziero Rech1, Rafael Massuti1, Rafaela Oliveira Leite2 (1) Hospital Geral de Caxias do Sul; (2) Fundação Universidade de Caxias do Sul Introduction: Rupture of the interventricular septum (RIVS) is a rare complication of acute myocardial infarction (AMI), which occurrence has decreased due to early intervention of AMI. Despite surgical advances, mortality of RIVS remains high, and such complications may be indicated by a new heart murmur and sudden hemodynamic impairment. Case report: A 70-years-old male with diabetes, dyslipidemia, systemic arterial hypertension, and previous coronary artery bypass surgery, was hospitalized with an acute coronary syndrome without ST-segment elevation. Physical examination demonstrated a holosystolic murmur and diffuse precordial fremitus, with no signs of left heart failure. The patient was stable and promptly referred for early intervention. The coronary angiography demonstrated a subtotal left main coronary artery (LMCA) injury, patent left internal thoracic artery with total occlusion of the anterior descending artery and first diagonal branch. Circumflex artery was dominant, and the first marginal branch had a subtotal injury at the origin and an occlusion of the saphenous graft. Ventriculography demonstrated RIVS on the inferior sept and preserved left ventricular systolic function, with drug-eluting stent implanted on the LMCA towards the circumflex-anterior descending artery, protected by the left internal thoracic artery. The RIVS percutaneous closure was scheduled for the following day. Transthoracic echocardiogram (TTE) identified the RIVS on the lower portion of the muscular sept, with an 11 mm diameter, mild hemodynamic consequences, a 55% Simpson ejection fraction and inferior akinesia. The patient evolved with cardiogenic shock on the day of the procedure, demanding volemic reposition and analgesia. Control TTE demonstrated an increase of the RIVS to 22 mm. We performed the RIVS occlusion as scheduled, through an arteriovenous strap from the right femoral vein to the left femoral artery. The introducer was placed anterogradely through the defect to the ascending aorta. The measurement of the VSD could be performed using a sizing balloon, but due to severe periprocedural instability, we decided on the use of a VSD prosthesis sized according to echocardiographic and angiographic controls. The procedure was successful, with a dramatic hemodynamic improvement after prosthetic placement. The patient was discharged after five days. Conclusion: RIVS percutaneous treatment after AMI is a therapeutic alternative for high-risk surgical patients. 108982 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CRISTIANE KOPLIN1, João Artur Amaral Guarienti2, Felipe Lisboa Frota3, Fabiana Amaral Guarienti4 (1) MD from Federal University of Rio Grande do Sul, Emergency Departament of Public Health Foundation of Novo Hamburgo, Brazil; (2) MD from University of Passo Fundo, Rio Grande do Sul, Brazil; (3) Professional in Physical Education, Physical Activities and Exercises for the Prevention, Treatment of Diseases and Health Promotion Postgraduate Program of Albert Einstein Hospital, São Paulo, Brazil; (4) Geriatrics and Gerontology Postgraduate Program of Pontifical Catholic University of Rio Grande do Sul, Brain Institute, Porto Alegre, Brazil Introduction: This report describes the case of a patient diagnosed with COVID-19 who presented with symptoms of myocarditis and pulmonary thromboembolism (PTE). Case description: Female patient, 35 years old, arrived at the emergency in June 2020 with flu-like symptoms. History of allergic rhinitis, asthma, grade II obesity (BMI: 37.2 Kg/m²) and miscarriage in 2017. Requested RT-PCR SARS-CoV-2 with positive result. The patient had a good evolution with symptomatic treatment. She returned after two months with burning chest pain and vomiting. On arrival she was hypertensive (BP: 190/120 mmHg) and the electrocardiogram showed no acute ischemic changes. However, myocardial necrosis markers, troponin T and CK-MB, were positive in three measurements. The case was initially managed as an acute myocardial infarction without ST-segment elevation. Coronary Angiography showed no coronary lesions. The Transthoracic Echocardiogram showed an LV with increased wall thickness (Fig 1). The patient also had increased inflammatory markers (C-Reactive Protein and ESV). Treated with analgesics and antihypertensives, she had an improvement of the symptoms. She was discharged with outpatient follow-up for suspected myocarditis. After three months the patient returned with dyspnea for 3 days. She had 82% O2 saturation on room air, BP 150/110 mmHg and no other findings. Pulmonary artery CT Angiography showed signs of PTE (Fig. 2). As she had a Pulmonary Embolism Severity Index (PESI) of 55, indicating a low risk of severe morbidity, she was released and maintained on oral anticoagulation. Conclusions: The pathophysiology of cardiovascular complications associated with COVID-19 is not completely understood. Myocardial viral infection and cytokine storm are etiological theories. This patient had typical signs of myocarditis with a history of recent viral illness, indicating an important relation between COVID-19 and myocardial damage. The thrombus tendency in COVID-19 may involve two mechanisms: a hypercoagulable state responsible for large vessel thromboembolism and direct endothelial injury responsible for microvascular thrombosis. In this report, PTE may have an etiologic link with previous SARS-CoV-2 disease. Interestingly, the relationship between COVID-19 and cardiovascular complications seems evident. Therefore, there is a need of additional studies to elucidate the pathophysiology of cardiovascular impairment related to COVID-19. 109004 Modality: E-Poster Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ROMERO HENRIQUE DE ALMEIDA BARBOSA1, Gabriela Santos Andrade1, Nayara Ribeiro Máximo de Almeida1, Poliana kalinne Simões de Melo Barbosa2, Adirlene Pontes de Oliveira Tenório1 (1) Universidade Federal do Vale do São Francisco; (2) Instituto Médico São Francisco Introduction: The subclavian vertebro steal phenomenon(SVSP) occurs when a stenosis or occlusion in the brachioencephalic trunk or subclavian artery proximal to the origin of the vertebral artery causes partial or complete reversal of blood flow from the ipsolateral vertebral artery. It usually behaves asymptomatically, in up to 80% of patients, but may evolve with symptoms of vertebrobasilar insufficiency. Atherosclerotic disease is the most common cause and the left side is more commonly affected in a 4:1 ratio with respect to the right side. Case description: I.M.S., female, 66 years old, Body Mass Index: 26, from Delmiro Gouveia-AL, diabetic, hypertensive, chronic renal failure on dialysis. Due to frequent dizziness and syncope, she had her first medical visit on 07/10/19, denying previous neurological and cardiologic symptoms. After suspension of beta-blockers and hydroelectrolytic and metabolic correction, Holter was requested, which showed advanced atrial ventricular block, being referred for definitive pacemaker implantation. After implantation, she returned for consultation on 24/01/20, hemodynamically stable, but with persistent vertigo. Doppler echocardiography of the carotid and vertebral arteries was requested, which revealed a totally reverse flow in the right vertebral artery, compatible with SVSP with permanente retrograde pattern. A computed tomography angiotomography of the cervical artery and venous was ordered on 01/21/2021, showing a mixed plaque located in the proximal right subclavian artery at the origin of the right vertebral artery, with an irregular surface, determining stenosis between 50 and 70%, with an extension of 2 cm. After evaluation by the vascular surgeon to analyze the need for the procedure, supra-aortic trunk angioplasty was indicated as treatment for SVPS. Conclusions: The SVSP in the right vertebral artery is of infrequent occurrence, about 10% of cases, and that in about 5% of patients present symptoms, the report of this case can contribute to the identification of such condition and differential diagnosis of vertigo and syncope. 109073 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR IMAGING PEDRO ANTONIO GALDEANO1, PATRICIA REGINA ALVES GALDEANO1, GABRIEL ANTONIO STANISCI MIGUEL1, FERNANDA NOGUEIRA BUENO RODRIGUES ALVES1 (1) CLINICORE; (2) CARDIOLIFE Identification: Patient 52 years old, female, white, natural from anápolis-goiás. Case report: patient presented in 2014 a complaint of palpitations with effort associated with episode of transient loss of movement in lower left left seeking medical assistance. Requested echocardiogram that demonstrates a large oval image (figure), regular outlines, measuring 03 × 03 cm, inside the left atrium with bulleting to the inside of the left ventricle adhered to the interatrial septum, and the hypothesis of left atrial mixoma being proposed. Patient after clinical stabilization was referred to heart surgery with complete removal and diagnostic confirmation by anatomological and pathological left atrial mixoma. In the year 2021 (07 years after surgery) in control examination with echocardiogram, a regular contour mass was again identified, measuring 07 × 07 mm adhered to the interatrial septum (figure) with probable diagnostic hypothesis of recurrence of left atrial mixoma. Discussion: Cardiac mixoma is the most common primary tumor of the heart and is mainly located in the left atrium. It predominates in the female sex and the age group around 50 years old, being rare in children. Symptomatology depends on the size, shape, mobility and location of the tumor. The treatment is surgical removal, which most of the times is curative. Conclusion: Recurrence of cardiac myxoma after surgical removal is uncommon. This is due to the excision of its implantation base in the heart walls. If this resection is not broad, there is a possibility of the tumor recurrence. In the present case, the patient was followed annually without presenting residual image to echocardiography. Mixoma recurrence in followed cases is uncommon, which aroused the interest of the authors. 109330 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR SURGERY DEINER PAULO MARTINS RESENDE 1, Alberto Rodolpho Hüning1, Giuliano Minor Zortéa1, Márcia Castilhos Puchalski1, Marcela da Cunha Sales1 (1) Santa Casa de Misericórdia de Porto Alegre (ISCMPA) Introduction: Benign intracardiac tumors are uncommon entities, among which blood cysts are even rarer. They are usually located in the heart valves or adjacent structures and have a congenital in origin, disappearing spontaneously with time and rarely found after the first year of life. These tumors can cause left ventricular outflow tract (LVOT) obstruction, valve dysfunction, pulmonary embolism, and coronary obstruction. From 1960 to 2021, only 54 cases of blood cysts located in the mitral valve have been reported. Case report: We present a case of a 36-year-old man, asymptomatic, who sought a cardiologist for routine evaluation. The electrocardiogram was normal. The transthoracic echocardiogram showed a rounded structure, with hyperechogenic edges, hypoechogenic center, measuring 2.1 × 1.9 cm (3,4 cm²), located in the chordae of the anterolateral papillary muscle of the mitral valve, without obstruction of the LVOT. Cardiac magnetic resonance imaging showed a sessile mass attached to the head of the anterolateral papillary muscle, 24 × 20 × 19 mm, hypointense and absence of contrast uptake. The patient was referred for cardiac surgery, where 2 cysts were identified, completely attached to the mitral valve and subvalvular apparatus. Resection of the anterior leaflet, chordae, top of the anterolateral papillary muscle and cysts were performed, followed by implantation of a bioprosthesis. Conclusion: Intracardiac blood cyst are more commom in newborns and extremely rare in adults. Surgery is usually indicated due to the risk of embolization and other potential complications, as well as to define the exact diagnosis. 109566 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY ANTONIO AUGUSTO XIMENES2, Carolina Thé Macêdo1, Jemima Araújo Silva Batista4, Marcela Machado Costa6, Tonnison de Oliveira Silva2 (1) Curso de Pós-Graduação em Cardiologia da SBC/INC/INCA; (2) Hospital Cardio Pulmonar, Rede D’Or; (3) Hospital São Rafael, Rede D’Or; (4) Universidade Federal do Vale do São Francisco, UNIVASF; (5) Faculdade Estácio de Juazeiro; (6) Escola Bahiana de Medicina e Saúde Pública, EBMSP Introduction: Hereditary transthyretin amyloidosis (ATTRv) is a severe disease transmitted by an autosomal dominant inheritance associated to more than 100 mutations in the transthyretin gene. The ATTRv phenotypic expression depends mainly, but not exclusively, on the type of mutation, with neurological and/or cardiological involvement most frequently. The improvement of the disease prognosis by the new drugs available relies on early diagnosis and treatment. The best strategy for the earliest diagnosis is based on knowledge about how the disease manifests itself in each mutation. However, due to the incomplete penetrance of mutations, detection of an initial stage of the disease is challenging. Val107 (Ile107Val) mutation is a rare form of ATTRv, characterized by neurological and cardiological involvement, with neurological impairment manifesting earlier and being more severe. We describe a case with cardiological alterations as the initial manifestation of the disease. Case: 20/12 Asymptomatic 58-year-old male with Val107 mutation. He is is part of a group of 27 carriers of the Val107 mutation, followed up by our group. The institution protocol for the asymptomatic carriers routinely includes cardiological tests. Normal physical examination. Bilateral carpal tunnel syndrome confirmed by electroneuromyography Protein electrophoresis – no monoclonal band. Echocardiogram (Echo) – Left Ventricular ejection fraction (LVEF) 65%/Global Longitudinal Strain (GLS) = –17%. Holter – rare supra and ventricular ectopy. Cardiac Ressonance – mild pericardial effusion, increased left atrium. 99 mTc-PYP bone scintigraphy – Perugini grade 3 cardiac uptake and H/CL ratio = 1.56. Tafamidis was introduced. 22/03 Echo: LVEF: 70%, GLS:–20%, apical sparing pattern. Conclusion: In the scarce literature available on Val107 ATTRv, the neurological impairment is the most common form of initial manifestation, being cardiac involvement observed later. However, this case points out that cardiological tests should also be included early in the monitoring strategy of asymptomatic Val107 carriers for diagnosis of ATTRv. 111119 Modality: E-Poster Researcher – Case Report Category: HYPERTENSION/RENAL DENERVATION ELIZABETH CARDOSO DOS SANTOS1, Vera Maria Lúcia Cury Salemi2, Fernanda Willemann Ungaretti1, Juliana Gil de Moraes1, Roberto Kalil Filho1 (1) Sociedade Beneficente de Senhoras Hospital Sirio Libanes; (2) Instituto do Coração Paragangliomas are rare tumors originating from cells from the neuroendocrine system, and like pheochromocytoma, they are catecholamine producers. However, they usually differ in their location, which is extra-adrenal. Regarding the clinical presentation, the classic triad consists of episodic headache, palpitations and sweating. Therefore, the objective is to describe an unusual clinical case and the general aspects of this entity. Case report: A 29-year-old man with previous retroviral disease and arrhythmia is admitted to the emergency room with headache and an episode of emesis, with blood pressure levels of 247 × 152 mmHg. He reports episodes of morning headache associated with nausea and palpitations for 3 weeks. He was evaluated by the neurologist, with normal eye fundus and cranial computed tomography, and was hospitalized for blood pressure control and investigation of secondary systemic arterial hypertension. Laboratory tests were performed, the results of which showed significantly increased levels of vanillylmandelic acid, catecholamines and urinary metanephrines, and complementary tests such as: abdominal computed tomography that showed a voluminous solid lesion with necrotic areas and heterogeneous post-contrast enhancement centered on the right adrenal store, measuring approximately 6.5 × 6.0 × 5.0 cm. The mass displaces the inferior vena cava, trunk and posterior right portal branch, without signs of vascular invasion, presents extensive contact with the upper pole of the right kidney, displacing it inferiorly, but maintains a cleavage plane, suggesting the possibility of pheochromocytoma. Abdominal magnetic resonance imaging showed a solid mass in the right adrenal, indeterminate, suggesting that, in addition to pheochromocytoma, we consider carcinoma as a possibility. Whole-body PET-CT showed marked glycolytic hypermetabolism in the right adrenal mass, suspicious for a neoplastic process. He underwent resection of a peritoneal mass, right hepatectomy and partial resection of the inferior vena cava with primary reconstruction. The immunohistochemical profile was compatible with succinate dehydrogenase deficient paraganglioma. After tumor excision, the patient evolved with good blood pressure control and is currently asymptomatic. Discussion: Once there is a clinical suspicion of pheochromocytoma/paraganglioma, laboratory tests and imaging tests should be performed, and surgical removal of the tumor remains the only curative treatment. 109606 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR SURGERY MARINA PINHEIRO ROCHA FANTINI1, Juan Mario Vaca Pereira Vrandecic2, Felipe Magalhães Becker1, Larissa Maria Ferrarez Faria4, Carlos Eduardo de Menezes e Souza Filho5 (1) Hospital Mater Dei; (2) Santa Casa de Belo Horizonte; (3) ECMO MINAS; (4) Faculdade Ciências Médicas de MG; (5) Faculdade Federal de Minas Gerais Introduction: Behçet Syndrome (BS) is an autoimmune systemic vasculitis of variable caliber and with different phenotypes. It classically courses with ulcerative, papulopustular and nodular erythema-like lesions on the skin, mucous membranes and genital region. Among its presentations, angio-behçet has venous and, rarely, arterial vascular involvement. These arterial presentations, despite affecting approximately 4% of patients, represent a unique characteristic of BS, whose aneurysms potentially affect peripheral, visceral and pulmonary arteries. This case report presents a 16-year-old boy with recurrent left ventricular pseudoaneurysms since 2020. Case Report: ELP, male, 16 years old. He was diagnosed with Behçet’s Syndrome in 2020 when he started presenting oral and genital ulcers, constitutional symptoms, hepatosplenomegaly, fever and night sweats. He underwent surgery to correct a left ventricular (LV) pseudoaneurysm on 5/29/20, did not improve and presented an aortic pseudoaneurysm and a new left ventricular apex pseudoaneurysm on examination on 8/28/20. As a result, he started pulse therapy with methylprednisolone and cyclophosphamide on 9/2/20 and surgical correction of ventricular and aortic pseudoaneurysms on 9/8/20. Treatment with pulse therapy was maintained, with 9 sessions with cyclophosphamide, until the finding of a new ventricular pseudoaneurysm on 10/01/21, when the corticosteroid was changed to infliximab. This new pseudoaneurysm had important dimensions (12.1 cm × 7.6 cm in its largest diameters), presence of LV shunt to the pseudoaneurysm, with a peak gradient of 64 mmHg and maximum velocity of 4.01 m/s, ejection fraction of (EF) 62.17% and preserved biventricular systolic function. He was admitted on 01/18/22 with decompensated heart failure with a warm and humid profile, associated with anasarca, EF of 37.5% and LV and right ventricle dysfunction. The conduct was clinical treatment and enrollment for urgent heart transplantation. Conclusion: This report discusses a rare case of recurrent left ventricular aneurysm in a 16-year-old boy, with recurrent cardiac manifestations despite immunosuppressive therapy and corrective surgery. The patient’s condition shows that Behcet’s Syndrome may present as an aggressive cardiac form with more severe manifestations. 109690 Modality: E-Poster Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARINA PINHEIRO ROCHA FANTINI1, Henrique Loiola Duarte5, Pedro Henrique Cavalcanti de Albuquerque Sá5, Leonardo Nunes Coelho Fantini1, Vinícius Loureiro2 (1) Hospital Mater Dei; (2) Santa Casa de Belo Horizonte; (3) ECMO MINAS; (4) Faculdade Ciências Médicas de MG; (5) Faculdade Federal de Minas Gerais Introduction: Anomalous coronary artery emerging from the contralateral sinus is a rare congenital anomaly. Some variations incur increased risk of adverse cardiovascular events, with sudden death often being the initial manifestation of the disease. This report presents a case of a patient with a variant of anomalous coronary artery with a poor prognosis, requiring surgical myocardial revascularization. Case Report: SASL, female, 59 years old, diagnosed with anomalous coronary artery in 2014. On that occasion, coronary angiography was performed after the atypical chest pain, which showed an anomalous origin of the left coronary trunk, without atheromatous problems. She also has arterial hypertension, dyslipidemia and hypothyroidism, and uses Olmesartan, Hydrochlorothiazide, Duloxetine, Levothyroxine, Rosuvastatin and Alprazolam. On 01/2022, she had angina pectoris and dyspnea on exertion. She underwent an exercise stress test, which was positive for ischemia. Coronary computed tomography angiography was performed, which showed the left coronary trunk with anomalous origin in the right coronary sinus, path posterior to the pulmonary artery and anterior to the aorta, calcified plaque in the ostium of the anterior descending artery, determining luminal reduction of 25 to 49%. After evaluation, The Heart Team of Hospital Felicio Rocho opted for surgical intervention. She was admitted electively on 03/03/2022 for myocardial revascularization by the construction of a bridge from the mammary artery to the anterior descending artery using cardiopulmonary bypass and under balanced general anesthesia and erector spinae plane block. Extubated while still in the operating room, she was referred to the intensive care unit (ICU) in good clinical condition. She was discharged from the ICU on the 2nd postoperative day (POD) and discharged from the hospital on the 7th POD. The postoperative echocardiogram showed preserved biventricular systolic function, mild aortic insufficiency, laminar pericardial effusion and left ventricular ejection fraction = 71%. She is currently asymptomatic and in outpatient follow-up. Conclusion: This report highlights the situation of a patient with an anomalous coronary trunk who had her cardiovascular prognosis positively modified by means of surgical myocardial revascularization. This demonstrates the importance of diagnostic investigation and surgical intervention in these patients, in order to avoid negative outcomes such as sudden death. 109701 Modality: E-Poster Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARINA PINHEIRO ROCHA FANTINI1, Felipe Becker Magalhães1, Carlos Eduardo de Menezes e Souza Filho5, Larissa Maria Ferrarez Faria4, Murilo Malta Batista2 (1) Hospital Mater Dei; (2) Santa Casa de Belo Horizonte; (3) ECMO MINAS; (4) Faculdade Ciências Médicas de MG; (5) Faculdade Federal de Minas Gerais Introduction: Anomalous coronary artery emerging from the contralateral sinus is a rare congenital anomaly. Some variations incur increased risk of adverse cardiovascular events, with sudden death often being the initial manifestation of the disease. This report presents a case of a patient with a variant of anomalous coronary artery with a poor prognosis, requiring surgical myocardial revascularization. Case Report: SASL, female, 59 years old, diagnosed with anomalous coronary artery in 2014. On that occasion, coronary angiography was performed after the atypical chest pain, which showed an anomalous origin of the left coronary trunk, without atheromatous problems. She also has arterial hypertension, dyslipidemia and hypothyroidism, and uses Olmesartan, Hydrochlorothiazide, Duloxetine, Levothyroxine, Rosuvastatin and Alprazolam. On 01/2022, she had angina pectoris and dyspnea on exertion. She underwent an exercise stress test, which was positive for ischemia. Coronary computed tomography angiography was performed, which showed the left coronary trunk with anomalous origin in the right coronary sinus, path posterior to the pulmonary artery and anterior to the aorta, calcified plaque in the ostium of the anterior descending artery, determining luminal reduction of 25 to 49%. After evaluation, The Heart Team of Hospital Felicio Rocho opted for surgical intervention. She was admitted electively on 03/03/2022 for myocardial revascularization by the construction of a bridge from the mammary artery to the anterior descending artery using cardiopulmonary bypass and under balanced general anesthesia and erector spinae plane block. Extubated while still in the operating room, she was referred to the intensive care unit (ICU) in good clinical condition. She was discharged from the ICU on the 2nd postoperative day (POD) and discharged from the hospital on the 7th POD. The postoperative echocardiogram showed preserved biventricular systolic function, mild aortic insufficiency, laminar pericardial effusion and left ventricular ejection fraction = 71%. She is currently asymptomatic and in outpatient follow-up. Conclusion: This report highlights the situation of a patient with an anomalous coronary trunk who had her cardiovascular prognosis positively modified by means of surgical myocardial revascularization. This demonstrates the importance of diagnostic investigation and surgical intervention in these patients, in order to avoid negative outcomes such as sudden death. 109781 Modality: E-Poster Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FRANCISCO ITALO DUARTE KUMAMOTO 1, Maria Alice Oliveira Magalhães Teixeira1, Maria Eliza Soares Gadelha de Oliveira1, Eduardo Franco Correia Cruz Filho1, Ana Carolina Navarro Henriques Cabral1 (1) Hospital Memorial São Francisco Introduction: Myocardial Infarction With Non-Obstructive Coronary Artery (MINOCA) is a special type of Acute Myocardial Infarction (AMI). It is considered a working diagnosis, because the disease behaves similarly to a conventional AMI until the Coronary CT Angiography is done, which does not show lesions greater than 50% of the lumen in a main epicardial vessel, and this possibility should be considered until no other clinically overt specific cause is proven. Compared to coronary obstruction, It most often affects young women without comorbidities, such as the case reported. Case Report: A 35 year old female, without comorbidities, entered in the ER in 2019 complaining of anginal chest pain, palpitations, intense sweating, drowsiness and high SBP of 180 mmHg. Based on the past medical history, the physical examination was normal and biomarkers were abnormal. A coronary CT Angiography showed an eccentric thrombus in the distal midsegment of the descendant anterior artery, which determined luminal stenosis below 50%. Changes in the left marginal branch, including left ventricle with subendocardic hypoattenuation of the anterior and septal walls of the apical region were also detected. According to the patient, the ECG was abnormal. The transthoracic echocardiogram showed an anterior and apical hypokinesis of the left ventricle. The cardiac catheterization presented an isolated lesion of the descendant anterior artery, which determined luminal stenosis below 50%. She was overtreated with aspirin, captopril, furosemide and low molecular weight heparine and was followed up by a cardiologist over 2 years. She remained asymptomatic until February 2022, when was admitted in the ER, reporting typical anginal pain. The cardiac troponin-1 was abnormal (2.8 ng/ml). She was admitted to the CCU. The electrocardiogram was normal. The coronary CT Angiography did not show any obstructive lesion and the cardiac catheterization was normal. The Magnetic Resonance Imaging (MRI) showed the presence of Late Gadolinium Enhancement (LGE) in subendocardial area, suggesting previous AMI. Today, she was discharged, with aspirine, statin and beta blocker. Conclusion: Young patient without classic cardiovascular risk factors with apical wall AMI confirmed by magnetic resonance imaging without obstructive lesion, which in our view is compatible with MINOCA. 109836 Modality: E-Poster Researcher – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ERIKA OLIVIER VILELA BRAGANCA1, Érika Olivier Vilela Bragança1, Graziela Oliveira de Avelar1 (1) RitmoCheck Introduction: 35-year-old woman with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) with non-sustained ventricular tachycardia, right ventricular enlargement, left ventricular dysfunction (LVEF 46%) and important cardiac fibrosis (45% of late enhancement in cardiac MRI) and an ICD was indicated to primary prevention. Preoperative tests showed a positive betahCG and obstetric consultation confirm the pregnancy. After sharing making decision against radiation exposure and sudden cardiac death, we decided to move on to procedure of implantation of an ICD. The ionizing radiation is absorbed by any part of the body and the pregnant woman receive protective apron against lead in her belly, neck, head and eyes. The procedure was performed using fluoroscopic view and a dual chamber ICD was implanted. The total time was sixty minutes, the radiation time was ninety seconds and the burden of radiation was 1.6rads and the acute electronic measurements were very good. The post operative period was suitable and no cardiovascular or obstetric complication was noted during the pregnancy. Betablocker has not been discontinued. At 38 weeks the patient went into labor and the route of delivery chosen was the vaginal one. The patient underwent epidural anesthesia and she gave birth a female newborn of 3,315g weight and an Apgar score of 9/10. Only ventricular ectopics beats was seem during this period of time and no device abnormality. The puerperium phase was uneventful. Discussion: There are a few studies of pregnancy with ICD managed and there are actually no guidelines for this issue. As regarding ARVC only sixty pregnancies were identified between 1968 and 2016 and only 17% having an ICD. ARVC is associated with a low rate of major cardiac events and vaginal delivery appears safe. X-ray exposure of less than 5 rads has not been associated with an increased chance for birth defects. Conclusion: The ICD is a relative contraindication during pregnancy, nonetheless the procedure and delivery should be carefully planned and protected. 109823 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY LARISSA ARLETE MOSKO1, Bruna Bozelli Leopoldino Barion1, Michael Malca Sepúlveda2, Sanderson Antonio Cauduro1, Celso Soares Nascimento2 (1) Hospital Erasto Gaertner HEG; (2) Hospital Angelina Caron HAC Introduction: Neuroendocrine tumors (NET) are neoplasms derived from enterochromaphin cells that produce neurotransmitters and neuromodulators. Its incidence is 2–5 new cases per 100,000 inhabitants/year. Skin flushing, diarrhea, telangiectasia and cardiac lesions are symptoms that characterize Carcoininoid Syndrom (CS). The Biopsy defines the diagnosis. Cardiac involvement is due to an increase in the right heart caused by tricuspid and pulmonary insufficiency. Valve replacement can improve functional capacity. Case Description: A 50-year-old male patient was admitted in 2017 to oncology hospital Erasto Gaertner with diarrhea, urticariform lesions and 15-kilo weight loss. He denied comorbidities and use of medications Upon examination, palpable mass in the abdômen was noticed. Hepatic nodulus were found on CT scan. A biopsy of these lesions revealed neoplasm of metastatic neuroendocrine pattern. Search for the primary site was carried out unsuccessfully. The initial echocardiogram showed an increase in the right chambers, with preserved right ventricular (RV) systolic function and severe tricuspid insufficiency. Despite the use of somatostatin analogs (SA), in 2018, the patient presented signs of right heart failure, hence a proposal for surgical approach, patient turned it down. Chemoembolization the liver lesions was performed to decrease serotonin hypersecretory. In 2021, he presented cardiac decompensation with RV dysfunction on echocardiogram. Back then, the patient agreed to tricuspid valve replacement, but in post-op even with the use of SA, the decompensation evolved to refractory cardiogenic shock due to RV failure, causing death. Conclusion: The carcinoid heart (CH) is a therapeutic challenge not only regarding risks of valvar surgical intervention, but also because of the systemic effects of CS itself triggered by surgical trauma. NGUYEN et al. evaluated 240 patients who underwent valve surgery. In this study, as well as in other analysis of smaller groups, most of the patients who were assessed received SA in pre and post op, modifying surgical evolution. Furthermore, early surgery improved short and long-term outcomes. In this case report, the patient refused to undergo early valve replacement surgery, and approach with RV dysfunction was performed, which probably contributed to the unfavorable outcome. Patient selection, the care of a multidisciplinary team and SA infusion protocols, seem to be the main points for success in the CH approach. 109832 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR SURGERY EDMILSON CARDOSO DOS SANTOS FILHO1, Diogo Alves Cardoso3, Simone Cristina Soares Brandão1, Manuela de Sousa Moura Fe1, Fábio Antônio Amando Granja1 (1) Hospital das Clinicas da Universidade Federal de Pernambuco; (2) Instituto do Coração de Pernambuco; (3) Faculdade Pernambucana de Saúde Introduction: Thoracic stent graft infections are devastating complications related to the treatment of aortic aneurysms. Definitive treatment requires a strategy of revascularization, explantation of the infected prosthesis, and debridement of all infected tissue. It represents a flaw in the minimally invasive approach to endovascular treatment and puts the patient at an increased risk of morbidity and mortality. Case description: A 42-year-old female patient, HIV + with an undetectable viral load, was admitted complaining of asthenia, cough, and intermittent fever (39–40°C) for 9 days. Hospitalized 6 months earlier due to COVID-19 infection, she evolved with pericardial effusion with purulent cardiac tamponade, and a pericardial window was performed. Had endovascular treatment for a traumatic descending aortic aneurysm 13 years ago. Despite an inconclusive investigation for an infectious focus, the patient initially presented a clinical and laboratory response to empirical ABX (cefepime + teicoplanin). After 10 days of treatment, she returned to present febrile peaks associated with an increase in WBC and CRP. PET-CT showed increased uptake and periprosthetic gas, suggesting infection with the presence of biofilm. The surgical approach was performed, through a sternotomy, an extra-anatomical by-pass with a Dacron prosthesis soaked in Rifampicin, between the ascending and descending aorta through of diaphragm, with re-implantation of the left common carotid, without the use of cardiopulmonary bypass (CPB), closed the sternotomy, left thoracotomy was performed with removal of the prosthesis and all the infected tissue. Conclusion: Infections in thoracic endoprostheses can be approached safely and with good results in a single procedure, without the need of CBP. 109991 Modality: E-Poster Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY SERGIO ALEXANDRE PEREIRA GONÇALVES1, Talita Nolasco Loureiro1, Jessica de Cassia dos Santos Peloso1, Alice Pereira Duque1, Raquel Tavares Boy1 (1) ¹Hospital Universitário Pedro Ernesto (HUPE/UERJ), Rio de Janeiro, RJ, Brazil. Introduction: Primary cardiac tumors are rare, with a prevalence range of 0.001%–0.03%. Rhabdomyomas are the most common type of cardiac tumors in children. They are often associated with tuberous sclerosis complex and can cause right or left ventricular outflow tract obstruction, severe heart failure and arrhythmias. Everolimus, an inhibitor of mammalian target of rapamycin (mTOR), is suggested to be a promising therapeutic strategy for the involution of rhabdomyomas. Case report: Male newborn (NB), diagnosed with multiple rhabdomyomas by fetal echocardiography, born by cesarean section, gestational age 39 weeks and 3 days, weight 3,405g, head circumference 36 cm, length 50 cm, APGAR 5/9. NB required positive pressure ventilation, orotracheal intubation in the delivery room and received care from neonatal intensive care unit. Thereafter, NB was extubated on the same day and maintained on non-invasive ventilation. On echocardiography: multiple rhabdomyomas, left ventricular (LV) enlargement with severe dysfunction and heart failure. During hospitalization, dobutamine, captopril and digoxin were administered. Everolimus 4.5 mg/m²/day beginning on third day (D3) after birth with clinical, laboratory and echocardiographic tumor involution follow-up and monitoring side effects. Hypochromic macules, presence of astrocytoma in fundoscopic examination and central nervous system tumors in magnetic resonance imaging determined the Tuberous Sclerosis Complex diagnosis. NB presented a 71.2% reduction of cardiac tumor in the LV (7.64 cm² vs. 2.20 cm²) and a 248% increase in ejection fraction (EF) determined by Simpson’s method, from D3 and after 12 weeks of Everolimus use, respectively. After hospital discharge, Everolimus was discontinued due to the restriction of purchasing high-cost drugs. After 13 weeks of Everolimus interruption use, the cardiac tumor showed an increase of 98% (4.37 cm²) but without reduction in EF (53.8%). Currently, the patient remains using captopril (0.08 mg/kg/day) and digoxin (0.01 mg/kg/day) daily and followed-up by pediatric cardiology. Conclusion: Despite being in a phase II clinical trial, Everolimus still improved the involution of cardiac tumors, providing clinical improvement of the heart failure condition and reducing the risks of an extended hospitalization. 110333 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES JEAN MAX FIGUEIREDO1, Tulio Rangel Cardozo2, Caroline Roberta Torquato2, Daniel Xisto Ferreira Lordani2, Otávio Henrique Correa Siqueira Chagas2 (1) Universidade Iguaçu; (2) Hospital Municipal de São João de Meriti; (3) Hospital Geral de Nova Iguaçu Male, 72 years old, with oppressive chest pain 2 hours before arrival at the emergency department. History of myocardial infarction 6 years ago, hypertension, smoking, and diabetes. ECG showing ST elevation on D3 without ST elevation on D2 or aVF, ST segment depression with terminal positive T wave in V4 and V5. V1 with ST segment higher than in V2. This electrocardiographic pattern was first described by Aslanger in 2020 and indicates right coronary occlusion in the presence of multivascular atherosclerotic coronary artery disease. The coronary angiography performed after one week showed occlusion of the right coronary artery and severe multivessel lesions including the left main coronary artery. We present here a case report in which the patient presented with occlusive myocardial infarction without the classic electrocardiographic expression of ST-segment elevation in two or more contiguous leads. Two questions need to be answered in the future: 1- In the emergency department, should patients with a clinical picture of acute coronary syndrome and electrocardiographic patterns highly suggestive of coronary occlusion, such as Aslanger or De Winter, be promptly thrombolysed when emergency coronary angiography is not available? And 2- Is it time to change the nomenclature from acute myocardial infarction with ST-segment elevation and acute myocardial infarction without ST-segment elevation to, respectively, occlusive myocardial infarction (OMI) and non-occlusive myocardial infarction (NOMI)? References 1-Aslanger E et al. J Electrocardiol. 2020. 2-Meyers HP et al. Int J Cardiol Heart Vasc. 2021. 110150 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM NÁGELA SIMÃO VINHOSA NUNES1, Joelma Dominato1, Paulo Roberto Benchimol Barbosa1, Alfredo de Souza Bomfim1 (1) Complexo Hospitalar de Niterói; (2) Hospital Universitário Antônio Pedro Background: Reflex syncope is the most common cause of syncope in all age groups and the classic triggers are orthostatic stress, pain, fear and phobia. Cough is an uncommon trigger and when it triggers vagal reflex with syncope it is classified as situational syncope. We report a case in which asystole followed by syncope was always triggered by cough or tracheal aspiration in a patient with critical COVID-19. Case: Female, 40 years old, hospitalized for 40 days with COVID pneumonia, tracheostomized and attached to respirator. She was on daily hemodialysis due to acute renal failure. The cardiology service was contacted to assess recurrent asystole followed by syncope that occurred during airway aspiration or coughing, without concomitant drop in oxygen saturation. Baseline electrocardiogram was normal. 24-hour Holter and continuous telemetry showed numerous sinus pauses of up to 25 seconds (Figure). Beta-blocker had recently been introduced and she was on continuous dexmedetomidine infusion. Staff was oriented about the need to avoid hypovolemia, suspend beta-blocker and dexmedetomidine and aspirate the airway with a transcutaneous pacemaker turned on demand, until the events were controlled. In the following days, after these measures, no new episodes of asystole were recorded. Discussion: The causal mechanism of the pauses in this case is compatible with the cardioinhibitory vagal reflex triggered by the Valsalva maneuver due to persistent cough or tracheal aspiration. In the initial phase of the Valsalva maneuver, there is an increase in intrathoracic pressure, that is reflected to vessels in the neck, in addition to a drop in venous return to the heart, which can cause both stimulation of vessel baroreceptors and cardiac mechanoreceptors (Bezold-Jarish reflex). The occurrence of overshoot pressure in the final phase of Valsalva maneuver also can trigger vagal reflex, leading to a reflex cardioinhibitory situational syncope. Recognizing the triggers (hypovolemia and Valsalva maneuver) followed by withdrawal of drugs that can potentiate both triggers and the vagal reflex (dexmedetomidine and beta-blockers) may be essential for the resolution of similar cases in critically ill patients infected with SARS-CoV2, during the COVID-19 pandemic. 110845 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES ASHOK TAHILYANI1, Dr Hannah Gower2, Dr. A. Kotecha2 (1) Royal Devon Universoty healthcare NHS foundation Trust.; (2) Royal Devon and Exeter Hospital Background: Spontaneous coronary artery dissection (SCAD) is an uncommon but important cause of non-atherosclerotic acute coronary syndromes (ACS) and sudden cardiac death[1]. There is very little data that exists in regards to patient clinical characteristics, risk factors, treatment and outcomes. SCAD can be easily missed, as patient characteristics and management differ substantially from typical ACS cases[1]. Results of revascularisation with percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are suboptimal1. Conservative management is the preferred option with spontaneous healing of the dissection in the majority of uncomplicated cases[1]. Case Presentations: We present an unusual case of an active 61-year-old male who presented to a local district general hospital with worsening continuous chest pain for 24 hours and arm stiffness, following a week of indigestion-like chest pain. He has noticed increasing fatigue and shortness of breath with exertion in the preceding 7 days. Coronary angiogram revealed triple vessel SCAD of the left anterior descending (LAD), left coronary circumflex (LCX) and right coronary artery (RCA) with long segments of severe, smooth, tubular narrowing, with distal vessels appearing to be normal calibre and disease free. No PCI was attempted. The diagnosis was confirmed with CT coronary angiogram (CTCA). Conclusion: This case highlights an unusual case of triple vessel SCAD, which emphasises the need to recognise SCAD as a cause for ACS presentations as the management differs from classical atherosclerotic ACS cases. It also highlights that whilst management is preferably conservative, revascularisation options may be indicated and PCI is the preferred revascularisation option. However, it is associated with significant challenges and has low reported success rates of <50%. 111456 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY LUIZ MARCELO CRUZ1, Davi Dorval Pereira Cordova1, Mariana Ribeiro dos Reis Arruda1, Márcio Costa Silveira de Ávila2, Alexandre David Ribeiro3 (1) Hospital Tereza Ramos; (2) Clínica Pulsar; (3) Clínica Neurocor Cardiac tumors are rare and 80–90% are benign in adults. About half are myxomas, especially in the fourth and sixth decades of life with a slight predominance in women (1.5:1). May be asymptomatic or it can present with respiratory and embolic symptoms such as pulmonary thromboembolism (PTE) if they are in right chambers and also sudden death. Woman, 43 years old, obese, smoker, pulmonary emphysema, deep vein thrombosis in 2016, COVID negative, hospitalized due to pleuritic pain, hemoptysis and hypoxemia one month ago. CT angiography showed involvement of the left pulmonary artery and its branches to the left lower lobe. Echocardiogram showed pulmonary hypertension, enlarged right chambers and supposed thrombi in the right ventricle (RV). Despite anticoagulation, there was no reduction in lesions in RV, neither with fibrinolysis with alteplase. Cardiac resonance showed RV insufficiency and isointense lesions, without enhancement, irregular contour, the largest with 24 × 12 mm in RV output, 28 × 8,5 mm in papilar musculature region of the tricuspid valve and 17 × 4,7 mm in septum, raising hypothesis of myxomas. PET-CT showed no contrast uptake by the lesion. Due to probable benignity of the lesion, the patient opted for conservative treatment and evolutionary control, discharged with home oxigen and warfarin. One year later, exams showed no lesions growth. Although rare and generally benign, cardiac tumors should be part of the differential diagnosis of PTE. 111538 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY DIEGO RAUL ROMERO CAWEN1, diego romero cawen1, eduardo schlabendorff1, vanessa santos dos santos1, euler roberto fernandes manenti1 (1) hospital mãe de deus; (2) instituto de medicina vascular mãe de deus One of the purposes of the cardio oncology team is to provide medical solutions for the oncological patient that enables him to receive the most indicated treatment for his pathology. Patients with heart disease also need optimal cancer treatment and cancer diagnoses. When cardiac surgery is indicated, hard decisions must be made and a multidisciplinary point of view promotes a qualified opinion. We present a case of a 50 year old male with a history of pain and paresthesia of upper and lower limb extremities, defined as sensory-motor non demyelinating polyneuropathy. He had a medical history of type 2 diabetes mellitus and hypertension. In April of 2018 he was hospitalized due to a severe aortic stenosis with surgical indication. Cardiac catheterization showed normal coronary arteries. In the preoperative evaluation a serum monoclonal peak was seen in proteinogram. Medullogram revealed 1.5% of non-clonal plasma cells. His whole body MRI showed an expansive lesion located in the proximal diaphysis of the right tibia, measuring 7.0 × 2.0 cm. Biopsy findings indicated a plasmacytoma. VEGF levels were 1294 ng/l indicating POEMS. He underwent radiotherapy and received dexamethasone, with a non satisfactory response. Sternum and fifth dorsal vertebra infiltration were detected in PET-SCAN. Systemic treatment was indicated and at this time patient was symptomatic from his AS. He suffered an acute pulmonary edema during mediastinal biopsy. His echocardiogram revealed a bicuspid aortic valve with severe aortic stenosis. After consultation with the heart team, the patient was judged to be an EuroScore II intermediate. We therefore decided to perform TAVI to reduce the interval between AS treatment and cancer treatment, preventing malignant tumor progression. In addition, benefits of TAVI have been reported in cancer patients because it does not require cardiopulmonary bypass. These include reducing the risk associated with tumor bleeding secondary to anticoagulant disorders and administration of anticoagulants, and reducing tumor dissemination with immunosuppressive and inflammatory effects of the cardiopulmonary bypass. After the procedure, the patient had a favorable evolution and a marrow transplant was performed. This point-of-care treatment was conducted due to the expertise of the multidisciplinary heart team. 111005 Modality: E-Poster Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT PAULO LEÃO ANDRADE1, Larissa Neto Espíndola2, Maria Cristina Costa de Almeida3, Maria Gabriela Costa de Almeida3, Fernando Carvalho Neuenschwander4 (1) Hospital São Vicente de Paulo, Itabirito-MG; (2) Hospital Santa Izabel, Salvador-BA; (3) Centro Universitário de Belo Horizonte; (4) Instituto Orizonti, Belo Horizonte – MG Case Presentation: A 17-year-old male, asymptomatic, amateur soccer player, sought a cardiologist after the sudden death of his maternal uncle at age 43 due to acute myocardial infarction, confirmed at necropsy. Apparently, he did not show any muscular, cognitive, cardiological or other system involvement. The electrocardiogram performed in the office met criteria for left ventricular (LV) hypertrophy and suggested the presence of ventricular preexcitation, such as the presence of delta wave and a short PR interval. Holter monitor showed preserved sinus automatism, ventricular preexcitation, infrequent ventricular extrasystole. Transthoracic echocardiogram showed hypertrophy of the middle and apical segments, suggesting the possibility of apical hypertrophic cardiomyopathy (HCM). He had normal LV ejection f n but incipient signs of systolic dysfunction (reduced longitudinal global strain(–13%). He underwent cardiac magnetic resonance which showed asymmetric HCM with areas of late enhancement in approximately 25% of the myocardial mass, in addition to increased thickness of the right ventricular free wall with small areas of late enhancement in its apical portion and signs of increased trabeculation. In view of the findings of HCM + ventricular preexcitation, we chose to perform a genetic test, which confirmed a mutation in the LAMP2 gene. Opted to implant an implantable cardioverter defibrillator for primary prophylaxis of sudden cardiac death. The patient is doing well, with no signs or symptoms of heart failure and no shock therapies to date. Discussion: Mutations in X-linked lysosome-associated membrane protein gene (LAMP2; Danon disease) produce a cardiomyopathy in young patients that clinically mimics severe HCM due to sarcomere protein mutations. However, the natural history and phenotypic expression of this rare disease is incompletely resolved and its identification may have important clinical implications. Early identification of Danon disease in male is extremely important for patient care because rapid clinical deterioration leading to cardiac death occurs in young men before 25 years of age, in general. Heart transplantation is the reliable treatment once the occurrence of heart failure and should be considered as early as possible due to its rapid progression. New hope is emerging with the possibility that gene therapy could change the course of this dreaded disease. 111024 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FABIO RODRIGO FURINI1, Carlos Antonio Abunader Kalil1, Anibal Pires Borges1, Oscar Ivan Lopez Leon1, Valter Correia de Lima1 (1) Santa Casa de Misericórdia de Porto Alegre Introduction: Pulmonary vein (PV) severe stenosis post atrial fibrillation (AF) catheter ablation is a rare event (<0.5% of treated veins) that can cause dyspnea, pneumonia, pleural effusion, hemoptysis, pulmonary hypertension and even death. Percutaneous stenting PV, instead of balloon angioplasty alone, is effective in order to reduce the chances of restenosis. Stents implanted with 8 mm or larger are related to better outcomes. Case Description: 60 y/o patient with AF since 2019 and two ablations of the arrhythmia. Three months after the ablation he presented with tiredness and CT scan which showed a pulmonary infiltration into the bronchus from the basilar left pulmonary trunk. The investigation ruled out neoplasia. A 3D reconstruction of the image showed the complete occlusion LIPV and a critical stenosis of the LSPV, with reference diameters of 15 mm on the last one. Therefore, we only address the LSPV. The procedure was performed in the cath lab by the interventional cardiology and electrophysiology teams. Invasive measurements of pulmonary pressures showed postcapillary pulmonary hypertension (mean arterial pulmonary pressure of 38 mm Hg and wedge pressure of 37 mm Hg) and the translesional pressure gradient measured by TTE Doppler US was 27 mm Hg on LSPV. Sequential dilatations were performed, and finally a 12 mm diameter balloon with a Palmaz Genesis XD 1019 was fully expanded at the lesion. Angiography showed an excellent angiographic result and the translesional pressure gradient by Doppler fell from 27 mm Hg to 5 mm Hg (See Figure). The patient was discharged from the hospital on the second day after the procedure. Conclusion: PV stenosis after AF catheter ablation is a rare and insidious event. Treatment can be challenging and require multidisciplinary expertise. 111030 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM INGRID LOUREIRO DE QUEIROZ LIMA1, RODRIGO FERNANDES CASTRO2, TALITA CARVALHO2, ANGELO BRUNO PAGOTO2, CARLOS ROBERTO ROHENKOHL EVANGELISTA SANTOS2 (1) UNIVERSIDADE FEDERAL DO AMAZONAS; (2) HOSPITAL DO CORAÇÃO FRANCISCA MENDES Background: Since the pneumonia outbreak in December 2019 in the province of Wuhan, China, the SARS-COV-2 infection has spread to hundreds of countries and emerged as a pandemic crisis. In addition to the fact that underlying cardiovascular conditions have a negative impact on the prognosis of these patients, the virus can also affect directly the cardiovascular system through myocardial injury, cardiac failure, arrythmia, myocarditis and shock. Hypercoagulability state is also described due to the SARS-COV2 infection. Series of patients with myocardial infarction without coronary artery disease were described and intracoronary thrombus was described as a possible cause. Case Report: A 58 y-o male was admitted in a public hospital in Amazonas/Brazil due to acute respiratory syndrome on May 11th, with low oximetry. He received oxygen support with nasal catheter O2 and remained stable for 8 days. On May 18th he presented a syncope without prodrome at the clinical ward. EKG showed a ventricular tachycardia, promptly reverted by electric cardioversion. The new EKG showed sinus rhythm and ST-elevation in inferior and lateral leads, immediately treated with thrombolytics. The coronary angiogram performed three days after the event showed coronary arteries without obstruction. Cardiac MRI evidenced transmural infarction in inferior and lateral walls. Discution: Myocardial injury is observed in some patients due to SARS-COV-2 infections and it can be caused not only by plaque rupture but also by inflammatory storm and massive citokyne release, coronary spasm, microthrombus or vascular an and endothelial direct lesion. The absence of coronary artery disease in the angiogram and the previous history of thrombolytic treatment of this patient points toward a possible intracoronary thrombus causing the myocardium infarction. The evidence of a transmural contrast enhancement in the cardiac MRI reinforces the diagnostic of myocardial infarction and helps differentiating it from a myocarditis. 112421 Modality: E-Poster Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS GISELLE LAURITZEN DUARTE1, Andre Sansonio de Morais1, Sergio Medeiros da Silva Junior2, Daniel Cavalcanti de Carvalho3, Flavio Roberto Azevedo de Oliveira4 (1) Hospital Dom Helder Camara (HDH); (2) Hospital Dom Helder Camara (HDH); (3) Hospital Dom Helder Camara (HDH); (4) Hospital Dom Helder Camara (HDH) Introduction: The central venous catheter is a device used in critically ill patients in emergencies and ICUs. Among the vascular complications, Pinch-off syndrome is related to puncture of the subclavian vein and occurs when the catheter is compressed between the clavicle and the first rib, causing laceration, fracture and embolization to the right chambers or pulmonary circulation, with high rates of serious complications, including death. We report the case of a patient with a finding of a foreign body in the coronary sinus during an echocardiogram performed in the context of heart failure decompensation. Case Description: SJF, male, 62 years old, with heart failure (HF) with reduced ejection fraction (EF = 29%), admitted to the hospital with decompensated HF profile B, underwent transthoracic echocardiography (TTE) with image being visualized irregular, mobile, hyperechoic in the right atrial cavity, measuring about 5 cm, which seemed to be attached to the interatrial septum, or coming from the coronary sinus, with mobile elements attached to the extremity – neoplastic mass? thrombus? catheter tip covered by thrombus? A chest tomography was performed and a linear image with high density located in the topography of the coronary sinus and right atrium was visualized, a nonspecific finding, which may be related to a foreign body (catheter fracture?). The case was discussed at a clinical meeting, and the diagnostic hypothesis of Pinch-off Syndrome was raised, with a probable etiology due to a fractured catheter in an internment performed 2 years ago in another Service. Anticoagulation was started and a hemodynamic approach was chosen. Before the procedure, a new TTE did not identify images suggestive of thrombi adhered to the catheter. The interventional procedure was uneventful, with rapid removal of the foreign body and confirmed to be a catheter tip. The patient evolved hemodynamically stable and was discharged asymptomatic with optimized treatment for HF. Conclusion: Pinch-off syndrome is associated with high rates of serious complications. The finding of an embolized catheter fragment on imaging is typical. The removal of the fragment is mandatory and the hemodynamic procedure is the first option. The thrombotic burden associated with the catheter initially made it difficult to define the nature of the fragment and data from the retrospective anamnesis were essential for the conclusion of the diagnosis. 111143 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA INÊS GONZÁLES1, Ana Inês Gonzáles1, Lucas Lemonie Zunino1, Josie Budag Matsuda1, Luis Otávio Matsuda1 (1) Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí (UNIDAVI) Introduction: Previous Systemic Arterial Hypertension in individuals affected by COVID-19 can lead to a worse prognosis, increase complication rates and reduce functional capacity. In view of this, exercise-based cardiopulmonary rehabilitation may be able to promote functional improvement. This study reports the case of a male patient, post infection by COVID-19, with a previous diagnosis of Systemic Arterial Hypertension, submitted to a cardiopulmonary rehabilitation program. Case Description: V.N.F, 24 years old, male, diagnosed with Systemic Arterial Hypertension prior to SARS-Cov-2 infection, referred to a cardiopulmonary rehabilitation program, due to fatigue and lack of physical conditioning in usual life activities 4 months post infection. Pre- and post-intervention assessment: Post-COVID-19 Functional Status Scale (PCFS), 1-minute Sit and Stand Test (SST1), 2-minute Stationary Gait Test (2MWT) and 6-minute walk test (6MWT). The rehabilitation program was carried out for 8 weeks, twice a week, based on: 1) aerobic exercises on a treadmill, 2) strength and muscular endurance exercises, developeded in a progressive and individualized way. Patient started the first session with continuous aerobic exercise for 10 minutes, at 3.5 km/h, without incline, reaching Borg 4, heart rate of 119 bpm (61%) and muscle strengthening exercise in upper limbs with an elastic band, reached Borg 3 for dyspnea. Patient completed 100% of the sessions performing, in the last session, aerobic exercise in running for 20 minutes, at 6.5 km/h, without incline, final Borg of 0.5 with heart rate of 148 bpm (75%) and 8 minutes in circuit associating exercise bike (load 4) interspersed with squat on a trampoline, with final Borg of 0. After 8 weeks, the following data were evidenced: PCFS (Grade 1 vs Grade 0), SST1 (18 repetitions/final Borg 5 vs 23 repetitions/Borg end 0), 2MWT (66 elevations/Borg end 5 vs 94 elevations/Borg end 0), 6MWT (510 m vs 552 m), respectively. The patient completed 100% of the intervention sessions. Conclusions: The results show that performing cardiopulmonary rehabilitation for 8 weeks was able to improve the functional status of a young adult individual with arterial hypertension and symptoms of post-covid syndrome. 111266 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR IMAGING ERIVELTON ALESSANDRO DO NASCIMENTO1, Fernando de Amorim Fernandes1, Zhuo He3, Weihua Zhou3, Claudio Tinoco Mesquita1 (1) Universidade Federal Fluminense – UFF; (2) Instituto Estadual de Cardiologia Aloysio de Castro – IECAC; (3) Michigan Technological University – MTU A 66-year-old female with ischemic heart failure had an ejection fraction (EF) value of 14%, with left bundle branch block, and was considered functional class IV despite optimized therapy. Subjected cardiac resynchronization therapy (CRT) with left ventricular (LV) lead positioning targeted by the last viable segment was determined by GATED SPECT. After CRT, the EF value was 57%. LV contraction was successfully analysed by radionuclide ventriculography at first, however, the addition of GATED SPECT phase analysis and its subsequent validation, has shown excellent potential in determining left ventricular mechanical dyssynchrony. Studies that report on the placement of the LV lead are consistent with the findings of the last contracting segment identified by GATED SPECT phase analysis and show significant clinical improvement. Recent VISION CRT data demonstrated that dyssynchrony improvement, but not target lead placement, predicts clinical outcomes in CRT. Parameters of the LV geometry obtained by GATED SPECT phase analysis were able to predict a super-response to CRT. This report demonstrates a super-response with a significant change in the LV eccentricity index and LV shape in both LV end-systole and end-diastole, denoting reverse remodelling after CRT target by GATED SPECT. 111276 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY VANESSA SANTOS DOS SANTOS1, Eduardo Schlabendorff1, Diego Romero Cawen1, Euler Fernandes Manenti1 (1) Hospital Mãe de Deus (HMD) Cardiac amyloidosis has been historically difficult to diagnose due to lack of specific symptoms and necessity of biopsy. In the oncologic population this becomes even harder, since there may be multiple confounding factors related to the essential neoplasm and its treatment cardiotoxicities. We present a case of a 72 year old male that developed symptomatic supraventricular arrythmias and congestive heart failure (CHF). He followed with repeated episodes with hemodynamic instability. Attempts to ablate the arrhythmia were unsuccessful and because he had a left atrial tachycardia, we chose to ablate the atrioventricular node and implant a dual chamber pacemaker in the deep interventricular septum. To rule out ischemic heart disease, he underwent a cardiac resonance which showed signs of global left ventricular systolic dysfunction due to diffuse hypokinesia with an ejection fraction of 35% and extensive areas of discontinuous delayed enhancement, predominantly mesocardial and subepicardial (non-ischemic pattern), diffusely compromising the left ventricle. These findings suggested an inflammatory process of the myocardium (post-infectious or drug related) or an infiltrative cardiomyopathy – presumably amyloidosis, although not a typical imaging presentation which usually appears with predominance in the subendocardial areas. Amyloidosis was pursued and after ruling out light chain amyloidosis by laboratory tests, he performed a pyrophosphate scintigraphy. The result was a heart to contralateral (H/CL) ratio of 1,7 (a ratio of >1,5 is considered to strongly suggest transthyretin amyloid deposition). He also performed genetic testing and we are waiting the identification of the mutation. His treatment for CHF was optimized and he was discharged with improved symptoms. His past medical history was notable for a non-Hodgkin’s lymphoma diagnosed in 1983 and treated with chemotherapy, which included an anthracyclic, and radiation therapy of the mediastinum. In 2013 he was diagnosed with a prostate cancer and underwent prostatectomy, followed by androgen deprivation therapy (ADT) with an GNrH antagonist, also with known cardiotoxicity. All of these aggressors may have contributed to the ventricular dysfunction and appearance of arrythmia, presumably being of multifactorial etiology. But we highlight the need to pursue the diagnosis of amyloidosis as the correct identification of this disease is crucial for the initiation of potentially life-saving treatments. 111305 Modality: E-Poster Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CARLOS MANOEL DE CASTRO MONTEIRO1, Laís Olivo Rossi1, Luísa Carvalho Benedito1, Raul Cordeiro Pessanha1, Mireya Mendonza Rodrigues1 (1) Hospital Samaritano Paulista Introduction: Heart failure (HF) is the end of many diseases that affect the heart, which explains its increasing prevalence. Restrictive cardiomyopathy is the least prevalent form of cardiomyopathies. This case report aims to discuss restrictive cardiomyopathy, addressing a rare etiology that is desmin deposit. Case description: Male patient, 26 years old, with cardiomyopathy diagnosed 3 years ago, evolving for 2 months, with swelling in the lower limbs and progressive dyspnea. He was admitted to our institution with dyspnea on minimal exertion and signs of low cardiac output, being diagnosed with HF profile C. Family history: father, aunt and paternal cousin have cardiomyopathy. The ECG showed atrial tachycardia with RBBB and the echo showed significant increase in both atria, slight increase in both ventricles and significant diffuse involvement of the RV and LV with EF = 45%. Magnetic resonance imaging (MRI) of the heart showed late non-ischemic enhancement. Given the clinical picture, family history, echocardiogram and “MRI”, genetic testing was indicated. The complete exome sequencing identified a heterozygous pathogenic variant in the “DES” gene related to a restrictive cardiomyopathy secondary to the accumulation of desmin, which is the most important intermediate filament of skeletal and cardiac musculature, functioning as a protein cytoskeleton. Cardiac manifestations can occur in the form of restrictive cardiomyopathy, conduction system disease, arrhythmias, and sudden death. The patient presented worsening heart failure, requiring vasoactive amines, “IAB” and “ECMO”, being listed on the heart transplant waiting list. Transferred to a transplant center, undergoing heart transplantation, however, in the postoperative period, he presented sepsis, progressing to death. In this clinical case, a pathogenic variant associated with autosomal dominant/recessive cardiomyopathy was identified. The genetic test elucidated the etiology of cardiomyopathy, the refractoriness of heart failure to clinical treatment and optimized hemodynamic support, included the indication for heart transplantation (HT). Conclusion: This clinical case reports the importance of genetic tests in the etiological diagnosis of cardiomyopathies and “HT” is considered a therapeutic option in patients with advanced and refractory HF. 111327 Modality: E-Poster Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MIGUEL MEIRA E CRUZ1, Mauricio Machado da Rosa1, Michele Gomes da Rosa1, Dario Acuña Castroviejo2, Amélia Feliciano3 (1) Centro Cardiovascular da Universidade de Lisboa, Lisbon School of Medicine, Lisbon, Portugal; (2) Centro de Investigación Biomedica, Universidad de Granada, Granada, Spain; (3) Faculdade de Medicina, Universidade Católica, Lisbon, Portugal Introduction: Daylight saving time (DST) imposes a twice-yearly hour shift. This circadian challenge has been associated to detrimental health effects including an increased incidence of acute myocardial infarction following the springtime transition and increased ischemic stroke following both DST transitions. While cardiovascular consequences of DST are still controversial, the possibility that DST may impact metabolic disorders was never discussed before. We aimed to investigate a possible relationship between cardiovascular and metabolic acute events and DST transitions. Sample and Methods: All acute cardiovascular and metabolic events occurring during the 3 days before (pre transitional period) and after (post transitional period) the spring and the autumn shift were extracted from the records of two main hospitals in Lisbon, Portugal. The variation between the number of cardiovascular and metabolic events observed in the post transitional and pre transitional periods from both ocasions were statistically compared. Results: From the total of 492 patients (47.2% males) recurring to the hospital either during 2018 spring time transition or same year autumn time transition, with a mean age of 70.36 ± 16.1 years old, 89% presented with cardiovascular events whereas 11% presented with metabolic events. The variation amplitude from the 3 preceding days to the 3 subsequent days was +8.33% for cardiovascular and +50% for metabolic events in the spring transitional period. The same variation in the autumn transitional period was +16.13% and –65%, respectively. Conclusion: Results from this preliminary study corroborate the belief that a conflict between DST and cardiovascular health could be expected in both DST transitional periods although with important seasonal differences. Further, a relevant impact on metabolic status was also showed for the first time. While these results may reflect an impact of the circadian misalignment on cardiometabolic functions, the clinical meaning of such interaction may rely on further complex interaction with circannual oscillations from seasonal timing systems for which future exploration is warranted. 111419 Modality: E-Poster Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ANA AMARAL FERREIRA DUTRA1, Isabelle Mendes Rodrigues Salomão1, Luanna Damasceno Amaral de Sousa1, Louise Freire Luiz1, Bruna Pereira de Mendonça1 (1) Hospital Pró Cardíaco Introduction: Spontaneous coronary artery dissection (SCAD) is a rare cause of acute coronary syndrome (ACS), which can lead to sudden death. SCAD is an important differential diagnosis of chest pain and should be considered especially in young women with few risk factors for coronary artery disease (CAD). Case report: Patient, 63 years old, female, grade IV obesity and arterial hypertension, and positive family history of CAD. Admitted to the emergency department of a quaternary hospital with hypertensive emergency (BP 230 × 92 mmHg) associated with typical chest pain, at rest, radiating to the back and upper limbs, with no improvement and/or worsening factor. She reported an intense episode of anxiety the night before the pain. Physical examination showed no changes in cardiopulmonary auscultation, blood pressure was similar in the upper limbs and no lower limb edema. A loading dose of acetyl salicylic acid (ASA) was performed and nitroglycerin infusion was started. The admission electrocardiogram showed: sinus rhythm, ST infra of DIII and aVF. First troponin of 312 ng/mL (ultrasensitive) and other tests without changes. Bedside echocardiogram with preserved biventricular systolic function, with no change seen in the ascending aorta. The patient evolved with chest pain, refractory to clinical treatment, and therefore, an emergency hemodynamic study was chosen. Coronary angiography (CAT) showed spontaneous coronary dissection (diagonal artery). We opted for conservative treatment with dual antiplatelet therapy (ASA and Clopidogrel), high-potency statin and strict control of the dual product (heart rate and systemic blood pressure). Discussion/Conclusion: The incidence of SCAD is higher in young women and presents as ACS, it is extremely important to make the differential diagnosis of chest pain. It may be related to fibromuscular dysplasia, pregnancy, puerperium and hormone replacement. The approach to patients should follow the normal ACS protocol, but it should be taken into account that CAT can aggravate the dissection. Recent studies have shown benefits with conservative treatment in low-risk DEAC, as most cases evolved with spontaneous healing of the intramural hematoma after 30 days. 111501 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY EDUARDO SCHLABENDORFF1, Vanessa Santos dos Santos1, Diego Raul Romero Cawen1, Marcelo Haertel Miglioranza1, Euler Roberto Fernandes Manenti1 (1) Hospital Mãe de Deus de Porto Alegre Introduction: Tumor thrombus is a rare complication of testis cancer and needs appropriate treatment including chemotherapy and surgery. Case report: A 23-year-old man was hospitalized in October 2019 with epigastric pain, dyspnea, tachycardia and tachypnea without hypoxemia. The EKG showed a S1Q3T3 pattern. Laboratory revealed mild anemia, leukocytosis, normal troponin and BNP levels. D-dimer levels were extremely high. Point-of-care echocardiogram followed by a 3d echocardiogram showed a large mobile mass compatible with a thrombus inside the right atrium that incursed towards the right ventricle in diastole. CT angiography revealed a probable tumor thrombus originated in the left renal vein, entering the inferior vena cava and ascending into the right atrium. There is also an increase in volume of the left testis and a voluminous expansive mass next to the left renal hilum most likely associated with lymph node conglomerate. Anticoagulation was started even though CT scan was inconclusive for pulmonary embolism. The next day, the patient presented clinical worsening with signs of low output. Due to the high chance of response with cytotoxic chemotherapy in testicular tumors, chemotherapy with Bleomycin, Etoposide and Cisplatin (BEP) was guided by the oncologist. The patient underwent a left radical orchiectomy. After 4 courses of BEP chemotherapy the tumor markers normalized. A surgical team composed of cardiovascular, urological and oncological surgeons performed a triple surgery at the same surgical moment to remove the tumor thrombus using extracorporeal circulation (figure above). It was also performed left nephrectomy and retroperitoneal and pelvic lymphadenectomy. Advanced testicular cancer was diagnosed with a clinical stage of pT2-Nx-MO-S3, which has a poor prognosis. The pathological examination revealed a mature teratoma. The patient has been disease-free since surgery. Conclusion: A multidisciplinary approach is necessary to treat patients with tumor thrombus secondary to advanced testicular cancer. 111659 Modality: E-Poster Researcher – Case Report Category: NURSING JOSELITO ADRIANO DA SILVA1, Nathalia Pereira1, Paola Begliomini2 (1) Allm Inc; (2) Boehringer Ingelheim Introduction: Acute Myocardial Infarction (AMI) has a high rate of morbidity and mortality in Brazil and worldwide. In 2017, DATASUS recorded that AMI was responsible for 10% of hospitalizations in the Unified Health System (SUS) and 7% of all deaths in the country. In view of the magnitude of the disease and the benefit of the treatment being time-dependent, in 2019 the Sprint Project was started: a public-private collaboration initiative aimed at optimizing care for patients who enter the service with AMI with supra of ST through the development of care networks in SUS units, promoting rapid diagnosis and the appropriate choice of therapeutic strategy in a timely manner. Methods: This is an experience report on the role of the nurse as a Scientific Educator in the Sprint Project, which acts as an interlocutor and facilitator of information within the public service of care and manager of the results that are obtained through the use of a mobile application. that allows communication between the services involved, for example UPA and hemodynamics. Results: Currently the project is in 4 states and 06 cities promoting the integration between emergency services and the reference hemodynamics center, as agreed with the municipal/state health departments. With a strategic role, the nurse who works as a Scientific Educator must have experience in the care of patients with AMI and will actively work with the leader (“Champion)” of the network and the services involved, supporting the structuring of the care network in all areas. the phases. Project actions include: project presentation, diagnostic evaluation of the service from the physical structure to the service flow, analysis of improvement opportunities, construction of a multidisciplinary plan, training and supply of tools according to needs, periodic monitoring of networks and management of quality of care indicators based on the European ST-supra AMI Guideline. Conclusions: It is concluded that the role of the Scientific Educator nurse is strategic and visionary, being considered by the care network as a partner to assist in the organization of the AMI care flow, strengthening the bond with the services involved, thus minimizing delays in treatment. and reducing mortality from AMI with ST elevation. The nurse’s role is, therefore, fundamental in the periodic maintenance of the project, reducing errors, engaging the professionals at the end and contributing to improve the journey of the patient with AMI. 111696 Modality: E-Poster Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CAMILA RICHTER1, Priscilla Vicente Lista1, Carlos Alberto Kenji Naashima1, Jessica Woehl1, Thammy Lethicia de Sousa Silveira1 (1) Hospital Angelina Caron Introdution: Infective endocarditis (IE) is a serious disease that requires treatment by a multidisciplinary team, involving cardiologist, infectious disease specialist and cardiovascular surgeon, as well as neurologists in cases of involvement of neurological events. Case Report: DB, 39 years old, male, no previous diseases. No vices. The patient was admitted in-hospital with fever, headache, evolving into mental confusion and decreased level of consciousness and strength in legs. Moreover, the cardiac auscultation presented a more audible systolic murmur in the mitral focus. The cranial tomography revealed right frontal and left temporal ischemia (figure 3). The sorologies were required and were negative for the hepatitis B, C, Human Immunodeficiency vírus (HIV) e syphilis. Meningitis was ruled out after liquor evaluation. Janeway lesions were observed in the toes (figure 4). A bedside investigation was performed with Phelcomâ portable retinograh and Roth spots were identified (figure 1). Blood culture was positive in less than 24 hours for Staphylococcus aureus. Transesophageal echocardiogram confirmed suggestive image of mitral vegetation on the atrial face (figure 2). The mitral valve was replaced by a biological prosthesis. Conclusion: This case report showed the critical course of the IE, presenting with the worst possible manifestations for the disease. The surgical treatment is the principal recommendation for this case. 111728 Modality: E-Poster Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CLAUDIO QUERIDO FORTES2, Paula de Medeiros Pache de Faria1, Ilan Gottlieb1, Luiz Antônio Carvalho1, Valdo José Carrera1 (1) Casa de Saúde São José; (2) Hospital Universitário Clementino Fraga Filho – Universidade Federal do Rio de Janeiro Introduction: Infective endocarditis (1E) with negative blood cultures represents a small percentage of the cases, however, in many of these episodes it is possible to identify the microorganism causative through serological and/or molecular methods. Case Report: A 65-year-old male with a bicuspid aortic valve, diagnosed with 1E with persistently negative hemocultures, complicated by aortic paravalvular abscess evidenced by transesophageal echocardiography (TEE) and by aortic angiotomography. Empiric treatment with ampicillin plus sulbactam associated with ceftriaxone was started and valve replacement by biological prosthesis was performed. After 6 weeks on antibiotics therapy, he was discharged. Three months later fever returns and after an extensive investigation PCR for Coxiella burnetii was positive in blood and in the specimen from hepatic biopsy and IgG phase I, II and IgM phase I was higher than 1;1024. Treatment with doxycycline and hydroxychloroquine was started. At that time TEE and angiotomography evidence an abscess which, in few days, resulted in pseudoaneurysm. As Eurocore was too high and there were technical difficulties for a reoperation it was chosen to use a vascular plug with which a successful closure of the communicating orifice was achieved. Patient was discharged with oral treatment and after 4 months of clinical and echocardiographic follow up he is still doing well without any sigh of recurrence. Conclusion: When the Euroscore is too high and the surgical procedure almost impossible to be performed, the use of devices such as the vascular plug might be a plausible alternative, as it was for the patient here reported. 111773 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY ROBERTO ABDALLA FILHO1, Hugo Bertipaglia1, Cledicyon Eloy da Costa1, Maurício Marson Lopes1, Gustavo Frigieri2 (1) Hospital Samaritano de Campinas, Campinas, SP, Brasil; (2) Brain4care, Brasil The connection between cardiac alterations and brain function is an area still under inquiry. The use of tools for neurological monitoring of patients with heart dysfunction may represent a possibility to achieve better outcomes. This report will discuss the use of a new non-invasive intracranial compliance monitoring tool for patients with heart dysfunction prior to and following transcatheter aortic valve implantation (TAVI). Case description: A 80-year-old patient with progressive fatigue with marked aggravation during the past year. The image tests showed aortic valve fibrocalcification with severe stenosis and severe atheromatosis in the aorta, that contraindicated de transfemoral access for TAVI and made the transapical access the eligible option. Intracranial compliance (ICC) monitoring was done prior to TAVI and post-procedure. Results: The patient showed improvement in the peak gradient, which went from 125 mmHg before the procedure to 14 mmHg after, and at the peak speed initially of 5.58 m/s and 1.88 m/s after TAVI. The ICC of the patient presented values altered before TAVI, with a Ratio of P2/P1 of 1.38 and time to peak of 0.27, values suggestive of intracranial hypertension. After the procedure, ICC was normalized, with a P2/P1 ratio of 0.71 and a time to peak of 0.14 (p < 0,001). Conclusion: The noninvasive system was able to show alterations in intracranial compliance with cardiac alterations, as well as improvement after TAVI. 111787 Modality: E-Poster Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES NINA NOVAES AZEVEDO1, Bruno Daniel Ferrari1, Fernanda Corrêa de Oliveira Lima2, Flávia Corrêa de Oliveira Lima2, Victor Sandi Mori Salvador2 (1) Curso Intensivo de Revisão em Cardiologia Clínica – Rio de Janeiro; (2) Hospital Regional de Presidente Prudente – HRPP Introduction: Aortoesophageal fistulas (AEF) are rare and require rapid diagnosis. They represent a major therapeutic challenge because of their high morbimortality; 2/3 of these are a consequence of thoracic aortic rupture, resulting from aneurysms or traumas. Case description: M.E.M.R., 87 years old, female, from São Paulo countryside, was admitted with hematemesis to the Emergency Room in a private hospital in the area. Personal history included arterial hypertension and diabetes mellitus. On physical examination, cardiac and pulmonary auscultation showed no findings, no carotid bruits, Glasgow 15, no focal neurological deficit. After clinical stabilization, the conduction of Upper Digestive Endoscopy showed esophageal aortic fistula, which was non-communicating, thrombosed and without active bleeding. The computed tomography of the chest showed a thoracoabdominal aortic aneurysm of approximately 70 mm in diameter, with the presence of extensive intramural thrombus from the abdominal aorta to the ascending aorta trunk, without signs of acute dissection, with esophageal compression. Assessment through vascular surgery indicated no intervention with endoprosthesis, due to her advanced age. Because of her hemodynamic stability, a second analysis showed that intervention was recommended, and she was inserted in the Vacancy Regulation Center (CROSS). The day before the procedure, a new bleeding led to her death. Conclusion: AEFs are rare and fatal presentations of thoracoabdominal aortic ruptures. The patient’s advanced age was a decisive factor in indicating the intervention, together with her hemodynamic stability. Despite the high morbimortality of AEFs, the effectiveness of the entire system of early diagnosis, stabilization, and treatment is essential for a complete patient care, often being successful, and sometimes unsuccessful. 111820 Modality: E-Poster Researcher – Case Report Category: CARDIO-ONCOLOGY FLÁVIA CRISTINA CARVALHO DE DEUS 1, Aurora Felice Castro Issa1, Claudio Domênico Sahione Schettino2, Carlos José Coelho de Andrade3 (1) “Curso de Pós-Graduação em Cardiologia da SBC/INC/INCA, Rio de Janeiro, RJ, Brasil”; (2) Hospital Pró Cardíaco; (3) Instituto Nacional do Câncer – INCA Introduction: Mediastinal masses with pericardial effusions constitute a great challenge that shows the importance of the Cardio Oncology specialty. Case Description: S.R.P., 73 years old, female. Admitted to the emergency room with progressive dyspnea. Stable vital signs, but dyspnea with saturation 88%. Performance Status of 2. Laboratory with anemia, hypoalbuminemia and elevated C-reactive protein. Echocardiogram showed preserved biventricular function, strain – 16, presence of a mass anterior to the mediastinum (15 × 13 cm), pericardial effusion without restriction and bilateral pleural effusion. Angioresonance showed right paracardiac mass that insinuates into the right atrium and extends to the aorta. It was requested an Oncologist Opinion. FDG PET scan showed hyper uptake in the right mediastinal mass with SUV 24 (Standardized Uptake Value). Values greater than 2 are suggestive of malignancy. CT-guided liver biopsy was suggestive of Sarcoma. Doxorubicin 75 mg/m2 was started. The electrocardiogram was monitored for the risk of QT interval prolongation and serial measurements of troponin and BNP were performed. Immunohistochemical panel result changed the diagnosis to GIST (Gastrointestinal stromal tumors) and Imatinib 400 mg orally once a day was started. Genetic biopsy test did not show mutations. Follow-up CT scans and echocardiogram showed no response to Imatinib. She presented with severe headache after the tomography and sudden coma with ventricular bleeding and unfortunately evolved to death. GISTs are sarcomas of cells of the gastrointestinal tract (cells of Cajal). They have CD 117 surface markers (C-kit) involved in tumor growth. They are rare tumors, diagnosed incidentally, occur between 50 years old and are most frequent in the stomach and small intestine. Only 5% in other locations. About 30% progress to malignancy. The final diagnosis is based on immunohistochemistry and genetic tests. The treatment is surgical resection and therapy with tyrosine kinase receptor inhibitors. The first choice is Imatinib at a dose of 400 mg per day. Early tumors have a 90% survival at 5 years, but tumors larger than 11 cm and metastatic have a worse prognosis. Conclusion: Mediastinal masses with cardiac involvement are of high risk for patients. Although rare, GISTs should always be considered. The union between Cardiology and Oncology is essential for the best treatment in these cases. 111924 Modality: E-Poster Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MIGUEL MEIRA E CRUZ1, Ana Brito2, Verona Mihai3 (1) Sleep Unit, Centro Cardiovascular da Universidade de Lisboa, Lisbon School of Medicine, Lisbon, Portugal; (2) Guy’s & St Thomas NHS Foundation Trust, London, UK; (3) Manchester University Foundation NHS Trust, London, UK Introduction: Insomnia and obstructive sleep apnoea (OSA) are the two most common sleep disorders globally, and co-exist in up to 50% of patients in clinic. The risk of cardiovascular and metabolic disorders (CMD) is often independently associated with insomnia and OSA, and such risk seems to be aggravated when these conditions are comorbid (COMISA). Recent data shows that collaboration of psychology and physiology-based Sleep Medicine strategies may help improve patient-centric care. Case description: 54 years old male patient with diagnosis of resistant hypertension, complaints of snoring and difficulties in initiating and maintaining sleep underwent sleep investigations in our department. The sleep study performed was consistent with the diagnosis of severe OSAS. The positive airway pressure (PAP) therapy was initiated and the patient was 100% compliant with the therapy. The response was excellent, with a residual Apnoea and Hypopnea Index (AHI) of 1.3/h and improved Epworth Sleepiness Scale from 11 to 5/24. Despite these good results, the patient still had problems initiating and maintaining sleep, presenting an Insomnia Severity Index (ISI) score of 13. He was at that point included in a Cognitive Behavioural Therapy program for insomnia (CBTI). After CBTI intervention, the patient presented a residual ESS of 4/21 and an ISI of 3, indicating no signs of clinical insomnia. The compliance with PAP remained excellent and the patient reported improvements on sleep quality, therapy management, as well as overnight awakenings. The blood pressure became normalised at 120/80 mmH2O. Conclusion: Despite the high co-morbidity and impact of COMISA, there are many unknowns concerning the pathophysiology and optimal therapeutic management of this condition. The optimal use of PAP and tailored CBTI showed significant results not only on resolving the OSAS and Insomnia but also by reduction of cardiovascular risk, with blood pressure being normalised. Therefore, the combination of sleep apnoea therapy and of behavioural sleep therapies, such as CBTI, can be the solution to address this complex condition. 111950 Modality: E-Poster Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT RAFAEL MODESTO FERNANDES1, Luciana Cunha Weber1, Vitor Queiroz de Castro Souza2, Loren Lacerda Rodrigues2, Mariana Baptista Guedes1 (1) D’Or Institute for Research and Education (IDOR), Hospital Aliança, Salvador, Brazil; (2) Bahiana School of Medicine and Public Health, Salvador, Brazil. Background: Described by Fountaine and Marcus in 1982, arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease characterized by progressive replacement of myocardial cells by fibrofatty tissue. This is a leading cause of sudden arrhythmic death in young people and athletes. Case: A 72-year-old man presented with heartburn, without relief after use of antacids. He denied chest pain, dyspnea, history of drinking or smoking. Past medical history was positive for stable coronary artery disease, non-specified arrhythmia, hypertension and hyperlipidemia. Physical examination was normal. The diagnosis of non-ST-elevation myocardial infarction (NSTEMI) was established based on T wave inversions in inferior and anterior leads at electrocardiogram and an abnormal ultrasensitive troponin level. A pharmacological stent was placed in the mid-segment of the right coronary artery to treat a sub occlusive during the invasive coronary angiography. Transthoracic echocardiogram (TTE) revealed preserved left ejection fraction but severely compromised right ventricle (RV), not corresponding to angiography. Cardiac magnetic resonance imaging corroborated the findings of the TTE, segmental changes and RV aneurysms, in addition to the reduced RV ejection fraction. These findings were supportive of ARVC. Holter identified frequent ventricular premature beats, however without complex arrhythmias. After discussion with the Heart Team, the patient was classified as low risk, despite the important involvement of the RV. He was discharged and kept under outpatient follow-up without the need for implantable cardio defibrillators (ICD). Further screening was advised for family members. Discussion: Patients with ARVC usually present symptoms during the second to fifth decade of life. Advanced age poses a challenge for the diagnosis of the disease, as symptoms could be disguised as those of NSTEMI. In order to prevent misdiagnosis and improve risk stratification, more information is needed on the natural history of these patients. In this case, the decision to implant the ICD is challenging, considering the patient’s age, degree of RV involvement and absence of complex arrhythmias. 112096 Modality: E-Poster Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM DANIELLA CUNHA BRANDAO1, BRUNA THAYS SANTANA DE ARAÚJO1, JULIANA ANDRADE FERREIRA DE SOUSA1, Maria Inês Remígio1, Armèle Dornelas de Andrade1 (1) Universidade Federal de Pernambuco Introduction: The main limiting symptoms in the post-COVID-19 period are dyspnea, fatigue and reduced exercise tolerance. In these patients, cardiovascular rehabilitation can improve functional capacity, reduce deconditioning after a prolonged stay in the intensive care unit, and facilitate return to work. Objective: To verify the effects of cardiopulmonary rehabilitation consisting of aerobic and continuous resistance training of moderate intensity on pulmonary function, respiratory muscle strength, maximal and submaximal exercise tolerance, fatigue and quality of life in post-COVID-19. Methods: This is a quasi-experimental study with a protocol of 12 outpatient intervention sessions in a reference service for COVID-19 in Pernambuco. Adults over 18 years of age (N = 26) diagnosed with COVID-19 and discharged from hospital at least 15 days before the first assessment were included. Participants performed continuous moderate-intensity aerobic and resistance training twice a week. Maximum and submaximal exercise tolerance, pulmonary function, respiratory muscle strength, fatigue and quality of life were assessed before and after the intervention protocol. Results: Cardiopulmonary rehabilitation improved maximal exercise tolerance, with an increase of 18.62% in peak oxygen consumption (VO2peak) and 29.05% in time to reach VO2peak. The VE/VCO2 slope reduced by 5.21% after the intervention. We also observed an increase in submaximal exercise tolerance (increase of 70.57 meters in the 6-minute walk test, p = 0.001), improved quality of life and reduced perceived fatigue after the intervention. Conclusions: In this work, cardiopulmonary rehabilitation improved pulmonary function, respiratory muscle strength, maximal and submaximal exercise tolerance, fatigue and quality of life in patients with limiting symptoms in the post-COVID-19 period. Aerobic and continuous resistance training of moderate intensity proved to be effective in the recovery of these patients. 112015 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LUIS AUGUSTO PALMA DALLAN1, Marcelo Harada Ribeiro3, Cristina Silveira3, Gabriel Tensol Rodrigues Pereira1, Guilherme Ferragut Attizzani1 (1) University Hospitals Cleveland Medical Center; (2) InCor – Instituto do Coração – HCFMUSP; (3) SOS Cardio, Florianopolis, SC, Brazil Introduction: Patient 70 years old male caucasian, with multiple comorbidities, including hypertension, diabetes, obesity, dyslipidemia, CKD 3B (GFR 37, Creat 1.8), S/P prostate cancer, and severe AS (LVEF 55%, mean grad 50, Vmax 4.6, DI 0.19) with NYHA Class II symptoms including dyspnea with minimal exertion, worsening fatigue over the past 3 months, and intermittent chest pressure. In summary, the patient met indications for AVR. Coronary angiography pre-procedure showing a severely calcified lesion in proximal/mid/mid-distal LAD with angiographic moderate-to-severe lesions (60%). Per Heart Team discussion, elective FFR with possible PCI to LAD and elective TAVR. Procedure: In this case, assessment through FFR, OCT and Intravascular Lithotripsy contributed to simplify a potential high-risk procedure. FFR confirmed the severity distally to sequential lesions and indication for PCI. The images evidenced by OCT completely changed the patient’s treatment. OCT was especially important to determine the anatomy of the lesion and adequate planning. Evidence of heavy proximal circumferential calcification and severe lesion in mid-distal LAD that would not be assessed through angiography. If the proximal lesion was not pre-treated, probably would have resulted in underexpansion of the stent with higher rates of stent failure. As per evidence of severe lesion in mid-distal LAD, it mostly would not be assessed only based on angiography. In addition, OCT also allowed for adequate treatment planning, with pre-dilatation and ideal stent sizing, with a satisfactory final result evidenced by excellent stent expansion and without signs of dissection of the stent edges. Intravascular Lithotripsy contributed to simplify a potential high-risk procedure such as a rotational atherectomy in proximal LAD in a patient with severe AS and no possibility of protected PCI with Impella due to severe aortic stenosis. Three days after discharge, the patient was submitted to elective TAVR successfully. Conclusion: We report the importance of physiology and intravascular imaging on a successfully OCT-guided PCI using intravascular lithotripsy: FFR confirmed the severity of the lesions and indication for PCI; OCT provided important information for adequate treatment planning and execution of the procedure, minimizing the risk of stent underexpansion; and Intravascular Lithotripsy contributed to simplify a potential high-risk procedure. 112041 Modality: E-Poster Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ERIVELTON ALESSANDRO DO NASCIMENTO1, Romero Ribeiro Duque2, Vinícius de Queiroz Aguiar2, Fabrício Vilhena de Castro Souza2 (1) Instituto Estadual de Cardiologia Aloysio de Castro – IECAC; (2) Centro Universitário de Volta Redonda – UniFOA Holt Oram Syndrome (HOS) is characterized by the presence of upper limb abnormalities associated with cardiac malformations and cardiac conduction disorders, being an autosomal dominant hereditary disease, present in 1 in 100.000 individuals. Cardiac malformations, in general, are related to defects in the septation of the heart, with interatrial communication ostium secundum and interventricular communication being the most common. Non-Compaction Cardiomyopathy (NCC) is a rare condition characterized by increased trabecculation in one or more segments of the ventricles, especially the left ventricle. NCC can be found isolated or related to multiple phenotypes of cardiac structural alterations and arrhythmogenic alterations, however, the association with HOS presents a low number of case reports in the literature. We report a case of HOS associated with biventricular NCC. A 17-year-old male student with HOS and deformities in both upper limbs and previous atrioseptoplasty, was referred to the arrhythmology ambulatory complaining of “weak beats” and suspected NCC after performing a transthoracic echocardiogram. In the evaluation by the specialist, the patient had a heart rate between 42 and 45 beats per minute, and a 24-hour Holter and cardiac MRI were requested to assess bradycardia and suspicion of NCC. The 24-hour Holter presented periods of atrioventricular dissociation with bradycardic junctional escape. MRI showed increased endocardial trabeculate in the right ventricle and increased subendocardial trabeculation in the left ventricle (LV), with diagnostic impression of LV dilation associated with criteria of non-biventricular compaction, with preserved systolic function and absence of local fibrosis. Given the diagnosis of bi-ventricular NCC, the patient is followed up by the arrhythmology ambulatory to evaluate bradycardia with serial examinations. HOS associated with NCC presents a reduced number of reports in the literature, with presentations commonly affecting only the left ventricle. Thus, the case reported describes a patient diagnosed HOS related with bi-ventricular NCC, manifesting with atrioventricular blockade. 112175 Modality: E-Poster Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT RAONI DE CASTRO GALVÃO1, João Paulo Velasco Pucci1, Ofir Gomes Vieira1, Edvagner Sérgio Leite de Carvalho1, Carlos Eduardo Duarte2 (1) Centro de Ritmologia de Brasília; (2) CARE: Centro Avançado de Ritmologia e Eletrofisiologia; (3) Hospital Santa Lúcia Cardiac resynchronization (CRT) has been an established procedure for over 20 years. Recently, direct stimulation of the cardiac conduction system has been gaining strength. Recent studies already show them with results similar to traditional CRT. We report a case submitted to CRT with direct stimulation of the left bundle branch followed by correction of the right ventricular depolarization delay with stimulation of its region with the greatest delay, in inferior wall activation. Case Report: Female 77y, idiopathic cmp, LVEF 23%, increased LV diameters, diffuse hypokinesia and absence of fibrosis in the LV walls. ECG with LBBB and QRS 170 ms. Evolved with decompensated HF, recent hospitalizations. After clinical compensation, a CRT was implanted on 04/2021. First ventricular lead was implanted in the deep interventricular septum, after mapping the HIS bundle. During threshold test, there was selective stimulation of the LBB, with a RBBB morphological QRS, axis at 80° and width of 130 ms. By direct pacing of the left bundle branch, we took advantage of the other ventricular lead to correct the RV conduction delay generated by LBB pacing. After finding a delay of 90 ms, we chose to fix the second ventricular lead in the inferior wall of RV. Joint ventricular pacing resulted in a QRS of 108 ms, QRS axis –20°. Transthoracic echocardiogram at 45 days post-op showed an increase in LVEF to 48%, LV dimensions at 55/40 mm. At the moment the patient remains in NYHA-I. A 6 month ECHO-TT showed a LVEF 54% and preserved LV dimensions, patient remaining in NYHA-I. Discussion: This report proposes a RV-CRT to correct a RBBB QRS morphology generated by direct stimulation of the LBB. About 30% with CRT are non-responders. Whether due to a non-ideal LV lead position, unfavorable coronary sinus anatomy, phrenic stimulation, etc. In this sense, CRT by direct stimulation of the HIS-purkinje system corrects many adversities in a simpler procedure to be performed. In this case, complete correction of previous LBBB encouraged us to use the other electrode to correct the RV activation delay generated by the LBB stimulation, resulting in a narrower QRS, allowing clinical response and an important and early LVEF increase. This report corroborates the literature by showing that stimulation of the conduction system is a real alternative to patients with indication for CRT. 112185 Modality: E-Poster Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LUIS AUGUSTO PALMA DALLAN1, Luis Augusto Palma Dallan2, Cristina Silveira1, Carlos Augusto Campos2, Michael Megaly3 (1) SOS Cardio, Florianopolis, SC, Brazil; (2) InCor – Instituto do Coração – HCFMUSP, São Paulo, SP, Brazil; (3) Department of Cardiology, Willis Knighton Heart Institute, Shreveport, Louisiana, USA A 78-year-old man with previous coronary artery bypass surgery (CABG) 10 years ago presented for right coronary artery (RCA) chronic total occlusion (CTO) percutaneous coronary intervention (PCI) for medical refractory angina after prior failed attempt. Coronary angiogram patent vein grafts to left anterior descending (LAD) and ramus arteries with early septal collaterals to occluded RCA (Figure 1, A). RCA occlusion was ostial and severely calcified (Figure 1, B). After crossing the Fielder XTa wire (Asahi Intecc, Japan), the lesion could not be crossed with different balloons or microcatheters. Therefore, we knuckled a RotaWire floppy (Boston Scientific, USA) subintimally beyond the proximal cap and performed rotational atherectomy (RA) with 1.25 mm followed by 1.5 mm burrs at 180,000 rpm (Figure 1, C). We then could advance a Finecross microcatheter (Terumo, Japan) along with a Pilot 200 wire (Abbott vascular, USA). We then crossed retrograde through septal collaterals and performed guide-extension assisted reverse controlled antegrade-retrograde dissection tracking and reentry (reverse CART) (Figure 1, D and E) followed by completion of PCI and good final angiographic result (Figure 1, F). Hospital stay was uneventful and the patient was free from angina at 6-month follow-up. Our case describes the modification of ostial CTO lesions with RA after DR, which has not been described before. Calcification remains one of the major challenges during CTO PCI.1 Different modalities can be used to modify calcium including RA. RA use after dissection-reentry (DR) techniques within the main vessel have been previously reported, but still carries a very high-risk of perforation and is advised against. The implementation of DR techniques has played a key role to make possible wire crossing even in severe calcified occlusions and increase the overall success rate. Although with DR, the pathway is easier as you go around calcium, the vessels might remain non-compliant requiring calcium modification. For ostial modification of CTO lesions after DR techniques, the authors recommend using high-speed (180,000 rpm) with very short runs and mainly in straight segments. Our technique represents a bailout strategy after failure of other techniques. It still carries a high risk of perforation or aortic dissection and benefits should outweigh the risks when making the decision to proceed. 112316 Modality: E-Poster Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION NATHALIA DUARTE CAMISÃO1, MIGUEL ÂNGELO RIBEIRO1, ANDRÉ PAZOS TEIXEIRA1, NATÁLIA DOMINGUEZ PAES LEME DE SOUZA1, JOSÉ HERBERT DA SILVA PALHANO1 (1) Hospital Norte D’or Introduction: Vasospastic angina is a variant form of angina pectoris in which angina occurs at rest with transient changes in the electrocardiogram and exercise capacity preserved. It is involved in many clinical scenarios such as stable angina, sudden death, acute coronary syndrome, arrhythmia or syncope. Smoking is a well-known precipitating factor in this clinical setting. Case Report: Male patient, 58 years old, systemic arterial hypertension, smoker (smoking history 25 years – pack), in outpatient follow-up due to long-standing chest pain but with significant worsening in the last two months. Transthoracic echocardiogram (TTE) with preserved left ventricular systolic function, no segmental changes, no intracavitary overloads. Electrocardiogram with alteration of repolarization in the antero septal wall. Myocardial scintigraphy without perfusion changes, without ventricular dysfunction. Guided lifestyle changes and smoking cessation. Admitted to the ER 6 months later with oppressive chest pain radiating to the left upper limb, profuse sweating, associated with dynamic ST-segment change in the high lateral wall and increase in myocardial necrosis markers (ultrasensitive troponin peak of 38520 ng/L). He continued smoking. TTE similar to that performed previously, without significant changes. Coronary angiography was performed: significant vasospasm in the circumflex artery reversed after nitroglycerin, no obstructive lesions, ventriculography with infero apical hypokinesia. A calcium channel blocker was introduced with good clinical evolution without a new episode of chest pain during hospitalization. Conclusions: Although the pathogenesis of coronary artery spasm has not been fully elucidated, different pathogenic mechanisms have been proposed, such as vascular smooth muscle cell hyperreactivity, endothelial dysfunction, vascular inflammation, altered autonomic nervous system response, and oxidative stress. These variables can be modified if it’s possible to detect early precipitating factors such as smoking in this case. 107810 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARINA PASSOS PIZZITOLA1, Vinícius Nasser de Carvalho1, Pedro Luiz Homem de Mello1, Paula Santiago Teixeira1, José Roberto Pineda Pietrobon Redini Martins1, Filippo Mancini Diotto1, Bruna Regina Cogo1, Adriano Henrique Pereira Barbosa1, Iran Gonçalves1, Pedro Ivo de Marqui Moraes1 (1) Universidade Federal de São Paulo – Unifesp Introduction: The risk of new cardiovascular events after an episode of ST elevation myocardial infarction (STEMI) conditions patients to a high-risk profile and reinforces the need to expand epidemiological knowledge about the long-term follow-up in this group. Objectives: To evaluate the rates of all-cause and cardiovascular mortality, reinfarction and ischemic stroke in the late follow-up of STEMI patients treated by the pharmaco-invasive strategy (PhIS). Methods: Data from a county network for the treatment of STEMI from January-2016 to December-2019 were analyzed, containing 1,170 consecutive patients undergoing fibrinolysis (97% tenecteplase) in county hospitals and systematically transferred to the tertiary center for cardiac catheterization and continuity of care, according to the PhIS. Post-discharge follow-up was performed on an outpatient basis or via telephone call to those who were absent, with approval by the institutional Research Ethics Committee. Numerical variables were described as median and interquartile range. Results: The baseline demographic profile of the 1,170 patients evaluated was 348 (29.7%) women, age 59 [51–66] years, 654 (55.9%) hypertensive, 324 (27.7%) diabetics, left ventricular anterior wall infarction in 559 (47.8%), door-to-needle time of 68 [41–110] minutes and fibrinolytic-catheterization time of 16.5 [7.3–23.1] hours. In-hospital mortality was 60 (5.1%) cases. Loss of post-discharge follow-up occurred in 251 (21.4%) patients who were not located, so 919 were analyzed with a median follow-up of 2.6 [1.7–3.2] years. The overall mortality rate was 50 (5.4%), with 34 of the deaths (68.0%) attributable to cardiovascular causes, and the rates of reinfarction and ischemic stroke were respectively 56 (6.1%) and 15 (1.6%). Conclusion: Although the elevated loss of follow-up constitutes a limitation for the analysis, patients from a county network for the treatment of STEMI based on PhIS had a high risk of recurrence of cardiovascular outcomes. 110502 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GIOVANNA MENIN DA SILVA 2, Marcelo Aguilar Puzzi1, Luciano de Andrade1, Amanda de Carvalho Dutra1 (1) Universidade Estadual de Maringá; (2) Universidade Federal de São Paulo Coronary artery disease (CAD) remains the leading cause of death in women. The negative impact of CADs can be reduced through preventive care with interventions that address from the population to actions that have an individual character. Thus, combining information regarding the occurrence of CAD in women with the spatiotemporal variation constitutes an instrument that enables the development of strategies to identify areas of greater risk for developing the disease, the increase in risk over time and proposition of policies that favor interventions to reduce morbidity and mortality. Objective: To analyze the spatiotemporal distribution of mortality rates of adult women with CAD in the State of Paraná – Brazil from 2008 to 2017. Methodology: This is a descriptive, observational, retrospective study with the purpose of analyzing the spatiotemporal distribution of inequalities, with the use of geospatial health statistics, based on secondary mortality data in this period. Results: There is a correlation between the annual and spatiotemporal rates, demonstrating that the analyzed covariates lead to municipal inequalities, mainly, income, education and access to a medical specialist, showing that comprehensive health care can disadvantage the survival of these patients. SMR greater than 1, correlates with higher income (p = 0.381), higher education index (p = 0.056), higher health index (p = 0.115), higher number of cardiologists (p = 0.232), higher rate of exercise test (p = 0.056) is considered high risk, corresponding to 75%, 50%, 25%. The correlation between greater accessibility, greater number of cardiologists, greater exercise testing and greater health index in Paraná with 75% of a SMR with greater risk. Conclusion: Public health policies that serve everyone with equity, observing their loco-regional particularities should be encouraged and taken as a goal in the management of conflicts that affect women unequally. 107823 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING ANNA PAULA ARPINI BOTELHO1, Júlia Passarelli Pereira1, Raiana Andrade Quintanilha Barbosa1, Aniele Moritz1, Marcelo Goulart2, Glauber Monteiro Dias1, Andrea De Lorenzo1 (1) Instituto Nacional de Cardiologia Background: Patient exposure to ionizing radiation has grown due to increases in medical imaging, raising concern about potential risks. Among cardiac imaging tests, myocardial perfusion scintigraphy (MPS) is vastly employed. Overall, the link between radiation from MPS and cancer has not been considered consistent; however, further evidence obtained from studies at the molecular level is desirable. Objective: To study the effect of ionizing radiation on the DNA of patients undergoing MPS using the comet assay, a method for detection of DNA damage, and the activation of repair genes in response to potential DNA damage. Methods: Twenty-nine patients ≥18 years without acute diseases, recent surgery, cancer or autoimmune diseases were studied. MPS was performed with Tc-99 sestamibi (15–20 mCi) in a 2-day protocol. Peripheral blood was collected immediately before radiotracer injection at rest and 60–90 minutes after injection for the analyses. Single-cell gel electrophoresis (comet assay) was performed with blood lymphocytes to detect strand breaks, which determine a “comet tail” of variable size, visually scored by 3 observers in a fluorescence microscope after staining (0: no damage, no tail; 1: small damage; 2: large damage; 3: full damage). A damage index was calculated as a weighted average of the individual scores. The expression of repair genes CDKN1A, BBC3, XPC, GADD45a was evaluated with RT-PCR (QuantStudio 5, Thermo Fisher Scientific). Results: After radiotracer injection, there was a significant, although small, increase of the damage index and of classes 1–3 of damage, even though most patients remained in class 0 (Figure A: top: percentages of damage classes before and after radiotracer; bottom: plot of DNA damage index from individual patients). There was no significant difference in the expression of CDKN1A, BBC3, XPC, GADD45a after radiotracer injection (Figure B). Conclusion: DNA damage was detected in lymphocytes after a single radiotracer injection for MPS, without activation of repair genes. This suggests that MPS with the current protocols may not result in relevant DNA damage. 107829 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY CUSTÓDIO JOSÉ GASPAR1, Miracelma de Assunção Pedro Alexandre1, Amélia Carlos Cazalma1, Jordão Augusto Trajanno2, Luciano Júlio Chingui1 (1) Universidade Metodista de Angola (UMA); (2) Casa de Caminho André Luís Introduction: Malaria is a multisystem, non-contagious infectious disease caused by protozoa of the species Plasmodium falciparum, vivax, P. maleriae, P. ovale Stephens. Worldwide, malaria is characterized as the main cause of morbidity and mortality, in Angola malaria is caused mainly by Plasmodium falciparum, that is, 87% of cases. Objectives: The objective of the present work is to evaluate and describe the effect of AMBAKA on the biological system of albino rats infected with Plasmodium falciparum. Material and methods: This is an experimental/animal study, developed at the Methodist University of Angola. Eighteen male albino rats, aged 3 to 4 months, were used. The animals were divided into 3 experimental groups of n = 6: Control group, malaria and malaria treated with Ammbaka. For the induction of malaria in animals, human blood infected with the protozoan Plasmodium Falciparum was used. Each animal received an intraperitoneal injection of 0.05 ml of infected blood. 15 days after infection, microscopy was performed to evaluate parasitemia, for treatment, Ammbaka was used for 3 consecutive days, each animal received a dose of 0.7 ml intraperitoneally. On the fourth day, microscopy was performed to assess the absence of the parasite. The results were submitted to the Kolmogorov-Smirnov normality test, followed by ANOVA and post Tukey test with a significance level of 5% (p < 0.05). Results: The results reveal that the animals developed malaria and Ambaka was able to eliminate plasmodium falciparum, the animals with malaria developed incomplete left bundle branch block, acute myocardial infarction and right atrial overload, both the malaria and the malaria group treated with Ammbaka caused insulin resistance, Ammbaka caused a reduction in adipose tissue. Conclusion: The proposed protocol was efficient for inducing malaria, Ammbaka can be used as a therapeutic model for malaria, Ammbaka did not change cardiac contractility, Ammbaka generated metabolic disturbance and depreciated the weight of periepididymal fat. The study revealed that malaria significantly compromises the functionality of the heart muscle, as these changes found can lead to death. 111453 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSICAL EDUCATION WILLIAM YANG ZHAO1, Li He2 (1) The George Institute for Global Health, China; (2) College of Physical Education and Sport, Beijing Normal University, China Background: Cardiometabolic multimorbidity is increasingly common and is associated with adverse health outcomes. Physical inactivity and obesity are associated with poor health and psychological well-being. This study aims to identify the longitudinal associations of physical inactivity, overweight and obesity on the development and worsening of cardiometabolic multimorbidity over time. Methods: Using a four-wave nationally-representative sample from the China Health and Retirement Longitudinal Study, we analyzed 17,708 participants ≥45 years with and without cardiometabolic multimorbidity. The development of Cardiometabolic multimorbidity was measured as the accrual of additional conditions over an 8-year period. Poisson-distributed Generalized Linear Models (GLM) were used to estimate the association of cardiometabolic multimorbidity with physical inactivity, overweight and obesity. Results: 22.48% of included participants had cardiometabolic multimorbidity at baseline and 35.28% at follow-up. 4,102 of 9,927 (41.32%) participants without multimorbidity and 819 of 2,879 (28.45%) with existing multimorbidity developed new condition/s. Physical inactivity was significantly associated with cardiometabolic multimorbidity (relative risk [RR] = 1.254, confidence interval [CI]: 1.136–1.385), complex multimorbidity (RR = 1.614, CI: 1.348 1.933), and worsened multimorbidity (RR = 1.214, CI: 1.032–1.427). Overweight & obesity was also significantly associated with cardiometabolic multimorbidity (RR = 1.705, CI: 1.574–1.846), and complex multimorbidity (RR = 2.156, CI: 1.852–2.510), and worsened multimorbidity (RR = 1.385, CI: 1.217–1.576). Conclusion: Physical inactivity, overweight and obesity are associated with the development and worsening of cardiometabolic multimorbidity over time. They support the recent National Institute for Health & Care Excellence (NICE) Guidance on multimorbidity that suggests that patients with multimorbidity should be identified and targeted for interventions to improve health outcomes. 107888 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY ALLANA MAYCHAT PEREIRA OLIVEIRA1, Bárbara Swarowsky Tabach2, Leonardo Silveira Nascimento1, Dannuey Machado Cardoso3, Silvio Augusto Ortolan1, Lara de Matos1, Júlia de Moraes Costa2, Francisco Coelho Lamachia1, Caroline Brand2, Lester Krann Motta1, Leonardo Dorneles de Souza1, Dulciane Nunes Paiva2 (1) Hospital Santa Cruz. Santa Cruz do Sul, RS, Brazil; (2) Universidade de Santa Cruz do Sul. Santa Cruz do Sul, RS, Brazil; (3) Centro de Ensino Superior Dom Alberto. Santa Cruz do Sul, RS, Brazil Introduction: Coronary artery bypass grafting is the most frequently used cardiac surgery (CS) for the treatment of symptomatic coronary artery disease. Because this is a major surgery, it is related to an increase in the inflammatory response in the postoperative (PO) period. Pain is one of the main complications of sternotomy. Objective: To verify the effect of colchicine on inflammatory markers, myocardial injury and pain control in the PO of CS. Methods: Randomized, double-blind, placebo-controlled clinical trial, carried out in a high-complexity cardiovascular hospital in southern Brazil. Patients undergoing CS and admitted to the intensive care unit (ICU) were evaluated. Patients of both genders, aged between 18 and 45 years and hemodynamically and neurologically stable, were included. The exclusion criteria were: emergency CS, patient with pacemaker, need for antibiotic therapy, presence of atrial fibrillation in the preoperative period and renal failure. Pain perception was assessed using the Visual Analogue Scale and troponin and C-reactive protein (CRP) levels. The patients were randomized into a control group, which received placebo, and a colchicine group, which received 0.5 mg 1x/day if body weight was up to 70kg or 0.5 mg 2x/day if body weight was over 70kg. Colchicine and placebo were administered preoperatively, restarted in the PO, and continued until outpatient reassessment (30 days after the CS). Patients were evaluated preoperatively, in the immediate PO, in the late PO (before discharge from the ICU) and 30 days after the CS. Results: The sample consisted of 43 patients (24 males and 19 females), of which 21 were allocated to the colchicine group and 22 to the control group. The mean age of the colchicine group was 63.9 years (±2.9) and 63.4 years (±3.0) in the control group. There was no statistical difference between the groups regarding variance in troponin, CRP levels and pain perception in the four assessments. However, in the analysis of individual prevalence, the colchicine group had 51.6% of respondents in relation to the decrease in CRP levels, which was observed in 48.4% of the control group. Conclusion: There was a higher individual prevalence of responders to colchicine on CRP levels compared to individuals who received placebo, with no difference in the prevalence of responders between the groups regarding myocardial injury markers and pain control in the PO of CS. 107849 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH NATANAEL ALVES DE LIMA1, Natanael Alves de Lima1, Thiago Emanuel Rodrigues Novaes2, Eduardo Pitthan2 (1) Universidade Federal da Fronteira Sul; (2) Universidade Federal da Fronteira Sul Introduction: Heart Failure (HF) is a complex clinical syndrome and represents the final stage of the natural history of heart diseases of multiple etiologies. The increase in survival has impacted the exponential increase in the elderly population. The elderly in Brazil have a high rate of incidence and prevalence of HF, morbidity and mortality and hospitalization, demanding pressure and high costs in the Brazilian Health System. Objective: To evaluate the trend of variation in crude mortality rates from HF in the elderly in Brazil, comparing age groups. Methods: Refers to an ecological, descriptive and quantitative study that used data available on DATASUS (https://datasus.saude.gov.br/informacoes-de-saude-tabnet/) on March 8, 2022 referring to to mortality in the elderly due to HF, in Brazil, from 2013 to 2019, divided into two age groups: 60 to 79 years and 80 years or older. Crude mortality rates were calculated in these variables per 100,000 inhabitants, based on the population variation in Brazil in the years chosen for the study, made available by the IBGE (https://www.ibge.gov.br/) in a consultation carried out in 08 March 2022. Results: The analysis of mortality from HF over 60 years, in 2013, showed a rate of 55.7/105 inhabitants, and showed a small drop in 2019, being 51/105 inhabitants. Making a drop of 8.4% per 105 inhabitants. On the other hand, in the same period the population of this age group increased by 94.4%. The analysis of mortality due to HF over 80 years, in 2013, showed a rate of 60.9/105 inhabitants and showed a small drop in 2019, making a drop of 1.5%. On the other hand, in the same period, the age population increased by 95.3%. Conclusion: The results of this study demonstrate that there has not been a significant change in the mortality rate from HF in the elderly in Brazil in recent decades. The trend is more pronounced in the range above 60 to 79 years. 108558 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY WANDA VIANNA MURY1, Wanda Vianna Mury1, Mariana Siqueira de Medeiros1, Marcela Anjos Martins1, Cláudia de Morais Sequeira1, Angela de Castro Resende1, Cristiane Matsuura1 (1) Universidade do Estado do Rio de Janeiro Introduction: Euterpe oleracea Mart. (açaizeiro) is a plant typical from Brazil, rich in polyphenols, which has vasodilator properties, prevents endothelial dysfunction and improves metabolic profile. However, its role on platelet function is not known. Platelets are essential for the maintenance of vascular hemostasis, but they may also participate in thrombus formation when hyperactivated, contributing to the pathogenesis of ischemic diseases. Thus, the objective of this study was to investigate the effects of açaí stone hydroalcoholic extract (ASE) on platelet aggregation. Methods: The blood from 15 healthy young men was collected, centrifuged and the isolated platelets were incubated with 10, 50 or 100 μg/mL ASE for 15 min (according to each experiment). Platelet aggregation was measured in platelet-rich plasma (PRP) and whole blood. In order to assess the effects of ASE on the platelet inhibitor nitric oxide (NO), we measured cGMP levels. Additionally, cAMP levels were quantified, the second messenger of platelet inhibitor prostacyclin. To compare the different ASE concentrations, the one-way ANOVA test with repeated measures was used. In case of significant F, a Holm Sidak post-test was performed. When only one concentration of ASE was tested, the paired t-test was applied. Statistical significance was considered when p ≤ 0.05. Results: ASE inhibited collagen-induced PRP aggregation induced by collagen (baseline, 82.7 ± 5.6, ASE50, 54.0 ± 7.2, ASE100, 31.4 ± 4.1%, p < 0.0001), ADP (baseline, 46.1 ± 7.5, ASE50, 32.1 ± 4.1, ASE100, 17.6 ± 2.6%, p < 0.0001) or thrombin (baseline, 103.4 ± 4.5, ASE 50, 77.2 ± 8.9, ASE100, 53.4 ± 7.1%, p = 0.0002), as well as in whole blood under stimulation with collagen (baseline, 28.0 ± 4.0, ASE50, 19.6 ± 4.4, ASE100, 14.8 ± 2.5 Ω, p = 0.008). ASE increase cGMP levels (baseline, 0.67 ± 0.19, ASE50, 1.50 ± 0.39, ASE100, 1.64 ± 0.49 pmol/108 cells, p = 0.020). Similarly, there was an increase in cAMP levels (baseline, 9.8 ± 1.8, ASE50, 13.9 ± 2.3, ASE100, 15.3 ± 2.6 pmol/108 cells. Concluison: These results indicate that ASE has a potent inhibitory effect on platelet aggregation. In this way it is possible that ASE has the potential to be used in the prophylaxis and treatment of diseases associated with platelet hyperaggregability, although the mechanisms underlying this process need to be fully clarified. 107969 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIEL SALIM SAUD DE OLIVEIRA 1, João Roquette Fleury da Rocha1, Bárbara Mariz Ferreira Passos1, Ismael Magalhães Matos de Faria1, Ana Carolina Rodrigues Lado1, Ricardo Antônio Correia Lima2, Pedro Paulo Noguères Sampaio2, Juliano Carvalho Gomes de Almeida1, João Mansur Filho2, Roberto Muniz Ferreira1, Lúcia Helena Alvares Salis1, Nelson Albuquerque de Souza e Silva1 (1) Federal University of Rio de Janeiro, Edson Saad Heart Institute, Brazil; (2) Samaritano Hospital, Botafogo, Rio de Janeiro, Brazil Introduction: Several biomarkers have demonstrated prognostic value in patients with Coronavirus Disease-2019 (COVID-19), primarily during hospitalization. Troponin I (TnI) has been widely studied in this context, though its association with outcomes after the initial hospitalization period remains unknown. Objectives: To determine the association between high sensitivity TnI elevation above the 99th percentile upper reference limit on admission in hospitalized patients with COVID-19 and long term survival among those who were successfully discharged. Methods: Medical records from consecutive patients with confirmed COVID-19 admitted to a single institution between March and July 2020 were retrospectively analysed. Clinical data and TnI values were collected and correlated with long term mortality after the index hospitalization. Results: Among 230 patients, 194 survived until hospital discharge, of which 149 had TnI values on admission and were included in the analysis. Median age was 65 years (52–78) and 56.4% were male. Cardiovascular disease (CVD) was present in 12%, and 36.2% were treated in the intensive care unit (ICU). Troponin elevation occurred in 21 patients (14.1%), and 9 (6%) died after a median follow up of 631 days (612–643). Troponin was associated with death in the univariate (OR 16.7; 95% CI 3.8–73.6, p < 0.001) and multivariate analyses (OR 8.6; 95% CI 1.5–49.6, p = 0.02), after adjusting for age, previous CVD, ICU admission, C-reactive protein and creatinine values. Figure 1 represents the Kaplan-Meier survival estimates after hospital discharge according to TnI elevation. Conclusions: Although most patients have a favourable outcome after hospitalization for COVID-19, cardiac injury on admission appears to remain predictive of long term survival after discharge. 107978 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS VÍTOR BONIATTI NEVES1, Vítor Boniatti Neves1, Raquel Melchior Roman2, Tiago Vendruscolo2, Gilberto Heineck2, Carlos Alberto Santos de Mattos2, Eduardo Ilha de Mattos2, Luiz Carlos Pereira Bin1, Karine de Lima Sírio Boclin3, Marcelo Fialho Roman1 (1) Faculdade Meridional (IMED), Passo Fundo, RS – Brazil; (2) Hospital de Clínicas de Passo Fundo (HCPF), Passo Fundo, RS – Brazil; (3) Universidade Estácio de Sá (UNESA), Rio de Janeiro, RJ – Brazil Background: The wide range of clinical presentations of acute coronary syndrome (ACS) makes it indispensible to use tools for risk stratification and for appropriate risks management; thus, the use of prognosis scores is recommended in the immediat clinical decision-making. Objective: To validate the Global Registry of Acute Coronary Events (GRACE) score as a predictor of in-hospital and 6-month post-discharge mortality in a population diagnosed with ACS. Methods: This is a prospective cohort study of consecutive patients diagnosed with ACS between May and December 2018. GRACE scores were calculated, as well as their predictive value for in-hospital and 6-month post-discharge mortality. The validity of the model was assessed by two techniques: discriminative power using the area under the receiver operating characteristic curve (AUC) and goodness-of-fit, using the Hosmer-Lemeshow (HL) test, at the 5% level of significance. Results: A total of 160 patients were included, mean age 64 (±10.9) years; of which 60% were men. The risk model showed to have satisfactory ability to predict both in-hospital mortality, with an area under the curve (AUC) of 0.76 (95% confidence interval [CI], 0.57–0.95; p = 0.014), and 6-month post-discharge mortality, with AUC of 0.78 (95%CI, 0.62–0.94), p = 0.002. The HL test indicated good-fit for both models of the GRACE score. Conclusion: In this study, the GRACE risk score for predicting mortality was appropriately validated in patients with ACS, with good discriminative power and goodness-of-fit. The results suggest that the GRACE score is appropriate for clinical use in our setting. 108053 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUIZ ANTONIO VIEGAS DE MIRANDA BASTOS1, Luiz Antonio Viegas de Miranda Bastos1, José Lucas Peres Bichara1, Paolo Blanco Villela1, Gabriela da Silva Nascimento1, Gláucia Maria Moraes de Oliveira1 (1) Universidade Federal do Rio de Janeiro UFRJ Introduction: Deaths from diseases of the circulatory system (DCS) and ischemic heart diseases (IHD) are declining, but slowly in developing countries, emphasizing its probable relationship with determinants of social vulnerability. Objectives: To analyze the temporal progression of mortality rates of DCS and from 1980 to 2019 and the association of the rates with the Municipal Human Development Index (MHDI) and Social Vulnerability Index (SVI) in Brazil. Methods: We estimated the crude and standardized mortality rates of DCS and IHD and analyzed the relationship between the obtained data and the MHDI and SVI. Data on deaths and population were obtained from the. The MHDI and the SVI of each federative unit were extracted from the websites Atlas Brasil and Atlas da Vulnerabilidade Social, respectively. Results: The age-standardized mortality rates of DCS and IHD showed a downward trend nationwide, which was unequal across the federative units. There was an inversely proportional relationship between the standardized mortality rates of DCS and IHD and the MHDI. The downward mortality trend was observed when the indices were greater than 0.70 and 0.75, respectively. The SVI was directly proportional to the standardized mortality rates of DCS and IHD. An upward mortality trend was observed with an SVI greater than 0.35. Conclusion: Social determinants represented by the MHDI and the SVI were related to mortality from DCS and IHD across the Brazilian federative units. The units with most development and least social inequalities had the lowest mortality from these causes. The most vulnerable die the most. 108060 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION ALINE DE OLIVEIRA SILVA1, Maria Carolina Guido1, Mauricio Tavares Costa1, Natalia de Menezes Lopes1, Amanda de Almeida Silva1, Priscila Oliveira Carvalho1, Raul Cavalcante Maranhão1 (1) Instituto do Coracao, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, SP, BR. Background: Systemic arterial hypertension is a major risk factors for cardiovascular diseases. Interstitial fibrosis is directly associated with ventricular dysfunction in spontaneously hypertensive rats (SHR). Incorporation of methotrexate (MTX), a folic acid inhibitor, into lipid nanoparticles (LDE) increases cellular uptake and decreases MTX toxicity. LDE-MTX showed potent anti-inflammatory and anti-proliferative effects in rabbits with atherosclerosis and rheumatoid arthritis. In rats submitted to acute myocardial infarction, LDE-MTX reduced infarct size and left ventricular (LV) dysfunction. Aim: To investigate the effect of LDE-MTX treatment on the prevention of cardiac remodeling that occurs in SHR rats. Methods: Three groups of 6-week-old male rats were studied: 1) Control (CT, n = 8), Wistar-Kyoto rats weekly treated with I.P injection of saline solution; 2) SHR-LDE (n = 8): SHR rats treated with injection of LDE only and 3) SHR-LDE-MTX (n = 8): SHR rats treated with LDE-MTX (1 mg/kg intraperitoneally, weekly, for 20 weeks). After the treatment period, echocardiography was performed and the animals were euthanized for LV morphometry and protein expression by Western blot. Results: Compared to CT, SHR group presented LV dilation, represented by increase in systolic and diastolic diameters; cardiac hypertrophy due to increased LV posterior wall thickness and relative heart weight. SHR also had reduced ejection fraction and LV shortening, indicating LV systolic dysfunction. In SHR-LDE, there was increase in collagen content in the interstitial region of the LV, possibly resulting from an increase in type 1 collagen, which indicates increase in fibrosis of LV. LDE-MTX had no effect on LV dilation, cardiac hypertrophy or LV systolic dysfunction in SHR rats. However, LDE-MTX decreased LV interstitial fibrosis. In order to identify possible mechanisms associated with LDE-MTX and the reduction of interstitial fibrosis, we quantified the expression of angiotensin II receptors type 1 (AT1) and 2 (AT2) in the LV of the animals. However, there was no difference in the expression of AT1 and AT2 between the 3 groups. Conclusion: LDE-MTX treatment reduced LV interstitial fibrosis in SHR rats, possibly by reducing type 1 collagen expression, despite having no effect on LV systolic dysfunction. It is possible that the reduction in LV fibrosis achieved by treatment with LDE-MTX reduces the damage of hypertension on cardiac function when the animal reaches more advanced. 108092 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOGERIATRICS MICHELE OURIQUES HONORATO1, Juscelio Trajano de Souza Filho1, Luiz Frederico Bezerra Honorato Junior1, Nathalia Watanabe1, Gabriela Machado Goulart2 (1) Hospital Sírio Libanês; (2) Universidade do Extremo Sul Catarinense Introduction: Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting about 2 to 4% of world population, and in patients hospitalized in intensive care units, this incidence can go up to 23%. Advanced age is a risk factor for the development of AF, and those above 80 years of age become more susceptible to its deleterious effects. The impact of AF in septic patients is reflected in worse clinical outcomes and the identification of the triggering factors can be a target of future prevention and treatment strategies. Objectives: To verify the relationship between the development of AF and the mortality in patients over 80 years of age included in the sepsis protocol and to identify the risk factors that contribute to the development of AF in this population. Methods: Retrospective observational study, with a review of medical records and inclusion of 895 patients aged 80 years or older, included in the sepsis protocol of a high-complexity hospital in São Paulo/SP, from January 2018 to December 2020. Results: The incidence of AF in the sample was 13%. After multivariate analysis, using multiple logistic regression, it was possible to demonstrate an association of mortality, in the studied population, with the SOFA score OR 1.21(1.09–1.35), higher values of C reactive protein – OR 1.04 (1.01–1.06), need for vasoactive drugs – OR 2.4 (1.38–4.18), use of mechanical ventilation – OR 3.49 (1.82–6.71) and mainly AF – OR 3.7 (2.16–6.31). Conclusion: In the great elderly patient (80 years of age and older), septic, the development of AF was shown to be an independent risk factor for in-hospital mortality. 108625 Modality: E-Poster Young Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE IEDA BARRETO MOURÃO BERTINI1, Sofia Gonçalves Tonoli1, Reinaldo José Gianini1, Emerson de Albuquerque Seixas1 (1) Pontifícia Universidade Católica de São Paulo (PUCSP) Introduction: Heart Failure (HF) is a disease where the heart is unable to properly pump blood around the body to support the metabolic needs. HF is associated with frequent underlying symptoms and complex treatments, which compromises a quality of life. The suffering can be lessened with a holistic approach focused on spirituality, which has been shown to affect the health and wellbeing of people with chronic diseases. Objective: To assess the clinical-hemodynamical profile of patients with HF to test if spirituality correlates with quality of life. Methodology: A cross-sectional study was undertaken, where two questionnaires were applied: i) Minnesorta Living with HF Questionaire (MLHFQ), to address the quality of life of patients with HF and ii) Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being (FACIT-Sp-12), regarding the spiritual wellbeing of chronic patients. The patient’s classification according to the New York Heart Association (NYHA) and the Stevenson hemodynamic profile were also used. Statistical tests were performed using STATA®, through Pearson’s correlation and linear regression. A total of 41 individuals ≥18 years old with HF were interviewed. Results: Most respondents were male (53%) and the mean age of respondents was 66 years old. The mean HF etiologies identified were hypertense (37%), ischemic (34%) and valvular (12%), with the majority of patients falling into NYHA class III (41%) and Stevenson category B (90%). According to the FACIT-Sp-12 classification suggested by the authors of this study, as there are no other classifications in the literature, 2.4% showed low levels of spirituality and 65.8% showed high levels. According to the MLHFQ, 24.4% of respondents have great quality of life and 22% have a bad one. When both questionaires were correlated, there was a significant r coefficient of –0.45, showing a significant inversely proportional relationship (p-value 0.003), meaning higher the levels of spirituality are associated with lessend symptoms in patients with HF. However, when FACIT-Sp-1 was correlated with both the NYHA and the Stevenson classification, no stastistically significant relationships were found. Conclusion: A holistic approach focused on spirituality in patients with HF could have a positive effects on their quality of life through lessened symptoms. However, there is no evidence to suggest that spirituality has an effect on either the functional status or hemodynamic profile of patients. 108163 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE CAROLINA RODRIGUES TONON1, Paola da Silva Balin1, Marina Gaiato Monte1, Danilo Malmonge Barbosa Luciano1, Natalia Fernanda Ferreira1, Marcos Ferreira Minicucci1, Paula Schimdt Azevedo1, Sergio Alberto Rupp de Paiva1, Leonardo Antonio Mamede Zornoff1, Marina Politi Okoshi1, Katashi Okoshi1, Bertha Furlan Polegato1 (1) Faculdade de Medicina de Botucatu Background: Cancer is one of the main causes of morbidity and mortality worldwide. Despite doxorubicin (DOX) being an effective chemotherapy drug, DOX-induced cardiotoxicity is the most severe side effect, limiting treatment and worsening quality of life. There is no effective treatment for preventing DOX-induced cardiotoxicity. Glucagon like peptide-1 (GLP-1) analogs such as liraglutide reduce oxidative stress and inflammation. Purpose: To evaluate the role of liraglutide in acute DOX-induced cardiotoxicity in rats. Methods: 62 male Wistar rats were allocated into 4 groups: Control (C), DOX (D), Liraglutide (L), and DOX + Liraglutide (DL). The animals in L and DL groups received a subcutaneous injection of 0.6 mg/kg liraglutide daily while C and D groups received a subcutaneous injection of an equivalent volume of saline daily, for 2 weeks. After 12 days, D and DL groups received an intraperitoneal injection of DOX (20 mg/kg). Rats were submitted to echocardiogram and isolated heart study 48 hours after DOX injection. Statistical analysis: one-way ANOVA. Results: In the first 12 days, L and DL had lower food ingestion and body weight than C and D. After DOX treatment, body weight was lower in D and DL than C and L and lower in DL than D. In isolated heart study, DOX decreased +dP/dt (C 4518 ± 1469; D 2083 ± 534; L 4125 ± 647; DL 2150 ± 399 mmHg/s; p < 0.001) and -dP/dt (C 2679 ± 886; D 1292 ± 504; L 2896 ± 496; DL 1413 ± 285 mmHg/s; p < 0.001), with no changes between DL and D. In echocardiogram, D and DL exhibited lower peak systolic annular velocity at tissue Doppler [C 3.7 (3.2–4.2); D 3.1 (3.0–3.5); L 3.8 (3.4–4.0); DL 2.9 (2.7–3.0) cm/s, p < 0.001], and posterior wall shortening velocity (C 39 ± 4.5; D 32 ± 2.5; L 41 ± 5.0; DL 30 ± 2.9 mm/s; p < 0.001) than C and L. Liraglutide did not change these variables. D and DL had decreased E waves (C 77.2 ± 10.5; D 56.6 ± 9.0; L 74.3 ± 7.4; DL 52.5 ± 9.6 cm/s; p < 0.001) and A (C 56.9 ± 13.8; D 46.7 ± 10.4; L 56.3 ± 16.6; DL 39.8 ± 10.3 cm/s; p = 0.002) than C and L. DL showed increased isovolumetric relaxation time normalized to heart rate (C 54 ± 4.9; D 58 ± 8.0; L 51 ± 6.1; DL 65 ± 8.8; p < 0.001) than C, L, and D. Conclusion: Liraglutide administration does not attenuate doxorubicin-induced acute cardiac dysfunction. 108578 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES RICARDO JORGE DOS REIS ALVES PINTO1, Miguel Martins Carvalho1, Tânia Proença1, Catarina Costa1, Ana Filipa Amador1, João Calvão1, Catarina Marques1, André Cabrita1, Luís Santos1, Mariana Paiva1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João, Porto, Portugal Percutaneous mitral commissurotomy (PMC) is a viable alternative to mitral valve (MV) surgery in the treatment of clinically significant mitral stenosis (MS). Our aim was to evaluate the long-term results of PMC in patients (pts) with rheumatic MS and to compare events concerning pulmonary hypertension (PH) presence. We analysed all pts between 1991 and 2008 with rheumatic MS undergoing PMC. MACE was a composite of all-cause mortality, MV re-intervention or cardiovascular hospitalization. A total of 124 pts were enrolled: 87% were female, mean age of 46 ± 11 years (yrs) and mean follow-up (FUP) of 20 ± 6 yrs. Before PMC, 34% were in NYHA class ≥ III, 81% had Wilkins score ≤8, all had preserved biventricular function, 83% presented PH; mean transvalvular gradient (TVG) and mitral valve area (MVA) were 12.8 mmHg and 1.0 cm2. Most of the procedures were successful (91%) and without major complications (94%), with mean MVA improvement of 0.9 cm2 and reduction of 8.5 mmHg in TVG and 9.7 mmHg in PASP after PMC. During long-term FUP, 42% of pts were re-interventioned and 24% died. In pts non-submitted to re-intervention, TVG and PASP remained similar throughout FUP, while MVA reduced over time, yet still statistically superior to baseline MVA (1.6 vs 1.0 cm2, p < 0.001). In time-to-event analysis, approximately 80% of pts kept uneventful after 10 yrs; after 30 yrs, >20% continued MACE-free and 50% alive. Regarding PH before PMC, there was no significant difference in MACE or mortality (p = 0,846 and p = 0.661, respectively). After long-term FUP, pts maintained reduction in TVG and PASP and a smaller but significative improvement in MVA. Most pts were MACE-free after 10 yrs and half were alive after 30 yrs. There was no difference in all-cause mortality or MACE concerning PH presence. 108271 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ERIC SANTOS RODRIGUES DE OLIVEIRA1, Larissa Araújo de Lucena1, Vitor Martinez Silva1, Antonio Lucas Arruda de Oliveira1, William Santos Rodrigues de Oliveira1, Nestor Rodrigues de Oliveira Neto1 (1) Hospital Universitário Onofre Lopes (HUOL) – Universidade Federal do Rio Grande do Norte (UFRN) Introduction: Rhythm disorders often present intermittently, making it difficult to record them. However, the electrocardiogram (ECG) performed during the event allows diagnosing the type of arrhythmia. Several studies have shown that portable devices can diagnose rhythm disorders, making these devices a practical alternative to the expensive and poorly available equipment. Thus, we hypothesize that our low complexity and cost device can monitor cardiac rhythm and diagnose cardiac arrhythmias with a single-lead ECG. Objectives: To evaluate a low-cost system’s accuracy and technical quality for recording ECG and diagnosing cardiac arrhythmias. Methods: This is a validation study where outpatient or hospitalized patients are selected based on continuous cardiac monitoring and standard 12-lead ECG. Patients who had cardiac arrhythmia underwent a 12-lead ECG, and then the heart rhythm was recorded with a lead similar to CM5 through the new device. The device comprises a biological signal amplifier associated with filters connected to the smartphone. We will categorize the records into six rhythm groups, which will give us its diagnosis accuracy by comparing the device and the 12 lead ECG registers. Results: Presently, there are 30 records made with the device. Of these, 29 were of satisfactory quality. 1 was excluded for artifact influence. The general profile of the patients from the study was the median age of 55; 15 were men, 13 women, 1 undetermined; 12 had no comorbidities, 1 case of hypertrophic cardiomyopathy, 4 congestive heart failure, 4 hypertension, 3 diabetes mellitus. The rhythm profile of the registers is 15 with sinus rhythm, 7 bradycardia, 5 atrial fibrillation, 1 supraventricular or ventricular ectopy, 1 narrow qrs tachycardia, no wide qrs tachycardia so far. The device diagnosis accuracy is undetermined as we have not compared the device registers and the 12-lead ECG yet. Conclusions: The partial results show a low-cost mobile device that records in satisfactory quality and can be a tool for diagnosing rhythm disorders. According to the initial analysis of 30 tracings, the quality was satisfactory in 96.67% of the cases. Our solution is an efficient lower price alternative to other existing devices. The main limitation of this study is the result’s initial state, which makes it challenging to verify the performance of the tested device. Thus we only accessed its ability to perform tracings of good quality. 108355 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY BARBARA PORTO VALENTE1, José de Ribamar Costa Junior1, Ibraim Masciarelli F Pinto1, Bruno Pereira Albuquerque Coelho1, Gabriela Carolina Santamaría Naranjo1, Luciano de Figueiredo Aguiar Filho1, Paul Salvador1, Tiago Senra1, Vivian Lerner Amato1, José Roberto Tuma da Ponte Junior1, Ricardo Pavanello1, Pedro Silvio Farsky1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: The potential benefits attributable to late reperfusion fall under the “open artery hypothesis”, in which the stunned peri-infarction zone after revascularization restores blood supply, improving its contractility. A study showed that late coronary recanalization after myocardial infarction (MI), irrespective of the viability status, improved left ventricular ejection fraction (LVEF) and myocardial contractility mainly in the segments adjacent and distant to the infarction. Purpose: To evaluate whether late recanalization in patients with STEMI without viability by Cardiovascular Magnetic Resonance (CMR) can reduce the reverse remodeling. Viable cases will be kept in the registry. Methods: This is a prospective randomized controlled trial, with at least 6 months follow-up. Patients with STEMI not reperfused between 24 hours and 28 days with IRA with lesion greater than 50% with segmental dysfunction and absence of viability on CMR are eligible for inclusion. Patients with previous MI, cardiomyopathy or clinical instability are excluded. Participants randomly assigned (1:1) to PCI and Optimal Medical Treatment (OMT) or only OMT. Expected outcome is the change on reverse remodeling of the end systolic volume at 6 months likewise the improvement in segmental contractility at 6 months. The sample size of 35 patients in each group provides 80% power with a two-sided significance level of 0.05. Descriptive statistics and statistical inferential methods are employed to measure changes between the groups. The trial has local ethical review board approval. Results: 28 patients have been already enrolled. Conclusion: This trial will provide insights into the potential benefits of opening the IRA in segments without viability, improving contractility of surrounding myocardium. 108396 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY LIDIANE MOREIRA SOUZA1, Bruna Brasil Brandao2, Luana Urbano Pagan1, Mariana Gatto1, Felipe Cesar Damatto1, Eder Anderson Rodrigues1, Patricia Aparecida Borim1, Gilson Masahiro Murata3, Leonardo Antonio Mamede Zornoff1, Katashi Okoshi1, Marina Politi Okoshi1 (1) Botucatu Medical School, Sao Paulo State University, UNESP; (2) Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA; (3) Institute of Biomedical Science, Sao Paulo University, USP Introduction: Skeletal muscle energy metabolism is commonly altered in heart failure patients, with a metabolic shift from oxidative to glycolytic muscle fiber. These changes contribute to reduced functional capacity. Sodium glucose co-transporter type 2 (SGLT2) inhibitors improve cardiovascular outcomes in both diabetic and non-diabetic patients, as well as those with and without heart failure. However, the effects of SGLT2 inhibitors on skeletal muscle during heart failure have not been established. The aim of this study was to assess the metabolic effect of empagliflozin (EMPA) on skeletal muscle of rats with myocardial infarction (MI)-induced heart failure. Methods: One week after MI induction or simulated surgery, male Wistar rats were divided into four groups: Sham (n = 10), Sham+Empa (n = 12), MI (n = 10), and MI+Empa (n = 09). EMPA was added to rat chow (5 mg/kg/day). Rats were supplied with ad libitum water and chow for 12 weeks. Infarct size was measured by histological analysis. Metabolic enzyme activity in the soleus muscle was assessed by spectrophotometry. Statistical analysis: ANOVA and Tukey, and Student’s t tests. Results: Only rats with infarction size greater than 35% of total left ventricle area were included in this study. Infarction size did not differ between infarcted groups (MI 41.8 ± 4.2; MI+Empa 40.7 ± 5.7 of total left ventricle area). In the MI soleus muscle, metabolic enzyme activity of glucose-6-phosphate-dehydrogenase, citrate synthase and beta-hydroxy-acyl-dehydrogenase was higher than the Sham group. These changes were not observed in the MI+Empa group. MI+Empa had lower hexokinase, phosfructokinase, and pyruvate kinase activity (glycolytic metabolism enzymes), and lower citrate synthase and glucose-6-phosphate-dehydrogenase activity than MI. Conclusion: Chronic treatment with SGLT2 inhibitor empagliflozin prevents metabolic abnormalities in skeletal muscle in infarcted rats. 108368 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS GABRIEL KANHOUCHE1, Jose Nicolau1, Talia Falcão Dalçoquio1, Mauricio Felippi de Sa Marchi1, Roberto R Giraldez1, Remo Furtado1, Luciano Baracioli1, Roberto Kalil Filho1, Fabio Sandoli de Brito Junior1, Alexandre Abizaid1, Henrique Barbosa Ribeiro1 (1) Heart Institute of Sao Paulo – InCor/HCFMUSP Introduction: About 5–10% of myocardial infarctions develop severe form of presentation, with cardiogenic shock (CS). Despite evidence showing higher in-hospital and short-term mortality in patients with STEMI complicated by CS, long-term follow-up data is lacking. Hypothesis: The aim of this study was to assess very long-term prognosis of survived patients with STEMI complicated by CS. Methods: STEMI patients (n = 1,444) were included (1998–2017) and followed for up to 17.6 years (median 5.4 [2.5–9.9] years). Long-term survival was assessed by Log-Rank test. Results: Compared to STEMI without CS, patients who presented CS (n = 118) had no significant differences regarding age (62.0 ± 11.8 vs. 60.7 ± 12.7; p = 0.31) and female sex (28.0% vs. 25.1%, p = 0.51), but had lower left ventricle ejection fraction (38.8 ± 13.0% vs. 49.7 ± 13.1%, p < 0.001) and higher mean baseline creatinine levels (1.4 ± 0.6 mg/dL vs. 1.2 ± 0.7 mg/dL, p = 0.002). Of note, CS patients presented higher in-hospital mortality (42.2%) compared to those without CS (OR = 28.5; 18.1–44.7, p < 0.001). Long-term survival was lower in patients with CS (10.7 ± 0.6 vs. 13.3 ± 0.2 years, log rank = 0.016). By multivariate analysis adjusted for baseline characteristics the main factors predicting mortality were age (HR = 1.06; CI95% 1.05–1.07, p < 0.001), previous MI (HR = 1.34;CI% 1.06–1.69, p = 0.01), history of heart failure (HR = 1.65; CI95% 1.15–2.37, p = 0.007), stroke (HR = 1.67; CI95% 1.12–2.47, p = 0.01), diabetes (HR = 1.41; CI95% 1.14–1.74, p = 0.001) and the presence of CS (HR = 3.65; CI95% 2.92–4.56, p < 0.001). Conclusion: In a very long-term follow-up after a STEMI, CS increased ~4-fold the mortality rates. After hospital discharge, CS remains an important independent risk factor for death in the follow-up. Our findings reinforce the importance of intensive clinical monitoring with optimal medical and non-pharmacological therapy for the remarkable risk factors in the very long-term post-STEMI complicated by CS. 108369 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS BARBARA PORTO VALENTE1, Maria Júlia Silveira Souto1, Manuela Gomes de Aguiar1, Gabriela Carolina Santamaría Naranjo1, Ismar Junior Peinado Lijeron1, Rodrigo Urdan1, Lucas Ferreira Marcondes Lemos1, César Henrique Morais Alves1, Kelvin Henrique Vilalva1, Maria Isabel Del Monaco1, Luciana Uint1, Ricardo Pavanello1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Late intervention of a chronic total occlusion (CTO) in stable patients is not routinely recommended by randomized trials. Previous studies have confirmed the negative effect of CTOs on prognosis. Purpose: To evaluate if a strategy of viability assessment (VA) to guide revascularization in the left anterior descendent artery (LAD) CTO can reduce 5-year clinical outcomes. Methods: Retrospective cohort with at 5-year follow-up of 223 patients with LAD with CTO without any other significant lesion. Patients with previous myocardial infarction, cardiomyopathy or clinical instability were excluded. The primary outcome was the composite end point of myocardial infarction (MI), death, new LAD revascularization, heart failure hospitalization (HFH) and severe arrhythmias. Results: We identified 223 patients with LAD CTO as a single lesion. Only 53 (23.7%) had VA to guide therapy compared to 170 (76.2%) with no VA (NVA). The mean ejection fraction was higher in the group with NVA (54.5 +/– 13.1 vs 41.5 +/– 11.8 with p < 0.001). The occurrence of angina was more common in the NVA group (64.7% vs 30.2% with p < 0.001), mainly due to Canadian Cardiology Society II (71.8%). The VA group was more frequently maintained in optimal medical therapy (OMT) (54.7%) compared to the NVA group (23.5%). NVA group underwent revascularization more often than the VA group, PCI was performed in 59.4% vs 41.5% and CABG in 17.1% vs 3.8% respectively. The primary outcome occurred in 5.7% in the VA group compared to 22.4% in the NVA (p = 0.056). Conclusion: VA is a feasible strategy to reduce the need of unnecessary interventions. There was a marginal reduction of the total number of events in the VA group in the 5-year follow-up, mainly with respect to reducing the need for new LAD revascularization. 108397 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY ALLA PAVLOVSKAYA2, Elena Lyasnikova1, Ekaterina Zhabina1, Maria Trukshina1, Victoria Galenko1, Larisa Prokopova1, Tatiana Lelyavina1, Marianna Wander1, Evgeniy Mikhaylov1, Maria Sitnikova1 (1) Almazov National Medical Research Centre; (2) Semashko Republican Clinical Hospital Introduction: Experimental data have showed the effect of cardiac contractility modulation (CCM) on the autonomic nervous system (ANS) activity. However, there are limited data regarding the prognostic value of ANS and its imbalance assessed by heart rate variability (HRV) in patients receiving CCM. Objective. To evaluate association of HRV with prognosis in heart failure patients with HFrEF and sinus rhythm undergoing CCM therapy. Methods: This retrospective study included 52 patients with HFrEF, II/III fc, being on optimal drug therapy more than 3 months (age 53 ± 10.4 years, 86% men, CAD 73%, CABG/PCI 54%, ICD 21%, sinus rhythm 100%, LVEF 25 ± 6%, peakVO2 16.8 ± 4.8 ml/min/kg, NtproBNP 1087.5 [706.6;1752.7] pg/mL), who underwent CCM device implantation with further followed by a team of heart failure specialists. HRV parameters were calculated using 24-hour ECG monitoring before device implantation. The relationship between pre-implant time-domain (SDNN) and frequency-domain (LF, HF, LF/HF ratio from 24-hour; VLF from a 5-min segment of the resting period) and the endpoint of 3-year all-cause death was analyzed. Results: During a mean 36 ± 2 months of follow-up, 11 patients reached the endpoint. All patients were divided into 2 groups depending of the SDNN duration. Initially, SDNN <100 ms was observed in 18 patients (34.6%). The Kaplan-Meier analysis using the log-rank test showed worse prognosis among patients with low SDNN (p = 0.01). LF, HF, VLF during 5-min were significantly higher in the subgroup of surviving patients (p = 0.02, p = 0.04 and p = 0.001, respectively) and low VLF (≤179 ms2) was associated with an increased risk of HF-death (p = 0,005). Conclusions: Our preliminary findings indicate that baseline ANS dysfunction (quantified by low HRV indices) in patients with HFrEF receiving CCM-therapy was associated with the all-cause mortality and cardiac events within 3 years. The future of risk stratification of events in HF patients undergoing CCM lies in the integration of HRV data with other prognostic factors, imaging methods and laboratory biomarkers. It might help in optimizing qualifications for this treatment. 108387 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH HILDEMAN DIAS DA COSTA1, Hildeman Dias da Costa1, Luiz Felipe Façanha Ramos2, Leo Christyan Alves de Lima3, Matheus Akira Suzuki de Oliveira1, Mateus Viana Osório de Barros1, Matheus Rodrigues Gomes3, Mathews Barbosa Santiago4, Milena Stephanie Alves Matos3, Júlia de Ávila Gutierrez5, Laura Jane França Lacerda3, Maria Beatriz Mourão8 (1) Universidade Federal de Rondônia; (2) Universidade Federal do Amapá; (3) Centro Universitário São Lucas; (4) Centro Universitário Uninorte; (5) Faculdades Integradas Aparício Carvalho Introduction: Myocardial infarction, or heart attack, is the death of cells in a region of the heart muscle due to the formation of a clot that interrupts blood flow in a sudden and intense way. Fast and efficient care proves to be of fundamental importance to reduce patient morbidity and mortality. Objective: To analyze the epidemiological profile of hospitalizations for acute myocardial infarction on an emergency basis in Brazil between 2010 and 2019. Methods: This is a cross-sectional epidemiological study, in which data were obtained from the Department of SUS Informatics – DATASUS. The variables researched were: total hospitalizations, sex, color/race, age group, deaths and mortality rate. The research period was delimited between the years 2010 and 2019. Results: 911,607 hospitalizations were recorded. Males reported 578,246 hospitalizations, and females 333,361. The white color/race recorded 372,948 hospitalizations. The most affected age group was 60 to 69 years old, with 266,378 hospitalizations. The total number of deaths was 109,295. The mean mortality rate was 11.99. Mortality rates in 2010 and 2019 were 13.46 and 10.37, respectively. Conclusions: The epidemiological profile of hospitalizations was characterized by male, white individuals aged between 60 and 69 years. Although the number of hospitalizations and deaths have increased, mortality rates from AMI have been decreasing over the years. 108400 Modality: E-Poster Young Researcher – Non-case Report Category: PSYCHOLOGY LEONARDO SANTOS DE SOUZA1, Leonardo Santos de Souza1, Silvia Maria Cury Ismael1 (1) Hospital do Coração de São Paulo Background: Considering the complex public health challenge of preventing tobacco-associated diseases as the main cause of morbidity and mortality, understanding the risky health behavior variables thus enabling their proper management. Few Brazilian studies have investigated this issue in the in-hospital setting and in supplementary health frameworks. Objectives: To characterize and assess the variables involved in the smoking behavior of in-patients with heart disease. Methods: Exploratory cross-sectional study approved by the Research Ethics Committee (CAAE nº44662921.0.0000.0060) that assessed 211 tobacco-addict patients with heart disease of which 77.7% were men, with a mean age of 60.5 years (±11.4), evaluated in the last five years by the psychology service of a private hospital in the city of São Paulo. Data collected retrospectively through the Smoker Profile Questionnaire, the Fagerstrom test, the Stages of Behavior Change and Reasons for Smoking Scale were submitted to descriptive analyses as well as non-parametric tests, Chi-Square and Fisher’s Exact tests, using the R Core Team software (2021) and setting a p-value <0.05 as statistically significant. Results: There was a prevalence (p = <0.001) of married men (72%) professionally active (82.3%) with higher education (75%). Consumption had been 20 cigarettes a day for 39.4 years (±13.4) with ineffective cessation attempts (M = 1.9 ± 1.8). Smoking was adhered to for pleasure (85.8%), stress reduction (81.5%) and physical dependence (62.1%), despite motivation to quit (82.9%). In addition, the respondents reported difficulty not smoking when they were sad (46.2%), angry (49.8%) or anxious (60.8%). There was an association (p < 0.001) between time of active smoking, age, hypertension and coronary artery disease, as well as cronic obstructive pulmonary disease (p < 0.001), overweight (p = 0.027), acute myocardial infarction (p = 0.022). Nicotine dependence was associated with chronic alcoholism (p = 0.012) and longer hospital stay (p = 0.032). Weight control (p = 0.003) and stress reduction (p = 0.018) were the main reasons for women with heart disease to keep smoking. Conclusion: Smoking in patients with heart disease has multiple functions and respondent-operant interactions, which makes smoking more resistant to change and demands a thorough functional analysis and comprehensive interventions still during hospitalization, for an efficient secondary prevention. 112369 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GIOVANNA BOLINI BRAZÃO1, ISABELLA ROCHA GONÇALVES1, RAISSA MARIA CHAVES LOBATO1 (1) Centro Universitário do Estado do Pará (CESUPA) Introduction: Cardiovascular diseases (CVDs) affects the heart and blood vessels, with the ability to compromise other vital systems, representing important causes of deaths worldwide. In Brazil, they are a major public health problem, being associated with several risk factors. To change this situation, it is necessary to know about the epidemiological profile of CVDs at regional level, in order to assist prevention and intervention in modifiable risk factors, through public policies and health actions. Objectives: To verify the prevalence of cardiovascular diseases in the Center for Medical Specialties of Cesupa – CEMEC outpatient clinics; to identify risk factors in the clinical history and their correlation with the development of CVDs; to identify the epidemiological profile of patients affected by CVDs. Methods: The prevalence of CVDs at CEMEC, in the metropolitan region of Belém-PA, during 2013, was evaluated through an observational and cross-sectional study, carried out from the analysis of medical records of the cardiology outpatient clinic in the referred period. Results: The study analyzed the medical records of 152 patients; 40.8% of these were male and the other 59.2%, female. In terms of the neighborhood of origin of these patients, Marambaia (16.4%) and Mangueirão (6.5%) stood out with the largest number of residents who are users of CEMEC’s cardiology outpatient clinic. The history of previous illness, medications in use, life habits, signs and symptoms at the time of consultation and other variables of each patient were analyzed, in order to record, create statistics and understand the needs of the outpatient clinic. Conclusion: Certainly, the poor filling of the medical records collected and the great demand for cardiac care in CEMEC draws attention. In addition, the close correlation between CVDs recorded with the risk factors prevalent in the study population suggests an important criterion in which future local policies can be developed. 108424 Modality: E-Poster Young Researcher – Non-case Report Category: DYSLIPIDEMIA URTE ALIOSAITIENE1, Zaneta Petrulioniene1, Egle Skiauteryte2, Dovile Gabartaite1, Emilija Meskene1, Juste Staigyte1, Viktoras Sutkus2, Rimante Cerkauskiene1 (1) Vilnius university faculty of medicine; (2) Vilnius university hospital Santaros Klinikos Background and aims: Familial hypercholesterolemia (FH) is highly underdiagnosed and undertreated congenital disorder worldwide. In this study we aim to establish an algorithm for most efficient FH detection and screening in Lithuanian population. Methods: Since 2016 more than 220 FH patients have been included in Lithuanian FH registry, which is based on Lithuanian High Cardiovascular Risk (LitHiR) primary prevention programme and established as a part of The European Atherosclerosis Society (EAS) Familial Hypercholesterolaemia Studies Collaboration (FHSC) global FH registry. Adults with clinically suspected FH (LDL-C ≥5 mmol/L) were referred to specialist lipid centre, where detailed personal and familial anamnesis, physical examination, evaluation of laboratory and instrumental tests have been performed and secondary causes of dyslipidemia have been excluded. The clinical diagnosis of FH according to Dutch Lipid Clinic Network (DLCN) criteria were determined. Patients with DLCN score ≥6 and/or LDL-C ≥6,5 mmol/L were referred to genetic testing. Cascade first-degree relatives screening were initiated if an index-case meets DLCN criteria for definite or probable FH. Results: During the period of 2016–2021 data of 220 patients (120 females, 100 males) were included in the analysis. Mean age at FH diagnosis were 45,3 (±12,6) years with significant difference between genders: 48,17 (±12,68) years in women and 41,73 (±11,5) in men (p < 0,001). According to DLCN criteria definite FH has been diagnosed for 56 subjects (25,5%), probable FH – 65 patients (29,5%), possible FH – 68 subjects (30,9%) and 31 patients (14,1%) has been included in unlikely FH group. Results of genetic testing have been received for 126 patients: no mutation or variant of unknown significance (VUS) had been detected in 83 patients (65,87%), LDL receptor mutation has been found in 24 patients (19,05%) and ApoB receptor mutation – in 19 subjects (15,08%). Conclusions: The algorithm we use for FH detection seems to be valuable tool for recognizing patients who are in the highest probability of having FH gene mutation. High percent of undetected mutations may be associated with a number of genetically tested patients who were included in possible and unlikely FH groups. Therefore, it could be assumed that in the first instance patients who meet criteria for probable and definite FH should be referred for genetic testing and initiation of cascade screening should be started at this point. 108432 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JOSE PESENTI1, Leticia Gonzalez2, Francisco Vargas4, Gonzalo Martinez5, Prakash Saha6, Marcelo E. Andia2 (1) School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile; (2) ANID-Millennium Institute for Intelligent Healthcare Engineering, Santiago, Chile; (3) Biomedical Imaging Center, School of Medicine, Pontificia Universidad Católica, Santiago, Chile; (4) Vascular Surgery Department, School of Medicine, Pontificia Universidad Católica, Santiago, Chile; (5) Cardiovascular Division, School of Medicine, Pontificia Universidad Católica, Santiago, Chile; (6) School of Cardiovascular Medicine & Sciences, King’s College London, London, UK Introduction: Atherosclerosis, deep vein thrombosis (DVT) and chronic venous insufficiency (CVI) have been seen as different entities, however thrombo-inflammation could be the link between them. In this study we explore this relationship in patients with DVT, CVI and controls. Methods: We recruited 7 patients with DVT and 9 with CVI from the Vascular Surgery Department of our University Hospital and 9 healthy controls matched for gender and age. We registered their cardiovascular risk factors and obtained blood tests for hs-CRP, Circulating Endothelial Cells (CEC), ICAM-1 and CXCL7 as biomarkers of inflammation, endothelial and platelets activation. Carotids plaques were classified according to the AHA MRI classification. We used Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) to study the combined effect of all variables. Results: There were no significant differences between the groups for inflammatory markers and cardiovascular risk factors (Fig.A). However, using PCA and HCA we identified 2 clusters: cluster 1 with all the controls, one DVT and three CVI patients; and cluster 2 with only DVT and CVI subjects (Fig.B). There was a significant difference between carotid plaque complexity between both clusters (Fig.C). Conclusions: Our study supports the link between inflammation, DVT and atherosclerosis, and contributes to consider CVI as another risk factor for atherosclerosis. Acknowledgment: FONDECYT 1180525. 108463 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION RAFAEL NAUFEL DE SÁ REBELO1, Rafael Naufel de Sá Rebelo1, Cibele Isaac Saad Rodrigues1 (1) Pontifícia Universidade Católica de São Paulo The goal of this study was to compare blood pressure (BP) control, pre-and post-educational and therapeutical interventions in kidney transplant recipients (KTR) obtained by office (OBP) and ambulatory blood pressure monitoring (ABPM) at baseline and 6 months later. It is an observational, and interventional cohort study with 33 adult hypertensive and non-diabetic KTR. Data collection included three medical appointments bimonthly. Statistical analysis included a paired t-test, Student’s t-test, and Fisher’s test. We observed that OBP presents mean values higher than those obtained by the ABPM (p < 0.05) and high prevalence of atypical BP curve, especially white coat hypertension (46.1%), and 90% showed abnormal nocturnal dipping. Association of drug adjustments plus lifestyle changes was the most needed intervention (88%). The diastolic OBP significantly decreased after 6 months (p = 0.040). Patients were more likely to follow the diet changing (69,3%) than the physical activity program (34.6%). Dietary modification was associated with a significant reduction of ABPM mean systolic BP. This study showed high prevalence of hypertension, detected different phenotypes using ABPM, and implemented personalized actions to achieved BP control. These changes proved to be effective after a 6-moths showing that ABPM is an important strategy for BP diagnosis and follow-up in KTR. 112340 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOGERIATRICS MARIANA GAMBOA ESPARZA1, Ana Mercedes Andrade Aguilar2, Arturo Moreno Pérez1, Natalia Capistrán Páramo1, Henry De Las Salas Pérez1, Carlos Alberto Guillén Rosaldo1, Luis Alberto Lasses y Ojeda1 (1) Instituto Nacional de Cardiología Ignacio Chávez INCICH; (2) Hospital General Regional No. 46 IMSS Introduction: Very old patients form an increasing population in cardiology consultation, then specialized geriatric care is needed. Objective: To describe cardiovascular and geriatric profile in patients older than 95 years with heart disease. Methods: We performed a cross-sectional study collecting data through clinical record and a telephone survey to complete a geriatric comprehensive assessment (GCA) and cardiovascular variables of patients older than 95 years attending a tertiary referral hospital during 2021. Results: Of a total 74 patients older than 95 years, 36 participated in this study. 19 were deceased and 19 were unreachable. Mean age is 97 ± 1.79 years, and 75% were female. Mean years attending Cardiogeriatric Clinic is 6.2 ± 3.46. The most common diagnosis was hypertension (75%) followed by arrythmia (61%). Atrial fibrillation was the most common arrythmia (40%). 36% of patients have pacemaker. Ten patients are anticoagulated and in reduced dose. Only 3 participants visited emergency room in the last year. None of them was for bleeding. Prefrailty is present in 42% and Frailty in 53% of patients. Sarcopenia screening was positive in 69%. 13.8% had 2 or more falls. All patients have caregivers, and the majority self-reported a good health status. Even thought, 33% have depression and 28% have dementia. Conclusions: Geriatricians trained in heart diseases may improve outcomes in this population. Implementation of a Cardiogeriatric Clinic in a tertiary referral hospital is preparing new physicians’ generations to design interventions for patients older than 95 years, especially in frailty, sarcopenia and falls. 108471 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH HILDEMAN DIAS DA COSTA1, Hildeman Dias da Costa1, Luiz Felipe Façanha Ramos2, Leo Christyan Alves de Lima3, Matheus Akira Suzuki de Oliveira1, Mateus Viana Osório de Barros1, Mathews Barbosa Santiago4, Júlia de Ávila Gutierrez5, Laura Jane França Lacerda3, Ayrison de Melo Sousa4, Erica Mayara Gama Pinheiro1 (1) Universidade Federal de Rondônia; (2) Universidade Federal do Amapá; (3) Centro Universitário São Lucas; (4) Centro Universitário Uninorte; (5) Faculdades Integradas Aparício Carvalho Introduction: According to the Brazilian Association of Organ Transplantation, the heart is the third most transplanted organ in Brazil, behind only the kidney and liver. Structural problems in the Brazilian health sector and the resistance of families to authorize donations are some of the main obstacles to performing surgeries. Objective: To analyze the epidemiological profile of heart transplant surgeries in Brazil in the last 10 years. Methods: This is a cross-sectional epidemiological study. Data were obtained from the Informatics Department of the Unified Health System – DATASUS. The variables studied were: total number of transplants, states that performed the most surgeries, average length of stay, deaths and mortality rate. The research period was delimited between 2012 and 2021. Results: 2,732 heart transplant surgeries were performed. In 2012 and 2021, 193 and 226 transplants were performed, respectively. São Paulo and Minas Gerais were the states that performed the most surgeries: 852 and 400, respectively. The average length of stay was 17.8 days. The total number of deaths was 311. The average mortality rate was 11.38. Conclusions: The epidemiological profile of transplants was characterized by surgeries that took place mainly in the states of São Paulo and Minas Gerais, with an average length of stay of hospitalizations of 18 days. The reduction in the number of surgeries in 2020 and 2021 may reflect the pandemic caused by COVID-19. 108481 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH VILMEYZE LARISSA DE ARRUDA1, Vilmeyze Larissa de Arruda1, Lúbia Maieles Gomes Machado1, Jaqueline Costa Lima1, Pãmela Rodrigues de Souza Silva1 (1) Universidade Federal de Mato Grosso (UFMT) Introduction: Heart failure (HF) is characterized by decreased cardiac output and/or high filling pressures at rest or on exertion. HF is considered the most prevalent cardiovascular disease and the main cause of hospitalization in the Unified Health System. Objective: To analyze the trend of mortality from HF in Brazilians aged 50 years and over, within 21 years. Method: Ecological study with time series analysis of mortality from HF in Brazil, according to regions and federation units, in individuals aged 50 years and older in the period from 1998 to 2019. Deaths that had HF as the underlying cause (International classification of diseases –10: I50), that occurred during the study period were included in the study. Statistical analyzes were performed using the Stata 11.1 program. The HF mortality rate per 100,000 inhabitants was calculated. In the trend analysis, the Prais-Winsten regression was used. Results: Between 1998 and 2019, 567,789 deaths from HF were recorded in adults over 50 years of age, which corresponds to an average rate of 75.5 per 100,000 inhabitants. The trend was decreasing by sex, regions and in 23 FUs. The highest observed mortality rates occurred at older ages in all regions of the country. Conclusion: The trend of mortality rates from HF in adults aged 50 years and over among the federation units and Brazilian regions was decreasing over 21 years. There was an increasing trend in mortality from HF in the northern region and in the category other health facilities. 108598 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION YASMIN LEMOS ROLLEMBERG CRUZ MACHADO2, Flávia Lopes Fonseca2, Rossana Ghessa Andrade de Freitas2, Pedro Pimenta de Mello Spineti2, Erika Maria Gonçalves Campana2, Roberto Pozzan2, Maria Eliane Campos Magalhães2, Dayse Aparecida da Silva1, Andréa Araujo Brandão2 (1) Roberto Alcântara Gomes Biology Institute, State University of Rio de Janeiro (UERJ); (2) Faculty of Medical Sciences, State University of Rio de Janeiro (UERJ) Introduction: Several genetic polymorphisms have been investigated as potential risk predictors of cardiovascular (CV) diseases and their comorbidities mainly in adults from genetically homogenous populations; although their CV complications correlated with the presence of obesity and high blood pressure (BP) since childhood. Objective: To evaluate the association of eight SNPs in genes related to the renin-angiotensin-aldosterone system, endothelial function and leptin-melanocortin pathway with high blood pressure (BP) and obesity in a sample of admixed Brazilian adolescents from a longitudinal study. Methods: 1,054 Brazilian adolescents aged 10–15 years, both genders, were genotyped for rs699 (AGT), rs1799998 (CYP11B2), rs1799983 (NOS3), rs9939609 (FTO), rs1137101 (LEPR), rs3746619 and rs3827103 (MC3R), and rs17782313 (MC4R) through customized Multiplex Minisequencing System. Results: Prevalence of high BP and obesity were 13.5% and 41.4%, respectively. Minor A allele of rs3746619 (MC3R) was more prevalent in black (P = .001), in hypertensive (P = .017) and correlated with increased systolic BP (P = .023). G wild-type allele of rs1799983 (NOS3) was more prevalent in obese (BMI, P = .002; WC, P = .027), correlated with increased BMI (P = .020) and was associated with obesity in non-adjusted (OR = 0.713, P < .001) and adjusted regression models (OR = 0.730, P = .001). Variants rs3827103 (MC3R), rs699 (AGT) and rs1799998 (CYP11B2) presented different genotypic distributions according to color, gender, WC and BP status (P < .05). No other SNP was associated with elevated BP, obesity or their quantitative traits. Conclusion: The G wild-type allele of rs1799983 (NOS3) was associated with increased BMI and risk for obesity in admixed Brazilian adolescents with high BP. 108492 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY STELLA DE SOUZA VIEIRA1, Bianca Kajimoto Magalhães1, Andressa Muzzo de Souza1, Marcelo Arruda Nakazone1, Lilia Nigro Maia1, Maurício Nassau Machado3 (1) Faculdade de Medicina de São José do Rio Preto – FAMERP; (2) Escola Paulista de Medicina – Universidade Federal de São Paulo – UNIFESP-EPM; (3) fundação Faculdade Regional de Medicina de São José do Rio Preto – FUNFARME Introduction: Although the use of the Cockcroft-Gault (CG) formula is no longer recommended, the ease of application still makes it widely used. Recently, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was updated to disregard the race statement, a non-biological construct. Objective: We evaluated the strength of agreement between the CG formula and the 2021 CKD-EPI equation and assessed the impact of disagreement between estimated glomerular filtration rates (eGFR) methods on outcomes. Methods: A total of 2,855 adult patients who underwent cardiac surgeries (isolated coronary artery bypass grafting or heart valve surgery) were analyzed. Linear regression, Bland-Altman plots and Cohen’s Kappa statistics were used to determine the strength of agreement between the eGFR determinations. Cumulative survival curves were constructed to demonstrate the impact of renal function categories on mortality. Results: Sixty-one percent (1,748) and 23% (658) of the patients were classified by both equations as eGFR ≥60 and <60 mL/min/1.73 m², respectively. Sixteen percent of the patients were classified as discordant eGFR. There was a significant positive linear correlation (Spearman’s rho, 0.817; P < 0.001) between the eGFR based on the CG formula compared with eGFR according to the 2021 CKD-EPI equation. The Bland-Altman plots showed that the mean difference between the methods was 2.5 with the 95% limits of agreement ranging from –30.8 to 35.8, but the one-sample T test of the difference showed disagreement between the eGFR methods (P < 0.001). Patients with prevalent eGFR 60 to <90 and 45 to <60 had modest strength of agreement (K = 0,389 and K = 0,365; respectively) and the percentage of agreement was 69.7% and 80.6%, respectively. There was only one agreement classified as good in the stage 5 (K = 0.729), but this group represented only 2.4% of our cohort. The Kaplan-Meier curves showed cumulative survival rates at 30-day of 95.8%, 91.5% and 83.9% for patients with eGFR ≥60, discordant eGFRs, and eGFR <60 mL/min/1.73 m² (P < 0.001). Conclusions: Despite of a positive linear correlation, the means of the differences between the two equations is statistically significant and the CG formula shows only modest strength of agreement with the 2021 CKD-EPI equation, limited to the prevalent strata of renal function. This finding suggests that the CG formula should be no longer used in cardiac surgery settings to estimate glomerular filtration rate. 108495 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH JOSÉ LUCAS PERES BICHARA1, José Lucas Peres Bichara1, Luiz Antônio Viegas de Miranda Bastos1, Paolo Blanco Villela1, Glaucia Moraes de Oliveira1 (1) Universidade Federal do Rio de Janeiro – UFRJ Introduction: Cardiovascular diseases (CVD) are the main causes of death in the world and in Brazil, and cerebrovascular diseases (CVD) are one of the main responsible for these statistics, a fact aggravated by the aging of the population. Previous studies have already suggested a relationship between the evolution of CVD mortality rates and socioeconomic indicators. Objective: To relate the evolution of CVD mortality rates and the sociodemographic index (SDI) from 2000 to 2019 in Brazil and in its federative units (FUs). Methods: Ecological time series study of deaths from CVD in Brazil. Crude and standardized mortality rate for CVD were analyzed by sex, age group and FU between 2000 and 2019. Data were correlated with the SDI. Deaths and population were taken from DATASUS to estimate crude and standardized mortality rates per 100,000 inhabitants (direct method with Brazilian population in 2000). The SDI for each UF was extracted from the Global Health Data Exchange website. Results: In the period, there were 1,925,765 deaths from CVD in Brazil, 50.54% of which were male. The national SDI ranged from 0.538 in 2000 to 0.64 in 2019, with constant growth in the period. At the same time, the age-standardized mortality rate for CVD decreased from 49.81/100,000 inhabitants to 30.98/100,000 inhabitants. In this way, CVD has become the second leading cause of mortality in the country. In the FUs, all states in the North and Northeast regions showed improveme. 108510 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM FERNANDA AREJANO VAUCHER1, Maria Antonia C. da Silva1, Maria Eduarda M. B. Busko1, Vanessa C. de Lima1, Thaísa R. F. Basagla1, Ana Karyn E. de Freitas1 (1) Irmandade Santa Casa de Misericórdia de Curitiba (ISCMC) Background: The infection caused by COVID-19 in patients undergoing cardiac surgery leads to higher mortality and clinical complications in the postoperative period. In addition, most patients are older and have several comorbidities, which are factors of worse prognosis for disease progression. Objectives: This study aims to identify the impact of COVID-19 infection on the mortality of patients undergoing cardiac surgery within a 30-day period, as well as the incidence of clinical complications. Methods: We retrospectively evaluated 213 patients undergoing elective cardiac surgery or in the context of urgency and emergency, from January to December 2020, in a tertiary referral center. Of these, those who tested positive for COVID-19 within 30 days before or after the procedure were compared with those who did not have the infection in the period, considered the control group. Results: Of the 213 patients included in the study, 27 (12.6%) were diagnosed with COVID and 186 (87.3%) did not have the disease. Of the total, 95 were women (45%) and 118 were men (55%). The mean age was 62 years. There was no significant difference between the groups regarding epidemiological data. Mortality was significantly higher in the COVID-19 group (33% vs 15% patients in the control group). Complications such as arrhythmias (37% × 19%), prolonged ICU time (8 ± 8 days × 7 ± 7 days), total hospital stay (25 ± 16 days × 16 ± 19 days), respiratory failure requiring orotracheal intubation (30% × 1.1%), mechanical ventilation time (3.23 ± 7 days × 1.83 ± 5.48 days) and venous thrombosis (11% × 1.6%) were also more prevalent in the COVID group –19. The others showed no statistically significant difference, although they were more frequent in the group of patients with COVID-19. Conclusion: COVID-19 infection in the perioperative period of cardiac surgeries is associated with higher rates of morbidity and mortality. Delaying elective surgeries in coronavirus-positive patients can help reduce the risk of complications, along with effective strategies to decrease in-hospital infection. 108514 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES JOÃO MIGUEL MARTINS DE CARVALHO1, Ricardo Alves Pinto1, Tânia Proença1, João Calvão1, Ana Filipa Amador1, Catarina Marques da Costa1, André Cabrita1, Catarina Marques1, Carla de Sousa1, Mariana Paiva1, João Carlos Silva1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João Percutaneous valve commissurotomy (PMC) is an established treatment in patients with significative mitral stenosis (MS). Although rheumatic MS incidence has decreased in the last century, it remains a prevalent pathology worldwide. The Wilkins score (WS) is a reference in echocardiographic assessment of MS; a score ≤8 is considered a predictor of treatment success and score between 9 and 11 is a “grey zone” (WGZ) in which doubts persists regarding PMC success. The purpose of our study was to evaluate the early and long-term results of PMC in patients with rheumatic MS and to compare long-term events between patients with WS ≤ 8 and patients in WGZ. We retrospectively analysed all patients between 1991 and 2008 with significative rheumatic MS undergoing PMC. Data were collected at baseline and during long-term follow-up. MACE was defined as a composite of all-cause mortality, mitral valve re-intervention or cardiovascular hospitalization. In our cohort, 124 patients were included. Most were female (87%), mean age at the time of repair was 46 ± 11 year-old and mean follow-up was 20 ± 6 years. Before the procedure, 81% had WS ≤ 8 and 19% were in WGZ. Both groups had similar baseline characteristics, namely age at first intervention, NYHA class and follow-up time. All patients had preserved biventricular systolic function, 83% presented PH, mean transvalvular gradient (TVG) and mitral valve area (MVA) were 12.8 mmHg and 1.0 cm2, respectively. Most of the procedures were successful (91%) and without complications (94%). Mean MVA improvement was similar in both groups [0.9 cm2 in WS ≤ 8 and 0.8 cm2 in WGZ, t(102) = 0.173, p = 0.863]; there was also no significative difference in TVG and PASP reduction after PMC. During long-term follow-up, re-intervention and mortality occurred in 40% and 23% in WS ≤ 8 and in 50% and 29% in WGZ, respectively, and none of these differences was statistically significant (p = 0.389 and p = 0.544, respectively). Concerning time-to-event analysis, approximately 80% of patients kept uneventful and >90% alive after 10 years in both groups and no significant difference in M ACE events and all-cause mortality between WS ≤ 8 and WGZ was observed (Log Rank, p = 0,419 and p = 0.950, respectively). PMC was safe and effective in clinically significant rheumatic MS in both WS ≤ 8 and WS 9–11, with similar MVA improvement. After 10 years, approximately 80% of patients were MACE-free and >90% alive in both groups. There was no difference concerning WS groups. 108515 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JOÃO MIGUEL MARTINS DE CARVALHO1, Ricardo Alves Pinto1, Tânia Proença1, Catarina Martins da Costa1, Ana Filipa Amador1, João Calvão1, Catarina Amaral Marques1, André Cabrita1, Sofia Cardoso Torres1, Cristina Cruz1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João (CHUSJ), Porto, Portugal The Fontan procedure (FP) is considered a palliative surgical technique used for complex congenital heart disease (CHD) patients not suitable for biventricular repair. Currently, these patients’ life expectation is extended but they experience high morbidity and mortality risks and their long-term management is challenging. Our aim was to evaluate the morbidity of these patients after a long-term follow-up. We collected a retrospective cohort of patients palliated with FP, that were followed in an adult CHD outpatient clinic born between 1980 and 2001. Clinical and echocardiographic data were collected. A time to adverse event analyses was performed. A total of 26 patients were enrolled, with a median follow-up of 17 years. The median age was 24 (IQR 22–30) years-old, 35% were female. As for the cardiovascular risk factors, none had hypertension or dyslipidaemia, 3,9% had diabetes and 11,5% were smokers or previous smokers. The main anatomic abnormalities that lead to the FP were single ventricle (53,8%), followed by pulmonary atresia (23,1%), double outlet right ventricle (11,5%), ventricular septal defect (3,9%) and Ebstein anomaly (7,7%). The majority of patients were previously submitted to a shunt (42,3% with a Blalock-Taussig and 19,3% with an atrial septostomy); the most prevalent surgical technique was the cavopulmonary connection (69,2% extracardiac, 15,4% intracardiac), followed by the atriopulmonary anastomosis (11,5%). The systemic ventricle was morphologically left in 84,6%. A fenestration or a residual shunt persisted in 30,8%. The mean basal oxygen saturation was of 95%, with 3,9% of patients being cyanotic. The majority of patients were asymptomatic (69,2%), with a normal ventricular function in those with a systemic left-ventricle (91,7% of patients), and a moderately impaired in those with a systemic right-ventricle (50% of patients); more than moderate AV valve regurgitation was present in 7,7%. Pulmonary hypertension was observed in 7,7%. Atrial arrythmias were present in 11,5%. Liver disease affected 50% of patients (1 patient with an hepatocarcinoma) and protein-losing enteropathy was present in 7,7%. One patient was submitted to a re-FP, due to a Fontan circulation obstruction; another patient was submitted to a heart transplant. Regarding the time-to-adverse-events analyses, more than 65% of patients were event-free during the first 15 years. However, after 20 years of FP more than 60% of patients presented with an adverse event. 108517 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BRUNNO LEMES DE MELO1, Brunno Lemes de Melo1, STELLA DE SOUZA VIEIRA1, Ednei Luiz Antonio1, Helenita A. Oliveira1, Ighor L. Teixeira1, Andrey Jorge Serra1, Paulo José Ferreira Tucci1 (1) Universidade Federal de São Paulo; (2) Faculdade de Medicina de São José do Rio Preto Introduction: Sleep is an active and heterogeneous physiological process, evolutionarily conserved, which plays a critical role in survival. It is presumed that sleep has the main function of restoring the physiological and biochemical balances that change during the waking period. It is also crucial for the tissue regeneration process and metabolism. It is known that sleep deprivation or restriction (SR) generates negative physiological effects on the body. Sleep deficit promotes patent neuroendocrine and metabolic changes in humans and laboratory animals, such as reductions in the levels of thyroid hormones, thyroid-stimulating hormones, growth hormones, and testosterone, as well as increases in adrenocorticotropin, corticosterone, adrenaline, noradrenaline, and dopamine. Added to this are the hyperactivation of the sympathetic nervous system and the stimulation of a pro-inflammatory state. These implications lead to obvious clinical repercussions, by which the cardiovascular system is also affected. Evidence indicates that the decrease in sleep hours is associated with an increased risk in the development and worsening of cardiovascular diseases (CD). Objective: To determine if SR enhances cardiac remodeling after Myocardial Infarction (MI) in male rats; to verify if the treatment with β-blocker can attenuate the damages resulting from the SR after MI. Method: Male Wistar rats were used, the animals were divided into 5 groups (SHAM:submitted to sham surgery; MI:submitted to MI; SR: submitted to SR; MISR: submitted to MI and SR; MISR+βB:IMRS treated with β-blocker), in the groups submitted to the IM technique, only animals that had infarction affected >37% of the LV. Results: The MISR group had pulmonary congestion; the MI resulted in an increase in the diastolic area (LVDA) and systolic area (LVSA) of the LV, in the SHAM and SR groups there were no significant changes. The MI group demonstrated a decrease in the LV cross-sectional area fraction (LVAF) and an increase in nuclear volume; increased LV end-diastolic pressure (EDP) values were identified in the MI and MISR groups vs. Sham and MI vs. SR. Conclusion: The use of β-blocker was effective in slowing down post-infarction myocardial remodeling under SR conditions. These effects were observed in the LVAF, PDF, and pulmonary congestion preservation data. Thus, it is concluded that the β-blocker administered under conditions of adrenergic hyperactivity caused by SR was effective in attenuating cardiac remodeling. 108525 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION THAIRINI DE SOUZA MIGUEL1, Letícia Machado Santos1, Jorge da Silva Pinho Junior1, Anna Paula Arpini Botelho2, Julia Passarelli Pereira2, Sergio Girão Barroso1, Glauber Monteiro Dias2, Andrea Cardoso de Matos1, Grazielle Vilas Boas Huguenin1 (1) UNIVERSIDADE FEDERAL FLUMINENSE; (2) INSTITUTO NACIONAL DE CARDIOLOGIA Introduction: Cardiovascular diseases (CVD) are the main causes of death in the world and hypertension is the main triggering factor of CVD. Some individuals are resistant to standard treatment for hypertension, and resistant arterial hypertension (RAH) is characterized. Cocoa has been shown to be a food with interesting repercussions on cardiovascular health, attenuating blood pressure, improving glycemic and lipid profile, body composition, in addition to possibly protecting against DNA damage. However, it is not yet known whether these effects are reproduced in the RAH. Objective: To investigate the effect of cocoa powder consumption on DNA damage, blood pressure, glycemic and lipid profiles and body composition in patients with RAH. Methods: Randomized, double-blind, placebo-controlled clinical trial. Twelve participants were randomized into a control group (CG = 6) or intervention group (IG = 6). The GI received 30g of cocoa powder for 60 days. The GC received 30g of maltodextrin, colored and flavored, for 60 days. Blood glucose, HbA1c, triglycerides, total, HDL and LDL cholesterol, weight, BMI, waist and hip circumference were analyzed at times T0, T30 and T60. DNA damage analysis was performed using the comet assay, at T0 and T60. Descriptive statistical analysis was performed using the GraphPad Prism software. When applicable, ANOVA test was performed. Results were presented as mean and standard error and the difference was considered significant when p ≤ 0.05. Results: Participants were 59 ± 8.27 years old and 58.33% were women. At T30, there was a significant decrease in HbA1c (p = 0.0531) in the IG (5.325 ± 0.3065%) compared to the CG (6.520 ± 0.3865%). We observed a trend towards attenuation of DNA damage at level 1 (T0 21.75 ± 14.73–T60 10.50 ± 5.18). This result is inverse in the placebo-supplemented group, where we observed a trend of increased damage (T0 7.17 ± 1.41–T60 14.33 ± 4.47). The other parameters studied did not change. Conclusion: Supplementation with cocoa powder, by individuals with RAH, was beneficial in reducing HbA1c levels, without altering fasting glucose. As for DNA damage analysis, cocoa supplementation seems to add some protection to DNA, at damage level 1. More studies need to be carried out to better elucidate the issues addressed in the present study. 108527 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BRUNO GIUDICE D’AVILA1, Bruna de Matos Bauer1, Rosane Hermann2 (1) Irmandade Santa Casa de Misericórdia de Porto Alegre – ISCMPA; (2) Atenção Primária, Prefeitura Municipal de Brusque-SC Introduction: Smoking is an important isolated risk factor for cardiovascular disease and the leading cause of preventable morbidity and mortality worldwide. Such relevance has demanded from the health systems the elaboration of strategies to face this problem. This article aims to describe the successful experience of a multidisciplinary team of a Brazilian Public Health Unit in the fight against smoking during a follow-up period of 21 months. Methods: The strategy used for smoking cessation treatment involved group activities and psychosocial interventions with an emphasis on the cognitive-behavioral approach. Drug treatment with bupropion and nicotine patches was used according to medical evaluation and in accordance with the guidelines of the Brazilian Ministry of Health. A survey was carried out to evaluate users motivated to quit smoking and interested in participating in the proposed activities, in addition to evaluating the existence of cardiovascular comorbidities. The limit of up to 15 participants per group was defined. The activities were carried out in 10 sessions, the first four being weekly and the follow-up sessions after 15, 30, 60, 90 and 180 days, from February 2017 to November 2018. The actions were carried out by a team composed of a doctor, a nurse, community health agents and with the support of a nutritionist, a psychologist and a physical educator. All the professionals involved had previously completed a training course for the development of actions according to the clinical protocols and therapeutic guidelines of the Brazilian Public Health System. Results: During the study period, 54 patients were selected to participate in the activities. In this group, there was a predominance of females, a prevalence of arterial hypertension of 59% and diabetes of 18%. 74% of individuals reported at least one previous unsuccessful attempt to quit tobacco use. At the end of the observation period, 70% of the participants remained abstinent. There were 4 evasions due to change of residence territory and 12 due to resumption of tobacco use. Conclusion: This study highlights the remarkable potential of primary care in controlling one of the main risk factors for cardiovascular disease. It is possible to observe that even low-cost interventions for the Health System, as long as they are planned considering local sociocultural factors, can provide satisfactory results. 108534 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUKA DAVID LECHINEWSKI1, Lucas Henrique Olandoski Erbano1, Bruna Olandoski Erbano1, Nicolle Amboni Schio1, Raisa Natalia Dotto1, Amanda Zanlorenzi1, Lucas Baena Carstens1, Rafaela Lima Camargo1, Fernanda Leticia Perez Moraes1, Márcia Olandoski1, José Rocha Faria-Neto1 (1) Pontifícia Universidade Católica de Curitiba Introduction: COVID-19 pandemic rapid spread caused pressure over health systems and impaired assistance to other diseases, as observed by the reduction of acute coronary syndromes hospital admissions. In Brazil the number of cases and deaths related to COVID-19 happened quite differently through the Brazilian states (BS). Objectives: Identify socioeconomic, demographic and health structure services indices that had association with a higher death rate on the initial months of the pandemic and propose a score to identify the most vulnerable states based on these indicators. Methods: This population based ecological study analyses data from the 100th days after the first registered death for COVID-19 in the 27 BS and correlate with socioeconomic, demographic and health structure indices. The vulnerability score (VS) of COVID-19 death was composed by the indices that presented a Pearson’s linear correlation >0,4. We separated the BS on 3 groups, according to the cumulative death rate/100.000 inhabitants at the 100th day after the first death in each state. Results: The indicators with higher correlation coefficient were: the number of hospital bed/100.000 inhabitants (0,61), the Social Vulnerability Index (0,52), percentage of population <40 years old (0,51), population with tap water (0,49), number of doctor/100.000 inhabitants (0,44) and life expectancy at birth (0,43); these indices built the VS. The overall correlation coefficient between the final score and the number of deaths/100,000 inhabitants on the 100th day after the first death in each state was 0.63. The correlation was 0.44 for states with higher deaths (group 1), 0.71 for intermediate profile (group 2) and 0.85 for states with lower death rates (group 3). Conclusions: Socioeconomic, demographic and mainly health structure services indices before the pandemic correlate with COVID-19 mortality. The VS proposed has good correlation with the cumulated death rate/100.000 inhabitants at the 100th day after the first death. As a underdeveloped country, identify the most vulnerable states is strategic to better allocate supplies and health investments. 108567 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY TÂNIA FILIPA GALVÃO PROENÇA1, Ricardo Alves Pinto1, Miguel Martins Carvalho1, Catarina Costa1, Carlos Xavier Resende1, Pedro Diogo Grilo1, João Calvão1, Ana Filipa Amador1, Gonçalo Pestana1, Ana Lebreiro1, Luis Adão1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João Pulmonary vein (PV) isolation is an established treatment for atrial fibrillation (AF). A contact force (CF)-guided ablation protocol respecting region-specific criteria of lesion contiguity and lesion depth (‘CLOSE‘ protocol) has been associated with high incidence of acute durable PV isolation and a high single-procedure arrhythmia-free survival at 1 year. Differences in ablation index (AI) targets exist between centers, and its optimal value remains unknown. In the present study, we sought to evaluate the safety and outcomes of our local ablation protocol. We retrospectively analyzed 37 patients with paroxysmal AF who underwent antral PV encircling using a CF–sensing catheter in a tertiary center from January 2018 to November 2019. Radiofrequency (RF) was delivered targeting interlesion distance ≤6 mm and ablation index (AI) ≥380 at posterior wall and ≥500 at anterior wall. Cavotricuspid isthmus (CTI) ablation was performed if previous typical atrial flutter (AFL) was documented. PV isolation was documented with entrance- and exit block and the use of adenosine. Recurrence was defined as any AF, atrial tachycardia (AT), or AFL >30 s on 24-hour Holter monitoring or 12-lead ECG; or symptoms recurrence. 37 consecutive patients (70% male, median age 53 years) underwent antral PV encircling. 30 were taking antiarrhythmic drug (AAD) and 12 were submitted to previous cardioversion (11 with successful CV to sinus rhythm). Procedure and fluoroscopy time were 100 +/– 19.6 min and 5 +/– 2.0 min, respectively. Mean AI values, CF and RF time per lesion, were as follows: anterior left PVs – 490 +/– 21, 9.5 +/– 1.9 g, 32.0 +/– 5.1 sec; posterior left- 383 +/– 11, 10.53.5 g, 28.8 +/– 5 sec; anterior right – 498 +/– 15, 12 +/– 2,9 g, 32.3 +/– 4.8; posterior right – 384 +/– 12, 10.3 +/– 2.2 g, 27.9 +/– 3.1 sec. Incidence of first-pass and adenosine-proof isolation were 97% and 95%, respectively. Touch up lesions were applied to ensure isolation, with 100% success at the end of the procedure. CTI ablation was performed sucefully in 22%. 76% were discharged on AAD and 24% maintained AAD at one-year follow-up. At 12 months, single-procedure freedom from recurrence was 89%. Only one patient had an acute complication, a femoral haematoma that solved with local compression. Our experience with “CLOSE” protocol with specific AI target supports that an ablation respecting the referred predefined criteria for lesion depth and contiguity results in safe and efficient outcomes. 108570 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM TÂNIA FILIPA GALVÃO PROENÇA1, Ricardo Alves Pinto1, Miguel Martins Carvalho1, Filipa Amador1, Catarina Costa1, João Calvão1, André Cabrita1, Catarina Marques1, Ana Pinho1, Luís Santos1, Paula Dias1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João Myocarditis and pericarditis are inflammatory diseases affecting 1 to 10 persons in 100 000 persons/year. Anti-SARS-Cov-2 vaccines are being widely used worldwide, and concerns about its association with a higher risk of cardiac inflammatory syndromes are being raised. We compare the prevalence and severity of myocarditis and/or pericarditis admissions before and after Covid-19 vaccination. We retrospectively analysed all patients admitted to a Cardiology Department due to myocarditis and/or pericarditis during COVID-19 pandemic and compared the clinical data between two 10-months periods, before and after Covid-19 vaccination. Pre-vaccination period (PVp) was defined from March to December 2020 and vaccination period (VCp) from January to October 2021. A total of 23 patients were enrolled, 12 in PVp and 11 in VCp, with a median age of 28 (IQR 22–43) year-old. Of total, 44% had perimyocarditis, 30% myocarditis and 26% pericarditis. In VCp there was significatively more man admitted (50% vs 90%, p = 0.019). Concerning clinical data, the two periods were similar. Median CRP was 68 mg/L and, in perimyocarditis or myocarditis, median high-sensitivity troponin I and BNP were 10 250 ng/L and 38 pg/mL, respectively. The majority of patients had ECG abnormalities, mostly ST-elevations (39%) and repolarization abnormalities (20%). At presentation, left systolic function was preserved in all patients in PVp group, while 27% had mildly to moderate reduced ejection fraction in VCp (p = 0.052); all of them improved to normal function. Of the 17 patients with myo or perimyocarditis, 14 were submitted to MRI (median 6 days after the event): 60% had high signal intensity in T2 weighted sequences suggestive of oedema and 73% had LGE enhancement compatible with myocarditis. Median left and right ventricular ejection fraction by MRI were both 61%. In VCp group, 55% of patients were previous vaccinated (3 patients with mRNA-1273, 1 with BNT162b2 and 2 with JNJ-78436735 vaccines). The median time between last vaccine administration and event was 73 days (2–112). In VCp group performing cardiac MRI, 40% of vaccinated patients had LGE enhancement (p = 0.038). There was no difference in the incidence of patients admitted. During two equal periods before and after Covid-19 vaccination, there was no difference in the incidence of patients admitted with myocarditis and/or pericarditis. Curiously patient with previous Covid-19 vaccination exhibited less LGE-enhancement on cardiac MRI. 108605 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING TAYNE FERNANDA LEMOS DA SILVA1, Larissa dos Santos Brandão1, Raquel Maria Alexandre da Silva1, Maria Tayná Silva Feitosa1, Monaliza Evelyn Pereira de Sousa2, Gislainy Thais de Lima Lemos1, Rodrigo Manoel do Nascimento2, Dara Stephany Alves Teodorio2, Hortênsia Paula Bernardino Ribeiro1, Renata Santos de Oliveira3, Dulcineide Gonçalo de Oliveira3, Simone Maria Muniz da Silva Bezerra4 (1) Enfermeira Especialista em Cardiologia pelo Pronto Socorro Cardiológico de Pernambuco – PROCAPE/UPE; (2) Residente de Enfermagem em Cardiologia Uniprofissional e Multiprofissional do PROCAPE/UPE; (3) Núcleo de Saúde da Secretaria Estadual de Saúde de Pernambuco – NET-SES/PE; (4) Coordenadora do Programa de Residência Uniprofissional e Multiprofissional em Cardiologia do PROCAPE/UPE Introduction: In Brazil, cardiovascular diseases account for 30% of all deaths, with about 1,000 deaths per day. In 2017, the Ministry of Health (MS) launched a projector called National Telediagnostic Offer, expanding the remote diagnosis service to the poorest areas of Brazil.² The Telecardiology Network of Pernambuco aims to meet the demands on the Clinical Management of Cardiovascular Diseases in its municipalities, with the issuance of online electrocardiogram reports (Tele-ECG) and teleconsultations. This network works in partnership with a multiprofessional residency program of a hospital recognized by the MS as one of the four cardiology reference institutes in Brazil, located in the state of Pernambuco. Objective: To describe the results of the activities carried out by nursing residents in cardiology in a referral hospital in the State of Pernambuco. Methods: This is a descriptive, observational study based on the activities carried out by nursing residents of a multiprofessional program in cardiology, from March 2020 to February 2022. Results: During the cardiology residency program, nurses had the opportunity to be pioneers in Teleassistance in cardiology services, in partnership with the Pernambuco State Health Department (SES-PE). The following were implemented: Telemedicine-based service for the follow-up and monitoring of patients treated with anticoagulant therapy and the Tele-ECG service in primary health care in the municipalities of Pernambuco. The residents established the outpatient department flowchart and carried out training for the multidisciplinary team. Up to now, 8,712 teleconsultations were performed from June 2020 to February 2022, and of these, 3,202 were performed by nursing residents. In the Tele-ECG service, from 185 municipalities, 56 (30.3%) are already issuing electrocardiogram reports and 52 (28.1%) are still in the implementation period. In total, 14,349 reports were issued (from October 2020 to March 2022), of which 14,121 had been classified as an elective electrocardiogram and 122 as an emergency report. Therefore, the joint work of the multiprofessional residency program and the SES-PE constitutes a success when considering the high productivity attained by the project so far and assuring the access of patients from the countryside of Pernambuco to specialized cardiology services. 108577 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY TÂNIA FILIPA GALVÃO PROENÇA1, Ricardo Alves Pinto1, Miguel Martins Carvalho1, João Calvão1, Ana Filipa Amador1, Catarina Costa1, Catarina Marques1, André Cabrita1, Cátia Oliveira1, Mariana Paiva1, João Carlos Silva1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João Rheumatic fever in childhood used to be the major cause of valve disease in women. Despite the reduction in its incidence, nowadays rheumatic mitral stenosis (MS) remains an important healthcare problem. We retrospectively analysed all women with clinically significant rheumatic MS submitted to percutaneous mitral commissurotomy (PMC) between 1991 and 2008 and compare long-term events between patients with and without pulmonary hypertension (PH). A total of 108 women were enrolled, with a mean age at the time of PMC of 46 ± 11-year-old and a median follow-up of 21 (IQR 17–26) years. Before the procedure, all patients had preserved biventricular systolic function; 86% presented PH, median transvalvular gradient (TVG) and mitral valve area (MVA) were 12.0 mmHg and 1.0 cm2, respectively, Most of PMC were successful (91%) and without major complications (94%), with a median MVA improvement of 0.9 cm2 and reduction of 9.0 mmHg in TVG and 8.0 mmHg in pulmonary artery systolic pressure (PASP). During long-term follow-up, 43% of patients were submitted to re-intervention (most of them surgically) and 25% died. In patients non-submitted to re-intervention, TVG and PASP remained similar with early post-procedure evaluation (p = 0.241 and p = 0.964, respectively), while MVA reduced over time, yet still statistically superior to baseline MVA (1.5 cm2 vs 1.0 cm2, p < 0.001). Concerning time-to-event analysis, approximately 80% of patients kept uneventful after 10 years; after 30 years, more than 20% continued MACE-free and almost 50% were alive. Regarding PH presence at time of PMC, there was no significant difference in MACE events and all-cause mortality (Log Rank, p = 0,711 and p = 0.689, respectively). PMC was safe and effective in clinically significant rheumatic MS in women. 108585 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY RICARDO JORGE DOS REIS ALVES PINTO1, Miguel Martins Carvalho1, Tânia Proença1, Catarina Costa1, Ana Filipa Amador1, João Calvão1, Catarina Marques1, André Cabrita1, Cátia Oliveira1, Cristina Cruz1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João, Porto, Portugal Introduction: Adult Congenital Heart Disease (ACHD) patients are an increasing population with known high risk for thromboembolic events. Validated scores as CHA2DS2-VASc and HAS-BLED are uncertain in this population. Although apparently safe, data is scarce about the use of non-vitamin K oral anticoagulants (NOAC) in this population. Its use remains off-label and more studies are warranted. Purpose: To evaluate all patients on-NOAC followed in an ACHD outpatients clinic and observe its safety and efficacy in thromboembolic prevention during a median follow-up of 34 months (IQR 7–60 months). Major bleeding was defined according to types 3 to 5 in Bleeding Academic Research Consortium (BARC) scale. Adverse event was defined as at least one of the follows: death, stroke, myocardial infarction, systemic embolism or major bleeding. Results: A total of 65 patients on NOAC were included, with a mean age of 52 ± 14 year-old, 66% were female. Most frequent ACHD were atrial septal defect (22%) and tetralogy of Fallot (TOF, 22%), followed by atrioventricular septal defect (17%) and transposition of great arteries (9%). Regarding cardiovascular risk factors, 37% had hypertension, 23% had dyslipidaemia, 9% had diabetes, 8% were smokers or previous smokers and 23% had obesity. Most patients had preserved biventricular function, 20% presented systemic ventricle systolic dysfunction and 12% subpulmonic ventricle systolic dysfunction. Atrial fibrillation or atrial flutter (AF/AFL) were the major reasons for anticoagulation (94% of patients); the remaining were on NOAC due to previous ischaemic stroke, intra-cardiac thrombus or deep venous thrombosis. At the time of NOAC initiation, 49% of patients had a CHA2DS2-VASc score ≥2 (median 1, IQR 1–3) and median HAS-BLED score was 0 (IQR 0–2); 43% were medicated with apixaban, 29% with rivaroxaban, 22% with edoxaban and 6% with dabigatran. During a median follow-up of 34 months, none of the patients had ischaemic complications or major bleeding and one patient died after pulmonic prothesis dysfunction surgery. Concerning time-to- adverse-event analysis, all patients kept uneventful after 2 years and more than 95% continued event-free after 8 years on-NOAC. Conclusion: In an ACHD population on-NOAC, the major reason for anticoagulation was AF/AFL. After a long-term follow-up, most patients maintained event-free after 8 years. This report highlights the safety and effectiveness of NOAC in ACHD patients. 108588 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE YASMIN LEMOS ROLLEMBERG CRUZ MACHADO2, Gizella da Cunha Rodrigues1, Rossana Ghessa Andrade de Freitas2, Maria Eliane Campos Magalhães2, Ricardo Mourilhe-Rocha2, Andréa Araujo Brandão2, Denilson Campos de Albuquerque3, Dayse Aparecida da Silva1 (1) Roberto Alcântara Gomes Biology Institute, Rio de Janeiro State University (UERJ); (2) Faculty of Medical Sciences, Rio de Janeiro State University (UERJ); (3) Department of Cardiology, Pedro Ernesto University Hospital/Rio de Janeiro State University (UERJ) Introduction: The role of genetic variants in multifactorial diseases has been widely investigated through genome-wide association studies, mainly replicated in genetically homogeneous populations. However, the replication of such associations in smaller assays requires an alternative molecular methodology more accessible to the scientific community as a whole. Customized Multiplex assays including informative genetic markers offer a single, low-cost and practical option. Aim: To develop a cheaper Multiplex methodology based on SNaPshot® technology for genotyping 21 SNPs clinically relevant in genome-wide studies and their application in clinical samples from an admixed population. Methodology: Four custom Multiplex Panels involving 21 SNPs in genes related to cardiovascular risk factors were constructed for genotyping 1,064 buccal swabs and 528 blood samples, differently preserved. The resulting genotypes were validated by Sanger sequencing. Results: The full genetic profile was achieved in 72–92% among the panels considering both swabs and blood samples. Marker’s performance ranged from 81–100%. Panels reached above 90% of confident genotyping calls in samples stored for 2 years or more, regardless of their type. The agreement rate between genotyping methods ranged from 97.5–100%. Conclusion: The customized Multiplex Panels represented an efficient molecular methodology to genotype SNPs with high sensitivity, efficiency, and low cost from different biological samples stored for years. 108608 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MATHEUS AKIRA SUZUKI DE OLIVEIRA1, Matheus Akira Suzuki de Oliveira1, Júlia de Avila Gutierrez2, Hildeman Dias da Costa1, Luiz Felipe Façanha Ramos3, Karen Tássia Façanha Ramos3, Mathews Barbosa Santiago5, Leo Christyan Alves de Lima4 (1) Universidade Federal de Rondonia; (2) Centro Universitário Aparício Carvalho; (3) Universidade Federal do Amapá; (4) Centro Universitário São Lucas; (5) Centro Universitário Uninorte Introduction: Acute myocardial infarction is a pathology that affects all age groups and should be prevented even in less common age groups, such as children and adolescents, in order to reduce morbidity and mortality rates. The points to be observed are care with the prevention of cardiac risk, and the adoption of healthy lifestyle habits throughout childhood. Objective: To describe the epidemiological profile of hospitalizations for acute myocardial infarction among children and adolescents in Brazil between 2012 and 2021. Methods: Cross-sectional epidemiological study. Data were obtained from the informatics department of the unified health system – DATASUS. The variables studied were: total number of hospitalizations, sex, color/race, deaths and mortality rate. The age group surveyed was individuals from zero to 19 years old. The period was delimited between 2012 and 2021. Results: 2,542 hospitalizations were recorded. Males reported 1,472 hospitalizations. The brown color/race registered 867 hospitalizations. The most affected age group was adolescents between 15 and 19 years old, with 1,182 hospitalizations. The total number of deaths was 172. The average mortality rate in the period was 6.77. Conclusions: The epidemiological profile of hospitalizations was characterized by brown adolescents aged between 15 and 19 years. The reduction in the number of hospitalizations in 2020 and 2021 may have been influenced by the COVID-19 pandemic. 108617 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES GUILHERME PINHEIRO MACHADO1, Fernando Luis Scolari1, Gustavo Neves de Araujo3, Alan Pagnocelli1, Angelo Chies2, Marco Vugman Wainstein2, Rodrigo Vugman Wainstein1 (1) Hospital de Clinicas de Porto Alegre (HCPA); (2) Universidade Federal do Rio Grande do Sul (UFRGS); (3) Imperial Hospital de Caridade Background: Incidence and mortality in cardiogenic shock (CS) remains high despite current management. Improvements in early diagnosis and risk stratification are warranted in order to prevent CS in ST segment elevation myocardial infarction (STEMI). Objectives: Our aim is to evaluate the association between pulmonary congestion evaluated by lung ultrasound (LUS), Society for Cardiovascular Angiography and Interventions (SCAI) shock classification and clinical outcomes in patients admitted with STEMI. Methods: Prospective cohort study of STEMI patients treated in a tertiary care hospital in Brazil. LUS was performed immediately before coronary angiography. Development of cardiogenic shock in the first 24 hours and in-hospital mortality were retrospectively evaluated. Results: A total of 582 included patients. Mean age was 61 ± 12 years and 373 (64.1%) were male. After 24 hours of admission, SCAI shock stage A was present in 361 (62%) patients, while 115 (19.8%) were class B, 44 (7.6%) class C, 58 (10%) class D, and 4 (0.7%) class E. There was an association between increasing number of positive LUS zones and the SCAI shock classification (P < 0.001). We also found strong association between number of positive zones in lung ultrasound and CS (OR = 1.4 (95% CI 1.3–1.5, P < 0.001), SCAI shock stages (OR 1.3 (95% CI 1.2–1.4, P < 0.001) and in-hospital mortality (OR 1.3 (95% CI 1.2–1.4, P < 0.001). Additionally, presence three or more positive LUS zones was associated with increased mortality, P (log-rank) < 0.001. Conclusions: Lung congestion evaluated by admission LUS was significantly associated with increment in SCAI shock stage, development of CS and in-hospital mortality in STEMI patients. 108636 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY THAYANNE MENDES DE ANDRADE1, Mariara Lopes da Costa Marques1, Sofia Almeida Guerra1, Thaís Rocha Salim1, Glaucia Maria Moraes de Oliveira1 (1) Universidade Federal do Rio de Janeiro (UFRJ) Introduction: Cardiac arrest (CA), in pediatrics, has high mortality and severe neurological sequelae. Information on the causes and mechanisms of death in children under 20 can provide theoretical support for improving health in childhood and adolescence. Objectives: To carry out a population analysis of mortality rates (MR) from basic and multiple causes of death, in children under 20 years of age, of both sexes, from 1996 to 2019, and to identify the frequency of description of CA in death certificates (DC) of these individuals and their places of occurrence, to promote strategies to improve the prevention of deaths. Method: An ecological time-series study, from 1996 to 2019, of children deaths under 20 years of age, in which MR and we evaluated proportional mortality (PM) due to underlying cause of death. We analyzed the percentage of CA description in the DC (any lines) and the place of occurrence. Also, we described MR per 100,000 inhabitants and PM due to underlying cause of death under 20 years of age, by sex, and age group. The percentages of death from underlying causes were calculated when CA was described in any line of parts I and II of the DC, by age groups, and the percentages of death from underlying causes, according to their place of occurrence. Data were taken from DATASUS, IBGE and SINASC. Results: In Brazil, from 1996 to 2019, there were 2,151,716 deaths in children under 20 years of age, with a MR of 134.38 per 100,000 inhabitants, with higher death rates among male neonates. Two hundred forty-nine thousand three hundred thirty-four had CA described in any DC line, corresponding to 11.6% of these deaths. We defined four patterns for the underlying cause of death when CA in the death sequence. In the neonatal period, perinatal causes; in children under five years, respiratory system diseases; 5 to 14 years, neoplastic and hematological; and in adolescents aged 15 to 19, external causes. The central place of occurrence of these deaths was in the hospital. Conclusion: The highest MR of underlying causes of death, in those under 20 years of age, in Brazil, from 1996 to 2019, was due to perinatal and external causes. When we evaluated multiple causes of death, the leading underlying causes of CA were respiratory, hematological, and neoplastic diseases, with higher in-hospital mortality. It is crucial to understand deaths sequence events and implement teaching strategies to pediatric cardiopulmonary resuscitation. 108644 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING LETÍCIA DE CARVALHO BATISTA1, Maria Do Perpétuo Socorro de Sousa Nóbrega1, Marina de Góes Savetti1, Rita de Cássia Gengo e Silva Butcher1 (1) School of Nursing University of São Paulo Objective: To evaluate the experience and acceptability of an educational and musical nursing intervention to reduce anxiety in patients undergoing unscheduled catheterization. Method: Qualitative study was carried out in a highly specialized cardiology hospital in the city of São Paulo. In the study, 15 patients diagnosed with the acute coronary syndrome were treated and submitted to unscheduled catheterization at the emergency unit. All patients received the nursing intervention, consisting of a musical and educational component called Education and Music Intervention to Reduce Anxiety (EMIRA), prepared according to the methodological framework of complex intervention by Sidani and Braden. The interviews were recorded, transcribed, and analyzed according to Bardin’s content analysis. Data collection took place from October to September 2021. Results: The analysis of the interviews allowed the grouping into three categories, 1) EMIRA Intervention: a new experience that helps reduce anxiety; 2) EMIRA intervention: an experience that generates satisfaction; and 3) EMIRA Intervention: anxiety alleviating experience. Participants have suggested using EMIRA to promote a feeling of relaxation and satisfaction. Conclusion: EMIRA seems to be an acceptable and potentially relaxing intervention for patients awaiting unscheduled catheterization in the emergency department. 108674 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LUAN OLIVEIRA MATOS1, Anna Engell Holm2, Laura C Gomes3, Alma Wegener2, Karine O Lima1, Molly Dam Kaagaard2, Isabelle V. M. Vieira1, Claudio Romero Farias Marinho3, Rodrigo Medeiros de Souza1, Tor Biering-Sørensen2, Odilson M Silvestre1, Philip Brainin1 (1) Universidade Federal do Acre; (2) Herlev-Gentofte Hospital; (3) University of São Paulo Background: B-lines by lung ultrasound (LUS) indicate presence of extravascular lung water and has been associated with reduced left ventricular ejection fraction (LVEF) in patients with heart failure. We aimed to describe the prevalence of reduced LVEF in a community sample without a history of heart failure and to evaluate the usefulness of B-lines by LUS in this setting. Methods: In a cross-sectional study we examined a random sample of adults (≥18 years) from a community in the Northwestern part of the Brazilian Amazon (June-December 2020). All participants underwent state-of-the art echocardiographic image acquisition and 8-zone LUS by a medical doctor. No patients had known heart failure, recent chest trauma or clinical signs of infectious disease. Reduced LVEF was determined by Simpson’s biplane method and defined as <45%. We assessed the mean of B-lines across all zones. Logistic regression models were applied to investigate reduced LVEF and B-lines. Results: A total of 551 participants were included (39% men, mean 41 ± 15 years) who had a mean LVEF of 57 ± 5%. From this group 16 (3%) had LVEF <45%, corresponding to a prevalence of 29/1000 adults with reduced LVEF. Participants with reduced LVEF were older, had higher blood pressure and more frequently smoked. Number of B-lines by LUS was significantly higher among participants with reduced LVEF compared to those with normal LVEF (mean B-lines 4 vs 1, P = 0.002. In logistic regression models, adjusted for clinical and cardiovascular risk factors, presence of a single B-line was associated with 1.18 higher odds of having reduced LVEF (95%CI 1.06–1.31, P = 0.002). Conclusion: The prevalence of reduced LVEF was 29/1000 adults in a community from the Amazon Basin without a known history of heart failure. B-lines by LUS were significantly more present in participants with reduced LVEF. As LUS is feasible in resource limited settings, may be conducted by non-medical personnel and by the use of handheld devices, this may be useful to identify patients with reduced LVEF in rural communities where echocardiography is not available. 108747 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE STIJN VAN BRUGGEN1, Sirima Kraisin1, Jore Van Wauwe1, Paolo Carai1, Thilo Witsch2, Kimberly Martinod1 (1) Centre for Vascular and Molecular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium; (2) Department of Cardiology and Angiology I, Heart Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany; (3) Department of Cardiology, University Hospital Basel, University of Basel, Basel, Switzerland Increasing age is the highest risk factor for cardiovascular disease and heart failure (HF) development, and HF is a leading cause of death in the elderly. In our study, we aimed to investigate the specific role of neutrophils and neutrophil extracellular traps (NETs) on cardiac remodeling and decreasing function with increasing age. For this, neutrophil-specific PAD4 deletion (Ne-PAD4-/-) mice were created and aged for 2 years together with their littermate controls (WT). After 2 years we could clearly identify a decrease in both systolic (LVEF) and diastolic (E/A ratio) cardiac function in WT mice, while this age-dependent cardiac dysfunction was absent in Ne-PAD4-/- mice. In addition, cardiac function of old Ne-PAD4-/- mice was comparable to young controls. In order to explain this decreased function, hearts were evaluated histologically which showed an increase in cardiac collagen deposition in old WT as compared to old Ne-PAD4-/- mice. Additionally, we found increased levels of circulating CXCL-1, a chemokine responsible for neutrophil recruitment to sites of inflammation in old WT mice, as well as increased levels of plasma IL-6. From this we can conclude that neutrophil PAD4, and in extension likely NETs, are essential for age induced cardiac remodeling, possibly through the increased recruitment of additional neutrophils to the heart, in response to an elevated state of chronic inflammation, which is the case during natural aging. 108681 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LILLIAN LIZE ENGELBRECHT (MARRIED SURNAME: GILES)1, Nasief van der Schyff3, Mark Engel1 (1) University of Cape Town (UCT); (2) Groote Schuur Hospital (GSH); (3) Victoria Hospital Wynberg (VHW) Introduction: Sub-Saharan Africa (SSA) is experiencing a change in its pattern of disease prevalence, and it has been projected that non-communicable diseases (NCD) will overtake infectious diseases by the year 2030. Despite the fact that more than 80% of deaths from NCD’s worldwide are estimated to occur in low-and middle-income countries, the majority of available research is from first world countries. Due to SSA’s own rich genetic, geographic, social and cultural diversity, this available first world research is not applicable to our context, highlighting the need for more relevant local studies. Objectives: To better understand the clinical and epidemiological characteristics of patients admitted with cardiac disease in our district, in order to inform and guide relevant clinicians and policymakers. Methods: We conducted a retrospective records review of all patients admitted with a primary cardiac diagnosis to a district level hospital in South Africa between 1 September 2020 to 30 November 2020. Results: Our final analysis is still in progress, but we can report some preliminary findings at this time. During the three month period, we collected data from 236 cardiac admissions at our district-level, 52 medical bed, hospital. The age of patients ranged from 22 to 95, with a mean age (standard deviation) of 60 (±14.79) years. The length of stay ranged from under 24 hours, up to 26 days, with a median length of 3 days. Hypertension (76%), Smoking (55%), and Diabetes (42.7%) were the top 3 cardiovascular risk factors noted. More than a third of diabetic patients (36.2%) had a glycated haemoglobin (HbA1c) of greater than 10.1%. Acute decompensated heart failure (45.8%) and acute coronary syndromes (40.1%) were responsible for the vast majority of admissions. Admissions were most often precipitated by prior inadequate therapy (29%), defaulting medication (17.8%) or uncontrolled hypertension (14.8%). There was a 6.7% readmission rate noted during the three month period. The inpatient mortality rate was 7.8%. Conclusion: This study showed that cardiovascular disease (CVD) can affect a wide range of ages, and reiterated the well known link between CVD and hypertension, smoking and diabetes. Poorly controlled hypertension was the third most common precipitant for admission, and a third of diabetic patients had poor glycaemic control, highlighting important areas for intervention in order to reduce the readmission and mortality rate. 108688 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSIOTHERAPY SAMPATH KUMAR AMARAVADI1, G Arun Maiya1 (1) Department of Physiotherapy, Manipal College of Health Professions, Manipal Academy of Higher Education, Manipal, Karnataka, India; (2) Department of Physiotherapy, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates. Background: Impaired glucose control & Insulin resistance are reported to be risk factors for the development of cardiovascular diseases. In the Indian population, Insulin resistance is one of the strong reasons for altered glycemic control, even though exercise training is found to be effective in glycemic control along with standard medical care. There is a dearth of literature on the effectiveness of exercise training on insulin resistance and quality of life in patients with Type 2 diabetes mellitus. Objective: To determine the effects of a structured exercise program on insulin resistance, glycaemic control, functional capacity, and quality of life in patients with Type 2 diabetes mellitus. Design: Randomized, controlled trial. Setting: Diabetic Foot Clinic, Department of Physiotherapy & Department of General Medicine, Kasturba Hospital in Manipal, Karnataka, India. Participants: 160 participants aged between 30–65 years with Type 2 diabetes mellitus. Intervention: A set of structured exercise programs (aerobic, resistance, and combined) along with the standard hospital care was performed 3–5 times weekly for 12 weeks. Measurements: Primary outcome measures: Fasting Insulin Level, Homa-IR, Six-minute walk test (6MWT), and WHOQOL-BREF questionnaire at baseline and 12th week. Secondary outcome measures: Body composition analysis, Fasting Blood Sugar, Postprandial Blood Sugar, Glycated Hemoglobin (HbA1c), and GPAQ questionnaire at baseline and 12th week. Results: The mean age of the participants in the study group was 56.05 ± 8.77 and the control group was 53.90 ± 10.20, the mean duration of diabetes in the study group was 12.74 ± 8.67 years and the control group was 11.46 ± 5.11 years, mean body mass index 25.32 ± 3.16 in the study group and 26.27 ± 3.63 in the control group. Significant differences have been observed in Homa-IR (p = <0.001), Fasting insulin (p = <0.001), Fasting blood sugar (p = <0.001), Postprandial blood sugar (p = <0.001), Glycated hemoglobin (p = <0.001), WHOQOL-BREF (p = <0.001), Six-minute walk test (p = <0.001) in the study group when compared with the control group. Conclusion: In the present study, we found that a 12-week structured exercise training program is effective in decreasing insulin resistance and improving the quality of life in type 2 diabetes mellitus. We also found that a 12-week structured exercise training program is effective on functional capacity and glycemic control in type 2 diabetes mellitus. 108714 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY TADEU LIMA MONTAGNOLI1, Jaqueline Soares da Silva1, Bruna de Souza Rocha1, Bianca dos Santos Carlos do Nascimento1, Bruno Eduardo Dematte1, Gabriel Fonseca Gomide1, Carlos Alberto Manssour Fraga1, Gisele Zapata Sudo1 (1) Universidade Federal do Rio de Janeiro Background: Pulmonary arterial hypertension (PAH) is characterised by increased pulmonary arterial pressure and vascular thickening, which ultimately leads to right ventricle (RV) failure. Current therapies lack efficacy in preventing disease progression and lead to poor survival of patients at moderate-to-high risk. Rho-activated protein kinases (ROCK) play a key role in smooth muscle contraction and proliferation and cardiomyocyte hypertrophy and survival, and ROCK inhibitors are potential candidates for treatment of PAH. This study assesses the efficacy of a new ROCK inhibitor, LASSBio-2065, on cardiac and vascular function of monocrotaline (MCT)-induced PAH in Wistar rats. Methods: PAH was induced after a single intraperitoneal injection of 60 mg/kg monocrotaline (MCT). After 2 weeks, PAH was confirmed by Doppler echocardiography and rats were randomly divided in groups (n = 6) and treated with vehicle or LASSBio-2065 (60 μmol/kg/day, i.p.) for 14 days. Wistar rats which received a single saline injection and treated with vehicle were used as controls. At the end of treatment, hemodynamics and cardiac function were evaluated by echocardiography and RV catheterization and tissues were collected for histologic and morphometric analysis. Data was analysed by One-way ANOVA followed by Dunnett post-test and presented as mean ± standard error. Results: LASSBio-2065 reduced the pulmonary acceleration time-to-total ejection time ratio (26.7 ± 2.0 vs. 21.7 ± 1.6; p < 0.05) and increased RV output (64.2 ± 6.1 vs. 39.1 ± 8.0; p < 0.05), thus alleviating pulmonary vascular resistance and RV afterload. Arterial and cardiac remodelling were prevented by LASSBio-2065, as indicated by reduced medial wall thickness of distal pulmonary arterioles (60.7 ± 1.1 vs. 68.5 ± 3.9; p < 0.05), short axis RV-to-left ventricle (LV) internal area (85.8 ± 11.4 vs. 115.2 ± 17.8; p < 0.05) and RV Fulton index of hypertrophy (44.8 ± 2.3 vs. 55.6 ± 2.3; p < 0.05). The new ROCK inhibitor also improved the RV diastolic function, restoring both RV end-diastolic pressures (5.3 ± 0.8 vs. 11.1 ± 1.9 mmHg; p < 0.05) and Tau (16.9 ± 2.2 vs. 27.1 ± 2.8; p < 0.05) to control levels. Echocardiographic analysis revealed no significant changes in heart rate and LV diastolic and systolic functions between control and PAH groups (p > 0.05). Conclusions: The data demonstrate that LASSBio-2065 significantly improves RV function and cardiovascular histology in PAH. 108722 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JOSE MIGUEL CAYO MONTES1, JOSE MIGUEL CAYO MONTES1, JOSE CARLOS PACHÓN MATEOS2, JUAN PACHÓN MATEOS1, JUAN CARLOS ZERPA ACOSTA1, ENRIQUE I. PACHÓN MATEO1 (1) HOSPITAL DO CORAÇÃO; (2) SEMAP Introduction: Functional atrioventricular block (BAV) can generate pre-syncope and syncope to the point of compromising patients‘ quality of life, and pacemaker implantation does not resolve the condition in some cases, it is important to clarify the diagnosis, especially in young and without structural heart disease. Objective: To perform cardioneuroablation (CNA) of the parasympathetic cardiac ganglia to modulate the vagal influence on the heart and thus eliminate the cardioinhibitory response on the AV Node, treating syncope due to functional BAV, as an alternative therapy to pacemaker implantation. Methods: CNA was performed in 8 patients under 50 years of age, who had syncope due to BAV, normal electrophysiological study and positive atropine test. The mapping of the parasympathetic ganglia, the neuromyocardial interface and the mapping of AF nests was performed with the Navix-St-Jude system. These regions and those anatomically related to the 4 ganglia were treated with endocardial radiofrequency (RF) ablation. Extracardiac vagal stimulation (ECVS) is used for the end point of the procedure, demonstrating the absence of parasympathetic activity on the sinus node and AV node, proving vagal denervation in these structures. Results: Imediate and persistent increase in heart rate was observed after CNA (HR = 61, final HR = 78 bpm), Wenckebach point increase (PW:100->148 ppm) p: < 0.05. In the short and long term follow-up (after 05 years) the patients had no more syncope episodes. Holter monitoring performed at 03 months of follow-up in these patients did not record pauses or BAV, and the R-R variability was predominantly sympathetic (SDNN = 30 ms). Conclusion: Syncope and normal EPS may be caused by functional origin; Functional AV block may be treated by Cardioneuroablation without pacemaker implantation; Extracardiac vagal stimulation was fundamental to evaluate the CNA step by step. 108734 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GABRIELA STOCHERO1, Gabriela Stochero1 (1) Unimed Noroeste/RS Introduction: Contrast-induced nephropathy (CIN) represents a form of acute kidney injury that occurs in patients undergoing diagnostic and therapeutic medical procedures using iodinated contrast. In the last decade, CIN has been identified as the third leading cause of acquired acute renal failure (AKI) in hospitalized patients, reaching 12% of cases. CIN is defined as an absolute increase in serum creatinine ≥0.5 mg/dL or a relative increase of 25% from baseline creatinine within 24 to 72 hours after exposure to the contrast agent and in the absence of an alternative cause. Objective: To determine the prevalence of CIN in patients with heart disease undergoing diagnostic and/or treatment angiographic procedures. Method: Prospective, quantitative study carried out in the hemodynamics department of a large hospital located in the northern region of Rio Grande do Sul, Brazil. To determine the sampling of this research, a sample size calculation was performed, with an error of 5%; confidence level 90%; population 253 patients, maximum percentage of CIN 12%, totaling 79 individuals. Results: The sample consisted of 52 (65.8%) men and 27 (34.2%) women. The mean age was 65.9 ± 9.52 years. No patient in the study underwent dialysis therapy, at least 72 hours after contrast use. The prevalence of contrast-induced nephropathy was 30.38%, totaling 24 patients. It was identified that 51 (64.6%) patients who underwent contrast-enhanced procedures did not receive prophylactic measures related to the prevention of CIN, while 28 (35.4%) received hydration with saline solution (SF) 0.9% intravenously. The volume of contrast used ranged from 50 to 500 mL, with a mean volume of 171.97 ± 91.27 mL. Comparing patients with and without CIN, patients who developed nephropathy were more hypertensive, had a higher percentage of reduced left ventricular ejection fraction and a higher incidence of heart failure. Patients who developed CIN had more complications after the procedures, corresponding respectively to 25% (n = 6) versus 3.6% (n = 2); (p = ≤ 0.001). Conclusion: A high prevalence of contrast nephropathy was evidenced, despite the patients having few risk factors, which highlights the need for preventive measures and reduction of the volume of contrast. 108749 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ANA PAULA DELGADO BOMTEMPO BATALHA1, Josiane Aparecida de Almeida3, Isabela Coelho Ponciano3, Dara Marília Damasceno2, Ana Carolina Oliveira Pierangeli Vilela2, Tamara Rafino de Castro2, Ana Carolina Silva d’Ornelas2, Angélica Jesus de Assis2, Camila Alves Quintino de Souza2, Samantha de Jesus Carolina2, Mariana Balbi Seixas1, Lilian Pinto da Silva1 (1) Graduate Program in Physical Education, Faculty of Physical Education and Sports, Federal University of Juiz de Fora, Juiz de Fora, Brazil; (2) Faculty of Physical Therapy, the Federal University of Juiz de Fora, Juiz de Fora, Brazil; (3) Graduate Program in Rehabilitation Sciences and Physical-Functional Performance Faculty of Physical Therapy, Federal University of Juiz de Fora, Juiz de Fora, Brazil Background: Although it is widely known that physical exercise contributes to blood glucose control and reduces cardiovascular risk factors, adopting and maintaining an aerobic exercise routine (AER) is still a challenge for people living with diabetes as it requires overcoming several barriers. Therefore, it becomes relevant to identify these barriers and correlate them with adherence to AER. Methods: Participants of a randomized pilot study involving a 12-week exercise intervention aiming to maintain an AER with at least 150 min/week in moderate-intensity were follow-up for six-month post-intervention. The follow-up objective was to support the participants in maintaining the AER through monthly structured phone calls; in the five calls, they answered a Likert scale about the impact of barriers on AER (No impact – 0 and extreme impact- 10) and reported minutes AER/week. The ten pre-established barriers are presented in Table 1. The participants were divided into two groups: G1) participants with AER ≥ 150min/week and G2) AER < 150min/week on average. The median for each barrier was calculated, and the Mann-Whitney test was used to compare the barriers between G1 and G2. The Spearman test analyzed the association between the interest variables. Results: During the six-month follow-up, 29 participants (55% male, 58.5 ± 9.0 yr, 10.3% prediabetes, 20.7% Type 1 diabetes, and 69% Type 2 diabetes) answered the phone calls. The average time of AER/week was 178 minutes, and 58% of participants reported AER ≥ 150 min/week during the follow-up. Table 1 shows the barriers comparing G1 and G2. Fatigue and lack of: company, space, and equipment had less impact on AER in the G1 than G2. A negative and moderate correlation was identified between fatigue, lack of time, lack of company, lack of space, and lack of equipment with the average time of AER/week (r = –0.50; –0.41; –048; –0.54; –0.55; P ≥ 0.05, respectively). Conclusion: Although some barriers were related to a short average time of AER/week, most participants could maintain more than 150 min/week of AER in moderate-intensity six months after a 12-week exercise intervention. 108759 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ANA PAULA DELGADO BOMTEMPO BATALHA1, Josiane Aparecida de Almeida3, Juliana Gomes Soares2, Bárbara Faria Filgueiras2, Isabela Coelho Ponciano3, Ana Carolina Silva d‘Ornelas2, Angélica Jesus de Assis2, Camila Alves Quintino de Souza3, Samantha de Jesus Carolino2, Tamara Rafino de Castro2, Mariana Balbi Seixas1, Lilian Pinto da Silva1 (1) Graduate Program in Physical Education, Faculty of Physical Education and Sports, Federal University of Juiz de Fora, Juiz de Fora, Brazil; (2) Faculty of Physical Therapy, the Federal University of Juiz de Fora, Juiz de Fora, Brazil; (3) Graduate Program in Rehabilitation Sciences and Physical-Functional Performance Faculty of Physical Therapy, Federal University of Juiz de Fora, Juiz de Fora, Brazil Background: Some studies have shown that diabetes patients with higher disease knowledge are more likely to adhere to the treatment and achieve behavior change, which is critical for effective glycemic control. Therefore, this study aimed to compare diabetes knowledge between patients with type 1 (T1D) and type 2 diabetes (T2D) and investigate the correlation between this outcome and glycated hemoglobin (A1c) levels. Methods: Participants from different phases of the Diabetes College Brazil Study (NCT03914924) – (1) validation of questionnaires, n = 106; (2) pilot randomized trial, n = 18; (3) feasibility study, n = 7) – whose glycemic control has been assessed by A1c levels measured up to three months before been enrolled in one of the phases of Diabetes College Brazil Study were included in the present study. The diabetes knowledge was assessed by DiAbeTes Education Questionnaire (DATE-Q) total and each item scores. Mann-Whitney test was used to compare DATE-Q total scores between patients with T1D and T2D, and the Spearman test analyzed the association between DATE-Q total score and A1c level. Results: One-hundred and thirty-one diabetes patients (55 ± 14 yr; 59% female; 74% T2D; 48% with effective glycemic control (A1C < 7%); 23.6% elementary school non-concluded, 23.6% high school concluded and 17% postgraduate; average house income of 4 Brazilian minimum wages) participated in the study. The median and interquartile range of A1C level and DATE-Q total score were 7.2% [6.2–8.3] and 14 [12–16], respectively. Patients with T1D presented higher DATE-Q scores compared to those with T2D (total score: 16 [14–18] vs. 14 [11–15], P < 0.01; item scores: item 2, P = 0.01; item 3, P < 0.01; item 4, P = 0.03; and item 12, P = 0.03). There was no significant correlation between A1C levels and DATE-Q total scores as well as between A1C levels and DATE-Q each item scores. Conclusion: The result of higher diabetes knowledge in patients with T1D is predictable since it is recommended to provide at least some education as part of this disease’s primary care. Although the non-association between DATE-Q scores and A1C levels found refutes our study hypothesis, it reinforces findings from other studies considering that diabetes control is related to disease type and treatment and psychosocial conditions rather than diabetes knowledge. 108807 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY BRUNA DE SOUZA ROCHA1, Jaqueline Soares da Silva1, Júlia Galvez Bulhões Pedreira1, Eliezer Jesus Barreiro1, Gisele Zapata-Sudo1 (1) Universidade Federal do Rio de Janeiro (UFRJ) Introduction: Systemic arterial hypertension is a multifactorial condition with structural and/or functional changes in target organs, heart, brain, kidneys and vessels. The search of new strategies for the prevention and treatment of cardiovascular diseases, led to the design and synthesis of new N-acylhydrazones derivates, to identify agents with vasodilator activity and antihypertensive effect. New compounds containing selenium, which has antioxidant properties, which could provide reduction of the oxidative stress associated with hypertension. Objectives: To investigate the vascular and antihypertensive effects of new N-acylhydrazones. Methods: Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro n. 017/19. Vascular reactivity was evaluated using isometric tension recording of pre-contracted thoracic aorta from male Wistar rats (220–250 g) after exposure to increasing concentrations of each derivate (0.1–100 µM) and was calculated the half maximal effective concentration (EC50). Two compounds (LASSBio-2062 and LASSBio-2063) were selected to investigate the antihypertensive effect in spontaneously hypertensive rats (SHR, 12–14 weeks old), through the measurement of mean blood pressure (MBP) and heart rate (HR) after intravenous administration of 10 µmol/kg. Results: The Table 1 shows EC50 of the compounds for the vasodilator effect in aorta in the presence and absence of endothelium. All derivates tested demonstrated vasodilator activity. LASSBio-2062 and its N-methylated analogue, named LASSBio-2063, were more potent than the prototype and LASSBio-2063-induced vasodilation occurs regardless of endothelium integrity. LASSBio-2062 and LASSBio-2063 (10 μmol/kg; n = 5, each) reduced MBP in SHR from 132.0 ± 9.2 to 82.1 ± 12.6 mmHg and from 129.7 ± 4.5 to 96.0 ± 14.6 mmHg, respectively (p < 0.05). LASSBio-2062 but not LASSBio-2063 reduced HR from 248.1 ± 12.1 to 146.5 ± 21.0 bpm. Conclusion: The vasodilator activity induced by the new N-acylhydrazones could explain their antihypertensive effect. 108809 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT AHMAD GILL1, Omar Al-Taweel1, Dalia Hawwass1, Chowdhury Ahsan1 (1) University of Nevada, Las Vegas (UNLV) Introduction: There is limited research on patients with combined systolic and diastolic heart failure. Objective: In this study, we compare the short-term outcomes of patients with combined systolic and diastolic heart failure to patients with diastolic heart failure. Methods: This was a retrospective cohort study. We queried the National Inpatient Sample database from 2016 to 2018 and identified adult patients admitted with a principal diagnosis of combined systolic and diastolic heart failure or diastolic heart failure. We analyzed the categorical and continuous variables by Pearson’s chi-squared and Student t-test respectively. Multivariable logistic regression, adjusted for age, gender and comorbidities, was used to compare mortality. The comorbidities adjusted for included coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, hypertension, obesity and type 2 diabetes mellitus. Results: 598,250 patients met our inclusion criteria. Patients with combined systolic and diastolic heart failure had 1.54 times higher odds of suffering in-hospital mortality compared to patients with only diastolic heart failure (aOR 1.54, 95% CI: 1.43–1.66; p < 0.001). When separated by race, combined heart failure patients had higher mortality rates amongst White (3.9% vs 2.7%, p < 0.001), Black (1.9% vs 1.6%, p < 0.001) and Hispanic (3.0% vs 1.8%, p < 0.001) patients. Patients with combined heart failure were predominantly male (57.6% vs 38.2%, p < 0.001), had longer hospital stays (5.8 days vs 5.1 days, p < 0.001) and increased hospital costs ($56,544.60 vs $41,666.02, p < 0.001). These patients also had higher rates of comorbidities such as coronary artery disease (46.2% vs 35.1%, p < 0.001) and prior myocardial infarction (12.9% vs 7.0%, p < 0.001), but decreased rates of hypertension (28.4% vs 33.2%, p < 0.001), obesity (16.5% vs 22.6%, p < 0.001) and type 2 diabetes mellitus (24.0% vs 26.2%, p < 0.001). Conclusion: Patients with combined systolic and diastolic heart failure had higher odds of suffering in-hospital mortality compared to patients with only diastolic heart failure. Our findings illustrate that combined heart failure patients require close monitoring in the inpatient setting. Additional studies should explore if specific races are at increased risk for prolonged hospitalization and mortality. 108813 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION TAISSA LORENA DOS SANTOS1, Taissa Lorena dos Santos1, Tomás de Souza Mello1, Ana Rachel Buchar Cervasio1, Bruna Gopp Botelho1, Carlos Augusto Parente Macedo Moura1, Karine da Silva Guimarães1, Ana Cristina Tenório da Costa Fernandes1, Daniela Fiuza Gomes Monteiro1, Elizabeth Silaid Muxfeldt1 (1) Universidade Estácio de Sá, School of Medicine, Campus Vista Carioca. IDOMED Background: Recent guidelines have recommended out-of-office blood pressure (BP) measurement to improve the accuracy of the diagnosis of hypertension (HTN) but there are few studies evaluating its use in the younger population and in the scope of primary care. Objective: To assess the degree of agreement between office BP and HBPM, evaluating their use in a young population assisted in primary care. Methods: This is a cross-sectional population study with adults between 20–50 years old registered in the Family Health Strategy. Sociodemographic, anthropometric data and CV risk factors were recorded. Office BP was the mean of 2 measurements and HBPM followed the 7-day protocol. HBPM < 135 × 85 mmHg and office BP < 140 × 90 mmHg were considered normal, identifying the 4 phenotypes: Normotension (controlled office BP and HBPM); white-coat HTN (uncontrolled office BP and controlled HBPM); masked HTN (controlled office BP and uncontrolled HBPM) and sustained HTN (uncontrolled office BP and HBPM). Results: 475 individuals were included (male sex: 38%; mean age: 37.6 years ±8.8 years, of which 93 (20%) had their diagnosis modified after HBPM (43 with white-coat and 47 with masked HTN). In multiple logistics regression, male gender (OR 3.87: 95%CI 1.70–8.82) was independently correlated with white-coat HTN, while obesity and prehypertension increased the risk of masked HTN by 2 and 5.5 times, respectively. Uncontrolled office BP has high specificity (89%) and low sensitivity (49%) for detecting sustained HTN, with a low concordance in the diagnosis of HTN (kappa = 0.388) Among men with uncontrolled office BP, 55.6% had a diagnosis of white-coat HTN and among women, 37.1%. Conclusion: HBPM was a useful procedure in the diagnosis of hypertension in a young and apparently healthy population in primary care, especially in those with uncontrolled office BP to identify white-coat HTN and in those with high normal office BP, especially if they are male or obese for early diagnosis of masked HTN. 108820 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION TAISSA LORENA DOS SANTOS1, Taissa Lorena dos Santos1, Carlos Filipe dos Santos Pimenta1, Victor Margallo1, Hugo Farah Affonso Alves1, Lucca Hiroshi de Sá Kimura1, João Gabriel Rega do Nascimento Vallaperde1, Vitor de Melo Nolasco1, João Gabriell Bezerra da Silva1, Bernardo Chedier1, Elizabeth Silaid Muxfeldt1 (1) Universidade Federal do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho – ProHArt. UFRJ – HCUFF Introduction: Refractory hypertension (RfHTN) is defined as uncontrolled blood pressure despite the use of 5 or more anti-hypertensive drugs. It is considered the extreme phenotype of resistant hypertension (RHTN). Objectives: To characterize prevalence and clinical profile of patients with RfHTN among a historical cohort of patients with RHTN in 2 moments: before and after the introduction of spironolactone. Design and Methods: A cross-sectional analysis was performed during the pre-spironolactone period (before 2005) with 1,048 participants with RHTN (72.3% women, average age: 61.2 ± 11.3 years). All of them were submitted to a standard protocol including clinical and laboratory tests, 24-hour ABPM, echocardiogram, and pulse wave velocity. In a second analysis, refractory patients were evaluated after the introduction of spironolactone (post-spironolactone period – after 2005). The statistical analysis included bivariate comparisons between patients with RHTN and those with RfHTN, as well as logistic regressions to assess independent correlations of RfHTN pre- and post-spironolactone. Results: In the initial cross-sectional analysis (pre-spironolactone), RfHTN prevalence was 14%. Age < 60 years, smoking, obesity and left ventricular hypertrophy (LVH) were independently correlated with RfHTN. After the introduction of spironolactone, prevalence increased to 17.6%. It was observed that refractory patients using spironolactone presented less aortic stiffness, lower prevalence of LVH, cerebrovascular disease and peripheral arterial disease (PAD), what might indicate a reduced cardiovascular risk despite the lack of blood pressure control. Refractory patients have also presented higher magnitude of white-coat effect, reflecting an exacerbated sympathetic activity. Age < 60 years and lower prevalence of PAD were correlated with RfHTN in the post-spironolactone period. Conclusion: The use of spironolactone seems to reduce cardiovascular risk despite the lack of blood pressure control. 108831 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION FERNANDA MARCELINA SILVA1, Luana Giatti Gonçalves1, Luisa Campos Caldeira Brant1, Maria de Fátima Haueisen Sander Diniz1, Alvaro Vigo2, Maria de Jesus Mendes da Fonseca3, Sandhi Maria Barreto1 (1) Universidade Federal de Minas Gerais, UFMG; (2) Universidade Federal do Rio Grande do Sul, UFRGS; (3) Escola Nacional de Saúde Pública, ENSP/Fundação Oswaldo Cruz, Fiocruz Introduction: Changes in dietary patterns related to food processing have been associated with an increase in the incidence of several noncommunicable diseases (NCDs). Greater consumption of ultra-processed foods (UPF) was associated with a higher risk of death from all causes and cardiovascular diseases (CVDs), but results are controversial, especially for CVDs mortality. Objectives: To investigate whether the consumption of UPF is associated with a higher risk of death from NCDs and CVDs in Brazilian adults after eight years of follow-up. Methods: A total of 14,011 adults from the baseline (2008–2010) of the ELSA-Brasil cohort study, aged between 35 to 74 years and free of CVDs were included. Food consumption at cohort insertion was obtained using a food frequency questionnaire and the NOVA classification was used to estimate the contribution of UPF, in grams/day, on the total dietary. The following baseline variables were used for adjustments: sex, age, investigation center, schooling, physical activity, smoking, excessive alcohol use, total energy intake, body mass index (BMI), and reported diagnosis of diabetes, hypertension, and use of lipid-lowering drugs. The association between UPF consumption and time to death from NCDs and CVDs was investigated using Cox regression models, after adjustments. Restricted cubic spline regressions were used to test nonlinearity. The participants were followed from the first year of the cohort until the date of death, lost to follow-up, or December 31, 2018, whichever came first. Results: After a mean follow-up of eight years, 331 deaths from NCDs and 116 from CVDs occurred. The proportion of UPF in the diet was 16.1% (10.5–23.4). After adjustments, the results suggested that a 10% increase in UPF consumption increased the risk of death from NCDs by 15% (95% CI: 1.03–1.29). These results were maintained even after adjustments for total energy intake and BMI and for comorbidities. However, no association was observed between UPF consumption and CVDs mortality. Conclusions: An increase in UPF consumption was associated with a higher risk of death from NCDs. These results support public policies aimed at reducing UPF consumption and encouraging the consumption of unprocessed foods for NCDs prevention. However, a longer follow-up period may be necessary to assess the association between UPF consumption and cardiovascular mortality. 109110 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM VICTOR LUIZ ROCHA PIRES1, Murilo Sousa Ramos1, Beatriz Rihs Matos Tavares1, Quelvin Claiton Souza Costa1, Grasiely Faccin Borges1 (1) Universidade Federal do Sul da Bahia (UFSB) Introduction: Children affected with COVID-19 are mostly asymptomatic, in this way, they do not belong a group of risk for this disease, due to mild symptoms, a better prognosis and low lethality. Recent studies describe that some children were affected with Multisystem Inflammatory Syndrome owing to COVID-19 and facilitated complications, such as mitral regurgitation, vasculitis, myocarditis, valve regurgitation, pericardial effusion, and electrocardiographic changes, thus increasing hospitalization and care intensive. Objective: To analyze the incidence of cardiovascular changes in pediatric patients associated with COVID-19. Methodology: This is a systematic literature review study, conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology. Researches and studies were performed in SciELO, LILACS, PUBMED, MEDLINE and BVSalud. Original articles on cardiovascular changes in pediatric patients, whose ages ranged from 0–18 years old, with complications repercussions for COVID-19, in the period from 2020 to 2022, were included. Results: Of the 40 studies found, three were included, one research has pointed out that a third of pediatric patients who had COVID-19 had coronary changes; Kawasaki disease and the multisystem inflammatory response stand out, which occurred in most patients, followed by cardiogenic shock and other shocks. In the imaging tests, it was observed 53% had left ventricular dysfunction, 27% had right ventricular dysfunction, and the others evolved with low normal systolic function and pericardial effusion. Laboratory standards showed an increase in the mean level of partial thromboplastin time and C-reactive protein, as well as hydroelectrolytic repercussions, which were related to prognosis and mortality. Conclusion: There are a few studies that analyzed children affected by COVID-19, and these indicate the possibility of pediatric patients to develop serious cardiovascular complications after infection by SARS-CoV-2. 110853 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION AMANDA SAMPAIO STORCH1, Helena Naly Miguens Rocha1, João Dario Mattos1, Larissa Lírio Velasco1, Juliana Mentzinger1, Gabriel Fernandes Teixeira1, Rebeca Lial Rosado1, Antonio Claudio Lucas da Nóbrega1, Ronaldo Altenburg Odebrecht Curi Gismondi1, Natália Galito Rocha Ayres1 (1) Fluminense Federal University Introduction: Patients with treatment-resistant hypertension (TRH) usually present mental stress (MS)-induced impairments in the endothelial dysfunction. We suggest that the decrease in nitric oxide (NO) bioavailability evoked by MS may aggravate vascular function in those patients. Objective: To determine the effects of increasing NO bioavailability during MS through inhibition of phosphodiesterase-5 on vascular response in those patients. Methods: It is a cross-sectional, randomized, crossover, double-blind, and placebo-controlled protocol (CAAE: 79958017.3.0000.5243). In two experimental sessions, postmenopausal women with TRH (61 ± 5 years) underwent a randomized oral administration of PDE-5 inhibitor (iPDE5; sildenafil 50 mg) or placebo (PL). After 30 minutes, the patients were submitted to MS (modified Stroop Color-Word Test) for five minutes. Flow-mediated dilatation (FMD; vascular ultrasound) and pulse wave analyses (applanation tonometry) were measured at baseline, immediately after (MS), and 30 minutes after MS (MS30). Blood samples were collected at baseline, during MS, and MS30 to measure coagulation (coagulometry), oxidative stress (colorimetry), and nitrite/nitrate concentration (chemiluminescence). Heart rate (HR; electrocardiogram) and BP (photoplethysmography) were continuously monitored throughout the protocol. Data were presented as mean ± standard deviation. Two-way repeated-measures ANOVA was used to compare baseline, MS, and MS30 measurements between sessions. Paired Student’s T-test was performed to compare the delta values, followed by effect size Cohen’s D test. Results: Preliminary results showed that MS increased the HR and systolic BP (p < 0.05) in both sessions. Baseline FMD was higher in the PL session (p = 0.02). However, there was a reduction in FMD at MS and MS30 in the PL session (p < 0.01 vs. baseline), while FMD was higher at MS30 in the iPDE5 session (p = 0.05 vs. baseline; p < 0.01 vs. PL). Additionally, the FMD response at MS30 was higher in iPDE5 compared to PL session with a medium effect size (p = 0.02; Cohen’s D:0.64). Nitrate concentration was higher at baseline and during MS in the iPDE5 session (p = 0.05 vs. placebo). No differences were observed throughout the protocol or in the response at MS30 in pulse wave analysis, coagulation, oxidative stress, and nitrite measurements. Conclusions: Present data suggest that the PDE-5 inhibition attenuates the deleterious effects of MS on endothelial function in patients with TRH. 108864 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM HANNAH CARDOSO BARBOSA1, Maria Auxiliadora Parreiras Martins1, Manuela Furtado Sacioto2, Jordana Cristina de Jesus3, Karina Cardoso Meira3, Luiz Guilherme Passaglia1, Milena Soriano Marcolino1, Carísi Anne Polanczyk4 (1) Universidade Federal de Minas Gerais UFMG; (2) Faculdade Ciências Médicas de Minas Gerais FCMMG; (3) Universidade Federal do Rio Grande do Norte UFRN; (4) Institute for Health Technology Assessment (IATS/CNPq) Background: Cardiovascular complications of COVID-19 represent an important aspect of the disease’s pathogenesis and prognosis. Troponin levels are commonly abnormal in hospitalized patients with COVID-19 and they are associated with poorer prognosis. Evidence on the prognostic role of troponin and myocardial injury in COVID-19 in-hospital patients from Latin America are still scarce. This study aimed to fill this gap by evaluating myocardial injury as an independent predictor of in-hospital mortality and mechanical ventilation requirement in COVID-19 hospitalized Brazilian patients. Methods: This cohort study is a substudy of the Brazilian COVID-19 Registry, conducted in 31 Brazilian hospitals in 17 cities. Consecutive patients admitted with confirmed diagnosis of COVID-19 between March and September 2020 were included. Medical records were reviewed to collect data regarding patients’ demographic and clinical characteristics; COVID-19 symptoms; clinical assessment upon hospital presentation, third and fifth days of hospitalization; laboratory, imaging, and electrocardiographic data; in-hospital medications, treatment, and patient outcomes. Models for the primary outcomes were estimated by Poisson regression with robust variance. For the multivariate analyses, two predictive models were estimated to evaluate the role of elevated troponin on the primary outcomes: in-hospital mortality and mechanical ventilation requirement. Results: Overall, 2,925 patients were included in this analysis. The median age was 60 (IQR 48–71) years old. The proportion of patients with comorbidities was higher in the group of people with cardiac injury. Laboratory tests brain natriuretic peptide, creatine phosphokinase, lactate dehydrogenase, N-Terminal pro-brain natriuretic peptide and C-reactive protein presented higher median values in patients with myocardial injury and this condition was a significant independent predictor for risk death and mechanical ventilation requirement. Patients with myocardial injury represented by elevated troponin levels, presented a higher risk of death relative risk (2.03 [1.60–2.58]) and mechanical ventilation requirement relative risk (1.87 [1.57–2.23]) when compared to controls. Conclusion: Cardiac injury showed to be an independent predictor of in-hospital mortality and need of mechanical ventilation in hospitalized COVID-19 patients. The design of strategies involving the continuous monitoring of troponin levels as risk biomarkers in thes. 109466 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GUSTAVO NEVES DE ARAUJO3, Guilherme Pinheiro Machado1, Marcia Moura4, Anderson Donelli da Silveira1, Pedro A. Lemos5, Alexandre Schaan de Quadros4, Marco Vugman Wainstein2 (1) Hospital de Clinicas de Porto Alegre (HCPA); (2) Universidade Federal do Rio Grande do Sul (UFRGS); (3) Imperial Hospital de Caridade; (4) Instituto de Cardiologia do Rio Grande do Sul (IC-FUC); (5) Hospital Israelita Albert Einstein Introduction: Coronary drug-eluting stents are continuously developing, and the current gold standard consists of new metal alloys with thinner struts and bioresorbable polymers. Our aim was to compare a new ultrathin strut, sirolimus-eluting stent (Inspiron®) with other third-generation drug-eluting stent platforms in patients with ST-elevation Myocardial Infarction (STEMI) submitted to primary percutaneous coronary intervention (PCI). Methods: The present work assessed clinical outcomes from a STEMI multicenter registry, from two centers in southern Brazil. Patients were considered for inclusion if they were admitted with STEMI and were submitted to primary PCI. All patients included received third generation DES, and Inspiron® was compared with five other stent platforms. A propensity score matching (PSM) adjusted for age, diabetes, admission Killip classification, cardiac arrest and creatinine was computed to generate similar groups in relation to clinical and procedural characteristics. Results: From January 2017 to January 2021, 1711 patients have undergone primary PCI and 1417 patients matched our entry criteria (709 in the Inspiron and 708 in the other third generation DES). Rates of hypertension (60 vs. 65%, p = 0.042), chronic kidney disease (2.7 vs 6.6%, p < 0.001), admission cardiogenic shock (5.2 vs. 9.5, p = 0.002) and cardiac arrest (1.0 vs. 7.2, p < 0.001) were lower in Inspiron group. After PSM, the study sample was comprised of 706 patients (353 in the Inspiron and 353 in the other third generation). Differences in baseline characteristics described above have lost significance. The rates of new revascularization (OR 0.52, CI 0.21–1.34, p = 0.173), stent thrombosis (OR 1.00, CI 0.29–3.48, p = 1.000), new revascularization (2.0 vs. 3.7%, OR 0.52, CI 0.21–1.34, p = 0.173), mortality (HR 0.724, CI 0.41–1.27, p = 0.257) and MACE (OR 1.170, CI 0.77–1.77, p = 0.526) were similar between groups after a median follow up of 17 months. Conclusion: Our findings support that Inspiron is safe in patients with STEMI, with similar outcomes after propensity score adjustment compared to well stablished third generation drug-eluting stents in the treatment with primary PCI at a long-term follow-up. 108891 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LUCAS CAUNETO SILVEIRA1, Patrícia Pereira de Almeida2, Marselha Marques Barral1 (1) Hospital e Maternidade Therezinha de Jesus – HMTJ; (2) Universidade Federal de Juiz de Fora – UFJF Introduction: Excess body fat is a modifiable risk factor associated with chronic non-communicable diseases, including systemic arterial hypertension (SAH). Regarding the assessment of nutritional status, anthropometric measurements are inexpensive; among which the neck circumference (NC) has been used as an indicator of cardiometabolic risk, as it estimates the accumulation of fat in the upper segment of the body. In addition, NC is considered an accessible measurement, it does not change with food intake and can be measured without the need to remove clothes. Objectives: To identify the presence of altered neck circumference among hypertensive patients treated in primary health care. Methods: Cross-sectional study conducted from September 2019 to March 2020 with adults and elderly treated in primary health care in the municipálity of Guidoval-MG. We collected socioeconomic, lifestyle, health history data through a questionnaire and took anthropometric measurements. We measured neck circumference with an inelastic tape; with the participant standing and the head in a straight position, the tape was positioned at the midpoint of neck height. For the classification of NC, we used the cutoff point proposed by Zanúncio et al (2017), which considers NC>39.5 cm for men and NC>33.3 for women associated with cardiometabolic risk. The diagnosis of SAH was self-reported and confirmed in the electronic medical record. The study was approved by the Research Ethics Committee of the Federal University of Viçosa, under protocol number 3,189,427. We analyzed data using descriptive statistics, with calculation of mean, standard deviation and prevalence. Results: The sample consisted of 361 individuals with a prevalence of systemic arterial hypertension of 40.2% (n = 145). Among hypertensive patients (n = 145), the mean age was 58 ± 12.6 years and 91 individuals (62.8%) were identified with altered neck circumference, the majority being female (n = 74). Thus, there was a high prevalence of hypertensive patients with altered neck circumference measurements. Conclusion: Our findings are in agreement with the literature, which states that NC constitutes an indicator of cardiometabolic risk and its alteration is associated with other chronic diseases such as SAH and diabetes. Therefore, due to its facility and low cost, NC measurement can be adopted in health services, especially in places where there is a scarcity of human and financial resources. 109084 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JOÃO DARIO MARTINS DE MATTOS1, João Dario Mattos1, Eduardo Schaustz1, Andréa Silvestre de Sousa2, Emiliano Horacio Medei3, Denílson C. de Albuquerque1, Olga Ferreira de Souza1, Fernando A. Bozza1, Gabriel C Camargo1, Juliana Ferreira4, Ronir Raggio Luiz3, Renata J Moll-Bernardes1 (1) D’OR Institute Research & Education (IDOR); (2) National Institute Of Infectious Diseases (Oswaldo Cruz Foundation); (3) Federal University of Rio de Janeiro; (4) Hospital Copa D’OR Background: Myocardial injury (MI), detected by elevations in cardiac troponin is common in hospitalized patients with COVID-19, and has been associated with adverse outcomes and mortality; however, there is still some controversy regarding the role of this biomarker, and routine measurement of troponin levels to increase risk stratification is not recommended. Aim: To explore the association between MI with mortality and complications in hospitalized patients with COVID-19. Methods: Consecutive adult hospitalized patients with COVID-19 were included prospectively in a multicenter registry (n = 3246). Troponin levels were normalized to the 99th percentile upper reference limit (URL) and presented as ratios. Myocardial injury was defined by troponin >1 × URL. Troponin ratios were categorized as normal (≤1x URL), mildly elevated (>1 to ≤3x URL), moderately (> 3 to ≤10x URL) and severely elevated (>10x URL). Clinical data was collected from electronic medical records. Patients were prospectively followed until hospital discharge or in-hospital death. Results: Myocardial injury was associated with increased in-hospital mortality (22.7% vs 5.5%) and most cardiovascular adverse outcomes, such as thromboembolic phenomena (4.0% vs 2.6%; p = 0.047), myocarditis (4.8% vs 1.2%; p < 0.001), heart failure (6.5% vs 1.3%; p < 0.001), myocardial ischemia (9.1% vs 1.4%; p < 0.001), sepsis or septic shock (20.1 vs 9.2%; p < 0.001), acute renal failure (21.8% vs 7.4%; p < 0.001) and respiratory failure requiring mechanical ventilation (36.5% vs 10.8%; p < 0.001). From a total of 3246 patients in our cohort, 1770 (54.5%) did not have troponin measurement during the first week of hospitalization. In-hospital mortality was higher in patients with no troponin measurement compared to patients with no myocardial injury (11.9% [95%CI, 10.5–13.5] vs 5.5% [4.3–7.0]), highlighting the negative predictive value of this biomarker. Some complications were also more prevalent in patients with no troponin measurement, compared to patients with normal troponin levels. Conclusion: Our findings suggest that troponin is an important biomarker that may improve risk prediction and resource utilization in hospitalized patients with COVID-19. 108920 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH XINPENG XU1, Jenifer Atala2, Andrea Z. Beaton3, Rosemary Kansiime2, Miriam Nakitto2, Emma Ndagire5, Haddy Nalubwama2, Emmy Okello5, Emily Chu6, Hui Miao7, David A. Watkins8, Yanfang Su8 (1) School of Public Health, Nanjing Medical University, Nanjing, China; (2) Department of RHD Research, Uganda Heart Institute, Kampala, Uganda; (3) Department of Cardiology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; (4) Department of Pediatrics, School of Medicine, University of Cincinnati, Cincinnati, OH, United States; (5) Division of Cardiology, Uganda Heart Institute, Kampala, Uganda; (6) Interlake High School, Bellevue, WA, United States; (7) Vanke School of Public Health, Tsinghua University, Beijing, China; (8) Department of Global Health, University of Washington, Seattle, WA, United States; (9) Department of Medicine, University of Washington, Seattle, WA, United States Introduction: Rheumatic heart disease (RHD) is prevalent in socially disadvantaged settings, placing a severe burden on patients and their households. Many studies have highlighted the relationship between social determinants and RHD disease development, but there is limited understanding about social determinants of healthcare utilization and related financial and time costs among RHD patients. Objective: To investigate social determinants of healthcare utilization and related costs among RHD patients in Uganda. Methods: This was a retrospective longitudinal study involving patients with RHD recruited from the Uganda National RHD Registry. Our survey included 87 patients from the registry stratified by 3 districts in Uganda. Between December 2018 and February 2021, data were collected. A random-effects model was used to examine the associations between social determinants and healthcare utilization, as well as financial and time costs. Results: Among the social determinants, increase of an additional person per bedroom is associated with the reduction of outpatient spending by 1.73 USD per visit (P < 0.05), owing primarily to a reduction in direct medical spending (1.25 USD; P < 0.01). RHD patients under the age of 18 had 3.2 more outpatient visits annually (P < 0.01), and their per-visit time costs and those of caregivers were 2.5 hours (P < 0.05) and 4.9 hours (P < 0.05) more than those of adult RHD patients, respectively. More educated individuals spend 2.5 USD less on nonmedical cost per visit (P < 0.05). In Western Region, which is underdeveloped in Uganda, the number of annual outpatient visits by patients with RHD were 4.8 higher than those in the Central Region(P < 0.01); RHD patients living in western region faced 3.8 USD higher direct medical costs per outpatient visit (P < 0.1) and more missed work hours (1.9 hours, P < 0.1). Conclusions: RHD patients underutilized RHD secondary prevention and experienced high healthcare expenditures. Crowding, age, and geographical characteristics all have significant correlation with RHD patients’ healthcare utilization and costs. Targeted measures, such as expanding housing construction and improving housing maintenance, reducing barriers in RHD secondary prevention among adult patients, ensuring the supply of medicines in economically worse-off areas, should be taken to ensure that equitable and affordable medical services are available to those RHD patients who are most in need of secondary prevention services. 108924 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT AHMAD GILL1, Emily He2, Omar Al-Taweel1, Dalia Hawwass1, Chowdhury Ahsan1 (1) University of Nevada, Las Vegas (UNLV); (2) Loma Linda University (LLU) Introduction: Although there is extensive literature present on hypertrophic obstructive cardiomyopathy (HCOM), there is limited data on HCOM patients with systemic lupus erythematosus. Objective: In this study, we investigate the short-term, in-hospital outcomes of HCOM patients with and without systemic lupus erythematosus. Methods: We queried the National Inpatient Sample database from 2016 to 2018 to identify adult patients admitted with a principal diagnosis of HCOM and a secondary diagnosis of systemic lupus erythematosus. We analyzed the categorical and continuous variables by Pearson’s chi-squared and Student t-test respectively. Multivariable logistic regression, adjusted for age, gender, comorbidities, hospital bed size, hospital region and hospital teaching status was used to compare mortality. The comorbidities adjusted for included atrial fibrillation, coronary artery disease, chronic kidney disease, heart failure, hypertension, obesity and type 2 diabetes mellitus. Results: 9395 patients met our inclusion criteria, of which 35 patients had both HCOM and systemic lupus erythematosus. Patients with HCOM and systemic lupus erythematosus had 7.08 times higher odds of suffering in-hospital mortality compared to patients with solely HCOM (aOR 7.08, 95% CI: 1.21–41.32; p = 0.03). When separated by race, HCOM and systemic lupus erythematosus patients had a higher mortality rate in White patients (21.7% vs 1.6%, p < 0.001), but decreased mortality rates in Black (0% vs 2.9%, p = 0.67) and Hispanic (0% vs 2.0%, p = 0.73) patients. Patients with both HCOM and SLE were predominantly female (85.7% vs 58.6%, p = 0.15) and had lower median household income (71.4% vs 23.6%, p = 0.02). These patients also had higher rates of comorbidities such as coronary artery disease (42.9% vs 28.0%, p = 0.05), chronic kidney disease (14.3% vs 0.8%, p < 0.001), chronic obstructive pulmonary disease (42.9% vs 9.8%, p < 0.001) and prior myocardial infarction (14.3% vs 5.0%, p = 0.01), but decreased rates of atrial fibrillation (0% vs 28.1%, p < 0.001), heart failure (14.3% vs 28.4%, p = 0.06) and hypertension (28.6% vs 42.8%, p = 0.09). Conclusion: Patients with HCOM and systemic lupus erythematosus had higher odds of suffering in-hospital mortality compared to patients with solely HCOM. Our findings illustrate that patients with HCOM and systemic lupus erythematosus are a vulnerable population that require additional medical support in the acute care setting. 108930 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE STELLA DE SOUZA VIEIRA1, Brunno Lemes de Melo1, Ednei Luiz Antonio1, Fabio Navarro Marques2, Daniele de Paula Faria2, Paulo José Ferreira Tucci1, Andrey Jorge Serra1 (1) Laboratório de fisiologia e fisiopatologia cardíacas. Universidade Federal de São Paulo (UNIFESP); (2) Instituto de radiologia, Hospital das clínicas da FMUSP, Universidade de São Paulo Introduction: Stem cell (SC) therapy is a promising approach to improve post-myocardial infarction (MI) cardiac remodeling, but the proinflammatory microenvironment may lead to SC loss and, therefore, may have a negative impact on therapy. It appears that exercise training (ET) improves myocardial microenvironment for SC transplantation. Objective: We tested the effect of ET on post-MI retention of adipose-derived SC and its combined effects on the inflammatory microenvironment. Methods: Female Fisher-344 rats (12 weeks) were randomized: SHAM; MI (MI); MI with SC (sMI); MI with ET and SC (esMI). Rats were trained 9 weeks prior to MI, followed by SC transplantation. Results MI led to left ventricular (LV) dilation and dysfunction; myocardial hypertrophy and fibrosis and increased expression of pro-inflammatory genes compared to SHAM rats. On the other hand, SC rats (sIM and esIM) exhibited better LV morphology and function; inhibition of hypertrophy and fibrosis; and attenuation of pro-inflammatory cytokines in the myocardium compared to MI rats. A positive correlation is observed between the expression of cytokines (interleukins 1β and 10, tumor necrosis factor α, and transforming growth factor β) and retention of SC. There was a correlation between cytokine expression and myocardial ADSCs retention. The. ET enhanced the beneficial effects of ADSCs in infarcted myocardium, which was associated with higher ADSCs retention. Conclusion: ET enhanced the beneficial effects of SC post-MI, which was associated with higher SC retention. Cytokine analysis suggests improvement in ET-linked myocardial microenvironment based on its anti-inflammatory action. 108953 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY IURI SCHWAAB1, Felipe Borsu de Salles2, Lucas Krieger Martins1, Rafael de Oliveira Ceron1, Marisa Fatima dos Santos1, Diogo Ferrari Centenaro1, Ari Tadeu Lirio dos Santos1, João Carlos Vieira da Costa Guaragna2, Renato Abdala Karam Kalil3 (1) Instituto de Cardiologia de Porto Alegre; (2) Hospital Divina Providência; (3) Hospital Hospital Moinhos de Vento Aortic Root replacement is the standard treatment for Aneurysm in Marfan syndrome, despite the morbidity of this kind of surgery. Personalized External Aortic Root Support (PEARS) is a novel approach for this condition, which consists in applying an pre-tailored external prosthesis. Initial reports demonstrate low in-hospital morbidity and good mid-term aortic-specific complication prevention.¹ We present the first three cases of PEARS in America. Patients were 2 male and 1 female, aged 30–41 years, with previous Marfan syndrome diagnosis and Aortic Root Aneurysm (ranging 47–52 mm). All patients had normal ventricular and kidney function, with no other systemic disease. All patients received standard aenesthesic monitorization and median sternotomy. Case #1 was submitted to PEARS and mitral valve repair (quadrangular resection and complete ring annuloplasty). He is a 33 years old male patient, with 52 mm aortic root aneurysm, mild aortic valve regurgitation and severe mitral regurgitation. Postoperatively Aortic Root size reduced to 42 mm, valvar function improved to only trace aortic and mitral valve regurgitation. Other 2 cases were submitted to PEARS off pump and were extubated in the operating room after the procedure. Case #2 is 41 years old, male, with 48 mm aortic root diameter and mild aortic valve regurgitation. After surgery aortic size was reduced to 43 mm and had complete correction of aortic regurgitation. Case #3 is 30 years old, female, with 47 mm aortic root diameter and trace of aortic valve function. Postoperative echo-cardiogram demonstrated reduction in aortic root aneurysm to 43 mm and stable valvar function. There was no surgical adverse event nor in-hospital clinical complication. Patients didn’t require vasoactive drugs nor had significant postoperative bleeding or laboratory abnormalities. Intensive care unit stay was 2–3 days and in-hospital stay was 4–8 days. Postoperative echo-cardiogram demonstrated normal ventricular function, general reduction in aortic diameter and improvement of valvar function. PEARS demonstrate feasibility and safety in this initial surgical series. Long-term results may support the inclusion of PEARS as a therapeutic alternative for patients with Marfan syndrome and aortic aneurysm. REF: Van Hoof L, Rega F, Golesworthy T, et al. Personalised external aortic root support for elective treatment of aortic root dilation in 200 patients. Heart 2021; 107: 1790–1795. 108962 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY BIANCA DOS SANTOS CARLOS NASCIMENTO1, Jaqueline Soares da Silva1, Bruna Rocha1, Tadeu Lima Montagnoli1, Eliezer de Jesus Lacerda Barreiro1, Rodolfo do Couto Maia1, Gizele Zapata-Sudo1 (1) Universidade Federal do Rio de Janeiro – UFRJ Introduction: Treatment of acute myocardial infarction (AMI) is based on strategies to relief pain and increase survival. This work investigates the effects of a new agonist of adenosine receptor (AR) named LASSBio-1860, in experimental AMI, in order to improve cardiac function. Methods: Protocols were approved by the Ethics Committee for the Use of Animals at Universidade Federal do Rio de Janeiro (n°103/17). Experimental AMI was induced by ligation of the anterior descending coronary artery in male Wistar rats (180–200 g) which were randomly treated orally with either vehicle (DMSO) or 70 μmol/kg of LASSBio-1860. After 7 days of treatment, hemodynamic parameters were measured by catheterization and hearts were collected for evaluation of cardiac inflammation (TNF-alpha, p38 MAPK and iNOS) and remodeling (ERK-1/2 phosphorylated) markers using Western blot and immunohistochemistry analysis. Results: Left ventricle thickness was increased after AMI from 0.11 ± 0.04 to 0.29 ± 0.02 cm (p < 0.05) and reduced to 0.21 ± 0.01 cm after treatment with LASSBio-1860. AMI impaired ejection fraction to 45.2 ± 1.3% (Sham: 91.5 ± 0.5%, p < 0.05) which was partially reversed to 60.1 ± 10.3% after treatment. The agonist of AR normalized the increased filling pressure of AMI animals from 31.3 ± 6.3 (Sham: 18.6 ± 3.7) to 15.9 ± 3.9. Increased LV end systolic pressure was detected after AMI, changing from 83.8 ± 12.5 (Sham) to 104.7 ± 1.1 mmHg, and was recovered to 95.3 ± 21.4 mmHg by LASBio-1860. Elevated collagen deposition from 3.6 ± 0.9 to 31.5 ± 4.29% was observed in AMI hearts, which the treatment attenuated to 26.8 ± 1.6%. AMI increased the ratio of phosphorylated-to-total ERK-1/2 from 0.78 ± 0.01 to 0.89 ± 0.01 while LASSBio-1860 reduced to 0.79 ± 0.01 (p < 0.05). Similarly, cardiac content of p38 increased to 1.58 ± 0.36 after AMI but reduced to 1.17 ± 0.43 after LASSBio-1860. AMI also promoted increased p38 nuclear labeling from 9.9 ± 3.9 to 40.8 ± 3.3%, which was decreased to 29.9 ± 1.7 after treatment. Expression of iNOS increased in AMI from 9.0 ± 4.1 to 25.3 ± 3.9 and LASSBio-1860 improved to 14.2 ± 0.6%. Conclusion: Activation of AR by LASSBio-1860 led to improvement in AMI-induced cardiac remodeling and cardiac function. 109017 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH PEDRO TOSCANO PAFFER1, Vitória Maria Terra Lopes1, Camila Santiago de Castro1, Kamille Didier Melo Almeida1, Silvio Hock de Paffer Filho1 (1) Faculdade de Medicina de Olinda (FMO) Introduction: Despite the number of syphilis cases has decreased in recent years due to the advent of antibiotics and public policies for the prevention of Sexually Transmitted Diseases, cases of cardiac syphilis are still reported worldwide, especially in developing countries. The cardiac sequelae of syphilis appears approximately 10 to 20 years after the initial infection. Understanding the relation between syphilis and its cardiovascular repercussions, its important to analyze the epidemiological indicators of syphilis in Brazil and its potential risk, with a view to preventing these complications. Objective: Analyze the epidemiological indicators of syphilis in Brazil and its potential risk of cardiovascular complications Methods Epidemiological, descriptive study with data obtained by SINAN (Information System on Notifiable Diseases). The platform was used to search for cases of acquired syphilis between 2010–2021, specifying by region, age group and gender. Results: Between 2010 and 2021, 911179 cases were reported in Brazil. The majority of the cases were reported in the South-east region, with 471767 cases (51%); followed by the South region, 203969 (22%); North-east region 121524 cases (13%); Mid-west region 63057 (6%) and northern region 50862 (5%). 549201 patients were male (60%). The age group with the highest incidence was between 20–39 years old, with 518970 cases reported (56.96%). Among people aged 60 years or older, 75368 cases (8.27%) were reported. In the year 2021 alone, 64279 cases were reported Discussion A total of 911179 cases of acquired syphilis were reported between 2010 and 2021, with its peak in 2018. Males had the highest incidence, with 549201. Syphilis infection has been linked to certain behavioral and social factors, including incarceration, multiple or anonymous sexual partners, sexual activity related to illicit drug use, and other high-risk sexual network dynamics. Given this large increase in the number of cases, there is a risk that in the future cases of cardiovascular involvement will increase. The most significant cardiovascular complication of cardiovascular syphilis is syphilitic aortitis, which leads to aortic aneurysm formation in most cases, and aortic valve insufficiency and coronary ostial stenosis in a minority of patients. Conclusion: The high incidency and cardiovascular implications the diseases carries, requires prevention, monitoring and adequate treatment, with awareness policies for the risk groups 109033 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY RENATA PINHEIRO MARTINS DE MELO1, Isabela Aragão Colares1, Mariana Salles Ballalai1, José Levi Tavares Cavalcante1, Gabriel Sousa Santos1, Helena Raquel Nogueira de Oliveira1, Marcelo Brito Cavalcante1, Gabriel Coelho Brito Dias1, Weiber Silva Xavier1, Sandra Nívea dos Reis Saraiva Falcão1, João Luiz de Alencar Araripe Falcão1 (1) Universidade Federal do Ceará (UFC) Introduction: Congenital anomalies are the fourth leading cause of neonatal death and, according to the World Health Organization (WHO), the most common and serious congenital disorders are heart problems, neural tube defects, and chromosomal abnormalities. In this sense, despite all advances in obstetrics and improvements in prenatal care for pregnant women, the prevention of mortality in neonates due to defects in the circulatory system remains a challenge to be overcome. Objective: Analyze, during the period from 1999 to 2019, the proper performance of prenatal care, correlated to the occurrence of deaths from cardiovascular defects in neonates. Methodology: Descriptive analysis, obtained from the DATASUS platform, of the frequency of prenatal care, the frequency of births with congenital anomalies and how many of these disorders are related to defects in the cardiovascular system and mortality rate in children under 1 year old due to diseases of the circulatory system. Results: During the period from 1999 to 2019, in Brazil, 8702 neonates died due to defects in the circulatory system and 3,443,087 women did not perform prenatal care or did it inappropriately. In addition, 30,734 children, whose mother didn’t perform prenatal care or it was performed inappropriately, were born with a certain congenital anomaly, which represents 21% more than those who had congenital disorders, but prenatal care was being performed. Furthermore, of a total of 170,392 cases of births with anomalies, 17.3% were characterized by defects in the cardiovascular system. Conclusion: From the information above-mentioned, it is possible to correlate the inadequate performance of medical attendance during pregnancy and the occurrence of congenital defects, such as disorders in the circulatory system, because, with the performance of prenatal, the pregnant woman can be treated for a certain pathologies, such as hypertension and diabetes, to avoid harmful consequences to the fetus and to prevent the occurrence of premature birth, since premature neonates are twice as likely to have heart abnormalities. In addition, in case the fetus is already with the disturb, the prenatal allows to early diagnosis and treat the condition, increasing the survival percentage. In resume, performing prenatal care correctly, is essential for the prevention or the successful treatment of defects in the circulatory system in neonates and for the reduction of mortality in children under 1 year old. 109057 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING JULIAN HOEVELMANN1, Charle André Viljoen3, Stephen Jermy4, Jacqui Cirota5, Sarah Kraus5, Karen Sliwa2, Ntobeko AB Ntusi4 (1) Department of Internal Medicine III, Cardiology, Angiology and Intensive Care Medicine, Saarland University Hospital, Homburg (Saar), Germany; (2) Cape Heart Institute, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.; (3) Division of Cardiology, Groote Schuur Hospital, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.; (4) Cape Universities Body Imaging Centre, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; (5) Department of Medicine, Groote Schuur Hospital, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. Introduction: Peripartum cardiomyopathy (PPCM) is characterised by left ventricular (LV) dilatation and systolic dysfunction developing towards the end of pregnancy or in the first five months postpartum. Cardiovascular magnetic resonance (CMR) allows for comprehensive evaluation of myocardial structure, function, and tissue characteristics. There is a dearth of studies investigating utility of CMR in PPCM. Purpose: To evaluate diagnostic benefit of multiparametric assessment of myocardial oedema, fibrotic burden, and strain impairment in PPCM using CMR. Methods: Eighteen consenting women with newly diagnosed PPCM and 20 female, age-matched healthy controls (HCs) underwent CMR imaging on a 3T MR scanner. A comprehensive, contrast-enhanced CMR protocol was used. Images were evaluated qualitatively and semi-quantitively for the presence of late gadolinium enhancement (LGE). Results: Patients with PPCM (median age of 34.5 years [IQR 25–38]) presented with severely impaired LV ejection fraction (LVEF) of 31.4% (IQR 19.6–37.9) and reduced right ventricular (RV) ejection fraction (RVEF) of 37.2% (IQR 21.6–51.7). LGE was present in 13 (81.2%) PPCM patients and included linear or circumferential mid-wall, patchy and diffuse patterns (LGE mass 19.1g [IQR 15.0–26.5] vs. 11.4g [8.8–13.2] in HCs, p < 0.001). Patients with PPCM had significantly higher T1 times (1369.3 ms [IQR 1343.7–1409.7 vs. 1207.8 ms [IQR 1194.8–1241.3], p < 0.001) and ECV (36.5% [32.7–37.0] vs. 27.5 [26.3–28.5], p < 0.001) compared to HCs. RV dysfunction (present in 61.1% of PPCM cohort) was associated with significantly higher ECV (37.0% [IQR 36.5–38.4] vs 33.4% [IQR 28.5–37.0], p = 0.05 and higher T1 (1409.0 ms [IQR 1349.0–1443.0] vs. 1311.3 ms [IQR 1299.3–1369.3], p = 0.015) compared to those with preserved RV function. LV fibrosis was not significantly different between PPCM patients with and without RV dysfunction. LGE mass correlated negatively with LVEF and RVEF (r = –0.540, p = 0.001; r = –0.568, p < 0.001), respectively. There was a strong positive correlation between LGE mass and native T1 (r = 0.619, p < 0.001), LGE mass and GLS (r = 0.638, p < 0.001) and moderate correlation with ECV (r = 0.528, p = 0.001). Conclusion: For the first time, we report a high prevalence of myocardial fibrosis in well-phenotyped patients with newly diagnosed PPCM. Increased LGE mass was associated with severe impairments in LV strain, LVEF and RVEF. RV dysfunction was associated with significantly higher ECV and native T1 times. 109059 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JULIAN HOEVELMANN1, Mark E Engel3, Elani Muller2, Ameer Hohlfeld4, Michael Böhm1, Karen, Sliwa2, Charle André Viljoen5 (1) Department of Internal Medicine III, Cardiology, Angiology and Intensive Care Medicine, Saarland University Hospital, Homburg (Saar), Germany; (2) Cape Heart Institute, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; (3) Department of Medicine, Groote Schuur Hospital, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; (4) South African Medical Research Council, Cape Town, South Africa.; (5) Division of Cardiology, Groote Schuur Hospital, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa Introduction: Peripartum cardiomyopathy (PPCM) remains a major contributor to maternal morbidity and mortality worldwide. The disease is associated with various complications mainly occurring early during its course. Reported adverse outcomes include decompensated heart failure (HF), thromboembolic complications, arrhythmias and death. Purpose: We aimed to systematically summarize the outcomes of women with PPCM across different geographical regions. Methods: We performed a comprehensive search of all articles published between January 2000 and June 2021 on a number of electronic databases. All cohort, case-control and cross-sectional studies, as well as control arms of RCTs reporting on the in-hospital complications and 6- and/or 12-month outcomes of PPCM were considered eligible. Results: Forty-seven studies (4875 participants across 60 countries) met the eligibility criteria. Hemodynamic and echocardiographic parameters were similar across all continents. In-hospital mortality was reported as 1.9% [95% CI 0.5–4.0] across all regions. About 10% of patients received invasive ventilation, 21.5% inotropic and 3.1% received mechanical support, respectively. Left ventricular (LV) thrombus complicated 9.0% [95% CI 6.5–11.9] of patients and all-cause embolic events occurred in 6.1% [95% CI 3.8–8.9]. All-cause mortality was 8.0% [95% CI 5.5–10.8, I2 = 79,1%) at 6 months and 9.8% [95% CI 6.2–14.0], I2 = 80.48%) at 12 months respectively. Overall, 44.4% ([95% CI 36.2–52.8], I2 = 91.7%) of patients recovered their LV function within 6 months and 58.7% ([95% CI 48.1–68.9], I2 = 75.8%) within 12 months, respectively. The lowest rate of LV recovery was reported by studies conducted in the Middle East (13.6% [95% CI 9.5–18.1], 3 studies), whereas the highest rate of LV recovery was reported for patients from Europe (56.8% [95% CI 38.1–74.7], 6 studies, I2 = 93.3%). All-cause mortality was highest in Africa and Asia/Pacific. Europe and North America reported the highest prevalence of LV recovery. Frequent prescription of beta-blockers, ACE-I/ARB and bromocriptine treatment was associated with significantly lower all-cause mortality and better LV recovery. Conclusion: We identified significant global differences in prescribed treatment, prevalence of in-hospital complications and 6- and 12-month outcomes. Frequent prescription of guideline-directed HF therapy was associated with better outcomes. Timely initiation and up-titration of HF therapy should be strongly encouraged. 109066 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS IGOR SAMUEL DOBE1, Neide Canana1, Simon Stewart4, Ana Olga Mocumbi2 (1) Instituto Nacional de Saúde; (2) Hospital Geral de Mavalane; (3) Universidade Edurdo Mondlane; (4) Torrens University Australia, Australia Introduction: Stroke is an often-deadly condition responsible for substantive great physical, social and economic limitations for survivors and their family globally. In LMIC, Unfortunately, there is a paucity of data describing its economic impact in sub-Saharan Africa. Our study aimed to evaluate the cost of stroke-related management and care during and post-hospitalization in Mozambique. Methods: We performed a prospective, cost-of-illness study (from the provider and patient perspective) of acute presentations of stroke to a first referral urban public hospital in Maputo, Mozambique. From June to December of 2019, 50 consecutively admitted patients were enrolled, data were collected from medical files and through interviews to patients or caregiver using semiquantitative questioner during hospitalization and by home visits in 28-days after discharge. Applying a bottom-up approach, direct and indirect costs were calculated. Data were presented as median, interquartile intervals, except financial data presented also as medians and ranges. All available costs were computed for the year 2019, the year of analysis. Results and discussion: In total 80 patients with acute stroke were admitted to ward during the study, of those, we consecutively recruited 50 patients comprised by 28 women (56%), a median age of 60.5 (38.3; 68.4) years, of whom 22 (44%) were discharged with diagnostic of hemorrhagic stroke. Median hospital stay was 7.0 (4.0; 8.0) days. 10 (20%) patients died within 28-days after discharge. Direct cost (Hospital stay, Medication, laboratory tests), for all cases was US$ 35 302,52 and the average direct cost per patient was US$ 706.05. Indirect cost (travel to hospital, family visits) during hospitalization and within 28-days post-discharge was US$ 22.00 and US$35,58, respectively, loss of productivity was very high in the informal sector, quality of life in all five dimensions were seriously compromised. The cost of stroke in Mozambique is particularly high considering, the relatively young age of affected cases. Conclusion: This study highlights the importance of understanding the costs of stroke for the system and for the patient. Routine register-based data can be used for accurate productivity cost estimates of health shocks. 109098 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSIOTHERAPY JÉSSICA GONÇALVES DE LIMA1, Victoria Maria Garcia de Medeiros1, Fernando Gomes de Jesus1, Ana Gabriella Arena de Sá1, Thaísa Sarmento dos Santos1, Mariana de Oliveira Carvalho1, Lucas Araujo de Carvalho1, Marcus Vinicius de Souza Amaral1, Juliana Rega1, Claudia Rosa1, Mauro Felippe Felix Mediano1, Luiz Fernando Rodrigues Junior1 (1) National Institute of Cardiology Introduction: Non-invasive ventilation (NIV) has been used for prophylaxis/treatment of pulmonary/respiratory complications during postoperative period (PO) of cardiovascular surgery. However, the indiscriminate use of prophylactic NIV may burden health systems due to the exacerbated use of human and material resources, resulting in a worsening of the standard of care provided. Objective: To identify the predictors for NIV indication in patients in the postoperative period of cardiovascular surgery. Methods: This was a retrospective cross-sectional study. Data from 614 patients submitted to cardiovascular surgery at the Instituto Nacional de Cardiologia (Rio de Janeiro-RJ), from October/2018 to March/2020, was obtained from the Physiotherapy Service database, stored on the Research Electronic Data Capture platform (REDCap) and complemented based on the review of physical records. Preoperative variables such as demographic data, comorbidities, functional class, cardiac function and risk assessment for cardiac surgery were collected. Intraoperative variables were: type/complexity of surgery, fluid balance, time on cardiopulmonary bypass (tCPB), time of aortic clamping and surgical complications. Variables evaluated in the PO were: level of consciousness, pain, water balance, imaging, laboratory and blood gas tests, tracheobronchial secretion, mechanics and pulmonary/respiratory function before and after extubation, extubation attempts and failures, and use of NIV after extubation. The univariate and multivariate logistic regression model (P < 0.05 considered significant) was used to verify the association of possible predictors with the NIV indication in the postoperative period of cardiovascular surgery. Results: The preoperative: age (OR:1.05; P = 0.005); the intraoperative: tCBP (OR:1.01; P = 0.046); and the postoperative variable: partial pressure of carbon dioxide (PCO2) before extubation (OR:1.08; P = 0.045) were independently associated with the need for NIV in the postoperative period of cardiovascular surgery. Conclusion: Age, tCPB and pre-extubation PCO2 are independent predictors of NIV need in patients in the postoperative period of cardiovascular surgery, so that the higher age, tCBP or PCO2, the greater the chances for the patient needing NIV, a finding that may encourage the choice of patients eligible for early intervention with NIV, and the creation of a score/calculator for supporting bedside decision about the use of prophylactic NIV. 109101 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING ISADORA SUCUPIRA MACHADO1, Tereza Cristina Pinheiro Diógenes1, Marilia Esther Benevides de Abreu2, Ana Gardenia Liberato Ponte Farias2, Marcia Maria Carneiro2, José Sebastião de Abreu1 (1) Clinicárdio Métodos Diagnósticos; (2) Cardioexata de Fortaleza Background: Adverse effects and serious complications can occur during dobutamine stress echocardiography (DSE) in a conventional protocol (CP). Objective: To assess the safety of DSE using a modified protocol (MP). Methods: Prospective collection of data from patients undergoing DSE, administering dobutamine in up to four stage. Atropine could be administered from the fourth stage in the CP and, as a routine, concomitant with the beginning of the third stage in the MP. At the end of the exam or to control arrhythmia, metoprolol was administered to the CP and esmolol to the MP. In the event of typical angina, the examiner defined the therapy in the CP, while in the MP, if necessary, a nitroglycerin solution could be infused over a period of three to twelve minutes. A p-value <0.05 was considered statistically significant. Results: Among 17,811 DSEs, 9,121 were included in the MP. Hypertensive peak (1% vs 0.4%; p = 0.0001) and non-sustained ventricular tachycardia (0.6% vs 0.1%; p = 0.0001) were more frequent in CP, while supraventricular tachyarrhythmia (1.9% vs 3%; p = 0.0001) and atrial fibrillation (0.8% vs 1.3%; p = 0.003) in the MP. These arrhythmias reversed spontaneously or with medication. Nitroglycerin was administered in 76 MP cases. Ventricular fibrillation and acute coronary syndrome occurred in CP. There was no acute myocardial infarction, sustained ventricular tachycardia, ventricular fibrillation, cardiac rupture, asystole, or death with MP. Conclusion: The MP for the DSE is a safe option in the application of this methodology. 109102 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MATHEUS AKIRA SUZUKI DE OLIVEIRA1, Matheus Akira Suzuki de Oliveira1, Júlia de Ávila Gutierrez3, Karen Tássia Façanha Ramos2, Luiz Felipe Façanha Ramos2, Leo Christyan Alves de Lima4, Laura Jane França Lacerda4, Hildeman Dias da Costa1, Mathews Barbosa Santiago5, Ayrison de Melo Sousa5 (1) Universidade Federal de Rondônia; (2) Universidade Federal do Amapá; (3) Faculdades Integradas Aparício Carvalho; (4) Centro Universitário São Lucas; (5) Centro Universitário Uninorte Introduction: Rheumatic heart disease is caused by damage to the heart valves and muscles that arise from inflammation and scarring caused by rheumatic fever. Rheumatic fever mainly affects children in developing countries, especially where poverty is widespread. About 2% of deaths from cardiovascular diseases in the world are related to rheumatic heart disease. Objective: To describe the epidemiological profile of hospitalizations for chronic rheumatic heart disease among children and adolescents in Brazil between 2010 and 2020. Methods: Cross-sectional epidemiological study. Data were obtained from the information technology department of the Unified Health System – DATASUS. The variables researched were: total hospitalizations, sex, color/race, age group, deaths and mortality rate. The age group surveyed was individuals from zero to 19 years old. The research period was delimited between the months of January 2010 and December 2020. Results: There were 5,909 hospitalizations in the studied period. In 2010 and 2020, 627 and 290 hospitalizations were recorded, respectively. Males reported 3,127 hospitalizations, and females 2,782. The brown color/race registered 2,115 hospitalizations. The most affected age group was 15 to 19 years old, with 2,136 cases. The total number of deaths was 194. The average mortality rate was 3.28. Conclusions: Hospitalizations for chronic rheumatic heart disease show a decreasing curve in recent years in Brazil. The epidemiological profile of hospitalizations between 2010 and 2020 was characterized by male adolescents, brown, aged between 15 and 19 years. The number of hospitalizations in 2020 may have been influenced by the pandemic caused by COVID-19. 109103 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOGERIATRICS CAROLINA JERONIMO MAGALHAES1, Matheus Dantas Soeiro3, Sabrina Barreto Braga Pires3, Maria Eduarda Borges Matias3, Ellen Beatriz Sobral3, Carolina De Carvalho Moury Fernandes3, Jessica Myrian de Amorim Garcia2, Francisco Bandeira1 (1) UNIVERSIDADE DE PERNAMBUCO; (2) HOSPITAL AGAMENON MAGALHAES; (3) FACULDADE PERNAMBUCANA DE SAÚDE Introduction: Diabetes mellitus (DM) and heart failure (HF) often co-exist, and the number of people with both diseases is increasing due to ageing and better treatment. DM contributes to the progression of HF, commonly associated with worsening of symptoms. Quantifying the quality of life (QOL) in HF is extremely important especially in the elderly, in order to maintain good health and personal satisfaction through life. Objectives: Evaluate the QOL of elderly based on the Kansas City cardiomyopathy questionnaire (KCCQ) associated with DM, pre-DM and normoglycemia in a large tertiary center. Methods: A single-center analysis of in-patients aged ≥65 years presenting with HF from August 2020 to January 2022 in a Brazilian cardiology teaching hospital. KCCQ was performed considering QOL 15 days prior to admission. Scores scaled 0–49 represented poor health status, and 50–100 good health status. Based on glycated haemoglobin (HbA1c) the elderly were diagnosed with DM, pre-DM or normoglycemia. Results: A total of 182 patients were studied. The mean age was 73 ± 6 years, 54.3% were men. Study population had 88.8% prevalence of hypertension and 51.5% of chronic kidney disease. The mean level of HbA1c was 6.6 ± 2.0%, minimum of 4.1% and maximum of 14.7%. Based on HbA1c, 45.8% had DM, 23.6% pre-DM and 30.6% normoglycemia. Mean score for KCCQ was 54.6 ± 19.65. QOL was considered good in 52% patients and bad in 48%. There was no association between HbA1c levels and poorer QOL in the elder patients. Conclusion: We found no differences in QOL specific evaluation for HF in elderly patients with or without DM or pre-DM. 109114 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY VINÍCIUS PEREIRA DANTAS1, Rafael Alves da Silva1, Ademar Alexandre de Morais1, Rosiane Viana Zuza Diniz1, Kelton Dantas Pereira1, Kelson Kemuel Confessor de Sousa1 (1) Hospital Universitário Onofre Lopes – HUOL UFRN Introduction: Considering the aging of the population and a common occurrence of risk factors, it is increasingly probable that a patient may have both cancer and cardiovascular disease. The cardiotoxicty is one of the main and most feared complications of cancer treatment. It can manifest itself in several ways including, with heart failure (HF) being the most frequent and most severe. So, oncologist and cardiologists should undertake the therapeutic option for a cancer patient to minimise cardiotoxicity without compromising anticancer efficacy. Aim: Identify the cardiotoxicity risk in cancer outpatients referred to the cardio-oncology clinic in a tertiary hospital and determine the treatment plan at the referral time. Methodology: Cross-sectional, observational and descriptive study, conducted between September 2018 and September 2021, involving 60 adults, at the first visit in a cardio-oncology clinic of a tertiary hospital. Results: The majority of patients were women (68.3%, n = 41), white (35%, n = 21). The mean age was 56 ± 15.5 and the most frequent aetiology was breast neoplasia (40%, n = 24). The levels of high to very high for cardiotoxicity risk were prevalent (71.6%, n = 43). At the first visit, 45 patients (75%) had already started chemotherapy and/or radiotherapy treatment against cancer and 38 (63.3%) of the patients were diagnosed with heart failure. Conclusion: The risk for cardiotoxicity in this population was high, as well as the presence of heart failure and use of anti-neoplastic treatment prior to consultation in a specialized outpatient clinic. The optimal timing and mechanisms for referral need to be studied and implemented. Referral to the specialist in cardio-oncology allows an early recognition of subclinical signs and symptoms, detecting and preventing CV side effects in cancer patients. 109139 Modality: E-Poster Young Researcher – Non-case Report Category: DYSLIPIDEMIA GISELI CASARINI1, Natan Alevato Donadon1, Gabriel Tamanaha Pacheco1, Rafael Ferreira e Bringel1, Sara Regina Alcalde Domingos1, André Árpád Faludi1, Daniel Branco de Araujo1, Natasha Soares Simões dos Santos1, Adriana Bertolami1, Luiz Paulo Bastos Schmidt1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: High-dose of potente statins are first-line and mandatory therapy to reduce LDL-c among very high cardiovascular risk patients. The Brazilian Guideline on Dyslipidemia and Prevention of Atherosclerosis of the Brazilian Society of Cardiology (SBC) recommends a target of LDL-c below 50 mg/dL or non-HDL cholesterol (NHDLc) <80 mg/dL. Objective: Evaluate the amount of very high-risk patients using the initial treatment recommended by the brazilian guideline that achieved the LDL-c and NHDLc therapeutic goals. Methods: This is a cross-sectional observational study that included 1122 very high cardiovascular risk patients (significant atherosclerotic disease with or without clinical events or obstruction ≥50% in any arterial territory), treated in outpatients clinics between January and March of 2022, in a tertiary care hospital in Brazil, using atorvastatin 40 mg or 80 mg daily. Exclusion criteria were: use of atorvastatin in a dose lower than 40 mg/daily or use of other statins. Data from the electronic medical record were collected regarding lipid profile, such as LDL-c and NHDLc, age and sex. RESULTS A total of 1122 patients were evaluated and 1012 were included. Mean age was 68.8 years (SD 9.2), 634 (62.4%) were men. Regarding statin use, 613 (60.6%) patients used atorvastatin 80 mg/daily and 399 (39.4%) used atorvastatin 40 mg/daily. Average LDL-c was 83.1 mg/dL (SD 29.5) and NHDLc was 113.5 mg/dL (SD 35). The mean TC was 152 mg/dL (SD 29.5), HDL-c was 39.5 (SD 11.8) and of TG was 154.9 (SD 85.6). Ninety two (9%) patients had LDL-c <50 mg/dL and 133 (13.14%) patients had NHDLc <80 mg/dL. Conclusion: The low amount of patients in this population that achieved LDL-c and NHDLc target shows that with high-intensity statins monotherapy may not be sufficient. These data suggest that in very high-risk patients, combined lipid-lowering therapy, in the initial phase, should be considered. 109153 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION MILENA SORIANO MARCOLINO1, Maria Cristina da Paixão1, Leonardo Bonisson Ribeiro1, Paulo Rodrigues Gomes1, Breno Max Horta Melo1, Gabriela Miana de Mattos Paixão1, Antonio Luiz Pinho Ribeiro1 (1) Centro de Telessaúde do Hospital as Clínicas da Unniversidade Federal de Minas Gerais Introduction: Cardiovascular diseases are the main cause of death in Brazil and in many developing low- and middle-income countries (LMIC). Access to specialized cardiac care remains precarious in most of these places. Objective: To describe the experience of the Telehealth Network of Minas Gerais of developing and implementing the Brazilian Nationwide Service of Tele-Electrocardiography. Methods: Remote points, located primarily on primary healthcare units, received a standard computing system including a computer, connected to the internet, and a digital electrocardiogram (ECG). ECG signal is captured by the computer, using in-house developed software, and uploaded to a central server, where they are pre-processed, including automatic ECG measurements. A web-based interface is available for the cardiologists to analyze and interpret ECG exams, including tools for magnifying and re-measuring the ECG waves and intervals and a support system for improving the quality of the report. Quality is regularly audited. This system, initially implemented in the state of Minas Gerais in 2006, has been implemented in other Brazilian states since 2017. Results: The service is available 24 × 7. Emergency exams or those with extreme abnormal automatic findings are prioritized and reported in less than 10 minutes; the others are analyzed in less than 2 hours. Since 2006, over 6 million ECGs have been analyzed and the system is now available in 1199 cities in 12 Brazilian states. The majority of the remote points are in primary care centers, but some are in small hospitals and emergency care units. The system is cost-effective, having generated economy of over R$ 345,000,000 with the reduction of referrals to other cities, and allows access to a basic cardiological exam in places where it was not available. More recently, the dataset of ECGs was organized and linked to mortality data (the CODE study) and is being used in epidemiologic and artificial intelligence studies. Conclusions: Telehealth tools can be used in LMIC countries to provide cost-effective access to high quality ECG diagnosis in large scale. This kind of service can also help to build datasets for relevant research for local communities and the development of advanced artificial intelligence. The availability of these tele-ECG systems may have impact in cardiovascular care in LMIC. 109170 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING SOMLUCK PUNAWAKUL1, Pairoj Chattranukulchai2, Monravee Tumkosit3, Yongkasem Vorasettakarnkij2 (1) Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; (2) Cardiac center, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand; (3) Department of Radiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Background: Adenosine stress CMR is playing an important role in diagnosis of coronary artery disease (CAD). Objectives To assess the prognostic value of adenosine stress CMR at 3 Tesla to predict clinical outcomes during two years of follow-up in patients with known or suspected CAD. Methods: A retrospective cohort study in patients who underwent adenosine stress CMR at 3 Tesla between 1 Sep 2017–31 Aug 2019 at a tertiary hospital in Bangkok, Thailand. The primary outcome includes composite outcomes of CAD-related death, non-fatal MI, and revascularization. Results: Total of 633 patients were analyzed (age 66.21 ± 12.60 years, 53.7% male, 31.9% known CAD). The result of CMR was positive in 215 cases (33.97%), and 118 patients (2.7%) had myocardial scar. We observed 5 CAD-related deaths, 10 non-fatal MI, and 94 revascularizations within 2 years of follow-up. Positive adenosine stress CMR demonstrated a significant association with both primary outcome (43.4% vs. 2.9%, p = <0.001), and secondary outcome (61.4% vs. 25.4%, p = <0.001). The severity of perfusion defect was significantly associated with the primary outcome in both patients with and without myocardial scar groups. Among the patients without scar, primary outcome in the no ischemia, mild/moderate, and severe ischemia group are 2.38%, 16.28%, and 53.03% (p = <0.001), respectively. Among the patients with scar, the primary outcome were 14.29%, 22.73%, and 70.21% (p = <0.001), respectively (Figure 1). Conclusions: In this cohort study, positive adenosine stress CMR at 3 Tesla is a powerful tool to predict clinical outcomes in patients with known or suspected CAD. The severity of myocardial ischemia and the presence of myocardial scar could help identifying high-risk patients. More study Further studies are warranted. 109179 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION JAVIER ELIECER PEREIRA RODRIGUEZ1, Jorge Lara-Vargas2, Fernando Rivera-Theurel3, Magalli Diaz-Bravo1, Eduardo Leyva-Valadez2, Luis Fernando Ceballos-Portilla1, Arizbeth De Jesus-Guerra1, Salvador Aguilar-Nava1, Sebastian De Marcos-Sanchez1, Guadalupe Montserrat-Pineda1 (1) Centro de Estudios e Investigación FISICOL. Colombia; (2) Cardio Fit. México; (3) University of Toronto. Canada Introduction: Heart Failure patients (HF) are characterized by decreased functional capacity and quality of life. The prevalence of depression of 25% in newly diagnosed HF and up 50% in advanced stages of HF. Anxiety prevalence in HF patients can up to 40%. Objetive: Determine the difference between strength training of upper versus lower limbs in the depression and anxiety scale of patients with heart failure with low functional capacity within a cardiac rehabilitation program. Methods and materials: Prospective randomized study with 412 cardiac rehabilitation participants comparing 2 groups (Aerobic exercise (60–80%) + Group 1: MMSS Strength (40–60%) and Group 2: MMII Strength (40–60%). An exercise stress test, a 6-minute walk, a 1-repetition maximum test, depression and anxiety with the HAD test were performed. Measurements were done after the 24 sessions of 60 minutes and in the absence of psychological intervention. In the statistical analysis, normality was evaluated by means of Kolmogorov-Smirnov and, subsequently, the analysis of variance ANOVA. Results: 411 finished the intervention and it was possible to determine that both groups improved the depression levels GE1: 21%vs11%; GE2: 27%vs7%; p = 0.001 and anxiety GE1:13%vs7%; GE2:15%vs4%; p = 0.001. Conclusion: Strength training combined with aerobic exercise reduces depression and anxiety in HF. 109183 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY FILIPA DANIELA COSTEIRA MACHADO1, Ana Afonso1, Filipa Machado1, Ângela Amaro-Leal1, Miguel Meira-Cruz1, Isabel Rocha1, Vera Geraldes1 (1) Faculdade de Medicina e Centro Cardiovascular da Universidade de Lisboa Doxorubicin (DOX) is a highly effective anticancer drug, but its effectiveness is offset by dose-related side effects, including autonomic dysfunction and cardiotoxic effects. Exercise training (ET) and β-blockers have been suggested to prevent cardiotoxicity. ET reduce sympathetic activity and increase parasympathetic activity. Beta-blockers reduce sympathetic activation and improve cardiac function. Despite extensive research it is not yet clear which therapeutic regimen is best suited to prevent or reduce DOX side effects. In our work, we compare the efficacy of two approaches: carvedilol (CVD) and a non-pharmacological intervention, an exercise protocol (ET) with treadmill training in an animal model of DOX. Female Wistar rats were divided into 4 groups: Doxorubicin (DOX; ip cumulative dose 16 mg/kg, 1 time/week, for 4 weeks), DOX with ET (DOX+EX;treadmill, 25 cm/sec for 30 min, 5 times/week), DOX with CVD (DOX+CVD:10 mg/kg, orally, 5 times/week, for 4 weeks) and controls (CTL ip with saline). At the end of the protocol, animals were anaesthetized, and blood pressure (BP), electrocardiogram, heart rate (HR) and respiratory rate (RF) were recorded. Baroreceptor and chemoreceptor reflexes were tested, and baroreflex gain and sensitivity, and chemoreflex sensitivity were calculated. HRV was determined to estimate sympathetic and parasympathetic activity. Our results show that DOX treatment induced a significant decrease in systolic BP (CTL:150.1 ± 5.338; DOX:103.1 ± 10.19 mmHg, p = 0.0127) and mean BP (CTL:129.0 ± 3.520; DOX 90.38 ± 10.08 mmHg, p = 0.0072), and in HR (CTL:379.5 ± 54.34; DOX:288.3 ± 53.11 bpm, p = 0.0284), compared with the CTL group. The ET protocol normalises systolic BP (122.4 ± 29.85 mmHg), mean BP (94.51 ± 20.08 mmHg) and HR (350.4 ± 22.18 bpm). Administration of CVD during DOX treatment significantly restored HR to near-physiological levels (368.6 ± 16.23 bpm p = 0.0359). DOX treatment resulted in an increase in baroreflex gain compared to the CTL group. CVD treatment, similar to the ET effect, decreased baroreflex gain compared with the DOX group (DOX:4.738 ± 2.771; DOX+EX:0.5046 ± 0.06773; p = 0.0473 and DOX+CVD:0.5516 ± 0.05672 bpm/mmHg; p = 0.0376) and also restored BEI, BRS and SDNN to normal values. No significant changes in chemoreflex sensitivity, RF and frequency domain indices were observed. Overall, these results suggest that CVD and ET improve baroreflex through HR modulation. However, ET can be considered the best strategy as it acts on both autonomic branches. 109234 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION JOÃO MOTARELLI1, Juliana Tieko Kato1, Marilia Andrade Papa1, Maria Cristina de Oliveira Izar1 (1) Universidade Federal de São Paulo Introduction: The reduction of body mass (BM) in obese individuals has been considered a modifiable risk factor for cardiovascular diseases (CVD) morbidity and mortality. However, the potential benefits of ketogenic diet in BM reduction may be compromised due to the reduction off strongh predictors of CVD such as lean mass (LM) and muscle strength, which may inadvertently affect the individual’s muscular functional capacity (MFC). Objective: To evaluate changes in body composition and MFC in physically inactive obese patients undergoing one of two different low-calorie diets over a 6-month period. Methods: We included 25 physically inactive patients with grade I or II obesity, of both genders, with a mean age of 40 years, followed for 6 months through periodic medical and nutritional care, allocated to the Dukan’s Diet (DD) group (n = 11) who received the nutritional recommendations specified by the Dukan Diet (ketogenic diet) or the Recommended Diet (RD) (n = 14), who received nutritional guidelines based on the 2014 Food Guide for the Brazilian Population. Double absorbance x-ray (DEXA) was used for analysis of LM, and Body Fat (BF); Biodex® Multi-joint System 3 isokinetic dynamometer was used for MFC analysis of the knee extensor muscles (Peak torque at 60°/s – PT and its variable corrected by BM – PT/BM). Food intake was quantified via 3-day dietary recall and ketosis analyzed through urine stripes. Analyzes were performed at baseline, 05, 30, and 180 days. Results: Significantly higher consumption of protein and lower consumption of carbohydrates and fiber were observed in the DD group compared to RD as the presence of ketosis in urine (in 05 and 30 days). At the end of the study, it was observed that both interventions promoted significant reductions in BM (DD P < 0.001; DR P < 0.001) and BF (DD P < 0.001; RD P < 0.001), however, only DD presented significant reductions of LM (P < 0.031), PT (P < 0.001), PT/BM (P < 0.001). Comparison between groups at 180 days showed significantly lower measures for BF (P < 0.031) in the DD group. Conclusion: DD promoted more expressive reductions in BF and LM at the expense of a loss of MFC even with a high protein intake. Longer-term studies are needed to understand whether the reduction in MFC could lead to functional impairment and/or increased risk for CVD in this population as a result of the practice of this diet. 109235 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION BIANCA FELDMAN1, Rodrigo Mazza1, Bruno Dias1, Gabriel Moraes1, Joao Magalhães1, Carlos Gil Ferreira3, Tatiane Montella3, Anderson Nassar2, Gustavo Nobre2, Marcelo Kalichsztein2, Leandro Toledo1, Fabricio Braga1 (1) Laboratório de Performance Humana(LPH); (2) Casa de Saúde São José (CSSJ); (3) Oncoclínica Background: The role of aerobic capacity as a predictor of surgical risk (SR) has been highlighted, especially in cancer patients. Cardiopulmonary exercise testing (CPET) is the gold standard in this assessment. Objective: To characterize and identify risk predictors among lung cancer (LC) patients evaluated with the CPET. Materials and methods: Cross-sectional analysis of LC patients who underwent CPET as part of the SR assessment for lung resection. Before CPET, all patients performed rest spirometry to determine forced vital capacity (FVC) and end-expiratory volume in the first second (FEV1). SR was classified by an algorithm based on peak exercise VO2 (pVO2) and the slope of the ratio between minute ventilation (VE) to carbon dioxide (VCO2) output (VE/VCO2 slope) in low, medium, high and very high SR. A multivariate logistic regression model (MLRL) was created to identify predictors of low SR. Results: Ninety-eight patients were analyzed (44.9% men, 71 ± 9.1 years). The mean pVO2 was 16.7 ± 4.2 ml/kg/min (77.9 ± 17% of the predicted values), and the mean VE/VCO2 was 36.6 ± 6.4. The FVC and FEV1 were 2.66 ± 0.8 L and 2.08 ± 0.65 L, respectively. According to the SR, 38 (38.6%), 31 (31.6%), 26 (26.5%) and 3 (3.1%) were classified as low, moderate, high and very high, respectively. Low SR patients were younger (67.8 +/– 9.3 vs. 73.1 ± 8.6 years [p = 0,005]), showed a higher FVC (2.87 ± 0.88 vs. 2.52 ± 0.73 [0.043]) and FEV1 (2.34 ± 0.70 vs. 1.89 ± 0.55 L [p = 0.001]), and trended to a lower COPD prevalence (25 vs. 43.5% [0 = 0.075]) than other risk patients. In the MLRL, age (OR = 0.93 [95% CI 0.88–0.98]) and FEV1 (OR = 24.4 [95% CI 2.2–266.3]) were independently associated with a low SR. Conclusion: (1) More than 60% of lung cancer patients are not at low risk when assessed by CPET; (2) advanced age and reduced FEV1 increased the odds of a higher SR. These data can guide prehabilitation strategies to reduce perioperative morbidities for LC patients. 110340 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY GABRIEL ODOZYNSKI1, Isabella Bianco3, Helcio G. Nascimento1, Andrei Lewandowski1, Maurício L. Spessatto1, Clóvis Froemming1, Grazyelle Damasceno1, Alexander Dal Forno1, André Pacheco Silva1, André d‘Ávila1 (1) Hospital de Cardiologia SOS Cardio; (2) Instituto de Cardiologia de Santa Catarina; (3) Universidade do Sul de Santa Catarina Introduction: Different electro-anatomic recurrence patterns were previous described. Yet, pulmonary vein (PV) electric reconnection to left atrium is the typical finding after atrial fibrillation (AF) catheter ablation recurrence. Aims and Method: We aim to evaluate AF ablation recurrence patterns in a cross-sectional, single-center study of recurrent AF patients who had underwent to a 2nd ablation procedure (REDO). Results: Of 47 enrolled patients to REDO, 31 patients had paroxysmal AF (≤7 days-PAF) and 16 patients persistent AF (>7 day ≤1 year –AF-Per). PV reconnection were confirmed in 21 (68%) and 16 (62%) patients of PAF and AF-Per. From 113 targeted PVs in PAF and 58 in AF-Per groups– 35 (30%) and 18 (31%) of PVs were reconnected, respectively. 10 (32%) PAF and 6 (37%) in AF-Per patients have maintained PV isolation during REDO procedure. 6 (19%) and 3 (18%) patients had 1 reconnected PV, 11 (35%) and 6 (37%) had 2 reconnected PVs in PAF and AF-Per groups. Only 2 patients in PAF and 1 in AF-Per have presented all PVs reconnected during REDO procedure (2 left common trunk). Right PVs were reconnected in 11(61%) of PAF patients. Cavo-tricuspid isthmus flutter ablation were performed in 6 patients in PAF group. Left atrial appendage was electrical isolated in 1 AF-Per patient during REDO procedure. At the first procedure, left atrium posterior wall ablation was performed in 6 (19%) and 8 (50%) of PAF and AF-Per. The posterior wall reconnection was confirmed in 2 AF-Per procedure. Left common trunk were reconnected in 2 (6%) and 2 (12%) in PAF and AF-Per. Conclusion: PV reconnection is the typical finding in patients with recurrent AF after catheter ablation. Right PVs reconnection was the common described pattern. 109269 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JULIANA TIEKO KATO1, João Henrique Fabiano Motarelli1, Andrey Santos2, Mario José Abdala Saad2, Maria Cristina de Oliveira Izar1, Carolina Nunes França1, Henrique Andrade Fonseca1, Francisco Antonio Helfenstein Fonseca1 (1) Universidade Federal de São Paulo; (2) Universidade Estadual de Campinas Introduction: There is clear evidence that the composition of the gut microbiota (GM) plays an important role, not only in extracting energy from food, but also as a key endocrine organ, producing metabolites that impact the pathogenesis of type 2 diabetes mellitus (DM). Despite advances in metagenomics techniques, it is still difficult to establish a microbial signature in DM due to numerous confounding factors, such as the association with other pathologies, diet, drug and geographic location. Objective: To identify changes in the GM composition in individuals with DM or without DM [prediabetic (PDM) and non-diabetic (NDM)] in the first 24 hours of acute ST elevation Myocardial Infarction. Methods: Eighty-five patients were included, metagenomic analysis was performed through stool using a technique that is based on DNA extraction and amplification of the 16S rRNA gene. Data were processed by the Miseq Illumina platform and analyzed using the Illumina 16S Metagenomics software. Other analyzes were performed using the SPSS version 21. The significance level was p < 0.05, and when necessary, the p-values of the fecal samples were adjusted using the False Discovery Rate (FDR) multiple comparison test. Subjects were divided into groups according to glycated hemoglobin values, <5.7% (NDM), 5.7 to 6.5% (PDM) and >6.5% (DM). Results: Relative abundance analysis showed significant differences between the phyla Firmicutes (P = 0.05) and Verrumicrobia (P = 0.02) and the genus Akkermansia (P = 0.03) between the NDM vs DM groups. When the values were adjusted by FDR, we found a significant increase in the phylum Firmicutes (P-FDR = 0.035) and class Clostridia (P-FDR = 0.041), as well as a decrease in the phylum Verrucomicrobia (P-FDR = 0.035) and its class Verrucomicrobiae (P-FDR = 0.045) in NDM when compared to DM. Conclusion: The present study showed that the composition of the intestinal microbiota differed between the NDM vs DM groups after acute myocardial infarction, with a duality in the results, where an increase (Firmicutes and Clostridia) and a decrease of bacteria considered beneficial (Verrucomicrobia) were observed. 109255 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANA LÍGIA VALERIANO DE OLIVEIRA1, Ana Lígia Valeriano de Oliveira1, Andressa Pimentel Afiune1, Jhenefr Ribeiro Brito1, Vitória Lorrane dos Santos1, Thais Aratk Marques Taia1, Danilo Borges de Sousa1, Anna de Paula Freitas Borges1, Andressa Morgado Parreira1, Lucas Eduardo Almeida França1 (1) Pontifícia Universidade Católica de Goiás Obstructive hypertrophic cardiomyopathy is a genetic disease capable of causing changes in the structure and function of the myocardium muscle. Hypertrophy results in left ventricular obstruction and symptoms of heart failure, exertional syncope, or sudden cardiac death. Radiofrequency myocardial septal ablation is a treatment option for symptomatic and drug-refractory cases. It is a less invasive method and can be used for septum reduction in patients at high risk for surgeries such as myectomy and alcohol ablation. The study seeks to evaluate the feasibility, efficiency and outcome of treatment with radiofrequency ablation in obstructive hypertrophic cardiomyopathy. This is a systematic review based on the methodological recommendations Preferred Reporting Items for Systematic Reviews and Meta-Analyses – PRISMA. The identification and selection of articles was conducted by two independent researchers between October and December 2021 on the platforms PubMed, Scientific Electronic Library Online and Latin American and Caribbean Literature. The filters used were “human” and “published in the last 10 years”. The included studies were in English and Portuguese. The descriptors used were obstructive hypertrophic cardiomyopathy, treatment and radiofrequency ablation that were associated with the Boolean operator AND. We found 66 articles, after applying the inclusion and exclusion criteria, we selected 10 articles that were used in this review. The studies analyzed showed a reduction in the left ventricular outflow tract gradient, septum thickness, mitral regurgitation volume and pro-BNP. Furthermore, ablation showed the ability to suppress ventricular tachycardia in all patients. There was improvement according to the NYHA functional class in four articles, with statistical significance. It was analyzed in a study that the procedure, combined with imaging techniques with a mixture of intracardiac echocardiography and CARTOSound, enabled a more effective mapping of the affected area and avoided damage to the conduction tissue. Finally, the radiofrequency septal ablation technique proved to be a safe and less invasive option, with a shorter hospital stay. Radiofrequency septal ablation allows a significant reduction in the left ventricular outflow tract gradient, in addition to improving the NYHA functional class, thus being an alternative to alcohol septal ablation and myectomy, but later studies comparing it with others invasive methods become necessary. 109270 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING RAFAELA BATISTA DOS SANTOS PEDROSA1, Natalia Balestra2, Roberta Cunha Matheus Rodrigues1, Adryel Vieira Caetano da Silva2 (1) Faculdade de Enfermagem da Universidade Estadual de Campinas; (2) Hospital Sírio-Libanês Introduction: Driveline infections are common in patients with left ventricular assist devices and an important cause of morbidity and mortality. Nurses play a fundamental role in the care of the driveline, however, scientific evidence regarding dressing standardization is lacking. Objective: To describe the categories of dressings used in the driveline of patients using the HeartMate (HM) device and the incidence density of local complications (infection, bleeding and pressure injuries) within 30 days of the postoperative period. Methods: This was a retrospective cohort of patients hospitalized in the Intensive Care Unit (ICU) after HM II and III implant surgery in a private hospital in the state of São Paulo, Brazil, between 2015 and 2021. The data source was the hybrid medical records of the participants. We performed a descriptive analysis and Fisher’s exact test and statistically significance level of 5% was considered. Incidence density was calculated for infection, bleeding and pressure injury. The study project was approved by the local Ethics Committee (No. 4,593,315). Results: The results showed a high variability of dressings with 22 different categories being recognized and for each participant, an average of 2.2 ± 1.0 dressings. The dressing categorized as C6 (Chlorhexidine, Excilon, Gauze and IV3000) was the most used (45.4%), followed by C13 – ChloraPrep, Biatain Ag, Gauze and IV3000 (31.8%) and C9 – Protosan, Excilon, Gauze and IV3000 (22.7%). Bleeding was more frequent in categories C6 (9.0%), C11 – ChloraPrep, Gaze, Excilon and IV3000 (9.0%) and C13 (9.0%), while pressure injury was identified in only two categories. Subjects using FlexiTrak stabilizer had the highest rate of bleeding (50.0%) and those using Hollister had the highest infection rate (61.1%) and pressure injuries (11.1%). The participants who presented bleeding had higher median values of cardiopulmonary bypass time (112.5 minutes) when compared to those without bleeding (100 minutes); p = 0.029. Infection incidence density was the highest, 43.6/1,000 person-days, mainly in men (55.5/1,000 person-days), using the Heartmate III (48.3/1,000 people-day) and who used two or more categories of dressings (47.2/1,000 person-days). Conclusion: The infection was the complication with the highest incidence density; the findings point to a standardization with the use of chlorhexidine, absorbent foam impregnated with silver and transparent film, as well as the use of a stabilizer. 109282 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JULIANA TIEKO KATO1, João Henrique Fabiano Motarelli1, Andrey Santos2, Mario José Abdala Saad2, Maria Cristina de Oliveira Izar1, Carolina Nunes França1, Henrique Andrade Fonseca1, Francisco Antonio Helfenstein Fonseca1 (1) Universidade Federal de São Paulo; (2) Universidade Estadual de Campinas Introduction: The study of the gut microbiota (GM) in coronary artery disease is an emerging area, because metabolites generated by GM seem to be involved in the progression of the disease, with the proatherosclerotic metabolite N-oxido-trimethylamine (TMAO) being the best described. Currently GM is considered a potential therapeutic target, but few studies have evaluated the changes and influence of GM composition in post-acute myocardial infarction. Objective: To identify changes in the GM composition of individuals in the first 24 hours (baseline), 30 and 180 days after ST-segment elevation acute myocardial infarction. Methods: 173 patients were included and 355 stool samples were sequenced, the metagenomic analysis was performed using the technique that is based on DNA extraction and amplification of the 16S rRNA gene, the data were processed by the Miseq Illumina platform and analyzed using the Illumina software 16S Metagenomics. Other analyzes of non-parametric variables were performed using Friedman test for paired samples, using SPSS 21 version. TMAO levels were measured in urine by liquid chromatography with a mass spectrometer from 23 subjects. The significance level was p < 0.05, and when necessary the p-values of the fecal samples were adjusted by the False Discovery Rate (FDR) multiple comparison test. Results: The analysis between times showed a more inflammatory profile at baseline when compared to 30 days, with a decrease in beneficial bacteria belonging to the phylum Bacteroidetes (P-FDR = 0.04) and genus Faecalibacterium (P = 0.04), in addition to the increase in Proteobacteria considered to be pro-inflammatory bacteria (P-FDR = 0.04). However, over time, GM showed an improvement in 180 days when compared to baseline, with a significant increase in bacteria producing butyrate, a short-chain fatty acid with a crucial role in human health, phylum Bacteroidetes (P = 0.01), family Lachnospiraceae (P-FDR = 0.04) and genus Faecalibacterium (P < 0.01), in addition to the increase in Prevotella (P = 0.02). Interestingly, other important analyzes of the composition of GM, showed a worsening of richness and diversity decreased at 30 and 180 days when compared to baseline, in addition to the increase of TMAO over time (P = 0.03). Conclusion: A more inflammatory profile was observed at baseline with improvement at 180 days after infarction, however TMAO increased over time, accompanied by a decrease in richness and diversity. 109304 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT NICHOLLAS COSTA ROSA1, Nichollas Costa Rosa1, Maria Antonieta Moraes1 (1) Programa de Pós Graduação em Ciências da Saúde: Cardiologia da Fundação Universitária de Cardiologia Introduction: The treatment of Heart Failure (HF) with diuretics substantially impacts this syndrome’s prognostic. Data suggest that patients at a higher risk of furosemide intake in the early stages of the disease can decrease the mortality rates. Objetivos: Monitor the time between the patient’s admission into the emergency room and were administered the first dose of diuretics among patients with HF and their risk of readmission within 30 days of hospital discharge using the LACE. Methods: Prospective and retrospective cohort study conducted with decompensated HF patients, supported by echocardiographic data, of both sexes with ≥18 years old, treated in a cardiology emergency (ES) from August 2018 to December 2018. 2019. The parameters used were diuretic infusion time, length of stay in the ES, and readmissions. The risk of readmission was classified using the LACE score as low (0 to 4), moderate (5 to 9), and high (>9) risk. The Chi-square test check for categorical variables. The Mann-Whitney test for the time spent in the SE and time of diuretic infusion. Results: We analyzed 361 medical records of mostly male patients (56%), aged 68 ± 12 years. There was a predominance of ischemic HF (49%), reduced ejection fraction (57%), functional class III (73%), and hemodynamic profile B (81%). After hemodynamic stabilization (60%) were discharged, and (39%) required 6 (4–12) days of hospitalization. The time between admission and infusion of the diuretic was 87 (60–128) minutes. Of these, 26.3% infused up to ≤60 and 73.7% in >60 minutes. The 40 mg dose of diuretic was used in 72% of them. There was a significant relationship between receiving a diuretic for ≤60 minutes and staying in the ES < 6 hours (early discharge), p < 0.05. The LACE score showed that 62% of patients had a moderate risk of unplanned readmission within 30 days. Patients at high risk for readmission (37%) were associated with altered pulmonary auscultation and the presence of jugular swelling (p < 0.05). After 6 months, 15% of the patients died, 9% lost contact and 75% were followed up. Of these, 37% were readmitted, 21% within 30 days and 46% within 180 days. Conclusion: The time between admission and intravenous infusion of diuretics in this population with acute HF was sub-optimized; however, patients who received the drug earlier were in the emergency department for a shorter time. The risk of readmission through LACE was effective in 34% of patients. 109307 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DIOGO MACIEL SILVA AZEVEDO1, Diogo Maciel Silva Azevedo1, Kelson Kemuel Confessor de Sousa1, Fabio Mastrocola1, Rosiane Viana Zuza Diniz1 (1) Hospital Universitário Onofre Lopes – HUOL Introduction: Cardiomyopathies are a heterogeneous group of structural and functional disorders of the heart muscle, with different etiologies and phenotypic expressions, in the absence of congenital, valvular, coronary diseases or arterial hypertension compatible with the myocardial alterations found. The diversity of clinical presentation makes the etiological definition difficult, and pathogenic genetic alterations are important in this definition. Objective: To determine the prevalence of genetic alterations in individuals with cardiomyopathies without defined aetiology in a tertiary hospital. Method: This is a retrospective, observational and longitudinal study involving 39 individuals with cardiomyopathy without an initially clarified diagnosis, inhospital and outhospital patients, follow-up in a tertiary hospital during the years 2020 to 2022, with identification of the genetic profile and phenotype of presentation assessed by echocardiographic parameters. Data were presented as means and proportions. Results: The mean age was 45.7 years+–14.5 years, with an even distribution regarding gender (51% men). The prevalence of positivity in the genetic test was 33.3% and the most frequent presentation phenotype was hypertrophic cardiomyopathy (53%), followed by dilated cardiomyopathy (33.3%). Among the 21 individuals with hypertrophic cardiomyopathy, 47% had a positive genetic test, with the MYBPC3 and MYH7 genes being the most frequently identified in pathogenic or probably pathogenic mutations, representing 70% of positive cases. Conclusion: The prevalence of genetic alterations in our population was high, especially in those with a hypertrophic phenotype, reinforcing the importance of determining the genetic profile and tracking family members in these patients. 109309 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING CAMILA GODOY FABRICIO1, Denise Mayumi Tanaka1, José Antonio Marin-Neto1, Luciano Fonseca Lemos de Oliveira2, Alessandra Arantes de Resende1, Fernando Fonseca França Ribeiro1, Alexandre Todorovic Fabro1, Sabrina Setembre Batah1, Jorge Mejia Cabeza3, Minna Moreira Dias Romano1, André Schmidt1, Marcus Vinícius Simões1 (1) Medical School of Ribeirao Preto – University of Sao Paulo, Sao Paulo, Brazil; (2) Physiotherapy Department – Federal University of Minas Gerais, Brazil; (3) Hospital Israelita Albert Einstein, Sao Paulo, Brazil Background: Chronic Chagas’ cardiomyopathy (CCC) have showed, in previous studies, a high prevalence of microvascular myocardial perfusion defects (MPD) that may participate in the myocardial damage process. However, there is a scarcity of studies correlating the histopathological (HISTO) changes with MPD in CCC. Purpose: We aimed at testing the hypothesis that MPD is more closely related to HISTO changes occurring in the endocardial layer in an experimental model of CCC in hamster. Methods: We investigated 23 female hamsters infected with 3.5 × 104 trypomastigote forms of T. cruzi, and 6 control non-infected. Animals underwent rest high-resolution 99mTc-Sestamibi SPECT myocardial perfusion scintigraphy, and rest transthoracic echocardiogram. The area of MPD was assessed through the calculation of polar maps by using Munich Heart® software. MPD was defined by areas with 99mTc-Sestamibi uptake <50% of the maximum pixel uptake value. After euthanasia, counting mononuclear cells for inflammation was performed by Weibel reticule in Hematoxylin-Eosin staining. Fibrosis was quantified by fraction area method using Red-Picrosirius staining. The analysis was topographically performed at three left ventricular myocardial wall layers: endocardial, mesocardial and epicardial. A total area of HISTO parameters was also computed, by summing up the respective areas of all myocardial layers. Results: Fourteen infected animals exhibited rest MPD (61%), and presented, in comparison to animals without MPD and to controls higher inflammation in total (1.09 ± 0.55, 0.93 ± 0.43, 0.41 ± 0.32, respectively; p = 0.03), endocardial (1.05 ± 0.50, 0.75 ± 0.44, 0.38 ± 0.22, respectively; p = 0.02), mesocardial (0.64 ± 0.44, 0.55 ± 0.33, 0.11 ± 0.08, respectively – p = 0.02) and epicardial layers (1.57 ± 0.97, 1.50 ± 0.73, 0.73 ± 0.89, respectively; p = 0.03). However, only the inflammation in endocardial layer presented positive correlation with individual values of MPD area (R = 0.55; p < 0.01). In addition, the total fibrosis area presented no difference between animal groups, but the endocardial layer presented a larger fibrosis area in the group with MPD (0.67 ± 0.18) compared to controls (0.45 ± 0.15; p = 0.03). Conclusion: Our results indicate a close relationship between MPD and HISTO changes in the endocardial layer of left ventricular myocardium, consistent with the notion that endocardial is the myocardial region more sensitive to the effects of myocardial ischemia in CCC. 109311 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY PAULINE ELOISE MARIANI1, Marco Antonio Vinciprova Dall Agnese2, Grasiele do Amaral Martins2, Maria Antonieta Pereira de Moraes1 (1) Instituto de Cardiologia/Fundação Universitária de Cardiologia – IC/FUC; (2) Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA Introduction: The functional capacity and degree of independence of patients undergoing cardiac surgery can be compromised by the length of stay in the Intensive Care Unit (ICU). Objective: To analyze the behavior of functional independence domains in patients undergoing cardiovascular surgery and its correlation with the length of stay in the ICU. Methods: Prospective cohort study, conducted with patients undergoing coronary artery bypass graft surgery and valvular surgery alone or in combination aged ≥50 years. Outcomes: self-care, work ability, mobility and eliminations were evaluated using the Barthel Index and the Karnofsky Scale at hospital discharge and at 90 days by telephone. Results: Preliminary analysis of 172 surgical patients showed a predominance of men (70%), hypertensive (58%), dyslipidemic (43%), smokers (37%), aged 63 ± 11 years. Coronary artery bypass graft surgery was the most prevalent (57.6%), with cardiopulmonary bypass time of 82 ± 31 minutes. The length of stay in the ICU among all patients was 6 ± 2 days. The Barthel index showed that the functional impairment was lower at discharge (86%) and 90 days (64%), that is, they showed greater independence right after the intervention and after 3 months they were a little more dependent. The Karnofsky scale showed that the degree of dependence was similar at discharge and at 90 days after the intervention, 76% and 78% respectively. The most prevalent domains of functional status decline were self-care 11.4 ± 1.6 and mobility 15.8 ± 2.6. However, a very expressive inverse correlation was observed (r –0.152 and p < 0.05) with statistical significance, regarding the functional impairment after discharge from the ICU and at the 90 days of follow-up. Conclusion: Partial results indicate that there is a reduction in behavior in all domains of functional independence and an inverse correlation between the length of stay in the ICU and the functional impairment of these patients. Thus, long periods of stay in the intensive care unit seem to be inversely associated with the development of functional impairment in relation to an individual’s basic daily activities. 109313 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION JEFFERSON FERNANDES EVANGELISTA1, Renata Alves1, Thaiane Lopes da Silva1, Juliana Silva Figueredo1, Cristiane Matsuura1 (1) Universidade do Estado do Rio de Janeiro Introduction: Beetroot is a functional food that has been gaining prominence due to its cardioprotective effects, in part attributed to its high nitrate content. Its effects are associated to the reduction of nitrate to nitric oxide, an important molecule involved in vascular homeostasis. Aims: To examine the effects of acute beetroot juice (BJ) ingestion on platelet aggregation, blood pressure (BP), and the expression of proteins involved in platelet activation in healthy adult men. Methods: Nine men (28 ± 6 yr old, 78 ± 8 kg, 1.77 ± 0.05 m), eutrophic and normotensive participated in the study. The experimental procedure consisted of only one visit in the morning. After 15 min of rest, BP was measured and blood was collected to measure platelet aggregation and protein expression by Western Blot. After that, the subjects ingested 500 mL of BJ (~400 mg of nitrate). After 2.5 h, a new measurement of BP was performed, as well as blood collection. Platelet aggregation was measured in washed platelets, platelet-rich plasma and whole blood using collagen (5 µg/mL) and ADP (10 µM) as agonists. Paired Student’s t test was used to compare pre- and post-BJ conditions. Results: Acute BJ ingestion reduced ADP-induced platelet aggregation in platelet-rich plasma (–29%), but was not altered when induced by ADP and collagen in whole blood and washed platelets, as well as collagen in PRP. Regarding BP, we observed decreases in systolic and mean BP (–10% and –6%, respectively) after 2.5 h. Protein expression of phosphorylated protein kinase B (Tyr 437/434) was lower after BJ ingestion (–19%), but the expression of phosphorylated-endothelial nitric oxide synthase (Ser1177) and vasodilator-stimulated phosphoprotein were not altered. All results are shown in Table 1. Conclusion: Our findings indicate that the acute ingestion of nitrate-rich BJ can be a strategy for controlling BP and inhibiting platelet aggregation in healthy individuals, although the topic still lacks further investigation, including an understanding of the its mechanisms and its effects in populations with increased cardiovascular risk. 109320 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MATHEUS TOSCANO PAFFER1, Pedro Toscano Paffer1, Rafaella Kelly Bello Wanderley1, Silvio Hock Paffer FIlho1 (1) Faculdade de Medicina de Olinda Introduction: Arterial stiffness is a new risk factor for cardiovascular diseases. It’s relation to aging is well known, being a condition to the development of hypertension (HTN). Objective: Evaluate the arterial stiffness of elderly patients and its correlation to HTN. Methods: 31 very elderly patients were submitted for evaluation of the central blood pressure via a Mobil O’Graph of IEM device. Results: 31 very elderly patients were analyzed (older than 80 years old), from which 78.1% were females. At the average age of 85.8 (+–5.20), and with average BMI of 25.96 (+–4.32). With an average arterial systolic blood pressure of 143.06 mmHg (+–22.71) and the diastolic of 74.83 mmHg (+–9.30). The average central systolic blood pressure of 155.19 mmHg (+–32.88) and the diastolic of 80.96 mmHg (+–20.79). The average PWV (Pulse Wave Velocity) was 13.34 m/s (+–1.44). From the analyzed patients, 67.74% had altered their PWV values. Discussion The prevalence of HTN increases the aging and the occurrence of arterial stiffness plays an important role in this process. Even though this finding is not common, it shouldn’t be considered a benign occurrence, because it’s the main cause of HTN development. It’s a condition prevalent in the elderly population, as observed in this cohort, but it can be reversed with the correct treatment for HTN. Conclusion: Arterial stiffness is a strong risk factor and important for the development of great cardiovascular diseases and its correlation with HTN is well known. Although the elderly population presents higher PWV rates, with an increase of arterial stiffness, it can be controlled and even reversed with optimized treatment of HTN. 109328 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JOAQUIM DE PAULA BARRETO FONSECA ANTUNES DE OLIVEIRA 1, Nestor Martins2, Sheila Tatsumi Kimura-Medorima1, Sofia Vitte1, Thiago Quinaglia2, Barbara Assato1, Otavio Rizzi Coelho Filho2, Jose Roberto Matos-Souza2, Wilson Nadruz2, Andrei Carvalho Sposito1 (1) Laboratory of Atherosclerosis and Vascular Biology, Cardiology Division, Unicamp; (2) Cardiology Division, Unicamp Introduction: The accumulation of Apo-B containing lipoproteins in the subendothelial layer is an initiating step of atherosclerotic cardiovascular disease. In the carotid artery, this event is assessed noninvasively by arterial layers width measurement using B-mode ultrasound. Due to technical limitations, the intima-media thickness (C-IMT), and not the carotid intima (C-IT) width, where atherosclerosis manifests, has been pursued as a marker of disease with a negligible incremental value when compared with traditional risk factors prediction models. Objective: We sought to determine the prognostic value of C-IT for cardiovascular outcomes. Methods: This was a predefined analysis of the Brazilian Diabetes Study, an ongoing, single-center, prospective cohort of T2D individuals aged 30 years or more. Carotid ultrasound was performed by two trained physicians in each common carotid artery to obtain three manual measurements of far-wall C-IT, media (C-MT), and C-IMT. C-IMT above the 75th percentile for age, gender, and race group, and C-IT or C-MT above the sample’s median were deemed as high. Cardiac CT was used to calculate the coronary artery calcium score, and a value above 0 Agatston was considered diagnostic of coronary artery disease (CAD). The primary outcome was major adverse cardiovascular events (MACEs) as a composite of death, myocardial infarction, stroke, myocardial revascularization, and limb amputation. Results: Among 402 individuals (Age: 58 + 7.6 years; 55% male; median T2D duration: 7.7 years) followed for 1676 days, 5 (3.1%) MACEs were reported in the low-IT, and 15 (10.1%) events were disclosed in the high-IT groups, with an HR of 3.11 (95%CI: 1.13, 8.58; p = 0.029). The prevalence of CAD was higher among high-IT than in low-IT group (58.4% vs 78.4%, p < 0.001), with an age-, and sex-adjusted OR of 2.29 (95%CI: 1.09, 4.85; p = 0.029). C-IMT and C-MT were not related to neither MACE nor CAD. Conclusions: C-IT, but not C-IMT nor C-MT, was related to incident MACEs and to coronary artery disease in T2D subjects. C-IT should thus be considered when assessing cardiovascular risk in T2D. 109345 Modality: E-Poster Young Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES CAMILA POLIS BELLOTT1, Gustavo Tavares Lameiro da Costa1, Matheus dos Santos Silva de Oliveira1, Cristiane da Cruz Lamas1, Renato Cerceau1 (1) Instituto Nacional de Cardiologia – INC; (2) Fundação Oswaldo Cruz – FIOCRUZ; (3) Universidade Federal do Rio de Janeiro – UFRJ Rheumatic Fever (RF) and Rheumatic Heart Disease (RHD) once again stood out internationally in 2018 in the panel proposed by the World Health Organization (WHO) held in Assembly, which set a deadline for its control and eradication. A document that guides public policies for RF was organized. Contains indicators for situational tracking of the national and subnational context. The worldwide incidence of RF is 19/100.000. It is estimated 30.000 new cases/year. There are about 33 million people with RC in the world, resulting in 300.000 deaths annually. In order to assess Brazil’s adherence to RF care, we applied the WHO tool. A search was carried out in public data and websites of medical societies. Data not found was requested in digital public services. It was possible to identify some national data, such as the number of cardiac surgeons (1.9/100.000 habitants), interventionists (0.46/100,000 habitants), echocardiography equipment (10572), cardiology services (40207). However, there is no mention of the value considered adequate. Overall mortality from RHD is low (0.16/100.000 habitants in 2014) but 3.9% of hospitalizations resulted in death, and 17% of those hospitalized for more than 90 days. As a comparison, in New Zealand, a reference country in the screening, control and treatment of RF, the annual mortality is 4.9/100,000 habitants. We were unable to quantify the acute cases and patients undergoing outpatient follow-up, there is only a record of hospitalization by international disease code involving RHD. It was impossible to count adherence to secondary prophylaxis, since there is no specific code for the application of Penicillin for RF. We saw that primary care is the most lacking in information. We highlight the absence of a code of procedure in the application of Penicillin for RF, non-accounting of acute cases and patients on secondary prophylaxis, recurrence of RF and mandatory notification of new cases. Is the incidence and prevalence of RF therefore falling? Or does the scarcity of data affect the measurement of the real epidemiological situation in Brazil? The Brazilian Society of Cardiology already highlights the importance of RF/RHD in our country. The socioeconomic impact of RHD must be reported to the government to include it in important public policies with the creation of a National Plan to combat and manage, training of health teams and guarantee of medication and adequate clinical follow-up to the patient. 109350 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION DAYANNA DE OLIVEIRA QUINTANILHA PALMER1, Pedro Gemal Lanzieri1, Ronaldo Altenburg Odebrecht Curi Gismondi1, Flavio Luiz Seixas1, Guilherme Schittine Bezerra Lomba1 (1) Universidade Federal Fluminense (UFF) Electronic health records (EHRs) are a great opportunity for big data research and professionals from the Coronary Care Unit (CCU) can benefit to support clinical practice. This study aims to analyze epidemiological information from patients admitted to the CCU, the readmission impact on mortality, and the factors associated with readmission. We retrospectively analyzed patients 18 years or older, admitted to the Beth Israel Deaconess Medical Center, Boston, between 2008 and 2019. Data were accessed through the Medical Information Mart for Intensive Care, a public data bank. Patients without discharge data were excluded. Demographic data, vital signs, and lab were evaluated. Posteriorly, patients were divided into two groups: one of the patients who were readmitted and one of the patients who weren’t. Factors associated with readmission were evaluated by statistical methods and machine learning. From an initial group of 53,150 patients, 8,015 were selected. The CCU mean period of stay was 2.8 days and the readmission rate was 7.42%. The mortality rate was 28.73% among those readmitted, while among those not readmitted, it was 18.61% (P-value < 0,001). The readmission rate was mainly influenced by the CCU length of stay, hospital length of stay, respiratory rate, creatinine, hematocrit, chloride, sodium, anion gap, temperature, and calcium. EHRs research can help define best practices and resource management. Machine learning was able to identify more relevant variables. Limitations include the need for longer analyzed periods and a broader data collection. 109362 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ALFREDO AURÉLIO MARINHO ROSA FILHO3, Lucas Brandão Cavalcante2, Alice Wanderley Rosa2, Briane Alcântara Vieira Pasini3, Alfredo Aurélio Marinho Rosa1, Edvaldo Xavier Ferreira Júnior1 (1) Santa Casa de Misericórdia de Maceió – SCMM/AL; (2) Centro Universitário Tiradentes – UNIT/AL; (3) Hospital Universitário Professor Alberto Antunes – HUPAA/UFAL Background: Coronavirus infection preferentially affects the upper airways and mainly the lung parenchyma, however, due to systemic infection, some patients have atrial fibrillation even in the absence of myocarditis or myocardial damage. Atrial fibrillation in most patients does not respond to drug treatment. Objective: To present the results of cryoablation for electrical isolation of pulmonary veins in patients with persistent atrial fibrillation after coronavirus infection. Material and methods: Between March 2020 and March 2022, 53 cases of cryoablation were performed for the treatment of atrial fibrillation, with 15% (8) of patients having atrial fibrillation after coronavirus infection. In all patients, atrial fibrillation did not respond to pharmacological therapy. In these patients, the echocardiogram showed a normal left atrium size, age ranged from 46 to 67 years with a mean of 59 years. The patients did not have structural heart disease or pulmonary sequelae of COVID-19. Of the 8 patients, 62.5% (5) were male and 37.5% (3) were female. All patients had a serological marker of previous infection assessed by IgG serology. Patients were referred to the electrophysiology laboratory, under venous sedation, submitted to transseptal puncture and then an angiography was performed to locate the pulmonary veins and introduction of a balloon catheter where an application of 04 minutes in each pulmonary vein was performed, temperature ranged –51 to –62 with an average of –55 degrees Celsius. In the trans and postoperative period, no complications were recorded. Results: In 100% (8) of the patients, all four pulmonary veins were isolated. There were no complications such as esophageal and/or phrenic nerve injuries and all patients returned to sinus rhythm. Conclusion: The disease caused by the coronavirus can lead to serious cardiac complications, such as uncontrolled atrial fibrillation, refractory to optimized drug treatment. In this sample, the experience of Cryoablation of pulmonary veins proved to be a safe alternative for patients with atrial fibrillation after coronavirus infection. 109380 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING YASMINN BENEVIDES ADBA1, Anne Caroline Lisboa Marinho2, Andressa Teoli Nunciaroni1, Bruna Ribeiro Almeida Magalhães1, Caio Victor Chagas das Virgens1, Clauanny Caroliny Bicego Reis Nogueira1, Débora Novais Lopes1, Judson Vieira de Carvalho Pereira1, Juliana Resende Correa Lima1, Luan Wesley Marques Máximo3, Renata Flávia Abreu da Silva1, Yasmin Rocha de Almeida1 (1) Federal University of the State of Rio de Janeiro – UNIRIO; (2) Federal University of Rio Grande do Norte – UFRN; (3) State University of Piauí – UESPI Introduction: In the context of high prevalence of Cardiovascular diseases (CVD), conditions faced by nursing students such as high curricular workload, stress, lack of time, long commute to university, access to food with low nutritional value, deserve attention in view of primary prevention. Objectives: To identify the cardiometabolic risk factors and its knowledge among nursing students. Methods: Cross-sectional, descriptive and quantitative study. An online self-instructional questionnaire was used for sociodemographic characterization and identification of cardiometabolic risk factors. The knowledge of cardiovascular risk was assessed from five statements about: modifiable risk factors, prevention of CVD, protective behaviors implementation and the contact way with the topic. Nursing Students from a Federal University were enrolled. Data collection occurred from September, 2021 to March, 2022. Data was analyzed through RStudio Software. The study was approved by the local Ethical Committee. Results: The study included 117 nursing students with 24.5(±5.7) years on average. Most(75.4%) were female, single(92.9%), with family income between two and four minimum wages*(59.6%), living with relatives(87.5%). Most of the students were attending 5th and 6th semester(22% and 20%, respectively). Regarding the time spent commuting to the university, 63.4% reported between 30 to 120 minutes. The risk factors identified in the sample are reported on Table 1. 74.6% declared CVD family history. Results relating to knowledge are presented on Figure1. *1 minimum wage = US$253.35. Conclusions: The study shows cardiometabolic risk factors among undergraduate nursing students, even when knowledge about the topic is reported. Knowledge does not mean implementing protective behaviors. 109410 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSIOTHERAPY VICTORIA MARIA GARCIA DE MEDEIROS1, Jéssica Gonçalves de Lima1, Fernando Gomes de Jesus1, Ana Gabriella Arena de Sá,1, Thaísa Sarmento dos Santos1, Cristianne Rafael Campos,1, Lucas Araujo de Carvalho1, Marcus Vinicius de Souza Amaral1, Juliana Rega1, Claudia Rosa1, Mauro Felippe Felix Mediano1, Luiz Fernando Rodrigues Junior.1 (1) National Institute of Cardiology Introduction: Patients in the postoperative period (PO) of cardiovascular surgery (CS) may have reduced functional status during hospitalization. However, little has been studied regarding preoperative, intraoperative and PO factors that may be related to better functionality at the time of discharge from the intensive care unit (ICU). Objective: To identify pre, intra and PO predictors of functional status of patients under PO of CS. Methods: Retrospective cross-sectional study. Data from 614 patients was obtained from the Physiotherapy Service database (stored in REDCap) and reviewed in medical records of the Instituto Nacional de Cardiologia (Rio de Janeiro-RJ), from October/2018 to March/2020. Preoperative variables such as age, sex, body mass index (BMI) and comorbidities were collected. Intraoperative variables were: type/complexity of surgery, fluid balance, time on cardiopulmonary bypass, time of aortic clamping, and surgical complications. The variables evaluated in the PO were: level of consciousness, pain on admission, fluid balance, imaging tests, blood gases, laboratory tests, mechanical ventilation and pulmonary function before and after extubation, peripheral muscle strength, extubation attempts and failures. Functional status was quantified at ICU discharge using Functional Status Score for the ICU (FSSICU). The univariate and multivariate linear regression model (P < 0.05 considered significant) was used to verify the association of possible predictors of functional status. Results: The PO variable: obesity (BMI ≥ 30 kg.m–2; β = –2.8; CI: –5.4 –0.3; P = 0.026) was negatively associated with functional status in the ICU; while the postoperative variables: pain on admission (β = 11.2; CI: 1.8–20.5 P = 0.02) and peripheral muscle strength at discharge (β = 0.3; CI: 0.2–0.4; P = 0.001) were directly and independently associated with functional status at discharge; Furthermore, muscle strength at the time of extubation were directly and independently associated with strength at discharge (β = 11.1; CI: 0.1–0.2; P = 0.007). Conclusion: Pain intensity on admission and peripheral muscle strength at ICU discharge predict functional status at discharge, while muscle strength at the time of extubation was evidenced as an independent predictor of muscle strength at discharge, and a suggestive intervention target for preventing functional status loss in adults submitted to cardiac surgery. 109426 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GUILHERME PINHEIRO MACHADO1, Gustavo Neves de Araujo3, Arthur Cabareira Baptista4, Mariana Rabolini4, Luiza Atanazio4, Alan Pagnoncelli1, Angelo Chies2, Wagner Azevedo2, Rodrigo Wainstein1, Marco Vugman Wainstein1 (1) Hospital de Clinicas de Porto Alegre (HCPA); (2) Universidade Federal do Rio Grande do Sul (UFRGS); (3) Imperial Hospital de Caridade; (4) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) Introduction: In patients submitted to elective percutaneous coronary intervention (PCI), treatment with small stents diameters (SD) has been associated with worse outcomes. However, the impact of small SD on outcomes in patients with acute myocardial infarction in scarce. Methods: This was a prospective cohort study that included patients with STEMI submitted to pPCI admitted to a tertiary university hospital between April 2011 and December 2021. Patients were categorized into groups based on SD. Small SD was considered <2.50 mm. Patients who underwent implantation of multiple stents were assigned to the study group according to the smallest stent size used. The primary clinical outcome was major adverse cardiovascular events (MACE) defined by death, in-hospital MI, stroke, and stent thrombosis and target vessel revascularization. Secondary outcomes included MACE and each individual outcome at in-hospital, 30 days and long-term period. Results: From 1458 Patients admitted with STEMI in the study period, 1238 were included and 468 (34.4%) were women. Mean age was 63.4 ± 12.8 years in small stent diameter vs 60.4 ± 11.7 years (p = 0.009). Patients with small SD had a higher prevalence of diabetes (36.9 × 25.3%, p = 0.003) and previous acute myocardial infarction 11 × 17%, p = 0.04). In multivariate analysis, stent diameter <2.5 mm remained independent predictor of MACE (odds ratio [OR] 1.6 95% confidence interval [95% CI] 1.08–2.42; p = 0.018); mortality (OR = 1.78, 95%CI = 1.092–2.878; p = 0.018, and target vessel revascularization (OR = 1.963; 95%CI = 1.019–3.612; p = 0.036). Conclusion: In this prospective cohort study of patients with STEMI treated with PCI, a small stent diameter <2.5 mm was associated with increased rates of MACE, mortality and target vessel revascularization. 109427 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE JANETE CORRÊA CARDOSO1, Amanda Rangel Madureira1, Vinicius Valois Pereira Martins1, Ramon dos Santos1, Suellem Torezani Sales1, Ana Paula Lima-Leopoldo1, André Soares Leopoldo1 (1) Universidade Federal do Espírito Santo (Ufes) Resistance to Obesity is associated with the complex interaction of stringent and environmental factors, conferring the ability to gain mass gain and body fat deposition, even when eating high-calorie diets. Considering that there are numerous gaps in the literature on the metabolic processes that explain Resistance to Obesity, specifically in relation to oxidative stress, the purpose of the study was to investigate whether obesity-resistant rats develop elevated reactive oxygen species in cardiac tissue. Wistar rats (n = 71), aged 30 days, were initially randomized into two groups: a)Standard diet (n = 35) and b)High-fat diet (n = 36). The experimental protocol consisted of two moments: obesity induction (4 weeks) and characterization of resistance to obesity (10 weeks). After obesity detection, the animals were redistributed into three groups: Control (C), Obese (Ob) and Obesity-Resistant (ROb). Was analyzed: Nutritional profile, metabolic changes, cardiac mass and Oxidative stress. The comparison of the experimental groups was performed using ANOVA, complemented with Tukey’s multiple comparisons test. The level of significance considered was 5%. Body mass showed a significant difference between the standard diet and high-fat diet groups in the 4th week of the experimental protocol, characterizing obesity. In the 4th week, after the characterization of Resistance to Obesity, there was a significant difference in body mass between groups C, Ob and ROb. Ob and ROb groups showed a significant increase in caloric intake (p < .001; p = .0005) in relation to the C. Ob group showed a significant increase in final body mass (p < .0001; p = .03), in retroperitoneal fat pad (p.0001; p = .006), sum of corporal fat deposits (p < .0001; p = .02) and in reactive oxygen species (p = .001; p = .0006), in relation to groups C and Rob. The area under the glycemic curve (p = .02), insulin resistance index (HOMA-IR) (p = .02) and basal glucose (p = .01) were elevated in the Ob group in relation to the C. Resistance to Obesity also promoted an increase in HOMA-IR when compared to C. Total cardiac mass (p = .0004 and;p = .01), right (p < .0001; p = .002) and left ventricles (p = 0.002; p = 0.03) the cross-sectional area (p < .0001; p < .0001) and cholesterol levels (p = .0001; p = .001) in were significantly elevated in the Ob and Rob groups compared to the C group. In conclusion, Resistance to Obesity promotes metabolic alterations and cardiac hypertrophy without changes in reactive oxygen species. 109458 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION KAROLINI ZUQUI NUNES1, Dalton Valentim Vassallo1, Julia Antonietta Dantas da Silva1 (1) Universidade Federal do Espírito Santo – UFES Introduction: It is a known fact that diabetes mellitus is associated with several cardiovascular abnormalities such as increased arterial stiffness and endothelial dysfunction. In addition to these changes, enhanced plasma copper concentration is also characteristic in individuals with diabetes. Although there are theories that the increase in plasma copper concentration and the development of cardiovascular problems are related, to date the mechanisms that explain this correlation are still unclear. Objective: The present study seeks to understand the effects of chronic copper overload on vascular reactivity in isolated segments of the thoracic aorta of diabetic rats. Methodology: This is an experimental study, in which about 44 12-week-old Wistar rats were used, weighing approximately 200g, obtained from the central vivarium of Federal University of Espírito Santo (UFES). The experimental protocols were approved by the university’s animal use ethics committee No 22/2019. The animals were divided into four experimental groups: Control (CT); Copper (Cu); Diabetes Mellitus (DM); Diabetes Mellitus + Copper (DM+Cu). Type 1 diabetes was induced via a single injection of streptozotocin 50 mg/kg/i.v., and the animals were treated with twice the recommended daily dose of copper (1.8 mg/kg). They were then killed after 30 days of treatment, and the thoracic aorta was removed for vascular reactivity experiments. Statistical tests, one-way and two-way ANOVA were used to examine the outcomes. Results: The main findings show a reduction in vascular reactivity in the DM+Cu group: Using an inhibitor of nitric oxide synthase (L-NAME), an increased bioavailability of nitric oxide was suggested, explaining the vasodilation; employing an inhibitor of NADPH oxidase activity (Apocynin) and an enzyme that degrades hydrogen peroxide (Catalase) we showed the participation of hydrogen peroxide as a vasodilator factor; the potassium channels blockade (tetraethylammonium) suggests the participation of the channels for potassium as vasodilators; and the use of selective angiotensin II AT1 receptor antagonists (Losartan) suggests that angiotensin II could be acting on AT2 receptors as vasodilators. Conclusion: Copper overload in diabetic rats causes a reduction of the aorta vascular reactivity of, which can be explained by the action of nitric oxide, hydrogen peroxide, and potassium channels, which are potential vasodilators, thus explaining the mechanism of our main discovery. 109470 Modality: E-Poster Young Researcher – Non-case Report Category: PSYCHOLOGY JOÃO GABRIEL REGA DO NASCIMENTO VALLAPERDE1, Bianca Botelho Viegas1, Fernanda Oliveira de Carvalho Carlos1, Victor Margallo1, Vitor de Melo Nolasco1, João Gabriel Bezerra da Silva1, Bianca Zattar de Mello Barreto1, Marcus Vinicius Serejo Borges Vale da Silva1, Daniela Fiuza2, Karine Guimarães2 (1) Universidade Federal do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho – ProHArt; (2) Universidade Estácio de Sá – UNESA Introduction: Symptoms of depression and anxiety may interfere in the quality of life of individuals diagnosed with hypertension. Objectives: To investigate the association between anxiety, depression and quality of life among pre-hypertensive and hypertensive patients. Materials and methods: A cross-sectional study including 89 pre-hypertensive (n = 15) and hypertensive (n = 74) participants. All of them were submitted to a standard protocol with collection of sociodemographic data, cardiovascular risk factors, BP measurement and mental health evaluation. For depression symptoms, it was used the Patient Health Questionnaire-9 (moderate/severe depression ≥10 points); for anxiety, the Generalized Anxiety Disorder Assessment-7 (moderate/severe anxiety ≥10 points); for quality of life, the World Health Organization Quality of Life instrument. Bivariate analyses were performed to compare participants’ characteristics according to the presence of anxiety, depression, and quality of life. Logistic regression assessed the main variables independently associated with moderate/severe anxiety and depression and reduced quality of life. Results: The average age was 50.2 ± 11.2 years old and 52.8% of the participants were female. Blacks and browns were the majority (53.9%). 21% of the participants have low education (Elementary School) and 64% have family income ≤3 minimum wages. Sedentary lifestyle (43.8%) and dyslipidemia (47.2%) were the most common risk factors among this population. Moderate/severe depression was found in 46.6% of the participants and moderate/severe anxiety in 43.8%, with no differences between men and women. The mean score of quality of life was 87 [63–111]. The presence of moderate/severe depression, moderate/severe anxiety and worse quality of life was not associated with sociodemographic conditions nor traditional CV risk factors, but they were associated with each other. Moderate/severe depression increased elevenfold the risk of worse quality of life (OR = 10.9; IC95% 2.81–41.65; p < 0.001). Moderate/severe anxiety (OR = 6.41; IC 95%; p < 0.004) and worse quality of life (OR = 11.19; IC 95%; p < 0.001) were independently associated with moderate/severe depression. The independent variable associated with moderate/severe anxiety was the presence of moderate/severe depression (OR = 6.37; IC 95%; p < 0.004). Conclusion: The presence of depression and anxiety symptoms were associated with each other and with worse quality of life of individuals evaluated. 109473 Modality: E-Poster Young Researcher – Non-case Report Category: DYSLIPIDEMIA SARAH FAGUNDES GROBE1, Maurício Tavares1, Thauany Tavoni1, Pedro Gabriel Senger Braga1, Raul Cavalcante Maranhão1, Luis Henrique Wolff Gowdak1 (1) Instituto do Coração da Faculdade de Medicina da USP – InCor HCFMUSP Introduction: HDL particle plays an important role in atherosclerosis for its complex metabolism and relationship with other pro-atherogenic lipoproteins. Patients with refractory angina (RA) usually presente advanced atherosclerosis of the coronary arteries. So far, no biochemical markers have been found to explain the greater atherosclerotic involvement in patients with RA compared with patients with non-limiting angina (NLA). Objective: To compare the activity of paraoxonase 1 (PON1) and the HDL particle diameter in patients with RA or NLA with healthy individuals (HI). Methods: 35 patients (15 RA and 20 NLA) and 20 HI were included. Plasmatic concentrations of lipids were determined using commercial kits and ApoA-1 and ApoB by the turbidimetric method. The HDL particle diameter was measured by light scattering method and PON1 activity by the spectrophotometric method. One-way ANOVA was used for statistical analysis, and a P-value < 0.05 was considered statistically significant. Results: There were no differences in age, sex, and body-mass index between groups. The lipid profile was similar between groups, except for higher levels of HDL-cholesterol and ApoA-1 in HI (see Table). There were no differences in ApoB concentrations, HDL particle size, and PON1 activity. Conclusion: As expected, patients with coronary atherosclerosis had lower ApoA-1 and HDL-cholesterol levels than HI. However, the HDL particle size and the PON-1 activity were similar among patients with RA or NLA. The search for markers of plasma lipid metabolism able to discriminate between patients with RA and NLA is of interest. They may mirror metabolic alterations related to the genesis of RA and become therapeutic targets. 109481 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ALFREDO AURÉLIO MARINHO ROSA FILHO3, Lucas Brandão Cavalcante2, Alice Wanderley Rosa2, Briane Alcântara Vieira Pasini3, Dandara Dias Gomes Cunha3, Alfredo Aurélio Marinho Rosa1, Edvaldo Ferreira Xavier Júnior1 (1) Santa Casa de Misericórdia de Maceió – SCMM/AL; (2) Centro Universitário Tiradentes – UNIT/AL; (3) Hospital Universitário Professor Alberto Antunes – HUPAA/UFAL Ablation of manifest anomalous pathways in the mitral annulus through transradial access with early hospital discharge due to the COVID-19 pandemic. Introduction: Ablation of anomalous pathways on the left or in the mitral annulus can be approached by transseptal puncture, by retrograde aorta approach or through the transradial approach. Due to the shortage of infirmary beds during the Covid-19 pandemic, we chose to perform transradially due to early hospital discharge. Material and methods: Between January 1998 and December 2021, 3,535 catheter ablations were performed. In 29,9% (1059) patients were treated for manifest anomalous pathways, of these 54.3% (575) were male, with ages ranging from 03 to 75 years and a mean of 31.24 years, success was obtained in 94, 9% (1005) of cases. Our service had previously performed 16 cases of transradial ablation. During the COVID-19 pandemic and the difficulty for the hospitalization of patients, between May 2020 and July 2021, 8 cases of ablation of manifest anomalous pathways manifested in the mitral annulus were performed, 62,5% (5) were anterolateral and 37,5%(3) Left postero-septal. 62,5% (5) patients were male and the age ranged from 24 years to 56 years with a mean of 40 years. The approach was made through the right radial artery, under local anesthesia, the patient was submitted to venous sedation, introduction of a 7Fr introducer and placement of only the exploratory catheter. Mapping performed always during sinus rhythm and after ablation performed tests with IV adenosine. The catheter was removed and a local pneumatic dressing was applied for 2 hours and replaced with a conventional dressing for 24 hours. All patients were selected after performing Doppler imaging of the radial and ulnar arteries. COVID-19 test were negative for all patients. Results: In the 8 patients ablation was successful (100%), the procedure time ranged from 40 minutes to 80 minutes with an average of 55 minutes. No complications were recorded during or after the procedures, all patients were discharged from hospital 3 hours after the intervention. At outpatient follow-up, all patients are asymptomatic and have an ECG without ventricular pre-excitation. Conclusion: Faced with the COVID-19 pandemic and lack of hospital beds for elective admissions of patients with left manifest anomalous pathways, the transradial approach was a safe option providing early hospital discharge. 109502 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DÉBORAH ESTEVES CARVALHO1, Déborah Esteves Carvalho1, Anna Laura Lima Larcipretti2, Aline Dias Bedetti2, Julia Regina Assis da Rosa2, Bárbara Alves de Abreu Rocha2, Leonardo Brandão Barreto3 (1) Universidade Tiradentes, Aracaju, Brasil (UNIT); (2) Escola de Medicina, Universidade Federal de Ouro Preto, Ouro Preto, Brasil (UFOP); (3) Departamento de Clínica Pediátrica e Adulto, Universidade Federal de Ouro Preto, Ouro Preto, Brasil (UFOP) Introduction: The Coronavirus Disease 2019 (COVID-19) pandemic has placed a drastic strain on healthcare services, requiring the reallocation of resources, and possibly affecting the quality of stroke monitoring and care. Objective: This is an epidemiological, descriptive, and retrospective study to analyze the profile of stroke in Brazil from January 2018 to December 2021. In addition, we aim to investigate the impact of the pandemic on stroke monitoring, including the possible reasons for the underreporting of cases. Methods: The results were obtained by collecting data from the SUS Hospital Information System (SIH/SUS). Also, a review of the literature on this topic was performed, using the Pubmed database. Results: Stroke is one of the main causes of mortality and morbidity worldwide. Although an increased number of stroke cases was expected due to cardiovascular events caused by the SARS-CoV-2 infection, the data show otherwise. Between 2018 and 2019, there was an increase of 6,116 in the number of cases. In contrast, a drop of 12,889 and 5,490 in the number of cases between the years 2019 and 2020, and 2020 and 2021, respectively, was observed. A significant decrease in the number of cases was observed in the Northeast region and in the elderly population. The literature describes many possible reasons for the underreporting. The most present hypothesis was fear of infection by the virus, which may have prevented many from seeking emergency medical care, impacting directly on the notification of stroke cases. Moreover, studies indicate that social distancing makes the recognition of stroke signs more difficult since the latter is facilitated by person-to-person interaction. Also, the pandemic requires the reallocation of healthcare resources to be used for COVID-19 care, it can create gaps in stroke care, affecting its quality. Conclusions: Thus, the decreased number of stroke cases observed between 2019 and 2021 may be a result of underreporting, which could be a direct impact of the COVID-19 pandemic on stroke care and monitoring. 109510 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY GABRIEL ODOZYNSKI1, Isabella Bianco3, Helcio G. Nascimento1, Andrei Lewandowski1, Maurício L. Spessatto1, Clóvis Froemming1, Grazyelle Damasceno1, Alexander Dal Forno1, André d‘Ávila1 (1) Hospital de Cardiologia SOS Cardio; (2) Instituto de Cardiologia de Santa Catarina; (3) Universidade do Sul de Santa Catarina Introduction: The use of contact force (CF) sensing catheters aims to provide safety and efficacy to atrial fibrillation (AF) ablation by permanent pulmonary veins electrical isolation. Objective and Method: This study sought to evaluate the AF catheter ablation outcomes (recurrence rate and adverse events) in a single-center, prospective cohort of 380 patients underwent to first-ever procedure using CF sensing technology. Data were analyzed based on digital AF ablation database/SysCardio®. Recurrence was defined as any atrial arrhythmia documented – episodes of 30s or longer after blanking period (90 days). Results: Of 380 enrolled patients, 290 patients had paroxysmal AF (≤7 days-PAF) and 90 patients persistent AF (>7 day ≤1 year–AF-Per). The mean age was 63 ± 11 and 67 ± 10 years, respectively. 68% and 80% of patients were men in the PAF and AF-Per groups. There was no difference among PAF and AF-Per groups regarding, hypertension and diabetes mellitus, coronary artery disease and previous stroke (16 patients). The CHA2DS2-VASc was 1.8 ± 1.4 vs 2 ± 1.5 in PAF and AF-Per, respectively. After the first procedure, 44 patients (15%) and 22 (24%) had recurrence in PAF and AF-Per groups (Fig.1). Considering a sequential redo procedure, these rates decrease to 5 and 8% in PAF and AF-Per, respectively. There were 15 minor primary adverse events from 427 procedures (7 inguinal hematomas, 6 pseudoaneurysms [1 surgical] and 2 pericardial effusions, no surgical drainage need). There were no major complications. There were no deaths related to the procedure. Conclusion: Initial outcomes using CF sensing technology catheter to AF ablation demonstrate a cumulative recurrence rate of 5% and 8% in patients with PAF and FAP-Per. The procedure is safe, no major adverse events were recorded in our study. 109527 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY NATÁLIA ALBERTIN DOS SANTOS1, José Cícero Stocco Guilhen1, Omar Alonzo Pozo Ibanez1, Vivian De Biase1, Renata Burini Chaccur1, Jaime da Conceição Padeiro Junior1, Luciana Fonseca Martins1, Wanda Teixeira Moreira do Nascimento1, Wagner Knoblauch Santos1, Maria de los Angeles García Andrade1, Daniela Lago Kreuzig1, Simone Rolim Fernandes Fontes Pedra1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Ebstein’s anomaly occurs due to failure in the delamination of the septal and posterior leaflets with apical displacement of the functional annulus, dilatation of the atrialized portion of the right ventricle, thinning of the free wall, dilatation of the true annulus and redundant anterior leaflet with multiple fenestrations. The “Cone” reconstruction of the tricuspid valve, described by Silva et al, results in complete coaptation of the valves with excellent results in the medium and long term. Objectives: In this study, clinical and echocardiographic data of patients undergoing surgical repair using this technique was reviewed. Methods: Pre and post-operative (PO) records and echocardiograms of 8 patients operated on from 2018 to 2022 were reviewed. Results: The median age of the group was 22.5 (2 to 42 years). In the pre-operative 3 patients had Wolf-Parkinson-White ventricular pre-excitation and 6 had atrial septal defect associated, 6 had moderate to severe dyspenia, with only one being cyanotic. All had severe tricuspid regurgitation (TI) and 6 had moderate to severe right ventricular dilatation. In the immediate PO, moderate TI was detected in 2 and mild in 6. In 1 patient, Glenn anastomosis was added to the Cone procedure due to hemodynamic instability and hypoxemia immediately after bypass. In the post-operative follow-up time, median of 11.5 months, all patients were asymptomatic and TI increased from mild to moderate in only one case. One patient required pacemaker implantation due to complete atrioventricular block. Conclusion: In this small cohort, the Cone procedure resulted in significant improvement of the tricuspid regurgitation, no mortality and low morbidity with significant improvement of the clinical status. 109533 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LEONARDO LEÃO BARROS1, Ludmila Oliveira dos Reis2, Mariana Lassance Maya Palheta1, Maria Luiza do Socorro Alves Lucas3, Juliana de Sousa Tavares1 (1) Universidade do Estado do Pará – UEPA; (2) Universidade Federal do Pará – UFPA; (3) Centro Universitário do Estado do Pará – CESUPA Introduction: The chronic rheumatic heart disease describes a group of heart diseases that can occur as a result of rheumatic fever. It is the most common cause of heart failure in children and young adults. In Brazil, these disorders generate high costs for the health system, with large number of hospitalizations resulting from this pathology. Besides, in the North region there is a high prevalence of this comorbidity. Objective: To evaluate and analyze the epidemiological profile of patients undergoing hospitalization for chronic rheumatic heart disease, in addition to determining their incidence and prevalence, in the period from 2011 to 2021 in Northern Brazil. Method: This research is a cross-sectional, analytical, and retrospective study, with observation of the period from 01/2011 to 12/2021. The materials used as a source were collected by the informatics department of the Brazilian Unified Health System on hospitalizations for rheumatic heart disease in the North region of Brazil and three variables were analyzed: age group, sex and race. Results: There were a total of 3.891 hospitalizations. The male population corresponded to 47,87% (1.863) of the hospitalizations, the female population corresponded to 52,12% (2.028). The races: brown, “no information”, white, black, yellow and indigenous, corresponded respectively to: 60,62% (2.359), 30,40% (1.183), 5,98% (233), 2,18% (85), 0,56% (22) and 0,23% (9). For age groups, the following ranges in years: “< 19”, “20 to 59” and “60 and +”, corresponded respectively to: 12,02% (468), 64,53% (2511) and 23,43% (912) of the hospitalized population. Conclusion: Rheumatic heart disease (RHD) is a condition with a significantly larger prevalence in women. Different studies show the ratio to be 2:1. In this study, women indeed corresponded to a larger number of hospitalizations (52,12%). But, the ratio found was only 1,08:1. No relation between race and RHD was found in studies. The higher prevalence in the brown race (60,62%) may be a reflection of the ethnic distribution of the region. RHD has a higher prevalence in the 25 to 45 years range. This is consonant with the findings of this study, which showed that the adult population with age between 20 and 59 corresponded to 64,53%. 109554 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ALICE PEREIRA DUQUE1, Luiz Fernando Rodrigues Junior1, Humberto Batista de Macedo Junior1, Daniel Arthur Barata Kasal1 (1) Education and Research Department. National Institute of Cardiology. Rio de Janeiro, RJ, Brazil Introduction/Objective: Autonomic dysfunction, a commonly underdiagnosed clinical entity with important cardiovascular implications, can occur in post-COVID-19 Syndrome. However, the entire impact of obesity on this clinical entity is still unknown. Thus, we aimed to assess if obesity can be a predictor of autonomic dysfunction in post-COVID-19 patients. Methods: Cross-sectional study recruited employees from a public hospital in Rio de Janeiro. Inclusion criteria: age ≥ 18 years and previous diagnosis of COVID-19. Exclusion criteria: physical inability to perform the autonomic test. Clinical, anthropometric and sociodemographic variables were collected. Volunteers were asked about the presence of cardiovascular symptoms (lipothymia, syncope, palpitations, dyspnea or chest pain) after COVID-19, and classified according to body mass index (BMI) in non-obese and obese (BMI < 30 and ≥30 Kg/m², respectively). Autonomic dysfunction was defined as the presence of orthostatic hypotension (OHYPO) and/or orthostatic hypertension (OHYPER). Systolic (SBP) and diastolic (DBP) blood pressure variation was evaluated after 15min of rest followed by 3min of orthostasis. Variations of SBP ≥ 10 mmHg and DBP ≥ 5 mmHg were considered as alterations. The association between variables was assessed using Fisher’s exact test and logistic regression adjusted by sex, age and medications that may influence autonomic function. P < 0.05 was considered significant. Results: In 55 volunteers evaluated, none required hospitalization and only 10.9% (N = 6) had post-COVID-19 symptoms. The time between COVID-19 diagnosis and the evaluation was 405 [380–421]days. In total, 87.3% (N = 48) had autonomic dysfunction. Of these, 76.4% (N = 42) had OHYPER and 20% (N = 11) had OHYPO. In common to both groups, 9.1% (N = 5) had both alterations. Non-obese represent 82.5% (N = 33) of the participants who had OHYPER, against 17.5% (N = 7) obese volunteers (P = 0.001). Obese individuals represent 32% (N = 17) of our sample. Of the volunteers who had OHYPO, 72.7% (N = 8) were obese, against only 27.3% (N = 3) non-obese (P = 0.002). Obesity exhibited a positive association with the occurrence of post-COVID-19 OHYPO (OR = 12.4; P = 0.009). Conclusion: Obesity increases in 12 time-fold the risk of developing post-COVID-19 OHYPO. The high prevalence of post-COVID-19 OHYPO in obese individuals occurs regardless of the presence of symptoms and the infection severity, suggesting that obesity could be a predictor of post-COVID-19 autonomic dysfunction. 109640 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES GISELLE OLIVEIRA AZEREDO1, Renata Flávia Abreu da Silva1, Andressa Teoli Nunciaroni1, Vanessa de Almeida Ferreira Corrêa1 (1) UNIVERSIDADE FEDERAL DO ESTADO DO RIO DE JANEIRO (UNIRIO) Introduction: Training in Cardiopulmonary Resuscitation through Telesimulation is challenging, due to its theoretical-practical peculiarity. However, gaps in the knowledge of health professionals can be filled through this remote education strategy. It is worth mentioning the actions described in the Cardiopulmonary Resuscitation protocol, which, if they have any problems in their compliance, can negatively influence the patient’s clinical outcome. Objective: To analyze Cardiopulmonary Resuscitation procedures addressed through Telesimulation to health professionals. Methods: Descriptive, cross-sectional, retrospective and quantitative study. We included secondary data from a remote course database with the telesimulation strategy and application of pre and post-tests instruments. The questionnaires items were grouped into four thematic areas: Identification and management; analysis of cardiac rhythms; medication administration; leadership. Participants were invited to provide their data. Descriptive and inferential statistics were used for data analysis. The study was approved by the Research Ethics Committee, with number: 4,945,123/2021. Results: 35 groups were formed with the participation of 227 health professionals. The pre-test, which corresponded to a situational diagnosis, showed that the leadership action had the highest number of hits, with 93 (96.8%), 47 (97.0%) and 70 (90.9%) and the action referring to the administration of epinephrine had the highest number of errors, with 11 (11.4%), 5 (9.20%) and 13 (16.8%) correct answers. The answers corresponded to the questionnaires applied in the three telesimulated scenarios, namely, hospital scenario (n = 96), psychiatric scenario (n = 54) and emergency scenario (n = 77). However, there was a progressive increase in correct answers of 47% and 69% between pre-test and post-test 1, respectively. In addition to the significant increase in their overall hit mean from 4.66 (1.67) to 6.85 (1.69), respectively (p < 0.0001). As for post-tests 1 and 2, there was an increase from 69% to 80%, and an overall average of correct answers from 6.85 (1.69) to 8.04 (1.55), respectively (p < 0.0003). Conclusion: The Telesimulation method, via remote course, seems to contribute to Cardiopulmonary Resuscitation procedures learning, considering the number of correct answers in the sample and the contexts studied. 109654 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY VITHORIA VIDOTTI NEVES1, Julio Cesar Tolentino Junior1, Sérgio Luis Schmidt1, Alan Marques Joaquim1, Vithória Vidotti Neves1 (1) Hospital Universitário Gaffrée e Guinle Background: Depression is linked to increased cardiac mortality risk. Dispersion of QT interval is an indirect electrocardiography (ECG) expression of ventricular repolarization heterogeneity. Increased QT dispersion is related to a higher risk of arrhythmias and sudden death. Therefore, depression may be associated with increased QT dispersion even in cardiac-healthy patients. Objective: We conducted a meta-analysis to investigate whether QT dispersion could be increased in depressed patients without cardiovascular disease. Methods: A systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was performed by two independent reviewers. A systematic search of MEDLINE and EMBASE from 1990 to 2022 was performed. An all-fields search for index terms (“depression” OR “depressive” OR “depressed”) AND (“QT dispersion” OR “autonomic nervous system”) was done. We included all articles that evaluated the relationship between QT dispersion on electrocardiogram and depression in patients without cardiovascular disease. Articles of revision, meta-analysis, editorials, and case reports were excluded. Outcome measures were either crude QTD or heart rate-corrected QT (QTcD) dispersions. The effect model was analyzed by MedCalc statistical software. The percentage of variability in the set of effect sizes due to true heterogeneity was tested with the I2 statistic. Results: The reviewers identified 677 publications. Four articles were included in the final analysis, which encompassed 267 cardiac health subjects (131 depressed and 136 non-depressed). There were 198 (74.1%) women. Age means did not differ between depressed and control groups. All studies reported significantly higher values of QTD and QTcD in depressed patients. As I2 values for crude QTD and QTcD were 79.49% to 83.48%, the random-effects model was used in this meta-analysis. The standardized mean difference was 1.493 (95% Confidence interval: 0.786–2.201; standard error: 0.36; Z-value = 4.135, p < 0.001) for crude QTD (Figure1). For QTcD, the standardized mean difference was 1.441 (95% confidence interval: 0.739–2.143; standard error: 0.356; Z-value = 4.042, p < 0.001). respectively. Conclusion: Depression is associated with increased QT dispersion in healthy cardiac subjects. Our data suggest that depression could be associated with changes in cardiac electrophysiological properties, which may predispose these individuals to cardiac arrhythmias. 109666 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY BRUNA SANTI DOS SANTOS1, Eduardo Bartholomay1, Adriano Kochi1, Karina Andrade1, Bruna May1, Thaís Nicola1, Cristiano Jaeger1, Daniel Souto Silveira1, Eduardo Schlabendorff1, Cícero de Campos Baldin1, Euler Manenti1 (1) Hospital Mãe de Deus Background: Due to advanced maternal age, cardiovascular risk factors, and the successful management of congenital heart disease conditions, the prevalence of cardiovascular disease complicating pregnancy is rising. When arrhythmias are present, they may increase fetal and pregnant morbidity and mortality. The use of medications during pregnancy requires a thoughtful approach, balancing fetal and maternal risk. Therefore the management of tachyarrhythmias during pregnancy including drug safety should be a core knowledge area for all physicians who work with emergency, obstetrics or cardiology. Objective: Our purpose is to facilitate the approach of tachycardias in pregnant women by presenting a new algorithm based on the current evidence regarding the management of this pathology including drug safety in this group. Methods: Formulation of a new algorithm for tachyarrhythmias’s management during pregnancy including drug safety. Results: The new algorithm proposed is presented in the figure. Conclusion: Besides rising prevalence of cardiovascular disease complicating pregnancy, any arrhythmia’s treatment must be carefully evaluated, aiming at the safety of the fetus and the mother, preventing both from taking unnecessary risks. We expect that the new algorithm presented considering drug safety in pregnant women facilitates the approach of tachyarrhythmias in these patients. 109674 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING ALYNE SANTOS BORGES1, Alyne Santos Borges1, Ana Carla Dantas Cavalcanti1, Flávio Luiz Seixas1, Paola Pugian Jardim1 (1) Universidade Federal Fluminense This study developed and evaluated an application for clinical decision-making in the care of patients with heart failure, registered at the National Institute of Industrial Property. As a general objective to develop and evaluate a mobile application based on the classification of nursing diagnoses from NANDA International Inc. for clinical decision-making in the care of patients with heart failure. As specific objectives: to map the defining characteristics of priority nursing diagnoses; develop an application for mobile devices for clinical decision making, and; to evaluate the usability of the application for clinical decision-making in the care of patients with heart failure. Method: This is a methodological study, carried out in three phases. In the first phase, the conceptual definition was carried out from the survey of the nursing diagnoses of the previously validated Nursing Consultation Instrument of the Heart Failure Clinic Coração Valente. A scope review was carried out with the objective of mapping the nursing diagnoses of patients with heart failure. In the second phase, the Nursing DiagnosIC mobile application was developed through meetings of a scientific committee composed of researchers from Nursing and Computer Systems. In the third phase, an evaluation of the usability of the application was carried out by expert nurses and Information Systems undergraduates, using a predictive and prospective technique. Results: From the scope review, 41 articles, 14 nursing diagnoses and 147 defining characteristics of patients with heart failure were mapped. The application has 19 screens, 205 defining characteristics, 27 nursing diagnoses, subdivided into 11 domains, using the version (2018–2020). The evaluation of the application was carried out by two groups containing a total number of 16 participants. The Nursing DiagnosIC was considered to have good/excellent usability (75.3%). The evaluated heuristics received 55 points considered high usability. Conclusion: With the developed and validated application, it is expected to impact the care of patients with heart failure through a technology for clinical decision-making, with easy access to the accurate judgment of nursing diagnoses of patients with heart failure. 111070 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY LUIS FELIPE RODRIGUES1, Bruno Rocha de Avila Pelozin1, Edilamar Menezes Oliveira1, Tiago Fernandes1 (1) School of Physical Education and Sport (EEFE), University of São Paulo (USP). Introduction: Cardiovascular diseases followed by cancers are the leading causes of death in the world. The bidirectional relationship between the diseases may potentiate cardiac damage and tumor progression, however little is known about their association. Our work aimed to investigate the impact of the association between arterial hypertension (AH) and cancer (CA) on physical capacity, tumor progression, cachexia percentage, and cardiac morphology in rats. Method: Wistar Kyoto (n = 18) and SHR (n = 18) male rats at 12 weeks old were assigned into 4 groups: Wistar Kyoto control (WKY), control tumor (WKY-T), SHR control (SHR), and SHR tumor (SHR-T). Walker-256 tumor cells (2.5 × 106 cells in 0.5 mL of phosphate-buffered saline) were subcutaneously injected into CA groups while control rats were injected with a vehicle. We assessed: tumor weight and growth, cachexia percentage (CP), grip strength, exercise tolerance, cardiomyocytes cross-sectional area (CSA), and left ventricular (LV) mass by LV: tibia length ratio. Results: SHR-T animals showed greater tumor growth (160%; p < 0.0001) and weight (96%; p < 0.05) compared to WKY-T group. In addition, SHR-T showed a higher CP (10 ± 1.96%; p < 0.001) compared to WKY-T group (2.84 ± 0.42%). The SHR showed a higher maximal muscle strength of hind limbs (2345 ± 81 g; p < 0.05) compared to WKY group (2033 ± 70 g). Otherwise, the association between AH and CA reduced the muscle strength in the SHR-T (1662 ± 94 g; p < 0.05) compared to the WKY-T group (1990.00 ± 35.29 g). Regarding the physical capacity, both groups with CA WKY-T (457 ± 44 m; p < 0.001) and SHR-T (297 ± 50 m; p < 0.001) developed exercise intolerance compared to WKY (695 ± 40 m); whereas the association between the diseases had even greater impairment in performance compared to CA alone. As expected, AH promoted cardiac remodeling by an increase in LV weight (23.9 ± 0.44 g/mm; p < 0.05) compared to WKY (21.96 ± 0.51 g/mm). However, both CA groups WKY-T (20.17 ± 0.44 g/mm; p < 0.0001) and SHR-T (20.54 ± 0.5 g/mm; p < 0.0001) showed a reduction in LV weight compared to WKY. This reduction in LV weight was accompanied by a reduction in cardiomyocytes CSA in the SHR-T (399 ± 11 µm2; p < 0.05) compared to SHR group (458 ± 12 µm2), highlighting that the combination of both diseases contributes to cardiomyocyte atrophy. Conclusion: Our data highlight the harmful effects of the bidirectional relationship between AH and CA on physical capacity, tumor progression, cachexia, and cardiac damage. 109709 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT PAOLA PUGIAN JARDIM1, Ana Carla Dantas Cavalcanti1, Bruno Bompet dos Santos1, Alyne Santos Borges1, Camila Achao Rosa1, Paula Vanessa Peclat Flores1 (1) Universidade Federal Fluminense (UFF) Introduction: Heart failure (HF) is considered a complex syndrome, characterized by a wide range of symptoms. Because it is a progressive chronic disease that limits life, there is a growing recognition of the important role of palliative care in HF. Symptom control is one of the goals of palliative care, but its assessment in HF remains a challenge. The Edmonton Symptom Assessment System (ESAS) scale has been used for this purpose in cancer patients, however, to our knowledge, there are no validated scales for use in monitoring the severity of HF symptoms. Objective: To adapt and validate the content of the ESAS for use in patients with HF in palliative care. Method: Methodological study divided into three phases: 1– Definition of content through a scoping review of the main signs and symptoms of HF in palliative care; 2– Adaptation of the ESAS content, based on the symptoms found in the scoping review and on the symptoms of the original version of the ESAS; and 3- Validation of the content through a committee of 30 expert judges. For analysis, the content validity index ≥0.80 was adopted. Results: The first version of the ESAS for use in IC (ESAS-IC) consisted of 33 symptoms. The ESAS-IC was evaluated by 16 nurses, 6 cardiologists, 4 physiotherapists, 2 psychologists, 1 nutritionist and 1 educator, 2 of them were palliative care professionals. Most specialists were female (83.3%), with a median age of 35 years old and an interquartile range of (32–41.5) years. The group consisted of 14 masters, 8 specialists, 7 doctors and 1 bachelor, with a median of experience working with HF of 7.5 years and an interquartile range of (3.75–10.25) years. After analysis, 23 symptoms were validated and after suggestions from experts, the second version of the ESAS-IC was composed of 20 symptoms (pain, shortness of breath, tiredness/weakness, sadness, sleep, anxiety, swelling/edema, cough, loss of weight, feeling of well-being, dizziness, urinary incontinence, numbness/tingling, weight gain, problems with sexual interest/activity, self-esteem concerns, mental confusion, Improved ability to perform activities of daily living, spiritual well-being and memory). Conclusion: This study adapted and validated the ESAS content for use in patients with HF. However, there is still a need for further validation. The use of the ESAS-IC can contribute to improving the health outcomes of patients with HF, in terms of quality of life, symptom control and readmissions, among others. 109726 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION STEPHANIE RODRIGUES SCHAFF1, Elise Souza dos Santos Reis1, Mario Augusto Cray da Costa1, Cassiano Ianke1 (1) Universidade Estadual de Ponta Grossa – Paraná Systemic arterial hypertension (SAH) is a public health problem and an important risk factor for cardiovascular diseases. Religiosity/spirituality (R/S) are practices that can interfere both in adherence and in the therapeutic goals of certain diseases. Objective: To evaluate the degree of influence of R/S in the drug treatment of SAH. Method: Study carried out at the cardiology outpatient clinic of a high complexity hospital (HAC) and a basic health unit (UBS) from September to December 2020 and August to December 2021, respectively. A total of 240 patients were evaluated using accredited scales to assess R/S and treatment adherence. Patients were divided into two groups: group with R/E and group without R/E. The p value was considered significant with p < 0.05. Results: In the general group there was a predominance of females (68.4%) in the group with R/S and males (54.1%) in the group without R/S (p: 0.006). White skin color prevailed in both groups with statistical difference (79.3% × 67%, p: 0.069). The other sociodemographic characteristics had no statistical difference between the groups. Regarding therapeutic adherence, the prevalence of high adherence was obtained both in the group with and in the group without R/S (82% × 69%, p: 0.026). The group with R/S from HAC achieved greater achievement of therapeutic goals (52.8% vs. 35.5%, p: 0.033). At the UBS, there was a high degree of adherence in both groups, with a statistical difference (83.3% × 63%, p: 0.011). Conclusion: Hypertensive patients with R/S had a greater adherence to treatment. 109730 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LUCIANA LONTRO ALVES1, Carolina da Silva Valentim1, Priscila Gomes Pereira1, Raíssa Leal de Carvalho dos Santos Cunha1, Kíssila Rabelo1, Bianca Torres Ciambarella1, Ana Lucia Rosa Nascimento1, Jorge José de Carvalho1, Jemima Fuentes Ribeiro da Silva1 (1) Universidade do Estado do Rio de Janeiro (UERJ) Renovascular hypertension (RH) is characterized by an increase in systemic blood pressure due to partial or total obstruction of the renal artery leading to overstimulation of the renin-angiotensin-aldosterone system. RH is potentially reversible and the system blockers are the ones chosen to control blood pressure. We aimed evaluate the antioxidant enzymes profile and tissue remodeling in the carotid artery of Wistar rats induced to HR by the two kidneys one clip (2K1C) model proposed by Goldblatt. Animals were divided in groups: SHAM; 2K1C; 2K1C+A. Treatment was administered via orogastric gavage with Aliskiren (10 mg/kg/day) for 30 days. Was performed electron microscopy and immunohistochemistry for collagens, metalloproteinases (MMPs), superoxide dismutase 1 (SOD1), catalase and glutathione reductase (GSH). Collagen I had greater staining in the hypertensive groups and aliskiren decreased your distribution. Collagen III was more expressed in the SHAM and the 2K1C+A present reduced expression. MMP2 show in the 2K1C an irregularity in the distribution and a more intense expression and the treatment decreased the marking. MMP9 2K1C showed more intense marking in concentrated areas, the treatment had no changes. Both catalase and GSH showed greater immunostaining in the SHAM group compared to the hypertensive groups. SOD expression was predominant in groups with hypertension. Oxidative stress enzymes showed no differences with treatment. Electron microscopy 2K1C generated alterations in endothelial cells, observed: pyknotic nucleus, poverty of organelles and decrease in their adhesion. We visualized fragmented and disorganized elastic lamellae. The treatment, these characteristics were not observed, indicating that Aliskiren contributed to the preservation of the carotid ultrastructural tissue. The SHAM group, on the other hand, presented ultrastructural characteristics expected for a control group. In conclusion, hypertension generated structural and ultrastructural changes in the carotid artery, that were reversed or preserved with the administration of Aliskiren. Oxidative stress had no effect with treatment. 109737 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING PAULA RODRIGUES DOS SANTOS PIRES1, Monaliza Gomes Pereira1, Renato Dias Barreiro Filho1, Renata Flavia Abreu da Silva2 (1) National Institute of Cardiology – INC; (2) Federal University of the State of Rio de Janeiro – UNIRIO Introduction: Technological advances and the increase in healthcare complexity have made it important to emphasize the concept of usability, which can be understood as the ability of a product to be used in the face of a specificity to users, objectives and context. The usability of high-risk medical devices, such as defibrillators, is highlighted due to their clinical importance in their proper management and their potential to cause harm to patients and professionals who operate them. Objective: To identify the usability of defibrillators focusing on the recognition of their model type, wave type and the ideal selection of the load to be used. Method: Descriptive study with a quantitative approach carried out with healthcare professionals in a public system hospital, located in the city of Rio de Janeiro (RJ), Brazil (BR), whose data collection took place between March and May 2021. The population of the study were healthcare professionals, namely, nurses, physicians, nursing residents and medical residents, allocated in care for adult patients and operating the cardiac defibrillator in clinical practice. There were no exclusion criteria. Sampling was done for convenience, with the sending of a semi-structured online questionnaire, which was divided between questions about the professional profile and about the operational usability and technical domain of the defibrillator in use in the subject’s sector. The study was approved by the Research Ethics Committee and received number 4,489,535 and 4,499,886. Results: 58 healthcare professionals took part, being 40 (69%) nurses, 14 (24%) doctors and 4 (7%) not identifying their professional category. When questioned, 34 (59%) healthcare professionals answered correctly about the defibrillator in their unit. The majority, 45 (78%), could not report the defibrillator wave in their sector. As for the energy load, 25 (43%) participants were unable to relate the manufacturer’s recommendation for the defibrillator in their unit and 28 (48%) subjects were not able to inform the American Heart Association’s recommendation, both in situations of shockable rhythms. Conclusion: Gaps were identified in the knowledge of healthcare professionals attending in the study regarding the use of the defibrillator, necessary for its handling, which may negatively influence its usability. 109904 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY PATRICIA FERREIRA DEMUNER1, Daniel de Magalhães Freitas1, Giulliano Gardenghi1, Silvio Roberto Borges Alessi1, Mauricio Lopes Prudente1, Fernando Henrique Fernandes1, Ellen Gonçalves Guimarães1, Débora Rodrigues1, Max Weyler Nery1, Ricardo Curado de Oliveira e Silva1, Larissa Xavier Alves de Oliveira1, Fernando Araújo Cintra Canedo1 (1) Hospital Encore Introduction: Transcatheter aortic valve implantation (TAVI) is one of the treatments of choice for patients with severe aortic stenosis. In the last decade, there have been technical and technological advances, but there has been no decrease in the number of cases requiring permanent pacemaker implantation (MPD): those with total atrioventricular block (TAVB) or second-degree Mobitz type II. One of the predictors of the need for MPD is the development of a left bundle branch conduction disorder, which can be diagnosed even in the subclinical form in an invasive way by measuring the his-ventricular interval (HV). Data on the impact of left bundle branch block after TAVI are scarce and treatment is individually tailored leading to marked differences in clinical management. Objective: To describe a series of 7 cases of patients undergoing TAVI simultaneously with the electrophysiological study (EPS), in order to immediately assess the need for MPD after implantation. Material and Methods: 7 patients were evaluated (4 males; mean age 80 years; mean BMI 23.3) whose data were collected in the Tasy® electronic medical record system and tabulated and analyzed by excel®. The EPS was performed with a quadripolar diagnostic catheter and positioned in the bundle of His to measure the HV interval, when the HV measurement exceeded 70 ms, MPD implantation was indicated. For TAVI, the technique used was the same in all cases, with transfemoral access, under sedation and local analgesia, Sapien®, Corevalve revolute R® and Evolut® prostheses, sizes ranging from 20 to 34 mm. Results: Among the patients analyzed, 4 evolved with left bundle branch block after TAVI. MPD implantation was indicated for 1 patient, the other patients underwent removal of the provisional MP while still in the hemodynamics room. The patient with an indication for MPD underwent implantation of the device during the same hospitalization, without intercurrences. The HV interval ranged from 46 to 58 ms (average 53,28) before the intervention, to 52 to 84 ms (average 62,8) after, approximately 18% positive variation. Only one patient died of an etiology unrelated to the procedure. No FES-related complications have been described. Conclusion: The FES strategy during TAVI seems to be a viable strategy to stratify patients for the risk of TAVB and 2nd degree Mobitz type II AVB in an early way, already referring them to appropriate treatment without demonstrating an increase in the risks related to the procedure. 109753 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MATHEUS TOSCANO PAFFER1, Matheus Vieira Cabral Figueiredo1, Carmen Beatriz Oliveira Silva Veras1, Francyelle Maria Barbosa Fonsêca1, Pedro Toscano Paffer1, Ramon Barbosa Barros1, Tatiana Maria Toscano Paffer1 (1) Faculdade de Medicina de Olinda Introduction: Hypertension is a highly prevalent disease, being one of the main risk factors for cardiovascular events. With just the measurement of blood pressure in the medical office, white coat hypertension can occur, clinical condition characterized by abnormal blood pressure values in the office, but with values considered normal by home blood pressure monitoring (HRPA). The aim of this study is to analyze a HRBP database from a private cardiology clinic, showing cases of white coat hypertension. Methods: Evaluate the results of HMBP exam of 1474 patients from a private cardiology clinic in Recife/PE, Brazil. The device used in all patients was the Omron 705 CP Blood Pressure Monitor. Results: A total of 1474 patients, of which 57.8% were women, with a mean age of 53.4 years (+–16.17). The mean office measurement of systolic blood pressure was 130.68 mmHg (+–18.54) and diastolic 81.83 mmHg (+–11.55), the overall mean systolic pressure of the HRBP exam was 123.99 mmHg (+–14.3) and diastolic was 76.5 mmHg (+–8.99). Among this total, 196 patients (13.3%) who were diagnosed with white coat hypertension were analyzed separately. Of these 196, 50.5% were men and the mean age was 56 years (+–15.15). Mean office systolic blood pressure averaged 148.5 mmHg (+–11.79) and mean diastolic blood pressure of 90 mmHg (+–8.78). The overall mean of systolic blood pressure was 125 mmHg (+–6.78) and diastolic pressure was 76.4 mmHg (+–6.44). Conclusion: White coat hypertension has a significant prevalence in the total number of patients evaluated, evidencing the importance of using HMBP for the diagnosis of arterial hypertension. Therefore, it is important to follow the guidance of the main hypertension guidelines around the world, such as the European one, which guide the use of this test in the diagnosis of arterial hypertension. 109764 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION ESTHER BOTELHO SOARES DA SILVA1, Conrado Roberto Hoffmann Filho1, Michele Tavares Mendonça1, Juliana Alzira Gonzales Oliveira1, Beatriz Vieira Roca1, Rafaela Louise Sales1, João Paulo Souza Brighenti1, Joao Paulo Panato Ribeiro1, Gabriel Erzinger1, Gilmar Sidnei Erzinger1, Jaqueline Barp1 (1) Hospital Regional Hans Dieter Schimidt Atrial fibrillation (AF) is the most frequent cardiac arrhythmia affecting 37.5 million worldwide. It’s a major risk factor for ischemic stroke and has strong associations with other comorbidities. Age is one of the greatest risk factors to develop AF. Life expectancy is increasing as does AF incidence and prevalence, affecting approximately 10% of subjects over 80 years. Effective anticoagulation reduces the burden of disabilities and death caused by stroke. Assess real-world data from our institution and provide tools to improve our effectiveness in diagnosing and treating this disabling disease. Retrospective study based on medical records. The inclusion criteria were AF and atrial flutter. From May 2021 to January 2022, we analyzed one thousand medical records of patients treated at our institution. Most of the population was male (58.4%), white patients (96.4%). The presence of comorbidities was frequent, especially arterial hypertension and heart failure (80.8% and 52.3%, respectively). Breathing disorders were present in 36.3% while diabetes corresponded to 32.8%. Coronary artery disease 28,1%, kidney disease 12,8%, cerebrovascular disease 8.7% and tobacco use 8.6%. 33.3% of patients had paroxysmal AF, 50.6% permanent, 8% persistent, 5.3% flutter and 2.8% indeterminate. The mean age for men was 66.9 years and for women 69.9 years. Male mean CHADSVASC was 3.2 HASBLED 1. Average female CHADSVASC was 4.4 HASBLED 2. The BMI for men was 28.4 kg/m² and 29.3 kg/m² for women, mean CKD EPI was 65. Comparing the number of strokes a greater rate was observed with warfarin versus DOAC (20.27% and 17.83% respectively). Regarding bleeding, the rate was 5.3% with DOACS and 6.37% with warfarin. The total death rate with DOACS was 10.36% and with warfarin 15.83% with an absolute risk reduction of 5.47% (p = 0,003). In the stroke analysis, patients treated with warfarin had a higher rate when compared with DOAC (p = 0,021). In this study of a public health care service, it was observed that a large percentage of patients were using DOAC therapies. It was also observed that the rate of bleeding and stroke was lower with the use of DOACs, however without statistical significance by chi squared test. The overall death rate was shown to be reduced in favor of DOAC therapy over warfarin. In the final analysis, comparing stroke patients, those treated with warfarin had a higher rate when compared with DOAC. One explanation could be the lack of adequate adjustment of the INR. 109789 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY GUSTAVO SANTOS PORFIRO1, Gustavo Santos Porfiro1, Leonardo Favaro Pereira1, Lucas Dalvi Armond Rezende1, Helena Alves de Andrade Ribeiro1, Fernando Luiz Torres Gomes1 (1) Universidade Federal do Espírito Santo Introduction: Cardiovascular diseases are the most common cause of death around the globe, with approximately 17.9 million people dying from heart attacks in 2021, representing 31% of global deaths. The rupture of atheromatous plaques is a major health problem and the main cause of heart attacks. Despite new technological advances for the diagnosis and treatment of coronary artery disease, early detection remains static, so that, commonly, myocardial infarction is the first clinical manifestation of this involvement. Thus, nanotechnology has been opening new horizons in the theragnostic perspective for the improvement and prevention of cardiac conditions such as coronary artery disease. Objective: To perform an integrative literature review to establish the main findings and relationships between coronary artery disease and nanoparticles. Methods: The method of integrative literature review was adopted, covering the period from January 2022, in the PubMed and Cochrane Library databases, using the descriptors: nanoparticles, coronary artery disease, and nanomedicine. Results: Sustained evidence was found about the miRNA of atherosclerotic plaque and the synthesis of nanoparticles, being characterized with low toxicity and anti-inflammatory properties, attenuating atherosclerosis, which would ease the cases of myocardial infarction due to atherosclerotic obstruction. Conclusion: The use of antibodies and peptides that recognize markers associated with cardiovascular disease has allowed the development of a more current generation of selective nano-systems. However, further studies on how nanoparticles can act in the intra-atheroma environment are needed to improve the understanding of nanomedications, generating important therapeutic contributions. 109790 Modality: E-Poster Young Researcher – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE STEPHANIE RODRIGUES SCHAFF1, Elise Souza dos Santos Reis1, Mario Augusto Cray da Costa1, Cassiano Ianke1 (1) Universidade Estadual de Ponta Grossa – Paraná Introduction: Systemic arterial hypertension (SAH) is a public health problem and an important risk factor for cardiovascular diseases. Religiosity/spirituality (R/S) are practices that can interfere both in adherence and in the therapeutic goals of certain diseases. Objective: To compare the degree of influence of R/S in the drug treatment of SAH in hypertensive patients who adhere to religious practices in a high-complexity hospital (HAC) and a basic health unit (UBS). Method: Study carried out at the cardiology outpatient clinic of a HAC and UBS from September to December 2020 and August to December 2021, respectively. A total of 155 patients were evaluated according to accredited scales to assess R/S and treatment adherence. Patients were divided into two groups: group with R/S from HAC and group with R/S from UBS. The p value was considered significant with p < 0.05. Results: There was a predominance of females in both the HAC and UBS R/S groups (60.7% × 78.8%, p: 0.016). Regarding marital status, there was a predominance of married people in both groups (53.9% × 68.2%, p: 0.003). The other sociodemographic characteristics had no statistical difference between the groups. Regarding therapeutic adherence, the prevalence of high adherence was obtained both in the group with R/S from the HAC and from the UBS (80.9% × 83.3%) with no statistical difference. With regard to therapeutic goals, both groups achieved goals in more than half of the interviewees (52.8% × 56%). Conclusion: Hypertensive patients with R/S from both HAC and UBS had greater adherence to treatment and achievement of therapeutic goals by more than 50%. 109796 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GABRIELA LAGO ROSIER5, Gleide Glícia Gama Lordello1, Patrícia Alcântara Doval de Carvalho Viana2, Luiz Eduardo Fonteles Ritt3, Gilson Soares Feitosa Filho4 (1) Bahiana School of Medicine and Public Health; (2) Hospital Santa Izabel, Santa Casa da Bahia; (3) Hospital Cárdio Pulmonar; (4) Hospital Aliança; (5) UniFTC Introduction: Inpatient rehabilitation is extremely important for patients recovering from cardiac surgery. Although a walking diary is routinely used in clinical practice, it has yet to be adequately tested and reported in the literature. Objectives: To establish whether use of a walking diary affects the number of steps taken following cardiac surgery and whether this is related to the patient’s level of cardiac anxiety. Methods: An open, controlled, randomized clinical trial was conducted at a referral hospital in cardiology with adult patients submitted to elective valve and/or coronary surgery, who had no motor impairment. Following discharge from the intensive care unit, all the participants used a pedometer to register the number of steps taken over five consecutive days. Twenty-nine individuals were randomized to an intervention group to use the walking diary as treatment strategy, while twenty-three were allocated to a control group. Statistical significance was defined as p < 0.05. A statistician blinded to patient allocation conducted the analysis on an intention-to-treat basis. Results: The groups were similar regarding their demographic, clinical and surgical characteristics. Mean age was 59.3 ± 13 years, most participants (76.9%) were male, and the most common type of surgery was myocardial revascularization (57%). There was no difference between the groups regarding the total number of steps taken: control group = 1,496 (477.5–2992.5) vs. intervention group = 1,468.5 (494.2–2,678) (p = 0.902). Conclusion: Use of the walking diary had no effect on the number of steps taken and was unassociated with the level of cardiac anxiety in inpatients following cardiac surgery. 109797 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION HEIGLON ESTEVÃO BONELLA DENTI1, Vítor Boniatti Neves2, Edimar Lima3 (1) Universidade Federal da Fronteira Sul – Campus Passo Fundo; (2) Faculdade IMED; (3) Hospital de Clínicas de Passo Fundo Transvenous temporary pacemaker (PM) is very useful for patients with severe bradycardia. Studies that analyzed its complications estimated that the rate of loss of capture is up to 43%. They have also found that the displacement of the PM cable requiring repositioning is one of the most common complications, occurring in 32% to 38% of the procedures performed. Cable displacement occurs because kits manufactured to install the devices do not have effective methods to lock in the pacemaker cable. This study proposes a simple, inexpensive and effective change in the temporary transvenous pacemaker kit (please see image attached). The original kit has a thread (blue color in the image attached) that locks the cable at the distal end of the contamination protection cap, but between this thread and the introducer the cable is relatively loose (it does not have a good fixation). Currently, the thread (in blue as seen in the image attached) is kept at the distal end of the protective cap as it is produced. The change this study proposes is to remove the blue thread from the distal end and to adapt this thread inside the plastic to the proximal end of the protective cap, securing it to the end of the introducer. Thus, it is possible to handle and to reposition the cable, if necessary, without losing the sterile environment. On the other end, instead of the thread, one could fix the plastic with micropore so there is no displacement of the protective cover over the cable which could accidentally reduce the sterile area. In this way, the cable is locked at the end of the introducer and does not allow displacements. With the proposed change, the sterile area of the cable is kept inside the protective cover, and it is still possible to loosen the cable if necessary and retighten after handling it because the blue thread will remain inside the protective cover (wrapped by sterile plastic). We concluded that this is an effective way to assemble the transvenous PM kit (from the attached photo) to avoid the loss of pacing due to cable displacement. This modification can be carried out when manufacturing the kit. 109798 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY LEONARDO TORREÃO BEZERRA CAVALCANTI1, Glaudir Donato Pinto Junior1, Ana Karolina Bento da Silva1, Lívia Farias de Holanda Furtado1, Manassés Almeida do Nascimento1, Gustavo Gomes Santiago1, Lucas Caetano da Silva1, Andressa Alves de Carvalho1, Wanessa Alves de Carvalho1, David Cesarino de Sousa1, Marcela Lukerli Araujo Paulina da Silva1, Evellyn Pereira de Melo1 (1) Federal University of Paraíba, UFPB Introduction: Transcatheter aortic valve replacement (TAVR) has emerged as an alternative treatment for patients with aortic stenosis, especially at high surgical risk (HSR). However, the procedure is not always available at lower complexity health centers. Further, controversies remain regarding the impact of TAVR on mid- and long-term mortality compared to surgical aortic valve replacement (SAVR). Aims: To compare TAVR versus SAVR mortality over the years after the procedure from randomized clinical trials (RCT) with patients at HSR. Methods: We searched Medline, Embase, LILACS and SciELO on March 27th, 2022, for TAVR versus SAVR RCTs. Were included studies that provided mortality data in follow-up for both interventions performed in HSR patients. Were excluded duplicates and studies that did not meet the inclusion criteria. An independent review was performed by two authors following the PRISMA protocol. Results: After applying the eligibility criteria, 20 studies remained to be reviewed. These studies included follow-ups from days up to 5 years. Considering mortality within 30 days, there was no significant difference between patients undergoing TAVR or SAVR. However, some studies marked a greater association of perioperative complications, such as bleeding and atrial fibrillation, linked to SAVR invasiveness. Still, major vascular events and even intraoperative deaths were seen in TAVR intervention. Regarding mortality from 1 to 3 years after the procedure, the studies diverged: while some showed no difference, others reported a reduction in mortality in the TAVR group. This result was also found in studies evaluating patients with diabetes and patients with chronic lung disease. Moreover, prosthesis-patient mismatch (PPM) was significantly lower in TAVR compared to SAVR, which in severe PPM was related to increased risk of death at 2 years. Nevertheless, paravalvular regurgitation was found to be more frequent in TAVR, which was associated with lower survival rates. Overall, at follow-ups of more than 3 years, there was no significant mortality difference between the groups. Conclusions: For 1 to 3 years follow-ups, it was outlined an advantage for TAVR, especially in the groups with diabetes and chronic lung disease. However, the complications arising from both interventions do not seem to be associated with statistically different mortality rates, especially at longer periods of follow up. 109984 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION EDUARDO FRANCO CORREIA CRUZ FILHO1, Eduardo Franco Correia Cruz Filho1, Ana Carolina Souza Diniz1, Fernando de Paiva Melo Neto1, Emanuel Francisco de Carvalho Pinto3, Lucas Vinicius Rafael Figueiredo1, Lorena Souza dos Santos Lima1, Laís Nóbrega Diniz3, Gustavo Nóbrega de Melo2, Gustavo Soares Fernandes1 (1) Centro Universitário de João Pessoa – UNIPÊ; (2) Faculdade de Ciências Médicas da Paraíba – FCM; (3) Faculdade de Medicina Nova Esperança – FAMENE Introduction: Anabolic androgenic steroids (AAS) are widely used for cases of osteoporosis, hypogonadism, Turner syndrome, hormone therapy in climacteric women, and are even used without indication as a form of treatment to increase physical performance. However, its use has several important adverse reactions in the cardiovascular system, often fatal to the individual, which make its use controversial and in most cases contraindicated. Objective: The present study aims to evaluate the cardiovascular risk of AAS use. Methods: A systematic literature review study, based on the PRISMA methodology, conducted with published articles on the Virtual Health Library and the United States National Library of Medicine (PubMed), between 2017 and 2022. The descriptors utilized were “Cardiovascular Disease” and “Anabolic Agents”, and their variations, associated with the Boolean operators “AND” and “OR”. Results: Initially, 57 articles were selected, which were chosen for manual selection based on their abstracts. In this way, duplicated articles and whose themes did not fit the objectives of the work were excluded, thus, 07 articles were chosen for the present work. Of the 07 articles involved, 05 are literature reviews, 01 meta-analysis and 01 case report. Among the studies evaluated, 100% highlighted the negative cardiovascular effects of steroid use, of which 85% evaluated these risks in the general population, while 15% focused on the risks for postmenopausal women using hormone therapy. As for the substance’s manifestations, the main alterations presented were: dyslipidemia (86%), coronary heart disease (86%), arterial hypertension (57%), cardiomyopathy (42%) and arrhythmia (42%). The meta-analysis that specifically evaluated the woman, compared tibolone with placebo and no treatment, especially evaluating its impact on lipoprotein (a) and the overall lipid profile. Conclusion: It is important to have well elucidated the cardiovascular risks due to the indiscriminate use and in supraphysiological doses of AAS, which in the long term can lead to negative and more severe outcomes. Furthermore, in order to evolve in this topic, more evidence-based therapeutic approaches are important, with clear objectives on the use of AAS and the risks of atherosclerosis, infarction and myocardial spasms and arrhythmias. 110051 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY EDUARDO FRANCO CORREIA CRUZ FILHO1, Eduardo Franco Correia Cruz Filho1, Camila Araújo Novais Lima1, Anna Julie Medeiros Cabral1, Gabriel Lucena de Carvalho Soares1, Waneska Lucena Nóbrega de Carvalho2 (1) Centro Universitário de João Pessoa – UNIPÊ; (2) Universidade Federal da Paraíba Introduction: Diabetes Mellitus (DM) type II is a metabolic disease with a high incidence, affecting 370 million people worldwide. DM II generates systemic changes, allowing a series of secondary disorders, among them, the increase in cardiovascular risk. Adequate therapeutic management is relevant, mainly related to cardiovascular protection. In this context, the use of agonists of the peptide similar to Glucagon I (GLP-1) has been increasingly highlighted, both in diabetics and in patients with heart disease. Objectives: To analyze major cardiovascular events in patients with type II DM using GLP-1 agonists. Methodology: This is a systematic review. The searches were performed using the PubMed database with works published in the last five years. The Health Sciences Descriptors used in the searches were: “Glucagon-Like Peptide-1 Receptor”, “Diabetes Mellitus, Type 2” and “Cardiovascular Diseases”, linked to the Boolean operator “AND”. To ensure a better structuring and organization of the results, the PRISMA recommendation was used. Initially, 116 articles were selected, which were manually selected based on their abstracts, excluding duplicate articles and whose themes did not fit the objectives of the work. This final selection resulted in 02 articles. Results: A meta-analysis using 360 articles and 56004 patients with type II DM evaluated the occurrence of non-fatal myocardial infarction, non-fatal stroke and death from cardiovascular or undetermined causes (Major Cardiovascular Adverse Effects – MACE). In that study, a significant 12% reduction in MACE was found for patients using GLP-1 receptor agonists. In addition, another cross-sectional study with 4076 patients with type II diabetes, which assigned patients to the use of efpeglanatide (2717) or placebo (1359), showed that major adverse cardiovascular events occurred in 189 patients (7.0%) using of efpeglanatide and in 125 patients (9.2%) on placebo. Conclusions: In summary, GLP-1 agonists are currently widely studied, with effects in more than one area of interest and no longer a glucocentric perspective. Therefore, the most recent studies have shown an important application of this drug in patients not only with diabetes, but also with heart disease. The main point of attention is the reduction of MACE, as it represents a reduction in cardiovascular risk in patients with type II DM. 109884 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY CATARINE BENTA LOPES DOS SANTOS1, Catarine Benta Lopes dos Santos1, Marcelo Bender Angst1, Sergio Ferreira de Ferreira1, Sheila Ouriques Martins1, Maurício Pimentel1 (1) Hospital de Clínicas de Porto Alegre Background: The stroke is a relevant cause of mortality and disability around the world. Electrocardiogram is part of the clinical examination of patients with stroke and its alterations are used for the etiological and prognostic evaluation of the patients. Changes in ventricular repolarization assessed by measuring the QT interval and its associated variables have been recently studied in stroke patients. Objectives: To evaluate the association of measures of the corrected QT interval (QTc), QTc dispersion, Tpeak-T-end dispersion (Tpe-d) and ratio Tpe/QT with mortality and neurologic disability (Rankin Scale) in ischemic stroke patients in the hospital discharge and within 3 months. Methods: Retrospective cohort study including patients admitted with acute ischemic stroke in a tertiary university hospital. The measurements of the QTc, QTc-d, Tpe-d and Tp-e/QT intervals were performed by experts physicians. The outcomes evaluated were: total mortality and the Rankin Scale at hospital discharge and within 3 months. The comparison between groups was performed using the Kruskal-Wallis test. Results: A total of 170 patients were included, predominantly female (53%), with a mean age of 64.4 ± 12.4 years. The length of hospital stay was 14 ± 21 days. Mortality during hospitalization was 11.17% and the total at 3 months was 14.1%. The results are shown in the table in milliseconds. Conclusion: This cohort showed that QTc was higher in stroke patients who died within 3 months and those with worse Rankin at hospital discharge. The ratio Tpe/QT was lower in those patients who died during hospitalization. The assessment of ventricular repolarization expressed by the QTc interval and associated measures can identify more severe patients who need implementation of optimized treatment. 109886 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING RAÍSSA GABRIELA VIEIRA DA CÂMARA BARROS1, RAISSA GABRIELA VIEIRA DA CÂMARA BARROS1, EVELINE BARROS CALADO1, ELLEN MAGALHÃES LISBOA ALVES1, MONICA DE MORAES CHAVES BECKER1, LUCIA MARIA VIEIRA DE OLIVEIRA SALERNO1, ROBERTO DE OLIVEIRA BURIL1, JOSÉ RENATO E MELO FIGUEIROA1, Eduardo Cavalcanti Lapa Santos1, TACITO CUNHA LIMA2, ROBERTO VIEIRA DA CAMARA2 (1) UNIVERSIDADE FEDERAL DE PERNAMBUCO- UPE; (2) UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE Aortic stenosis (AS) is the most common primary valve disease in the US and Europe. The gold standard diagnosis of AS is to perform a echocardiogram (ECO) and the measurements to classified the severity are: maximum jet velocity (Vmax), maximum and mean transvalvular pressure gradient (GM) and aortic valve area by the continuity equation. The miocardial strain assessment through the global longitudinal strain (GLS) is one of the most promising techniques for the early detection of reduced left ventricular ejection fraction (LVEF) and has become useful in AS scenario by correlating with the severity of AS. The GLS is a technique to assess the myocardial deformation suffered by myocardial fibers through Speckle tracking (normal value is below –18%). Several studies have already shown that the reduction in GLS precedes the decrease in LVEF, with relative reductions of 15% in GLS predicting a subsequent decrease in LVEF. This study aims to detect early changes in ventricular systolic function not only based on LVEF by Simpson’s method, but also through the assessment of the GLS in the AS and to correlate the pattern of changes in the GLS with the degree of stenotic impairment observed. General objective: Assess global and segmental LV systolic function through myocardial strain in aortic stenosis. Secondary objectives: Characterize the distribution pattern of the GLS in the AS; correlate the degree of AS with the myocardial GLS; correlate LVEF by Simpson’s method and GLS. Methods: This is an observational, descriptive, cross-sectional study. Results: A total of tirty patients were obtained, with a prevalence of 33% of major AS, 36.7% of moderate AS, 16.7% of mild AS and 13.3% of paradoxical AS was evidenced. An inverse relation between the severity of the AS and the GLS could be seen, that is, the more severe the stenosis, the lower the GLS value. We observed an average GLS in important AS of –17.6%, in moderate AS of –18.86%, in mild AS of –19% and in paradoxical AS of 16.85%. Regarding the pattern of segmental distribution of GLS values, there was a greater involvement of the mid-basal segments than the apical segments of the LV. Conclusion: The present study suggest a progressive impairment of LV systolic function assessed by the GLS as the severity of AS progresses, even though there is no direct impact on the LVEF measurement what can be primordial on the decision of early valve replacement despites the severity of AS founded on ECO. 109890 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANA LÍGIA VALERIANO DE OLIVEIRA1, Matheus Araújo Borges1, Gabrielly de Souza Correia1, Eduarda Tatico Lagares1, Lucas Eduardo Almeida França1, Isabela Castro Pereira1 (1) Pontifícia Universidade Católica de Goiás Introduction: Alcoholic septal ablation (ASA) consists of a selective infusion of high-grade alcohol in a septal branch that supplies the basal interventricular septum. It is a technique used as an alternative to surgical myectomy in obstructive hypertrophic cardiomyopathy (HOCM). The procedure seeks to create an iatrogenic infarction and, as a consequence, reduce left ventricular outflow tract (LVOT) obstruction. AAS has been improved over time, with benefits similar to myectomy. Objective: To analyze the benefit of treating hypertrophic obstructive cardiomyopathy with alcohol septal ablation as an alternative to myectomy. Methodology: This is a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses – PRISMA methodology. The literature search was carried out between January and February 2022, using the Scientific Electronic Library Online (SciELO), PubMed and Latin American and Caribbean Literature (Lilacs) databases. Thirty-three articles were selected, in English and Portuguese, excluding those that did not fit the objectives of this review and the methodological recommendations. The filters used were “human” and “published in the last 10 years”. The descriptors used were “obstructive hypertrophy cardiomyopathy”, “myectomy” and “alcohol ablation”. Results: Rigopoulos et al., found that threatening arrhythmic events seem be rare after ASS and occur more in patients with an estimated very high risk of sudden death. One study confirmed these findings, finding ventricular tachyarrhythmia or 30-day sustained ventricular fibrillation in only 7% of patients. ASS is a therapeutic alternative in patients with advanced age or comorbidities and showed shorter hospital stay and reduced post-procedure pain. Naidu et al., concluded that ASS results were similar to those of myectomy up to 8–10 years, in 90% of patients. showed to be able to reduce LVOT and relieve symptoms. Another relevant finding was the need for experienced services to perform SSA so that complications are less and the greatest therapeutic effects. Conclusion: AAS has become an alternative to myectomy that can be considered in carefully selected patients. Data show similar functional and hemodynamic benefit to myectomy, in addition to shorter hospital stay, reduced pain and associated complications. 109942 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM VITOR FEUSER DA ROSA1, Guilherme Pinheiro Machado1, Victor Henrique Ignácio de Souza1, Georgia Martina Chichelero1, Simone Magagnin Wajner1 (1) Hospital de Clínicas de Porto Alegre Introduction: The SARS-COV-2 pandemic had a great impact on the morbidity and mortality of affected patients. T3 has a direct impact on cardiomyocytes, causing myocardial injury in patients with SARS-COV-2. Clinical observations have revealed a relatively high (up to 64%) prevalence of sick euthyroid syndrome among COVID-19 patients, with some exhibiting a profound decrease in thyroid hormone levels. We know that low levels of T3 in sick patients occur early, correlate with disease severity, and that normalization of hormone serum concentrations is related to clinical recovery. Objective: The objective of the study is to describe the changes in thyroid hormones present in patients diagnosed with COVID-19. The primary outcome of the study is to assess the relationship between T3 levels and the degree of cardiac involvement. Methods: Samples from PCR positive patients for COVID-19 who were referred for admission to the ward or clinical ICU of a tertiary hospital were included. Serum samples stored in the COVID-19 biobank at Hospital de Clínicas de Porto Alegre were used. Half of the samples collected in the year 2020 and half collected in the year 2021 were requested. In the requested serum, T3 levels were measured on arrival at the hospital and around day 7 of hospitalization. Results: The number of patients enrolled in the study was 119. The mean age of patients was 61 ± 29 years. About 31.09% of the patients in the study died and 68.90% were discharged from the hospital. Among patients who died, T3 on the 1st day of hospitalization had a mean of 52.01 ± 16.48 and among patients who were discharged, the mean T3 on the first day of hospitalization was 62.25 ± 22, 14. On arrival at the hospital, among those who died, the mean troponin was 623 ± 152 and among those who were discharged was 176 ± 34.5. The T3 levels on the 7th day of the patients who died had an average of 42.66 ± 7.22 and of those who were discharged it was 60.98 ± 21.53. Conclusion: The study seeks to evaluate two tests that are relatively possible to obtain in patients admitted to hospitals of great complexity in the context of the COVID-19 pandemic, with the objective of helping medical teams in taking conducts and evaluating the prognosis of these patients. It is possible to infer that patients who have T3 levels below 50 have a higher risk of death associated with elevated troponin values, regardless of other clinical factors. 112331 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY PETER VAMOSI1, Marton Tokodi1, Patrik Toth1, Ferenc Komlosi1, Istvan Osztheimer1, Peter Perge1, Katalin Piros1, Zoltan Sallo1, Nandor Szegedi1, Bela Merkely1, Laszlo Geller1, Klaudia Vivien Nagy1 (1) Semmelweis University Heart and Vascular Center, Department of Cardiology Background: Monomorphic ventricular tachycardia (VT) is a life-threatening condition. Catheter ablation is an effective treatment method for many patients, but significant variability is observed in postprocedural mortality, due to the high burden of comorbidities and other factors. Therefore, there is a high need for an accurate risk stratification system. Aim: We sought to implement a machine learning pipeline to predict 1-year all-cause mortality in patients undergoing VT ablation. Methods: For 265 consecutive VT ablation patients at our center, we processed procedural, demographic and medical history data, their laboratory and echocardiographic findings (63 parameters). To predict 1-year all-cause mortality, several supervised machine learning models were trained and evaluated using 5-fold cross-validation with applying recursive elimination to identify the optimal subset of input features. We quantified their performance with the area under the receiver operating characteristic curve (AUC); we identified the most important predictors by Shapley values. Finally, we used topological data analysis to visualize patient subgroups with different mortality risks. Results: 57 (22%) patients died during the 1-year follow-up. The best predictive performance was achieved by a random forest model with 18 input features [AUC: 0.73 (95% CI: 0.68–0.78)], which significantly outperformed previously published risk scores (I-VT [AUC: 0.63 (95% CI: 0.55–0.70), p < 0.001], PAINESD [AUC: 0.63 (95% CI: 0.55–0.71), p = 0.009]). The most important predictors were mitral E-wave deceleration time, cardiac resynchronization therapy, age, electrical storm, and hemoglobin concentration. In the topological network based on the input features of the above model, we identified five groups with differing clinical characteristics and mortality rates (Figure). Conclusions: Our machine learning model could effectively predict 1-year all-cause mortality in VT ablation undergoing. Thus, it facilitates the identification of high-risk patients and the personalization of treatment and follow-up strategies, ultimately leading to improved outcomes. 109975 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CAROLINE TEIXEIRA BERNARDI3, Beatriz Sadigursky Nunes Cunha1, Rafael Fortes Locateli1, Giana Bevilacqua Schmitz1, Bárbara Müller Rettore1, Gustavo de Lemos Souza1, Sergio Rosas de Barros Neto1, Ângela Quatrin Campagnolo1, Lucca Corcini Biscaino2, Pedro Miguel Mariussi3, Sérgio Luís Pereira Furtado1 (1) Universidade Federal de Santa Maria – UFSM; (2) Universidade Franciscana – UFN; (3) Hospital de Clínicas da Faculdade de Medicina da Universidade de São Paulo – FMUSP Introduction: Left ventricular noncompaction (LVNC) is defined as a pattern of prominent trabeculae, intra-trabecular recesses, and a thin compacted layer on the left ventricular (LV) wall. It is an entity with yet unclear physiopathology, and may be congenital or acquired. The prevalence ranges from 0,014 to 1,3%. Objective: Summarizing LVNC’s main findings of clinical manifestations, etiology, diagnosis, prognosis and treatment. Methodology: Our research was conducted on PubMed with using “Isolated noncompaction of the ventricular myocardium”, and papers from the last 5 years were selected. 188 results were found and a selection was made from their abstracts and afterwards from reading the full article. The final number of selected articles for this review was 58. Results: LVNC’s clinical presentation may range from an asymptomatic entity to more serious manifestations, such as arrhythmias, heart failure and embolic events. Genetics play an important role in this condition, although a definitive correlation between genotype and phenotype has not yet been established. LVNC is similar to other cardiomyopathies, genetically heterogeneous and more frequently inherited as a dominant autosomal disorder or related to the X chromosome. There are no gold standard criteria for its diagnosis, which leads to many false positives. Transthoracic echocardiography, cardiovascular magnetic resonance and computerized tomography are the most frequently used imaging methods, allowing for morphologic and functional evaluation. Patients with LVNC present a significantly higher risk of fatal arrhythmias – requiring implantable cardioverter-defibrillator (ICD) – death, heart failure and cardiac transplantation. Patients with these presentations have an increased risk of death in 6 years of 47–75%. Furthermore, a higher prevalence of this pathology was observed in other genetic conditions, such as Ebstein’s anomaly (15%) and aortic coarctation (3%), among others. A definitive treatment is not presented in any guidelines, and the orientation is to avoid and treat heart failure, thromboembolism and arrhythmias caused by the condition. Conclusion: The lack of sufficient knowledge of its physiopathology, the still restricted understanding of the genetics and the diagnostic challenges are obstacles in fully understanding LVNC, and further studies are required to better characterize this pathology. 109976 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ANA KAROLINA BENTO DA SILVA1, Lucas Caetano da Silva1, Gustavo Gomes Santiago1, Leonardo Torreão Bezerra Cavalcanti1, Glaudir Donato Pinto Júnior1, Lívia Farias de Holanda Furtado1, Manassés Almeida de França1, Andressa Alves de Carvalho1, Wanessa Alves de Carvalho1, David Cesarino de Sousa1, Evellyn Pereira de Melo1, Marcela Lukerli Araújo Paulina da Silva1 (1) Universidade Federal da Paraíba Introduction: The association between atrial fibrillation (AF) and heart failure with reduced ejection fraction (HFrEF) increases the risk of cardiovascular complications. The Early Treatment of Atrial Fibrillation for Stroke Prevention Trial (EAST-AFNET 4) demonstrated that early rhythm-control therapy generated favorable outcomes in patients with AF. Objective: This study sought to investigate whether there is benefit in catheter ablation (CA) based rhythm-control in patients with AF and HFrEF compared to other strategies. Methods: Clinical trials and randomized clinical trials found in the Medline, Embase, Cochrane, and Scopus databases that analyzed the use of CA in early rhythm-control in patients with HFrEF and AF — paroxysmal, persistent or diagnosed in less than 12 months — between 2012 and 2022 were included. Articles that contemplated long-standing persistent AF and did not indicate left ventricular ejection fraction or New York Heart Association (NYHA) functional class were excluded. An independent review was performed by three authors following the PRISMA protocol. Results: Of the 41 articles found, 4 were selected for this review. Of these, 3 articles chose a similar primary outcome — composition of cardiovascular events, including death and hospitalization due to worsening of HF. Nevertheless, the results were discordant. In the CASTLE-AF trial, CA altered primary outcome and death and hospitalization for HF. In the CABANA trial, however, there was a significant variation only in isolated criteria. In a subanalysis of EAST-AFNET 4, Rillig et al., 2021 indicates that the early rhythm-control therapy positively impacted primary outcomes, but that CA alone showed no significant difference compared with the antiarrhythmic control strategy. Jones et al., 2013 chose to approach AF from a functional perspective — VO2max and impacts on quality of life after CA, with favorable results after 3 months of the procedure. Conclusion: Although CA is a promising alternative for early rhythm-control therapy in patients with AF, there is no precision on cardiovascular morbidity and mortality outcomes when HFrEF is associated. The variability of results found in this review indicates the need for even more robust future clinical trials in order to outline the possible benefit of the optimized indication of CA in this group. 109981 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION LAIZ TEIXEIRA PONTES1, Alice Cunha Darzé1, Pamela Castro Pereira1, ALmir Ferraz1, Ana Luíza Guimarães Ferreira1, Carlos ALberto Cordeiro Hossri1, Carolina Christianini Mizzaci1, Flavia Bernardes Morais1, Guacira Grecca1, Susimeire Buglia1, Ediele Carneiro Brandão1, Rica Dodo Delmar Buchler1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Given the advances in diagnosis and clinical-surgical therapies, congenital heart defects are increasing their prevalence in the adult population. Yet, there is still no consensus on the best way to follow up with these patients. Among the validated tools for monitoring patients with heart disease, the Cardiopulmonary Exercise Test (CPET) plays an important role. However, the use of this test for monitoring the population of Grown Up Congenital Heart (GUCH) still lacks studies. This study aimed to assess the correlation between ergospirometry variables and the severity of the GUCH population measured by echocardiographic aspects. Methods: A retrospective cohort with 248 GUCH (Table 1) over 18 years old, was referred to CPET, from 2015 to 2021, in a tertiary hospital in the state of São Paulo. Patients were sequentially included, and those in functional class IV or with contraindications to CPET were excluded. Ergospirometry variables were analyzed in association with ventricular function – estimated by echocardiogram in the same period. Results: Most patients were in functional class I and II (86.3%). In the echocardiographic findings, 40% had pulmonary hypertension and almost all had preserved left ventricle function. The CPET showed a median VO2 peak around 69% of predict. Other parameters are summarized in Table 2. CPET variables were able to stratify the severity of GUCH, mainly by pulmonary hypertension. Comparing CPET data and imaging diagnosis, VE/VCO2 slope >32 and OUES <60% were related to the presence of pulmonary hypertension. Conclusion: The CPET is an important resource for prognostic and diagnostic definition in the evolution of GUCH patients. 110000 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GUSTAVO GOMES SANTIAGO1, Ana Karolina Bento da Silva1, Leonardo Torreão Bezerra Cavalcanti1, Evellyn Pereira de Melo1, Marcela Lukerli Araujo Paulina da Silva1, Andressa Alves de Carvalho1, Wanessa Alves de Carvalho1, David Cesarino de Sousa1, Glaudir Donato Pinto Júnior1, Lívia Farias de Holanda Furtado1, Manassés Almeida do Nascimento1, Lucas Caetano da Silva1 (1) Universidade Federal da Paraíba Introduction: Systemic Arterial Hypertension (SAH) is a multifactorial condition defined by persistent elevation of blood pressure (BP). It is considered an important public health problem due to its high prevalence and an independent cardiovascular risk factor. Studies show higher morbidity and mortality from cardiovascular disease (CVD) among individuals with high BP. Between 2008 and 2017, 667,184 deaths attributable to SAH were estimated in Brazil and it is estimated that more than 30% of Brazilians have SAH. However, despite the high prevalence, it is considered that the control of SAH in Brazil is still poor. Objective: To assess the rate of control of SAH at the level of the Basic Health Strategy and the socioeconomic indicators of cardiovascular risk and mortality. Methodology: For the production of this integrative literature review, articles were searched in PubMed and SciELO databases, using the following descriptors: “Primary Health Unit” and “Hypertension”. The following inclusion criteria were used: articles from 1996 to 2020; studies in English and Portuguese. The following exclusion criteria were used: studies that did not correlate the two descriptors; published outside the period from 1996 to 2022; studies not available in English, Spanish and Portuguese. 732 articles were found, 5 of which included randomized double-blind studies, literature review and clinical trials. Results: BP values were considered controlled in most studies (systolic blood pressure lower than 140 mmHg and diastolic blood pressure lower than 90 mmHg). However, some studies have adopted different values for BP control and different BP targets depending on the target group that hypertensive patients are in (elderly, diabetic, post-infarction, post-stroke, etc). The rate of SAH control at the primary health care level in Brazil, according to the studies evaluated, ranged from 20 to 53.9%. There was a great diversity of techniques adopted to measure the control rate, such as repeated BP measurements by different devices (oscillometric, auscultatory, etc.), active search in medical records and in databases. There was variation in the BP control rate according to the Brazilian states. Conclusion: There is a wide variation in the frequencies of SAH control rates. Consequently, further studies are needed for a better care strategy and reduction of cardiovascular morbidity and mortality. 110036 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION PATRYCK ARAUJO DANTAS DA SILVA1, Carlos Eduardo Batista de Lima1, Igor Denizarde Bacelar Marques1, Victor Eulálio Campelo1, Maria Susane Filgueira Barreto Ferreira1, Lucas Cortez Macedo1, Lina Madeira Campos Melo1, Alessandro Aita1, Ginivaldo Victor Ribeiro do Nascimento1, Paulo Márcio Sousa Nunes1 (1) Hospital Universitário da Universidade Federal do Piauí Introduction: Teleconsulting activities in remote intensive care units can improve the quality of care and health indicators in regions with less access to specialists. In times of the Covid-19 pandemic, this technology has been increasingly used in assistance actions in health services. Objective: To evaluate the clinical-demographic profile and the remote coverage of the telehealth service in the studied population. Methods: The ICU telehealth system consists of a base at the HU-UFPI and a analysis system called Gestor Saúde that keeps the peripheral units connected in a teleconsultation platform, allowing simultaneous consultation with the on-duty physician in the remote unit. From August 2020 to February 2022, all data from teleconsultations in intensive care performed in the telehealth service of the Hospital Universitário of the Federal University of Piauí provided at the Floriano, Bom Jesus, São Raimundo Nonato and Parnaíba units were evaluated. Data on age group, sex, unit of origin of care and clinical diagnosis of patients were analyzed. Other variables included were the use of vasoactive drugs and the need for oxygen therapy. The results were tabulated in an Excel spreadsheet and presented in a descriptive way. Results: 2114 teleconsultations were carried out in the ICU, most of them in male patients over 80 years old, followed by the age group from 60 to 69 years old. Most diagnoses were sepsis, with a lower proportion of patients with Covid-19 infection and traumatic brain injury. Among the centers assisted, the one with the highest number of consultancies was the city of Floriano, which already had a link with the UFPI due to a similar service previously implemented. Patients from 68 cities in Piauí were assisted, demonstrating the coverage of the project. Conclusion: In this initial experience of the telehealth center in the ICU of the HU UFPI, we observed the shortcomings that places far from large health centers present, such as, for example, a shortage of specialists in intensive care, internal operational difficulties and complementary exams. The frequent contact with HU-UFPI specialists helped to improve processes and transmit the quality culture that has been proven to improve results in the ICU. We encountered technical difficulties during implementation, but they did not prevent the process from progressing in most centers. 110040 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSICAL EDUCATION RENATA MARIA BEGNI AFONSO1, Dáira Karoline Silva de Sousa1, Thiago Nascimento dos Santos1, Juliana Magalhães Santos2, Rafaela Rodrigues de Sousa2, Thaiane Campos Martins2, Luana Cristina Pereira Pacheco11, Lucas Heitor Moura2, Renato Luiz de Alvarenga3 (1) Instituto Carlos Chagas; (2) Cardioclin; (3) UFRJ Introduction: Resistance training in Cardiac Rehabilitation is still underused, even though the low strength and muscle mass levels of this population. This is due to the fear of a greater cardiovascular risk in relation to this model, although it is already understood in the literature that heart rate (HR) is more responsive to the time of muscle stimulus than the applied load, presenting higher mean values in resistance training than in resistance training. Objective: To compare the HR behavior of cardiopaths trained during resistance and endurance exercise. Methods: Eighteen cardiac patients (65 ± 12,9), practitioners of Cardiac Rehabilitation, clinically stable and medicated with beta adrenergic blockers, performed their usual training on a horizontal exercise bike Movement RT230 (Brazil), between 10 and 15 minutes and 40% and 80% of reserve FC. Soon after, they performed resistance training in the Buick leg extension (Brazil): 10 submaximal repetitions and after 2 min of interval, 5 more submaximal repetitions with the addition of 20% of the previous load. HR was measured using the Polar Verity Sense transmitter tape (Finland), before and shortly after each exercise model, and a ∆HR (final – initial) was calculated. For statistical analysis, the Friedman test and the Bonferroni post-test were used, with p < 0.05. Results: Table 1 shows the highest HR value for the group of exercise bike compared to the others, which was confirmed with p = 0.018 and p = 0.000 when compared to 10 repetitions and 5 repetitions, respectively. Conclusion: A greater spread of resistance training is encouraged in Cardiac Rehabilitation programs, in order to provide eligible cardiac patients with all the cardiovascular and peripheral benefits that resistance training makes possible. 110050 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION SAMANTA MATTOS CARDOSO1, Samanta Mattos Cardoso1, Michelle Rabello Cunha1, Marcia Regina Simas Torres Klein1, Mario Fritsch Toros Neves1 (1) Universidade do Estado do Rio de Janeiro Introduction: Obstructive sleep apnea (OSA) is known to be an independent cardiovascular risk factor. The presence of OSA and obesity may have synergistic effects on the progression of cardiovascular disease. Objective: To evaluate sympathetic tone and vascular disease in obese patients with moderate and severe OSA. Methods: Individuals of both sexes, aged 40–70 years and body mass index (BMI) ≥30 and <40 kg/m2, submitted to assessment of heart rate variability (HRV), central parameters by Mobil-O-Graph and carotid ultrasound. The sleep study was performed through a portable home sleep test device (WatchPAT). Results: Patients (n = 76) were divided into two groups based on the apnea-hypopnea index (AHI): mild-absent (MA) group (AHI <15 events/h, n = 30) and moderate-severe group (MS) (AHI ≥15 events/h, n = 46). The mean age was higher in the MS group (50 ± 6 vs 54 ± 8 years, p = 0.022) and BMI was similar (35 ± 3 vs 34 ± 3 kg/m2, p = 0.239). As expected, the MS group presented higher oxygen desaturation index (3.4 ± 2.2 vs 17.6 ± 12.7 events/h, p < 0.001) and respiratory disturbance index (12 ± 4 vs 33 ± 14 events/h, p = <0.001). Systolic blood pressure (119 ± 13 vs 127 ± 15 mmHg, p = 0.004), pulse pressure (41 ± 8 vs 46 ± 10 mmHg, p = 0.026), cardiovascular risk (6.4 ± 3.8 vs 11.9 ± 9.5%, p = 0.012) and cardiometabolic age (48 ± 6 vs 52 ± 9 years, p = 0.033) were significantly higher in the MS group. The SD2/SD1 ratio (1.4 ± 0.4 vs 1.7 ± 0.6, p = 0.064) and the low frequency/high frequency (LF/HF) ratio (0.85 ± 0.51 vs 1.53 ± 1.76, p = 0.051) were higher in the MS group, although not reaching statistical significance. The pulse wave velocity (PWV) (6.9 ± 0.7 vs 7.7 ± 1.2 m/s, p = 0.003), normalized PWV (7.1 ± 0.8 vs 7.7 ± 1.6 m/s, p = 0.046), PWV adequacy (–0.14 ± 0.42 vs 0.07 ± 0.37, p = 0.030), vascular age (47 ± 6 vs 53 ± 9 years, p = 0.005), mean carotid intima-media thickness (cIMT) (0.59 ± 0.08 vs 0.66 ± 0.13 mm, p = 0.008) and maximum cIMT (0.64 ± 0.09 vs 0.71 ± 0.15 mm, p = 0.015) were significantly higher in the MS group. PWV was significantly correlated with LF/HF ratio (r = 0.611, p = <0.001) in the MS group. Conclusion: In this sample of obese individuals, moderate to severe OSA was associated with sympathetic hyperactivity and evidence of accelerated vascular aging with arterial stiffness and subclinical atherosclerosis. 110944 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LIDIA EINSFELD1, Leticia Orlandin1, Jacqueline Kohut Martinbiancho1, Thalita da Silva Jacoby1, Simone Dalla Pozza Mahmud1, Nadine Clausell1, Livia Adams Goldraich1 (1) Hospital de Clinicas de Porto Alegre, Brasil. Background: The role of clinical pharmacists as part of multidisciplinary teams for solid organ transplant care has been discussed worldwide. Particularly, heart transplant recipients have a complexity of medication regimens, which greatly impact on both patient and transplant program outcomes. Aim of the study: To evaluate the participation of clinical pharmacists in the multidisciplinary heart transplant outpatient clinic through the identification of drug therapy problems (DTPs) and interventions in the pharmacotherapy based on pharmacist recommendations. Methods: In this descriptive pilot study, pharmacists provided direct patient care in the weekly heart transplant clinic from January to December 2021. Each patient seen at a pharmacist consult visit received a comprehensive assessment using a systematic drug therapy review process (Figure 1). Pharmacist’s activities were documented in electronic charts, and data, collected prospectively. DTPs and pharmacist interventions were classified according to the PCNE. Results: A total of 207 clinic visits to 70 patients were performed and 223 DTPs identified, leading to a median of 2 [0, 9] interventions per consult visit. In more than a third of the opportunities, patients received a dose adjustment recommended by the pharmacist (39.2%). Frequent interventions included laboratory monitoring (15.9%) and medication deprescribing (9.3%). Immunosuppressants were the drugs most commonly involved (35.6%), followed by electrolyte supplements and antihypertensive agents (15.1% and 13.7%, respectively). Conclusion: Our study was the first to describe pharmaceutical care of ambulatory heart transplant recipients in Brazil. Pharmacist-led interventions can potentially contribute to the management of challenging post-transplant pharmacotherapy. 110056 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DAYANNA MACHADO PIRES LEMOS1, Ana Paula Beck Da Silva Etges3, Nadine Oliveira Clausell2, Lívia Adams Goldraich1 (1) HOSPITAL DE CLÍNICAS DE PORTO ALEGRE; (2) UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL; (3) Instituto de Avaliação de Tecnologias em Saúde Background: The use of intravenous (IV) diuretics to treat congestion in outpatients with heart failure (HF) seems to be as effective in maintaining clinical stability as usual hospital therapy, and can potentially reduce costs. Objective: To assess the cost of a day hospital strategy for IV diuretic in patients with HF. Method: Cost study was carried out in a public and university hospital in Brazil. Time-driven Activity-based Costing was applied to calculate the total cost of services and their composition per patient, in addition to an exploratory analysis of a day hospital schedule for HF. Results: Data of 68 sessions of IV diuretic from 20 patients during 2020 were analyzed. On average, 80 mg of furosemide was administered in sessions lasting 39 minutes; 11 patients had a single session of IV diuretic, while 2 patients underwent 19 and 21 sessions each. The annual median cost per patient was US$ 34 (min. US$ 30; max. US$ 515) (Figure). Costs was attributed to professionals (58%), exams (23%), physical structure of the unit (10%), consumables (5%) and medicines (3%). A hypothetical schedule with 48 monthly sessions would cost US$ 1,464. Conclusions: This study contributes with novel information involving microcosting of a day hospital strategy for IV diuretic treatment in patients with HF. This data can generate subsidies for the incorporation of such potentially cost-saving strategies in a sustainable manner. 110183 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GIZELLA DA CUNHA RODRIGUES1, Felipe Neves de Albuquerque2, Gustavo Salgado Duque2, Ariene Brito do Amaral2, Yasmin Lemos Rollemberg Cruz Machado1, Marcelo Imbroinise Bittencourt2, Denilson Campos de Albuquerque2, Dayse Aparecida da Silva1, Ricardo Mourilhe-Rocha2 (1) Laboratório de Diagnóstico por DNA, Instituto Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil; (2) Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil Introduction: Heart failure (HF) is a clinical and multifactorial syndrome that affects approximately 26 million people around the world. HF is generally subclassified according to the left ventricular ejection fraction (LVEF) into 3 categories: HF with preserved ejection fraction (LVEF ≥ 50%), HF with mid range ejection fraction (LVEF 41%–49%), and HF with reduced ejection fraction (HFrEF, in which the LVEF is ≤40%). The pathophysiology of HFrEF is complex, and it is usually preceded by direct injury to the myocardium or a previous disease that leads to reduced ventricular contraction. The renin-angiotensin-aldosterone system (RAAS) is an important target to study the pathophysiological mechanisms involved in HF development. Objective: The main objective of this study was to determine the association of rs3097 (C>T) and rs3802228 (A>G) single nucleotide polymorphisms, present in CYP11B2 gene, with HFrEF. Ancestry informative markers of polymorphisms insertion/deletion were also determined to identify individuals’ ancestry and exclude spurious associations regarding the selected genetic targets. Methods: A total of 185 unrelated patients with HFrEF were selected, in addition to 124 unrelated volunteers without cardiovascular diseases. Data provided by HGDP-CEPH reference, containing profiles from African, European, and Native American populations were used to perform ancestry analysis estimations. Results: The control and patient groups were aligned according to gender (HFmen = 66% versus Cmen = 55%, p = 0.06) and age (HF = 50.6 ± 11.6 versus C = 48.5 ± 8.9, p = 0.09). A statistically significant association can be observed in relation to the genotypic frequencies of rs3097 C>T between HF group versus controls when using the codominance (p = 0.0031), dominance (p = 0.0007) and overdominance (p = 0.0035) models. There were not observed any association regarding the rs3802228 A>G polymorphism. The analyses involving the ancestral relationships between the groups are still being carried out. Conclusion: The preliminary results suggest a possible link between the T allele of CYP11B2 rs3097 C>T polymorphism and HFrEF. 110087 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSICAL EDUCATION EDNA SUSANA LOPES LICHUCHA1, Adjine Mastala Fumo1, Andrea Neves3, Euridsse Amade1, Keila Jamal4, Karen Sliwa5, Ana Mocumbi1 (1) Instituto Nacional de Saúde (INS), Maputo – Mozambique; (2) Universidade Eduardo Mondlane (UEM), Maputo – Mozambique; (3) Hospital Geral José Macamo, Maputo – Mozambique; (4) Mozambique Institute for Health Education and Research, Maputo – Mozambique; (5) Cape Heart Institute, University of Cape Town – South Africa Abstract Rheumatic Heart Disease (RHD) usually follows repeated episodes of acute rheumatic fever (ARF), which complicates untreated group A streptococcus (GAS) infections in susceptible individuals. RHD is a neglected disease that affects socially and economically disadvantaged young people in endemic areas, with a more malignant course in Africa, where it is an important cause of premature mortality, including indirect maternal mortality. We report an approach to increase awareness and promote health education of high-risk populations in a low-income setting. Methods: The activities were implemented in peri-urban Maputo, Mozambique from February/2019 to February/2020. We trained 21 maternal health professionals in RHD screening and implemented a bi-monthly joint cardio-obstetric clinic involving health education sessions run by six patients living with RHD (PLRHD) trained as peer-educators. At Matchikitchiki Primary School we painted outdoor RHD educational walls (September–October/2019) and trained 40 teachers to support RHD book coloring and video workshops. Results: The Cardiac-Obstetric Clinic assisted 127 women, of which 21 (17.3%) with RHD. Six trained PLRHD delivered 17 health education sessions to 127 patients. We trained 864 children (375 colored the book), 357 watched and discussed the educational video, and 232 were involved in outdoor activities using the RHD wall). Conclusions: Training of teachers, maternal health professionals and PLRHD allows creation of awareness and early detection of RF/RHD in high-risk populations. These strategies may foster decentralization of early diagnosis and prevention of RF/RHD in highly prevalent communities in Africa. 110129 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES RAYLANE MARQUES DE BARROS CRUZ1, Pollianna de Souza Roriz1, Daiane Dias de Jesus1, Marta Gabriela Moura Lopes1, Tatiana de Sena Leitão1, Rilary Silva Sales1, Shirley Casais Reis1 (1) Serviço de Atendimento Móvel de Urgência – SAMU The electrocardiogram (ECG) is essential for the stratification of Acute Coronary Syndromes (ACS), in which the finding of ST-segment elevation reflects coronary occlusion and indicates the need for reperfusion therapies. In order to optimize the door-to-ECG time, the team responsible for the evaluation of cases with high risk ACS in the city of Salvador-BA, entitled “Protocol-IAM” (P-IAM), developed the “ECG Protocol”, easy to apply in the risk classification – implemented in the Pre-hospitalar Emergency Care Units (UPA) of the municipality since July/2020. The time elapsed between the ECG and the P-IAM trigger (ECG-trigger) is also relevant as it starts P-IAM attending. Objective: To compare the door-to-ECG and ECG-trigger with high-risk ACS in patients at the UPA in the city of Salvador-Ba before and after promoting ECG Protocol in the city. Method: This is a cross-sectional and descriptive study; The P-IAM database was used, referring to users diagnosed with high-risk ACS treated at the UPA in Salvador, between 03/04/2017 to 12/31/2021. The analyzed data were the mean and median (minutes) of the Symptom-Admission, and ECG-Trigger times; total number of patients on reperfusion therapy. Results: A total of 1,679 patients were attended by the P-IAM accessing the public health system through the UPA during this period. Of these, 1,416 (84%) were diagnosed with ST-segment Elevation Myocardial Infarction (STEMI), with an increase of 58% comparing 2017 to 2021, followed by reperfusion therapies (171 in 2017 to 310 in 2021). Median times are seen in figure 1. Conclusion: After the disclosure of the ECG Protocol, there was an increase of P-IAM team activations; greater sensitivity of the emergency network in identifying patients with STEMI, as beneficiaries of the program. 110139 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION ANDRESSA TEOLI NUNCIARONI1, Gabriella Sena do Nascimento Santos2, Renata Flávia Abreu da Silva1 (1) Alfredo Pinto Nursing School, Federal University of the State of Rio de Janeiro – UNIRIO; (2) State University of Rio de Janeiro- UERJ Introduction: Among the drugs that make up the treatment of cardiovascular diseases, the class of oral anticoagulants (OAC) stands out. In Brazil, the main representative of the class is sodium warfarin, due to its low cost and distribution by the Brazilian Unified Health System. The treatment with OAC is complex and requires specific care related to prescription, guidelines for use, taking the drug, and monitoring of efficacy. Thus, adherence to treatment is necessary to ensure its full effectiveness and achieve positive prognosis. Objectives: To identify OAC adherence among outpatients with heart disease and to recognize strategies for increasing adherence among non-adherents. Methods: Cross-sectional quantitative study, conducted in the Anticoagulation Outpatient Clinic of a large Cardiology Federal Hospital, located in Rio de Janeiro, Brazil. Data collection occurred from March to July 2021. We included 76 patients in outpatient follow-up, older than 18 years of age, using OAC. Adherence to OAC was measured through the Instrument for Global Assessment of Medication Adherence (IAGAM), already validated for Brazilian culture and for the assessment of adherence among patients using OAC. Data was analyzed through RStudio Software. The research was approved by the local ethics committee under evaluation number 4.531.072 of 09/02/2021. Results: Considering only medication adherence rate, the participants had averages above 99% when asked about taking the medication on the day, week, and month prior to data collection. However, when associating the number of pills taken with the care required, only 86.84% implement adequate care. These numbers represent the overall adherence measured by IAGAM. Strategies reported by participants to increase adherence were use of an alarm clock; placing the prescription in a visible place, such as the refrigerator and bedside table; keeping the medicine box next to bed, with the schedule written on it; taking the medication associated with routine activities, such as before going to work; family members being responsible for administering the medication, thus remembering the schedule and the correct dose. Conclusions: The assessment of adherence to OAC should consider, in addition to taking the pills, the care needed to improve the drug efficacy. Strategies to enhance OAC adherence might target the underlying care. 110182 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION FILIPE FERRARI1, Henrique C. da Silva2, Artur H. Herdy3, Luiz G. M. Emed4, Felipe E. F. Guerra5, Haroldo C. Aleixo6, Guilherme D. Dilda7, Fernando Bassan8, Anderson D. da Silveira9, Ricardo Stein1 (1) Hospital de Clínicas de Porto Alegre (HCPA), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS – Brazil; (2) Universidade do Estado do Pará (UEPA), Belém, PA – Brazil; (3) Instituto de Cardiologia de Santa Catarina, Santa Catarina, SC – Brazil; (4) Hospital Cardiológico Costantini, Curitiba, PR – Brazil; (5) Clínica Biocardio, Natal, RN – Brazil; (6) Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG – Brazil; (7) Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP – Brazil; (8) Universidade Estadual do Rio de Janeiro (UERJ), Rio de Janeiro, RJ – Brazil; (9) Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS – Brazil Introduction: Afro-Caribbean athletes may present some peculiarities in resting 12-lead electrocardiogram, such as T-wave inversion (TWI) confined to V1–V4 preceded by ST-segment and J-point elevation. However, the prevalence of this finding in young Afro-Brazilian football players (YABFP) is unknown. Purpose: To compare the prevalence of ‘domed’ ST-elevation and TWI in V1–V4 among YABFP with young Ghanaian black football players (YGBFP). Methods: A visual analysis was performed, as the data from the YABFP were raw data and those from Ghana were aggregated data. A forest plot was constructed with the point estimate and 95% confidence intervals. Results: 668 YABFP (mean age: 21 years) and 159 YGBFP (mean age: 19 years) were evaluated and compared. The average height and weight were similar (178 cm and 73 kg for YABFP, and 175 cm and 68 kg for Ghanaian players). Ghanaians had a significantly higher prevalence of ‘domed’ ST-elevation and TWI in V1–V4 than YABFP (16.7% versus 2.4%, respectively). Conclusion: YABFP presents a low prevalence of ‘domed’ ST-elevation combined with TWI in V1–V4, and African players had an almost 8-fold prevalence of this finding compared to Afro-Brazilian players. 110181 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION FILIPE FERRARI1, Henrique C. da Silva2, Luiz G. M. Emed3, Artur H. Herdy4, Guilherme D. Dilda5, Felipe E. F. Guerra6, Haroldo C. Aleixo7, Fernando Bassan8, Frederico P. L. Coimbra9, Mateus F. Teixeira10, Anderson D. da Silveira11, Ricardo Stein1 (1) Hospital de Clínicas de Porto Alegre (HCPA), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS – Brazil; (2) Universidade do Estado do Pará (UEPA), Belém, PA – Brazil; (3) Hospital Cardiológico Costantini, Curitiba, PR – Brazil; (4) Instituto de Cardiologia de Santa Catarina, Santa Catarina, SC – Brazil; (5) Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP – Brazil; (6) Clínica Biocardio, Natal, RN – Brazil; (7) Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG – Brazil; (8) Universidade Estadual do Rio de Janeiro (UERJ), Rio de Janeiro, RJ – Brazil; (9) Hospital de Urgências de Goiânia, Goiânia, GO – Brazil; (10) Clube de Regatas Vasco da Gama, Rio de Janeiro, RJ – Brazil; (11) Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS – Brazil Introduction: A 12-lead resting electrocardiogram is a useful tool for diagnosing pathological conditions in athletes. The prevalence of electrocardiographic abnormalities in young Brazilian football players (YBFP) is unknown. Purpose: To describe the prevalence of abnormal electrocardiographic findings in YBFP based on the “2017 International Criteria for Electrocardiographic Interpretation in Athletes”. Methods: Cross-sectional/descriptive study. Intra-group differences were estimated by linear models or binomial and multinomial logistic regressions. Results: 3.490 athletes from 41 Brazilian clubs, aged 15–35 years (median: 19 years) were evaluated. 1.668 were Caucasians, 1.154 were Mixed-race (MR), and 668 Afro-Brazilians (AB). T-wave inversion in the inferior leads (4%), high lateral leads (0.5%), V5 (2.4%), V6 (2%), and V5–V6 (2%) were identified. Prolonged corrected QT interval (0.1%), QRS ≥140 ms (0.1%), premature ventricular contractions (0.2%), PR interval ≥400 ms (0.03%), Wolff-Parkinson-White pattern (0.06%), and a suggestive case of a type 2 Brugada pattern were also observed. Other abnormalities were not observed. Overall, 216/3.490 (6%) YBFP had electrocardiographic changes considered to be abnormal. Conclusion: This is the first large electrocardiographic cohort of YBFP described. In it, a prevalence of approximately 6% of abnormal findings was identified. Further evaluation in all these cases is indicated. 110364 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LUCIANNA SERFATY DE HOLANDA1, Maria Elizabeth Navegantes Caetano Costa1, Maria Talita Rodrigues Pinto Campos1, Laíse Braga Vieira1, Vitor Bruno Teixeira de Holanda1, Ivy de Almeida Cavalcante e Silva1 (1) FUNDAÇÃO HOSPITAL DE CLÍNICAS GASPAR VIANNA DO PARÁ Background: Studies on heart disease in pregnant patients show that it is the main indirect cause of deaths of pregnant women who are in the pregnancy-puerperal cycle. In addition, heart disease can lead to other problems that depend on the clinical and epidemiological state presented during pregnancy. Objectives: To describe the clinical and epidemiological behavior of pregnant with heart disease registered at Hospital das Clínicas Gaspar Viana, from January 2017 to December 2019. Methods: An epidemiological, retrospective and cross-sectional study, by analyzing the medical records of 133 pregnant women with heart disease admitted to HCGV in the aforementioned period. Excel 2007 and Bioestat 5.3 software were used for descriptive statistics; the results were evaluated using the categorical variables of the sample acquired by the G and Chi-Square Adherence Tests and G and Chi-Square Independence Tests. Results: There was no difference between the age groups of the patients, where 81.2% did not have a personal morbid history; the most frequent diagnosis among the others was atrial septal defect with 14.3%. 80.5% are aware of their heart disease and are followed up. In turn, the functional class with the highest proportion was NYHA I with 72.2%. Finally, 60.2% had no problems with pregnancy, childbirth and postpartum. At the time of delivery, a statistically significant majority (*p = 0.0003) of patients were between 35 and 39 weeks of gestation (66.9%). Cesarean delivery was the one with the highest proportion (70.7%) and statistically significant (*p = 0.0008), in relation to vaginal delivery (27.1%). The majority of live births were statistically significant (*p < 0.0001). Conclusions: This study is relevant because due to the scarcity of information about heart disease in pregnant woman in the State of Pará, considering that each one pregnant presents peculiarities, mainly on maternal and perinatal results or clinical and epidemiological profile of then. 110370 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY BRENDA GABRIELE SMANIOTTO RAULIK1, Mateus de Miranda Gauza2, Rodrigo Ribeiro e Silva2, Júlia Opolski Nunes da Silva2, João Pedro Ribeiro Baptista2, Caroline Figueiredo da Silva2, Marcelo Pitombeira de Lacerda2, Gibran da Costa Reis3 (1) Universidade Positivo; (2) Universidade da Região de Joinville; (3) Hospital Regional Hans Dieter Schmidt Introduction: In many cardiovascular diseases and procedures, statin therapy has been associated with a reduction in cardiovascular risk for future events. However, the effect of statin therapy for patients undergoing TAVI is not well documented and there are no randomized clinical trials evaluating this association. Considering the long-term mortality after TAVI not to be very satisfactory, it Is of great interest to find alternatives to reduce mortality after this procedure. Objetive: Evaluate the prognostic factor associated with statin therapy in patients who underwent TAVI. Methods: A systematic review was caried out using three data bases: Medline, Embase and the Cochrane library. We included observational studies that evaluated major adverse cardiovascular events, death and major bleeding in patients using statin therapy prior to TAVI. The terms used in the search strategy included “tavi”, “tavr” and “statin”. We found an initial number of 140 articles published until April 2022. After exclusion of duplicates and other studies that did not fit our objectives, 15 articles were included in our analysis. Results: In our review, 15 observational studies were included in the analysis. From them, 7 studies reported a reduction in all-cause mortality and cardiovascular mortality in patients using statin therapy. 3 studies reported a dose-dependent reduction in long-term mortality, with 1 paper indicating no reduction in the short term with any intensity. Only 1 case-control study found no reduction in mortality in the long-term nor the short term. The effect on mortality may even extend to octogenarian population. Conclusion: Statin therapy appears to be related with a reduction in long-term mortality rates after TAVI procedure, in dose dependent manner. 110416 Modality: E-Poster Young Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES NATHALIA LIMA SCHRAMM DOS SANTOS1, Nathalia Lima Schramm dos Santos1, Lucas Santos Silva1, Isabele Carolina Tokumoto1, Rebeca Magalhães Araújo1, Lucas de Almeida Santos Rocha Pereira1, Ana Layse de Sá Gonçalves Torres1, Pedro Emanuel de Jesus Ferreira1, Fernando Gassmann Figueiredo1, Vanessa Arata Figueiredo1 (1) Universidade Estadual de Feira de Santana (UEFS) Introduction: Rheumatic Fever (RF) is a multisystemic disease that, although preventable, still represents a common sequel of pharyngotonsillitis (PT) caused by group A beta-hemolytic streptococcus. In Brazil, there are an estimated ten million cases of PT per year, of which thirty thousand culminate in cases of RF. Its clinical importance is due to the sequelae of this condition, with biological impacts, as it is the main cause of mitral stenosis in the country and also socio-economic impacts, as it is related to a third of the heart surgeries in Brazil. Objective: To analyze the relationship between hospital admission rates for acute rheumatic fever per 100,000 inhabitants and the ratio between hospitalization costs of patients with pharyngitis and acute tonsillitis over the total number of cases of hospitalization for rheumatic fever in Brazilian states in the periodo of January 2008 to May 2021. Method: This is an ecological epidemiological study, whose data were obtained from the DATASUS database and from estimates from the Brazilian Institute of Geography and Statistics for the year 2020. The data were tabulated in the Microsoft Excel program, in which the rates and measurements of the linear regression test were calculated (Pearson’s correlation coefficient and p-value). Results: During the study period, there were 49,056 hospital admissions for acute rheumatic fever in Brazil. The age group from 40 to 69 years old stands out, which has the highest number of hospitalizations (n = 20,733). Among those under 20 years old, there is a range of 10 to 14 years old with 4,673 hospitalizations. After performing the correlation between hospital admission rates for acute rheumatic fever and the ratio of hospitalization costs of patients with pharyngotonsillitis by the total number of hospitalizations for RF, a significant negative correlation was observed (r = –0.4 and p = 0.03888). The analysis of the distribution of hospital admission rates by RF by state highlights Pernambuco with 68 admissions per 100,000 inhabitants; Goiás with 50 and Ceará with 41. The correlation between the mortality rate and costs per hospitalization was also negative, although negligible (r = –0.2 and p = 0.03893). Conclusions: The results show a relationship between the low investment in the treatment of acute pharyngotonsillitis and the higher rates of hospitalization for rheumatic fever. 110420 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING MARYANNA CRUZ DA COSTA E SILVA ANDRADE1, Ana Carla Dantas Cavalcanti1, Juliana de Melo Vellozo Pereira Tinoco1, Isabelle Andrade Silveira1 (1) Universidade Federal Fluminense – UFF Introduction: Nurses working in cardiology intensive care units need to improve their knowledge about diagnoses, interventions and nursing outcomes of cardiac surgery patients. Despite, there are no studies that have mapped these concepts in immediate postoperative period context. Objective: To map diagnoses, interventions and nursing outcomes for adult patients in immediate postoperative period of cardiac surgery. Method: Scope review based on Joanna Briggs Institute manual and guided by Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-SrC) carried out on bases: Biblioteca Virtual em Saúde, Cochrane Library, EBSCO, Epistemonikos, Embase, National Institute Heath and Care Excellence, PMC, Pubmed, Scielo, Science.gov, Scopus e Web of Science. Data were mapped considering Population of adult cardiac surgery patients with sternotomy, in the context of immediate postoperative period and with concept of interest in diagnoses, interventions and nursing outcomes. Results: 23 studies were identified for extraction in the end of selection process. 70 diagnoses, 57 interventions and 47 nursing outcomes using standardized language were mapped, summarizing an unprecedented way the three essential components for the nursing process of this clientele. Five scales for pain assessment, three for neurological assessment, one for predicting patient outcome and one for measuring severity were also mapped. Conclusion: The steps of nursing process were presented in systematic way for the patient in the immediate postoperative period of cardiac surgery through diagnoses, interventions and nursing results representing the affected needs of this clientele. Thus, main implication of this review for nursing care is to guide nurses‘ critical thinking. 110482 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUHANDA LEONORA CARDOSO MONTI SOUSA1, Bruno Mahler Mioto1, Nilson Tavares Poppi1, Julio Yoshio Takada1, Luís Henrique Wolff Gowdak1, Carlos Augusto Homem de Magalhães Campos1, Luis Roberto Dallan1, Amanda Mazetto1, Miguel Antonio Moretti1, Luiz Augusto Ferreira Lisboa1, LUIZ ANTONIO MACHADO CESAR1, Luciana Oliveira Cascaes Dourado1 (1) Instituto do Coração (InCor) do Hospital da Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) Introduction: The Coronary Heart Team (CHT) is based on shared decision-making, between of the non-interventional, interventional cardiologist and cardiac surgical. The focus of defining the best treatment in a individualized way, based in scientific evidence. It has class I recommendation, evidence level C guidelines. However, the impact of this intervention is still poorly understood. Objective: To assess the impact of the CHT decision: clinical treatment (CT) percutaneous coronary intervention (PCI) and coronary artery bypass graft surgery (CABG) in reducing primary composite outcomes: Acute Coronary Syndrome (ACS) and Cardiovascular Death (CV) and symptom improvement: angina class (CCS) and heart failure (HF) class in patients with chronic CAD according to the degree of agreement with the CHT. Methods: Prospective, single-center cohort study, mean follow-up of 41.1 ± 20.7 months. From a cohort of 500 patients discussed in CHT from 2015 to 2020, we evaluated 113 patients, whose final treatment (FT) was defined by the CHT of a tertiary hospital between august 2015 and november 2016. Fisher’s exact test or the chi-square test was used. Student’s t test for paired samples. The agreement was when estimating the Kappa coefficient, p < 0.05 indicated statistical significance. Stata/SE Program v.14.1. StataCorpLP, USA and SPSS 20.0. Results: The mean age was 62.6 ± 9.8 years, 62.8% were male, diabetic (63%) and a history of AMI in 53.1%. The mean EF was 49.7%. Angina CCS II–IV 57.2%, HF class II–IV 49.1%. They were multivessel in 65.6% and main left coronary >50% associated in 19.47%. There was agreement between the proposed treatment (PT) by the referral cardiologista and the CHT Ƙ = 0.19 and good agreement between the CHT and the FT Ƙ = 0.62. CABG was the predominant FT and with the highest agreement with the CHT 88.1%, followed by PCI (79.2%) p = 0.005. Total mortality was 10.2%, CV death 5.6% and ACS 27.7%. There was a higher incidence of primary composite outcome when PCI was performed in disagreement with CHT p = 0.001, no difference for the other strategies. In 72% of the cases, angina improvement occurred (p = 0.001). For HF class, only the cases in agreement with the CHT had an improvement of 74.4% p = 0.008. Conclusion: Final treatment in agreement with the CHT was associated with a lower incidence of primary composite outcome and significant improvement in long-term symptoms. 110487 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING SARA ABOURADI1, Abouradi Sara1, Bendahou Hajar1, Mackonia Noel1, Tamir Mehdi1, Bennouna Ghali1, Habbal Rachida1 (1) CHU IBN ROCHD CASABLANCA Background: Several factors may contribute to higher risk in patients with presumably moderate AS. Acceleration time (AT) and ejection time (ET) increase in parallel with AS severity, however AT/ET ratio in identification of high-risk subjects among asymptomatic mild-moderate AS is unknown The aim of the present analysis was to investigate the association between higher AT/ET ratio and outcomes in mild-moderate AS. Methods: Were included all patients with asymptomatic patients with presumably mild-moderate AS followed at the hospital of Ibn Rochd cardiology departement service in casablanca from October 2017 to June 2021, Patients were grouped according to the optimal AT/ET ratio threshold to predict cardiovascular death and heart failure hospitalization. Outcome was assessed in Cox regression analyses, and results are reported as hazard ratio and 95% CI. Results: The mean age was 65 ± 10. Higher AT/ET ratio was significantly associated with lower systolic blood pressure(p = 0,02), lower left ventricular ejection fraction(p = 0,01), low flow (p = 0,02), and with higher left ventricular mass (p = 0,03) and higher peak aortic jet velocity (p = 0,01). AT/ET ratio ≥0.34 had a higher risk of cardiovascular death and heart failure hospitalization (hazard ratio: 1.98 [95% CI, 1.50–3,8]). In patients with moderate AS, AT/ET ratio >0.36 was associated with higher rate for cardiovascular death (p = 0,01) and HF hospitalization (P = 0.035). Conclusions: In asymptomatic moderate AS higher AT/ET ratio was associated with increased cardiovascular morbidity and mortality. 110488 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES SARA ABOURADI1, SARA ABOURADI1, NOEL MACKONIA1, HAJAR BENDAHOU1, SOUKAINA ZAHRI1, ABDNASSER DIGHIL1, RACHIDA HABBAL1 (1) CHU IBN ROCHD CASABLANCA Introduction: Low myocardial energy efficiency (MEE) is undoubtedly associated with left ventricular dysfunction (LV) of the myocardium. the objective of this study was to investigate the effect of MEE in patients with asymptomatic aortic stenosis (AS). Methods: Were included all patients with asymptomatic aortic stenosis followed at the hospital of Ibn Rochd cardiology departement service in casablanca from October 2017 to June 2021. MEE was calculated from Doppler stroke volume/([heart rate/60]) and indexed to LV mass (MEEi). Outcome was assessed in Cox regression analysis and reported as HR and 95% CI. Results: The mean age was 60 ± 14 years mean ejection fraction: 50 ± 9%; mean aortic valve area: 1.04 ± 0.45 cm2; MEEi <0.35 mL/s per gram was associated with increased cardiovascular mortality [HR 1,98 (95% CI 1.60–3.28)] (both p < 0.02). This relationship persisted after multivariate adjustment and in the subgroups including more severe AS, higher body mass index, lower ejection fraction and presence of hypertension. (p = 0.01). Conclusions: In patients with asymptomatic AS, Low myocardial energetic efficiency is associated with increased mortality in aortic stenosis regardless of other associated factors. 110489 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES SARA ABOURADI1, SARA ABOURADI1, HAJAR BENDAHOU1, NJIE MALICK1, AMRI MERYEM1, AZZOUZI LAILA1, HABBAL RACHIDA1 (1) CHU IBN ROCHD CASABLANCA Background: Aortic stenosis is a very common pathology, several studies lean towards rapid surgical management, The aim of the study the objective of the study is to determine the appropriate attitude between rapid or conservative surgery. Methods: Were enrolled all patients with asymptomatic patients with severe aortic stenosis (defined as an aortic-valve area of ≤1 cm2 with either an aortic jet velocity of ≥4 m per second or a mean transaortic gradient of ≥40 mm Hg) followed at the hospital of Ibn Rochd cardiology departement service in casablanca from October 2017 to June 2021 Patients were classified according to early surgery or to conservative care according. The primary end point was death during or within 30 days after surgery. Results: The mean age was 65 ± 10 In the early-surgery group there was no operative mortality. The primary end-point event occurred in 3 patient in the early-surgery group (2%) and in 12 patients in the conservative-care group (14, 3%) (hazard ratio, 0.07; 95% confidence interval [CI], 0.03 to 0.24; p = 0.002). Death from another cause occurred in 7 patients in the early-surgery group (10,5%) and in 16 patients in the conservative-care group (19,06%) (hazard ratio, 0.22; 95% CI, 0.11 to 1,90, p = 0,02). Conclusions: Early surgical aortic-valve replacement has a significantly lower risk of operative mortality or death from cardiovascular causes during the follow-up period than conservative care among asymptomatic patients with severe aortic stenosis. 110490 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY MARCELLO FACUNDO DO VALLE FILHO1, Marcello Facundo do Valle Filho1, Odilon Pereira Velho Filho1, Vanessa Mendes Moreira1, Thalita Giovana Diniz Silva1, Giovanna Piva1, Lia Mizobe Ono2, William Hiromi Fuzita2, Monica Regina Hosannah da Silva e Silva1 (1) Faculdade Metropolitana (FAMETRO); (2) Oncologia Sensumed e Instituto Sensumed de Ensino e Pesquisa Ruy França (ISENP), Manaus-AM. Introduction: Heart disease and cancer are the main causes of morbidity and mortality in the industrialized world. Breast Cancer is a heterogeneous disease with different prognoses, therapeutic protocols and treatment outcomes. Identification of HER2+ on immunohistochemistry and/or FISH indicates a need for targeted therapy with HER2+ receptor blockers. However, its use may be related to cardiotoxicity, which may be manifested by an asymptomatic decline in left ventricular ejection fraction (LVEF) and/or by the occurrence of symptomatic heart failure, diagnosed through echocardiography. One of the therapies involves pertuzumab (a monoclonal antibody) associated with trastuzumab (which can also be applied alone) and docetaxel, with improved overall and progression-free survival. Considering normal LVEF above 55%, a fall below 10% may be completely reversible, while in those above 10%, reversal may occur in 91% of cases. Even in the presence of cardiotoxicity, 70% to 80% of patients continue to receive targeted therapy. Method: Cross-sectional, observational, descriptive and retrospective study of 697 patients diagnosed with breast cancer at Oncologia Sensumed in the city of Manaus-AM where 69 patients with HER2+ were selected. Results: Of the 69 patients, 45 (65%) received transtuzumab monotherapy, with a mean of eight doses (minimum of 3 and maximum of 17) at 21-day intervals, 16 (23%) received combined triple therapy. All ejection fractions measured quarterly by echocardiogram were within the normal range, ranging from 61% to 75%. Eight patients (12%) did not use any of the three drugs due to previously diagnosed medium and high-grade heart failure. Conclusion: The assessment of LVEF and the presence of heart failure (HF) before starting treatment for breast cancer with HER2+ influences the therapeutic protocol and treatment outcome. In addition, maintaining a quarterly evaluation elucidates data that indicate maintaining, discontinuing or establishing new options for treatment of both cancer and reduced LVEF and HF. Therefore, even with cardiotoxic effects, therapy directed at HER2+ in breast cancers can bring benefits to the prognosis, provided that they are monitored and evaluated quarterly through echocardiography and anamnesis to identify symptoms. 110496 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PEDRO ARTHUR RODRIGUES DE OLIVEIRA1, Pedro Arthur Rodrigues de Oliveira1, Marcello Vieira dos Santos1, Daniely Maués Beliqui1, Laura Coutinho Viana1, José Wilker Gomes de Castro Júnior1, Jéssica Lorena Alves2, Juliana Miranda Benício3, Juliane Costa Santos3, Lucian Herlan da Costa Luz Fernandes3, Lígia Maria dos Santos de Oliveira Vieira1, Amanda Carício Gomes1 (1) Centro Universitário do Estado do Pará (CESUPA); (2) Universidade Federal do Pará; (3) Centro Universitário Metropolitano da Amazônia (UNIFAMAZ) Introduction: Heart failure (HF) is a syndrome characterized by the inability of the heart to maintain sufficient cardiac output for the body’s metabolic needs, leading to ventricular dysfunction. In the pediatric population the most common cause of HF is congenital anomalies, such as cardiac malformation. Other less frequent causes are myocardiopathies, myocarditis, and arrhythmias. Objectives: For this reason, this paper aims to conduct an evaluation of the epidemiological profile of hospitalizations for heart failure iciency in Pará (Brazil) in the period from 2017 to 2021 in pediatric patients. Methodology: A descriptive, retrospective, quantitative study was conducted based on secondary data provided by the Hospital Information System (SIH) of the SUS Computer Department (DATASUS). The collected information was stored and tabulated in the Microsoft Office Excel™ program. Results: Among the 1,577 cases found after analyzing the evaluated period, there are the years 2017 with 327 cases (20.73%), 2018 with 379 cases (24.03%), and 2019 with 311 notified cases (19.72%), being the 3 most incident years of the investigated period. The municipalities with the highest number of hospitalizations for heart failure were: Belém (79.64%) followed by Breves (1.83%) and Santarém and Altamira (1.5% each). Moreover, it was identified that mixed race, with 1,130 cases (71.65%), female gender, with 798 cases (50.60%) and the age group of less than 1 year, with 578 cases (36.65%) are the most affected epidemiological variables. After evaluating the cases reported, it was noted that 104 cases (6.59%) died. Conclusion: It was observed, therefore, that patients under the age of 1 year had a higher prevalence of hospitalizations for heart failure, which may be associated with hypertrophic congenital heart diseases. Moreover, the considerable amount of deaths is evident, reflecting the need for attention to pediatric HF. 110500 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION CARLOS FILIPE DOS SANTOS PIMENTA1, Taissa Lorena dos Santos1, Camila Bello Nemer1, Gabriela da Silva Nascimento1, Hugo Farah Affonso Alves1, Lucca Hiroshi de Sá Kimura1, Bernardo Chedier1, Arthur Fernandes Cortez1, Elizabeth Silaid Muxfeldt1 (1) Universidade Federal do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho Introduction: Refractory hypertension (RfHT) is defined as an uncontrolled blood pressure (BP) despite the use of 5 or more antihypertensives, including spironolactone and it is considered an extreme phenotype of resistant hypertension (RHT). High BP levels lead to RAAS stimulation, sympathetic hyperactivity and endothelial dysfunction with consequent production of pro-inflammatory cytokines. Objective: To evaluate the relationship between inflammatory markers and refractory hypertension in a large cohort of patients with RHT. Methods: A cross-sectional study that evaluated 423 resistant hypertensives (30.5% male, mean age 63.9 ± 10.8 years), of which 62 (14.6%) diagnosed as RfHT. All of them were submitted to the dosage of inflammatory markers: TNF-alpha, MCP-1, E-selectin and PAI-1. Socio-demographic and anthropometric characteristics and cardiovascular risk factors (CV) were recorded. Variance analysis compared serum levels of the 4 inflammatory markers and bivariate analysis compared patients with RHT versus RfHT. Results: Patients with RfHT are younger, with higher prevalence of smoking, higher levels of albuminuria and higher prevalence of chronic cerebrovascular and renal disease stages 4 and 5. PAI-1 values (126 [108–162] vs 118 [94–153] were higher in those with RfHT, although they did not reach statistical significance. The other biomarkers evaluated showed no association with the diagnosis of RfHT. Conclusion: Among inflammatory markers, PAI-1 was the one that correlated most strongly with refractory hypertension. Our study is the first one to demonstrate PAI-1 association with treatment refractoriness. 110881 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION ALAYN KLEBER FREIRE DA SILVA JUNIOR1, Adelyanne Santos Barbosa1, Alayn Kleber Freire da Silva Junior1, Bárbara Maria Oliveira da Silva1, Guilherme de Azevedo Guedes1, Marco Antônio Mota Gomes2 (1) Centro Universitário CESMAC, Maceió, AL, Brazil; (2) Centro de Pesquisas Clínicas Dr. Marco Mota, Maceió, AL, Brazil Introduction: Systemic Arterial Hypertension is a chronic disease with a multifactorial condition characterized by blood pressure (BP) levels above or equal to 130 and/or 80 mmHg. The combination of BP measurements in the office and Home Blood Pressure Monitoring (HPBM) and Ambulatory Blood Pressure Monitoring (ABPM) made it possible to define BP standards, including White Coat Hypertension (WCH). HAB is characterized by the elevation of BP in the office during repeated visits, concomitantly with normal values of BP out of the office, verified through ABPM and HMBP. Objective: This study aims to identify and analyze evidence about cardiovascular events associated with White Coat Syndrome. Methodology: A literature search was performed using the descriptors: White Coat Hypertension, Cardiovascular and Events together with the Boolean operator “AND” in MedLine/PubMed, Lilacs and Scielo databases. The use of these descriptors allowed the retrieval and restriction of articles. The filter of the last 5 years was used. The inclusion criterion used was the presence of keywords in the title or abstract of the articles. The following were excluded: letter to the editor, dissertations and theses. Reading steps: titles, abstracts, case reports and full articles. Results: Six articles were selected, and it was possible to observe that in a significant part of them, patients with WCH, in general, presented a higher level of cardiovascular alterations when compared to the group of normotensive individuals. Although these alterations were smaller when compared to individuals with sustained hypertension, individuals who suffer from WCH may manifest several anomalies, such as increased LV mass, thickness of the interventricular septum, structural alterations in the carotid artery and greater LA diameter. Because of these changes, WCH has been associated with negative physiological changes, such as sympathetic hyperactivity, which is related to increased cardiovascular risks. Considering this, some studies proposed to analyze the effectiveness of treatments with antihypertensive drugs, but in most of these, there was no reduction in cardiovascular events. Conclusion: In view of the aforementioned arguments, it can be inferred that the cardiac and vascular damages of patients diagnosed with WCH are greater when compared to normotensives, and further studies are needed to develop effective ways to reduce the problems brought about by WCH. 110591 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY GUSTAVO PINHEIRO SANTANA1, Gustavo Pinheiro Santana1, Rodrigo Morel Vieira de Melo2, Tainá Teixeira Viana1, Ana Luísa de Aguiar Almeida Silva2, João Pedro Martins Moreira Granja2, Osvaldemar Regis Do Nascimento Junior1, Danilo Sousa Sampaio1, Thais Harada Campos1, Edmundo José Nassri Câmara1, Luiz Carlos Santana Passos1 (1) Hospital Ana Nery, Salvador – Ba (HAN); (2) Universidade Federal da Bahia (UFBA) Introduction: Rheumatic valve disease is still a health problem worldwide. Pulmonary hypertension (PH) may occur with the gradual progression of mitral stenosis and regurgitation in this condition. Whether or not PH is associated with early surgical mortality in this specific population remais controversial. Objective: In a population of patients undergoing cardiac surgery for rheumatic mitral valve disease, evaluate the impact of preoperative PH on early surgical mortality. Methods: This is a retrospective cohort carried out from January 1, 2017 to December 30, 2020. All patients over 18 years of age who underwent cardiac surgery to correct rheumatic mitral valve disease with functional tricuspid regurgitation in an echocardiogram performed up to 6 months before surgery were included. Systolic pulmonary artery pressure (sPAP) value was also defined by preoperative echocardiogram evaluation. The primary outcome was surgical mortality. Results: 144 patients were included. The mean age was 46.2 (±12.3) years with 107 (74.3%) female individuals, the median left ventricular ejection fraction was 61.0% (55–67) and sPAP was 55.0 mmHg (46–74), with 45 (31.3%) individuals with right ventricular dysfunction. The predominant valve disease was mitral stenosis (74.3%). The prevalence of severe tricuspid regurgitation was 47.2%. The total in-hospital mortality was 15 (10.4%) individuals. sPAP was independently associated with early surgical death RR 1.04 (1.01–1.07), p = 0.003. To determine a sPAP cut-off that indicates higher mortality and help decision making in clinical practice, we performed an analysis through the ROC curve (area 0.70, p = 0.012). The estimated value of 73.5 mmHg has the highest accuracy in our model for predicting early mortality. Conclusion: In patients with rheumatic heart disease who will undergo mitral valve surgery, pulmonary hypertension is associated with higher early mortality. Values above 73.5 mmHg predict higher risk and, in this part of the population, additional measures to control intraoperative and immediate postoperative pulmonary hypertension should be considered. 110617 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING FABRICIO MOREIRA REIS1, Maryanne Zilli Canedo Silva1, Nayrana Soares Carmo Reis1, Fabiana Lourenço Costa1, Caroline Ferreira da Silva Mazeto Pupo da Silveira1, Alejandra Del Carmen Villanueva Mauricio1, João Carlos Hueb1, Rodrigo Bazan1, Pasqual Barretti1, Luis Cuadrado Martin1, Silméia Garcia Zanati Bazan1 (1) Faculdade de Medicina de Botucatu – Universidade Estadual Paulista Introduction: The phase angle has been used as a nutritional marker and predictor of mortality in patients on peritoneal dialysis. The coronary artery calcium score has showed to predict the incidence of acute myocardial infarction and death from cardiovascular disease in these patients. However, the association between phase angle and coronary artery calcium score in patients on peritoneal dialysis is not well established. Objective: To evaluate the association between phase angle and coronary calcium score in patients on peritoneal dialysis. Methods: This was a cross-sectional study with patients on peritoneal dialysis, followed up at a University Hospital, between March 2018 and August 2019. Phase angle was evaluated by unifrequency bioimpedance. The coronary artery calcium score was calculated based on cardiovascular computed tomography, considering positive when greater than or equal to 100 Agatston and negative when less than 100 Agatston. Results: We evaluated 44 patients on dialysis, with mean age of 56 years and 11.7 months. In a univariate analysis, an association was observed between the coronary artery calcium score, the presence of atherosclerotic plaque in the carotid artery, and the difference in systolic blood pressure in the upper extremities with the phase angle. In the hierarchical multivariate logistic regression, only the coronary artery calcium score maintained an association with the phase angle in the final model. Conclusion: The phase angle is associated with a positive coronary artery calcium score in patients on peritoneal dialysis, and despite other factors, may be useful as a risk marker for coronary artery disease in this population. 110620 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING FABRICIO MOREIRA REIS 1, Maryanne Zilli Canedo Silva1, Nayrana Soares Carmo Reis1, Fabiana Lourenço Costa1, Caroline Ferreira da Silva Mazeto Pupo da Silveira1, Alejandra Del Carmen Villanueva Mauricio1, João Carlos Hueb1, Rodrigo Bazan1, Pasqual Barretti1, Luis Cuadrado Martin1, Silméia Garcia Zanati Bazan1 (1) Faculdade de Medicina de Botucatu – Universidade Estadual Paulista Introduction: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD) undergoing peritoneal dialysis (PD). The assessment of coronary calcification by coronary artery calcium (CAC) score has shown to predict the incidence of acute myocardial infarction and death by CVD in those patients. Left atrial volume has also been associated with acute myocardial infarction and cardiovascular events. Objective: To assess the association between left atrial volume and CAC score in PD patients. Methods: A cross-sectional study with PD patients, followed up between March 2018 and August 2019, including demographic, clinical and laboratory data collection; assessment of nutritional status by anthropometry and bioimpedance; protocol of residual renal function and adequacy of dialysis (Kt/V); doppler echocardiogram, carotid ultrasound, pulse wave velocity, ankle-brachial index and CAC. Patients were divided into two groups: positive or negative CAC score. Comparisons between groups were performed using the Chi-square, Student t or Mann-Whitney test (p < 0.05). Results: 44 patients on PDP, mean age of 56 years. Main cause of CKD was hypertension. Median time on dialysis therapy was 11.7 months. In the univariate analysis, a significant association of CAC score was observed with variables such as age, diabetes mellitus, over hydration, femoral pulse wave velocity, left atrial volume index, E/A ratio and left ventricular mass index, but in the multivariate analysis, only the left atrial volume index was related to a positive CAC score, odds ratio of 1.71. Conclusion: The left atrial volume index is associated with positive CAC score in patients with CKD undergoing PD, and it can be used as a risk marker for coronary artery disease in this population. 110669 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION VANESSA PROÊZA MACIEL GAMA1, Camilla Medeiros Macedo da Rocha4, Edna Massae Yokoo6, Amanda de Moura Souza7, Katia Vergetti Bloch7, Rosely Sichieri5 (1) Instituto Federal de Educação, Ciência e Tecnologia Fluminense – IFFlumnense, Av. Souza Mota, 350, Parque Fundão, CEP 28060–010, Campos dos Goytacazes, RJ, Brazil;; (2) Instituto de Saúde Coletiva, Universidade Federal Fluminense – UFF, Rua Marques de Paraná, 303, 3° andar, Centro, CEP 24030–210 Niterói, RJ, Brazil;; (3) Curso de Especialização em Nutrição Clínica, Instituto de Nutrição Josué de Castro, Universidade Federal do Rio de Janeiro – UFRJ, Av. Carlos Chagas Filho, 373 – bloco J 2° andar, Ilha do Fundão, CEP 21941–902, Rio de Janeiro – RJ, Brasil.; (4) Instituto de Alimentação e Nutrição, Centro Mutidisciplinar – UFRJ – Macaé, Universidade Federal do Rio de Janeiro, Av. Aluizio da Silva Gomes, 50, Novo Cavaleiros, CEP 27930–560 Macaé, RJ, Brazil;; (5) Instituto de Medicina Social, Universidade do Estado do Rio de Janeiro – UERJ, Rua São Francisco Xavier, 524, Pavilhão João Lyra Filho, 7° andar, CEP 20550–900 Rio de Janeiro, RJ, Brazil;; (6) Departamento de Epidemiologia e Bioestatística, Universidade Federal Fluminense – UFF, Rua Marques de Paraná, 303, 3° andar, Centro, CEP 24030–210 Niterói, RJ, Brazil;; (7) Instituto de Estudos em Saúde Coletiva, Universidade Federal do Rio de Janeiro -UFRJ, Avenida Horácio Macedo, s/n, Ilha do Fundão, CEP 21941–598, Rio de Janeiro, RJ, Brazil; Introduction: The consumption of foods and meals with high glycemic index (GI) and high glycemic load (GL) have been suggested as risk factors for chronic diseases, including hypertension. Population-based studies that have investigated this association in adolescence are still scarce. Objective: To evaluate the association between dietary indicators of glycemic response and changes in blood pressure among adolescents in the Study of Cardiovascular Risk in Adolescents (ERICA). Methods: A sample (n = 69,057) of students (12 to 17 years) from public and private schools, representative of Brazilian adolescents living in cities with 100,000 inhabitants or more was evaluated. Prevalence of hypertension was estimated, as well as mean systolic and diastolic blood pressures (mmHg) stratified by sex and age group and mean dietary indicators of glycemic response: daily GI, average GI, GI of the first meal of the day, daily GL, average GL and GL of the first meal of the day, according to sex and age group. Linear and logistic regression models stratified by sex and adjusted for age and body mass index were estimated to assess association between dietary indicators of glycemic response and systolic, diastolic blood pressures and hypertension. Results: The mean age was14.7 years and 56% female. The prevalence of hypertension was 9.1%, almost twice more frequent among boys (12.3%), compared to girls (6.5%). The mean systolic blood pressure was 110.6 mmHg and the mean diastolic blood pressure was 65.8 mmHg. Boys had higher mean dietary indicators of glycemic response (GI of first meal, daily GL, average GL, and GL of first meal) compared to girls (p-value < 0.05), except for mean GI and mean GL. There was a positive association between GI of the first meal with systolic blood pressure in boys. Conclusions: Factors related to carbohydrate metabolism and the nutritional value of the diet should include GI and GL of individual meals, beyond the global indicators of glycemic response such as daily GI and GL. Further studies are needed to deepen the relationship between the glycemic response from food intake. 110759 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM SRISAKUL CHAICHUUM1, Shuo-Ju Chiang2, Masao Daimon3, Su-Chen Chang2, Chih-Lin Chan2, Chu-Ying Hsu2, Ching-Li Tseng1 (1) Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan; (2) Division of Cardiology, Department of Internal Medicine, Taipei City Hospital Yangming Branch, Taipei, Taiwan; (3) Department of Cardiovascular Medicine, The University of Tokyo Hospital, Tokyo, Japan Background: Cardiac discomfort has been reported periodically in individuals who received COVID-19 vaccines. Transient cardiac symptoms are imperatively monitored to evaluate changes in heart function. Objective: This study aimed to evaluate the role of myocardial strains in the early assessment of the clinical presentations after COVID-19 vaccination. Methods: In total, 121 subjects who received at least one dose of a COVID-19 vaccine within 6 weeks were recruited. Serological markers and echocardiography were assessed. Two-dimensional speckle tracking echocardiography (2D-STE) was implemented to analyze changes in the left ventricular myocardium. Results: After vaccination, 66 individuals (55.4 ± 17.4 years) developed cardiac discomfort, such as chest tightness (n = 59, 89%), palpitations (n = 23, 35%), dyspnea (n = 21, 32%), and chest pain (n = 6, 9%). All had normal serum levels of creatine phosphokinase (93.0 U/L; IQR, 66.5–137.5), creatine kinase myocardial band (10.4 U/L; IQR, 2.3–14.7), Troponin T (6.0 ng/L; IQR, 4.0–8.5), N-terminal pro b-type natriuretic peptide (44.5 pg/mL; IQR, 20.2–79.5), platelets (222.0 × 103/𝜇L; IQR, 199.2–275.5), and D-dimer (0.3 mg/L; IQR, 0.1–0.4). Echocardiogram presented left ventricular ejection fraction in the symptomatic group (71.41% ± 7.12%) and the control group (72.18% ± 5.11%) (p = 0.492) were normal. However, the symptomatic group had a greater ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (e’) than the normal group (9.32 ± 3.56 vs 7.74 ± 2.34, respectively; p = 0.005), correlated with e’ (8.64 ± 3.96 vs 10.25 ± 3.58, respectively; p = 0.023). Use of 2D-STE showed that global longitudinal strain (GLS) and global circumferential strain (GCS) were reduced in the symptomatic group (17.86% ± 3.22% and 18.37% ± 5.22%) compared to the control group (19.54% ± 2.18% and 20.73% ± 4.09%) (p = 0.001 and p = 0.028). Conclusions: COVID-19 vaccine-related cardiac adverse effects can be assessed early by 2D-STE. The prognostic implications of GLS and GCS enable evaluation of the subtle changes in myocardial function after vaccination. 110780 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION MARCEL DE PAULA PEREIRA1, Eduardo Gomes Lima1, Fabio Grunspun Pitta1, Luis Henrique Wolff Gowdak1, Bruno Mahler Mioto1, Luiz Antonio Machado Cesar1, Henrique Trombini Pinesi1, Omar Asdrubal Vilca Mejia1, Fabio Biscegli Jatene1, Roberto Kalil Filho1, Francisco Carlos da Costa Darrieux1, Carlos Vicente Serrano Junior1 (1) Instituto do coração – Universidade de São Paulo Introduction: New post-operative atrial fibrillation (POAF) in the setting of coronary artery bypass graft (CABG) is an entity observed in up to 40% of procedures. Despite this, there is little evidence about the use of direct oral anticoagulants in this scenario. Purpose: Our aim was to compare cost-effectiveness of a strategy using rivaroxaban versus the standard therapy after POAF. Methods: This is a single center, randomized, prospective and open-label study comparing two anticoagulation strategies: Warfarin associated with bridging with enoxaparin versus rivaroxaban after POAF. Medications started during hospitalization and continued up to 30 days. Primary endpoint was the assessment of cost-effectiveness. Costs were analyzed from the perspective of supplementary health in Brazil and converted to US dollars. Quality-adjusted life year (QALY) were assessed using the SF-6D questionnaire. Safety endpoint was any bleeding using the international society of thrombosis and haemostasis. Results: In a tertiary hospital, 51 patients who underwent CABG and presented with POAF were randomized. Twenty-six patients were randomized for the warfarin group and 25 for the rivaroxaban group. Both groups had similar baseline. The median of the primary cost-effectiveness outcome was $ 1347,7 in the warfarin group and $433,0 in the rivaroxaban group (p < 0.001), as shown in Figure 1. The result of the SF-6D questionnaire showed no statistical difference between groups. Bleeding rates were 26% in warfarin and 12% in rivaroxaban group (p = 0.18). Conclusion: In this study with a 30-day follow-up, anticoagulation strategy with rivaroxaban in patients with POAF was more cost-effective the standard therapy using warfarin. 110933 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION MARCELLO VIEIRA DOS SANTOS1, Cassiane da Silva Portela Pinto2, Jessica Lorena Alves3, Pedro Arthur Rodrigues de Oliveira1, Izadora Avelar Neto2, Daniely Maues Beliqui1, Roberta Louise dias Rodrigues1, Adilson talis Ferreira dos Santos1 (1) Centro Universitário do Estado do Pará; (2) Universidade do Estado do Pará; (3) Universidade Federal do Pará Introduction: Thrombosis is formed from clots in the circulatory system, and is related to venous stasis, trauma, surgery, and others, and can evolve to more fatal conditions, such as pulmonary thromboembolism. To avoid this condition, it is necessary to start anticoagulant therapy early. This condition is recurrent in the postoperative period of plastic surgery, being aggravated by the non-use of elastic stockings, the stimulation of early ambulation, the use of anticoagulants, and the failure of risk analysis. Therefore, this paper discusses anticoagulants in the postoperative period of plastic surgery as a way to prevent thrombosis. Objectives: To analyze the use of anticoagulants as a prophylactic form for thrombosis formation in the postoperative period of patients undergoing plastic surgery. Methods: This is a systematic review of literature, based on the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyse – PRISMA. We used the term “(anticoagulants) AND (thrombosis) AND (disease prevention)” in the LILACS, PubMed and Scielo databases, obtaining initially 65 publications and after the PRIMAS guidelines, 7 remained for analysis. Results And Discussions: Throughout the studies, it was seen the presence of Virchow’s triad in the postoperative period of plastic surgery, characterized by venous stasis, endothelial injury and hypercoagulation. For this, it was observed, only the Caprini score as recommended for risk assessment in the postoperative period. Regarding anticoagulants, it was shown vitamin K antagonists, Factor Xa inhibitors (heparins), direct oral Factor Xa inhibitors (rivoraxaban) being the first line of choice, however there are still many discussions about the ideal propedeutics, leading to its irrational use, since its excess can bring bleeding and smaller doses do little effect. Conclusion: Thus, the diversification of methods for the evaluation of venous thromboembolism (VTE) were shown to be necessary in plastic surgery, since only one method of choice was analyzed, which ends up hindering the patient’s risk assessment. Similarly, the creation of a protocol by the Brazilian Society of Plastic Surgery, addressing VTE in the postoperative period could standardize chemoprophylaxis and make it more effective. Finally, greater follow-up of patients at higher risk by a hematologist also shows promise for improving the efficacy of therapy. 110782 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FERNANDO COUTO PORTELA1, Sayuri de Souza Yamamura1, Francisco Expedito de Albuquerque Filho1, Marcus Vinícius Alves Gomes1, Luis Felipe Mousinho Lima de Carvalho1, Marco Túlio Hercos Juliano1, Márcio Mesquita Barbosa1, Ana Cláudia Conrado de Oliveira1, Gustavo Travassos Gama1, Márcio Mendes Pereira1 (1) UDI Hospital Introduction: The valve prosthesis-patient mismatch (PPM) is characterized by a small effective orifice area (AOE) in relation to the body surface, resulting in high transvalvular gradients. The presence of MPP in the scenario of transcatheter aortic valve implantation (TAVI) has different characteristics according to the type of prosthesis (balloon-expandable or self-expanding). Knowing the real prevalence of PPM in a real-world sample is very important since such presence impacts survival, functional recovery, hospitalization for heart failure and valve durability. Objectives: To determine the prevalence of PPM in a sample of patients undergoing TAVI, using predicted AOE and AOE measured by the continuity equation. Associate the presence of PPM with several clinical, echocardiographic, tomographic variables and with the type of prosthesis used. Method: An observational study was performed with 26 consecutive patients undergoing TAVI in a private hospital. PPM was defined as present if AOE <0.85 cm2/m2 (<0.70 cm2/m2 if obese). The predicted PPM was obtained from data provided in the medical literature according to the size and prosthesis used. Results: All 26 patients who underwent TAVI within the 9-year period were included in the study. The mean age was 78.9 ± 8.3 years and 57.7% were male. The measured prevalence of PPM was considerably higher (41.7%) than predicted (15.4%). The mean residual gradient was 18.7 ± 4.4 mmHg in patients with PPM and 10.5 ± 4.5 mmHg in patients without PPM (p = 0.029). The rate of PPM in balloon-expandable prostheses was significantly higher than in self-expanding prostheses (42.9% vs. 5.3%, p = 0.018). Patients with PPM had a smaller area of the aortic annulus measured by tomography than those without PPM (3.34 ± 0.16 cm2 vs. 5.06 ± 1.95 cm2, p = 0.014). Conclusion: The use of the AOE calculated by the continuity equation overestimated the prevalence of PPM in patients undergoing TAVI. PPM was more prevalent in balloon-expandable prostheses than in self-expanding ones. Reduced aortic annulus was another factor associated with PPM. 111196 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM YURI CAVALCANTI ALBUQUERQUE TENORIO1, Priscila Alves da Silva2, Ana Lívia de Oliveira Barros2, Gabriel Noé Albuquerque Paffer Cruz2, Nicole de Lima Larré Barbosa4, Carolline Cavalcante de Melo3, Ana Clara Valente de Lima Melo3, Arthur Henrique Fernandes Rodrigues2, Edecio Galindo de Albuquerque1, Antônio Everaldo Vitoriano de Araújo Filho2, Francisco de Assis Costa2 (1) Hospital Veredas; (2) Centro Univesitário Tiradentes; (3) Universidade Federal de Alagoas; (4) Universidade Estadual de Ciências da Saúde de Alagoas Introduction: Covid-19 was declared a pandemic in early 2020 by the World Health Organizations (WHO) and it is a respiratory disease caused by SARS-CoV-2 virus. One of its main complications is the development of coagulopathies, given that the infection promotes increased thrombin formation and hypercoagulability state. The modified CHA2DS2-VASc score is used to estimate the risk of thromboembolic stroke in atrial fibrillation, as well as the thrombotic risk in other heart diseases. The abbreviation stands for congestive heart failure, hypertension, age, diabetes, previous stroke/transient ischemic attack. The risk factors that make up the m-CHA2DS2VASc score could also be recognized as risk factors for mortality in patients with COVID-19. The aim of this study is to identify the association between m-CHA2DS2VASc score in mortality and hospital staying in Covid-19 patients to use it as prognostic score of severe disease. Methods: Case-control study in a philanthropic hospital in the Brazilian state of Alagoas, selecting 103 hospitalized patients from 2020 to 2021 aged more than 18 years old and diagnosed with SARS-CoV- 2 infection. The statistical analysis was calculated using Fisher’s Exact test. Results: The mean m-CHA2DS2VASc score. was 1,74 (SD 1,53) and mean age was 53.81 years (SD 17.37). There was a significant relationship between CHA2DS2VASc score and mortality rate (p = .003) and hospital staying (p = .009). Discussion: The results lead us to associate m-CHA2DS2VASC score to risk of death (any cause). Another similar study with 864 patients also found this relation with m-CHA2DS2VASc score and death (p < .001). Other authors suggest that m-CHA2DS2-VASc score can be used to discern patients with higher thromboembolic risk. Also, higher m-CHA2DS2-VASc score is associated with a higher need for mechanical ventilation and a greater risk of death. Conclusion: There was a relationship between m-CHA2DS2VASc score and development of severe covid-19 and mortality according to the population analysed and other similar studies. 110794 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION YURI CAVALCANTI ALBUQUERQUE TENORIO1, Thayrone Romário da Silva Santos1, Francisco de Assis Costa2 (1) Hospital Veredas; (2) Universidade Federal de Alagoas Introduction: Epigallocatechin-3-gallate (EGCG) is a natural flavonoid present in green tea leaves with important therapeutical effects such as antioxidant properties, reduction of free radicals involved in numerous chronic diseases. The literature identifies a strong correlation between EGCG supplementation and the treatment and prevention of cardiovascular diseases; consequently, this flavonoid is a potential adjuvant for patients with several clinical conditions, such as arterial hypertension, cardiac heart failure (CHF), arrhythmias, chronic coronary disease, or acute myocardial infarction. Therefore, this paper aims to identify correlation between EGCG extracted from green tea leaves and treatment of CHF. Methods: A systematic literature review was developed selecting seven original articles, focusing on laboratory experiments in animal models. Results and discussion: The results found that EGCG supplementation promoted treatment of CHF and cardiac remodelling prevention from reducing heart weight (lower p was < .0001), increasing left ventricle ejection fraction (lower p was < .01), decreasing protein expression levels of hypertrophy markers, such as brain natriuretic peptide (BNP) in animal models (lower p was < .01), and reducing mortality in one study (p < .05). Conclusion: Therefore, it was possible to conclude that there is a positive relationship between EGCG supplementation with the treatment and prevention of CHF. However, although laboratory tests in animal models have positive results, multicentered randomized clinical trials in the population are necessary to define the actual association of this substance in humans. 110809 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY GISELE PLAÇA RODRIGUES OLSON1, Eduardo Schlabendorff1, Euler Manenti1, Jairo Lewgoy1 (1) Hospital Mãe de Deus, Porto Alegre/RS – Brasil Summary Base: Cardiovascular diseases (CVD) and cancer (CA) are the main causes of death in Brazil. Its incidences are associated with rising in life expectancy and various common risk factors. With the improvement of CA treatment and the increasing number of survivors, the event of CVD in these individuals has also been increasing, due to the impacts of the anticancer drugs in the cardiovascular (CV) system, which is called cardiotoxicity. Cardio-oncology becomes essential for early identification, treatment, and prevention of such complications. Our practice suggested a protocol capable of stratifying cancer patients according to the risk of cardiotoxicity. Purpose: The purpose of this study was to estimate the number of patients that were at elevated risk for cardiotoxicity, at their first oncology visit. And the amount of them were referred for an evaluation in cardio-oncology. Methods: We reviewed 104 medical records of patients who had their first appointment at the oncology department of Hospital Mãe de Deus, in Porto Alegre/RS, from June 1 to August 31, 2019. To stratify them, we filled out an appropriated protocol based on guidelines. Results: We have identified that 87 (83%) patients at elevated risk for cardiotoxicity, which should be evaluated by a cardio-oncologist. Only 15 (17.2%) were referred. The risk determinants are in the figure. Conclusion: We have shown that most patients seen at the first visit to an oncologist had a substantial risk of cardiotoxicity, but only few were actually referred to the cardio-oncologist department. This initiative encourages initial cardiologic evaluation routine in patients who will undergo therapies with potential cardiotoxicity. 110812 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS PEDRO EMANUEL DE PAULA CARVALHO1, Thiago Mamede Alencastro Veiga1, Gabriel Venturim Porto1, Felipe Sarsur de Lanna Machado1 (1) School of Medicine, Federal University of Minas Gerais Introduction: It has been recognized that diabetic patients with complex coronary artery disease have poor outcomes when undergoing Percutaneous Coronary Intervention (PCI) compared to Coronary Artery Bypass Grafting (CABG). However, among diabetic patients with unprotected Left Main Coronary Artery (LMCA) disease, the optimal method is not established. Objectives: Evaluate long-term outcomes after PCI with drug-eluting stents, as compared with CABG in diabetic patients with unprotected Left Main Coronary Artery (LMCA) disease. Methods: MEDLINE, ClinicalTrials, and Cochrane databases were searched for randomized clinical trials (RCTs) that reported outcomes after PCI with drug-eluting stents vs CABG in unprotected LMCA disease among the diabetic population. We chose a minimum follow-up period of 3 years. Trial level Risk Ratios (RRs) and 95% confidence intervals (CIs) were pooled by random-effects modeling. The risk of bias was estimated by assessing funnel plots. PRISMA-IPD guideline was followed to present the data. Results: Four RCTs including 1080 patients were identified, of whom 553 (51.2%) underwent PCI. The risk of composite endpoints of death, myocardial infaction, stroke or revascularization was higher after PCI compared with CABG (RR 1.30, 95% CI [1.09–1.56], P = 0.0037, I2 = 0%). There was no difference for individual endpoints of deaths, cardiovascular death and myocardial infarction (RR 1.32, 95% CI [0.94–1.85], P = 0.1131, I2 = 0%; RR 1.29, 95% CI [0.76–2.18], P = 0.3405, I2 = 0%; RR 0.94, 95% CI [0.61–1.45], P = 0.7881, I2 = 0%, respectively), however the risk of stroke was higher in CABG group (RR 0.41, 95% CI [0.18–0.94], P = 0.0351, I2 = 0%) meanwhile the risk of any revascularization was higher in PCI group (RR 1.99, 95% CI [1.44–2.75], P < 0.0001, I2 = 0%). There was no difference between groups with respect to graft occlusion or stent thrombosis (RR 0.66, 95% CI [0.19–2.31], p = 0.5162, I2 = 64,6%). Conclusion: A higher risk of revascularization was associated with PCI compared to CABG in diabetic patients with left main coronary disease. This finding affects the composite endpoint that favors CABG. However, the risk of stroke was greater in patients submitted to CABG and no significant difference was found between the other outcomes evaluated (MI, cardiovascular death, and total deaths) when analyzed individually. These findings could help with the decision-making between PCI and CABG in diabetic patients with unprotected LMCA disease. 110830 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GUSTAVO PAES SILVANO1, Rodrigo Pinheiro Amantéa1, Guilherme Pinheiro Machado1, André Luiz Theobald1, Alan Pagnoncelli1, Luiz Carlos Corsetti Bergoli1 (1) Hospital de Clínicas de Porto Alegre (HCPA) Introduction: Left main coronary intervention is increasingly used as a treatment option for unprotected left main coronary artery (ULMCA) lesions due to technical improvements in percutaneous coronary intervention (PCI), stent technology and new guideline recommendations. Objectives: To report the clinical profile, angiographic status, and procedural outcomes in patients undergoing ULMCA PCI. Methods: A prospective observational study including all consecutive patients with ULMCA who underwent PCI at a tertiary hospital in Southern Brazil between January 2017 and February 2022. Data including clinical-demographic profile, angiographic details and procedural complications were analyzed. Results: A total of 206 patients were investigated, of which 63.1% were male, with a mean age of 67.7 ± 11.4 years. Hypertension and diabetes were the most common risk factors, present in 81.1% and 44.2% of patients, respectively. Smoking, previous acute myocardial infarction, chronic kidney disease, heart failure and peripheral vascular disease were present in 43.9%, 22.3%, 22.3%, 15.5% and 11.7% patients respectively. Chronic stable angina was the most common mode of presentation (32.5%), followed by non-ST-segment elevation myocardial infarction (23.8%), ST-segment elevation myocardial infarction (16.5%) and unstable angina (15%). The percentage of patients with single-, double-, and triple-vessel coronary disease (in addition to left main stenosis) was 28.6%, 20.9%, and 39.3%, respectively. Isolated ULMCA lesion was present in 11.2% patients and left main bifurcation lesions in 70.9%. Majority of the procedures were performed via femoral approach (63.6%). The median stent number placed was 2 (1–3) and the median contrast volume used was 240 mL (175–300). Predilatation with balloon was performed in 90.8% of patients and postdilatation in 96.1%. Pre-PCI ultrasound was used in 31.2% of patients and 42.9% following stent implantation. Procedure related complications occurred in 19 (14.6%) cases. Most common among these were distal embolization (3.4%), side branch occlusion (3.4%), and no reflow phenomenon (2.4%). Procedure related mortality was 1.0% and occurred exclusively in acute coronary syndrome patients. Among the 18 patiens who died during hospitalization, 12 (66.7%) had cardiogenic shock at presentation. Conclusion: ULMCA PCI was safely performed and presented excellent results in this single-center prospective registry. 110843 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION HENRIQUE TROMBINI PINESI1, Eduardo Martelli Moreira1, Cibele Larrosa Garzillo1, Eduardo Bello Martins1, Fabio Grunspun Pitta1, Paula Martins Paulino Bolta1, Carlos Alexandre Wainrober Segre1, Desiderio Favarato1, Roberto Kalil Filho1, Fabiana Hanna Rached1, Eduardo Gomes Lima1, Carlos Vicente Serrano Junior1 (1) Instituto do Coracao, Faculdade de Medicina, Universidade de Sao Paulo Introduction: Incidence of cardiovascular events in patients with Chronic Coronary Syndrome (CCS) may vary significantly between geographical regions. Although populous, Brazil is often underrepresented in international registries. Objective: This study aimed to describe the quality of care and 3-years incidence of cardiovascular events and associated prognostic factors in CCS patients in a tertiary public health care center in Brazil. Methods: Patients with CCS (either previous revascularization, myocardial infarction, or stenosis >50% in at least one epicardial coronary artery) presenting for clinical evaluation were enrolled and followed for at least 3 years. The main endpoint was the composite of MI, stroke, or death. We also evaluated prescription, symptoms, and laboratory records. Results: 844 patients with mean age of 65 (±9.4) years, 31.3% women, were included. Previous MI was present in 60.9%, previous CABG in 28.8%, and previous PCI in 44.5%. Diabetes was prevalent in 49.6% and high blood pressure in 86.7%. The use of aspirin was prevalent in 90.9% of the patients, 21.2% with dual antiplatelet therapy, and only 7.1% with no antiplatelet agent. Statins were prescribed to 94.4% of the patients, with high-intensity statin therapy in 68.2%. At a median follow-up (FU) of 881 days, we recorded 70 events of primary composite endpoint, with a 3-year estimate event incidence of 8.3%. Age (1.54, 95%CI 1.32–1.90), stroke (2.62, 95%CI 1.18–5.84), LDL (1.10, 95%CI 1.03–1.18) and left ventricular function (0.84, 95%CI 0.76–0.93) were the main prognostic factors in multivariate analysis. At admission, angina was present in 29.1% of the patients. This percentage dropped to 16.3 after follow-up. On the other hand, there was no improvement in the LDL levels. Conclusion: CCS patients at our institution had a 3-year incidence of the primary composite endpoint of 8.3%, the prescription of antiplatelet therapy and statins were high, and LDL-cholesterol was the main modifiable risk factor of worse prognosis. 110847 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DIANA SEIXAS FERRÃO1, Joana Pereira1, Pedro Ribeirinho Soares1, Marta Soares Carreira1, Marta Amorim1, Catarina Elias1, Rita Gouveia1, Sérgio Madureira1, Ana Neves1, Jorge Almeida1, Patrícia Lourenço1 (1) Internal Medicine Department, Centro Hospitalar e Universitário de São João Introduction: Pulse pressure (PP) is the difference between systolic and diastolic blood pressure. Increased PP is a well-recognized cardiovascular risk factor but is less well established in HF, particularly with reduced ejection fraction (HFrEF). Objective: To study the prognostic role of increased PP in HfrEF. Methods: Retrospective cohort study of adult (>18 years) ambulatory patients with chronic HFrEF that were followed in a specialized HF clinic between January 2012 and May 2018. Patients with preserved ejection fraction and those with recovered ejection fraction were excluded. Patients with PP ≤50 mmHg and PP >50 mmHg were compared. The 50 mmHg cut-off corresponded to the median value of PP distribution and to the midst of the normal range. Patients were followed-up to 5 years and all-cause mortality was the endpoint. A Cox-regression analysis was performed to study the association of PP with 5-year mortality. Interaction between PP and age (cut-off 80 years) was tested. The analysis was further stratified according to age strata: <80 years and ≥80 years. Adjustments were made considering sex, hypertension, diabetes, atrial fibrillation, ischaemic aetiology, systolic dysfunction severity, heart rate, systolic blood pressure (SBP), diastolic blood pressure (DBP), renal function, B-type natriuretic peptide (BNP) and evidence-based therapy. Results: We studied 854 chronic HF patients. Mean age was 71 years and 65% were male. HF was ischaemic in 46.0% of the patients and 51.3% had severe systolic dysfunction. Median (Q1–Q4) PP was 51(40–64)mmHg. Patients with PP >50 mmHg were older, had higher SBP, less systolic disfunction and more often medicated with renin-angiotensin-aldosterone system inhibitors. During the 5-year follow-up, 391 (45.8%) patients died. Patients with PP >50 mmHg had no difference in death risk compared to those with lower PP: HR = 0.94 (95% CI: 0.77–1.15), p = 0.55. When the analysis was stratified according to age, in the subgroup of patients aged ≥80 years, elevated PP was associated with survival benefit, HR of 0.58 (95% CI: 0.37–0.91), p = 0.02. No association with mortality existed in the group of patients <80 years. Conclusions: Elevated PP was not predictive of mortality in patients with chronic HF and in the subgroup of patients aged ≥80 years, a PP >50 mmHg was associated with lower mortality, so it appears that age influences the impact of PP in mortality in HF. 110858 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM HENRIQUE TROMBINI PINESI1, Eduardo Martelli Moreira1, Melina de Oliveira Valdo Giugni1, Pedro Henrique de Almeida Marins1, Eduardo Gomes Lima1, Fabio Grunspun Pitta1, Ludhmila Abrahão Hajjar1, Juliana Carvalho Ferreira2, Carlos Vicente Serrano Junior1, Cibele Larrosa Garzillo1 (1) Instituto do Coracao, Faculdade de Medicina, Universidade de Sao Paulo, Brazil; (2) EPICOV Study group, Faculdade de Medicina, Universidade de São Paulo, Brazil Background: Studies have shown an association between cardiovascular diseases and higher rates of mortality and complications in patients with COVID-19. The aim of this study was to compare mortality and other events in critically ill cardiac and non-cardiac patients with COVID-19 in a referral center intensive care unit (ICU) in Brazil. Methods: We analyzed data from a prospective registry of patients admitted to a tertiary hospital ICU for COVID-19 between March and June 2020. 1501 patients were admitted and 221 had heart disease. Continuous variables were described as mean and standard deviation or median and interquartile range. Categorical variables were described as absolute frequency and percentage. To compare clinical and demographic characteristics, as well as laboratory tests between patients with and without heart disease, the Student’s T test and the Mann-Whitney U method were used. For the occurrence of events, Fisher’s exact test was used. Results: Patients with heart disease were older, and had a higher prevalence of diabetes and chronic kidney disease, compared to non-cardiac patients. On the other hand, the prevalence of cancer and obesity was higher among non-cardiac patients. Blood pressure, heart rate and oxygen saturation were similar between the groups, as well as the need of oxygen and ventilatory support. Patients with heart disease were more frequently using vasoactive drugs on admission. Patients with heart disease had worse renal function and higher cardiovascular biomarkers (troponin, NT-proBNP and D-dimer). Non-cardiac patients had higher values of CRP, leukocytes, lymphocytes and platelets. Surprisingly, in-hospital and ICU mortality were similar in both populations studied. Non-cardiac patients required more frequent orotracheal intubation and had more thrombotic events. In contrast, patients with heart disease had a higher occurrence of atrial fibrillation. Conclusion: In our center, the presence of cardiovascular disease in critically ill patients with COVID-19 was not associated with mortality compared with non-cardiac patients. 110863 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ALFREDO AURÉLIO MARINHO ROSA FILHO3, Lucas Brandão Cavalcante2, José Carlos de Souza Neto2, Sara Carolline Gomes de Araújo Lima2, Alice Wanderley Rosa2, Dario de Moura1, Virginia Barreto1, Marcelo Russo1, Fabian Fernandes1, Flavio Loureiro1, Alfredo Aurélio Marinho Rosa1, Edvaldo Ferreira Xavier Júnior1 (1) Santa Casa de Misericórdia de Maceió – SCMM, AL; (2) Centro Universitário Tiradentes – UNIT, AL; (3) Hospital Universitário Professor Alberto Antunes – HUPAA/UFAL Background: Persistent left superior vena cava (PLSVC) is a rare venous anomaly, however, in the thorax it is the most frequent venous alteration with a prevalence of 0.3 to 0.5% in the general population. The PLSVC is asymptomatic and in most cases is identified during implantation of the cardiac device. Objective: To present the technique used to implant a cardioverter-defibrillator (ICD) with biventricular pacing (BIV) in patients with PLSVC. Material and Methods: Between March 2007 and April 2022, 776 multi-site cardioverter-defibrillators (ICD-CRT) were implanted, of which 4 patients (0.5%) had SEPVC. All patients had symptomatic HF and refractory to optimized drug treatment with recurrent hospitalizations for HF. The 4 patients were male. They presented LBBB on ECG and ventricular tachycardia on 24h Holter. In all, the anomaly was identified during the procedure. They were then submitted to implantation of ICD associated with stimulation (BIV), during the puncture of the left subclavian vein, the presence of PLSVC was found. It was decided to implant the ICD through the right subclavian vein and introduce the left ventricular lead into the left posterolateral vein through the deflectable sheath. Access to the coronary sinus was obtained by femoral access. Results: In all, the procedure was performed successfully. The procedure time was around 90 minutes. All patients (100%) did not show displacement of the electrodes, being discharged 24 hours after the procedure and in the clinical follow-up, the patients showed clinical and ejection fraction improvement, with an average EF from 24% to 42%. Conclusion: Although PLSVC is a rare and complex anomaly, the implantation of the ICD was possible through the right subclavian artery, not compromising the final result of cardiac resynchronization. 110865 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ALFREDO AURÉLIO MARINHO ROSA FILHO3, Sara Carolline Gomes de Araújo Lima2, Lucas Brandão Cavalcante2, José Carlos de Souza Neto2, Alice Wanderley Rosa2, Marcelo Russo1, Gustavo Santiago1, Fabian Fernandes1, Lenine Angelo1, Flavio Loureiro1, Alfredo Aurélio Marinho Rosa1, Edvaldo Ferreira Xavier Júnior1 (1) Santa Casa de Misericórdia de Maceió – SCMM, AL; (2) Centro Universitário Tiradentes – UNIT/AL; (3) Hospital Universitário Professor Alberto Antunes – HUPAA/UFAL Background: Heart failure (HF) is considered the final stage of all heart diseases and with the increase in life expectancy of the population, octogenarian patients with HF can benefit from the implantation of a multisite cardioverter-defibrillator (ICD+CR- T). Objective: To present the results of the implantation of multi-site cardioverter-defibrillators in octogenarian patients with heart failure unresponsive to optimized clinical treatment. Material and Method: Between March 2010 and April 2022, 763 multisite cardioverter-defibrillators (ICD+CR-T) were implanted, of which 56 patients (7.34%) were octogenarians with 40 male patients (71.4%). Patients had HF with optimized drug treatment without adequate response, ejection fraction less than or equal to 32%, ventricular tachycardia and syncope. In this sample, they underwent implantation of the ICD+CR-T with access to the coronary sinus via the femoral route. Immediate success criterion coursing with QRS complex narrowing. Results: Of the 56 patients, forty-five (80.3%) responded satisfactorily to the therapy used, with a decrease in the number of hospitalizations and an improvement in the quality of life. Regarding the etiology of heart diseases: 38 (67.8%) were ischemic, 12 (21.4%) were chagasic and 6 (10.8%) had hypertensive heart disease. There were no intraoperative and postoperative complications and the main vein addressed for system implantation was the left posterolateral vein in 32 patients (57.1%). No displacement of electrodes was recorded. In the clinical follow-up, 22 pt (39.3%) received appropriate therapy for ventricular tachycardia. Survival was 100% at the end of 12 months in all patients undergoing ICD+CR-T implantation. Conclusion: It is therefore evident in the analyzed sample that the treatment of heart failure in octogenarian patients undergoing ICD+CR-T implantation showed a satisfactory response in terms of morbidity and mortality. 110870 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JOSE MIGUEL CAYO MONTES1, JOSE MIGUEL CAYO MONTES1, JUAN CARLOS ZERPA ACOSTA1, JOSE CARLOS PACHON MATEOS2, JUAN CARLOS PACHON MATEOS1, ENRIQUE INDALECIO PACHON MATEOS2, YVAN FLORES TARDIO1, CARLOS THIENE CUNHA PACHON2, CIBELE MATSUURA DE OLIVEIRA1 (1) HOSPITAL DO CORAÇÃO; (2) SEMAP Introduction: Pacemaker (PM) implantation is the treatment of irreversible bradyarrhythmias, normalizing the heart rate, right ventricular apical pacing (RVAP) has been traditionally chosen, but it can induce ventricular dyssynchrony, resulting in a widening of the QRS complex duration. (QRSd) and left ventricular activation time (LVTA) increase, leading to heart failure and/or arrhythmias. In order to prevent these adverse effects, right ventricular septal pacing (RVSP) and more recently left bundle branch area pacing (LBBAP) of the bundle of His were developed, which reproduce more physiological activation of the cardiac conduction system. Objectives: The aim of this study is to compare the electrophysiological parameters of ventricular synchrony (QRSd, LVAT) and other parameters such as QRS axis, R-wave amplitude, threshold and impedance of RVAP, RVSP and LBBAP, in a group of patients with pacemaker implantation indication. Methods: Prospective and controlled study, which included 25 patients with indication for bicameral pacemaker implantation. Using radioscopy, first, the electrode is positioned in the position of the RAVP, using the electrogram recording system and the PM device programmer, unipolar stimulation is performed at this point and the electrophysiological parameters are recorded; QRSd, LVAT in V5 or V6, QRS axis, R wave, ventricular lead threshold and impedance. Afterwards, the ventricular electrode was positioned in the region of RVSP and LBBAP and the same parameters were measured. Results: Electrophysiological parameters. QRSd: RVAP: 162 ± 25 ms, RVSP: 145 ± 20 ms, LBBAP: 109 ± 17 ms (p < 0,05) LVAT: RVAP: 80 ± 11 ms, RVSP: 70 ± 16 ms, LBBAP: 60 ± 11 ms. (p < 0,04). QRS axis: RVAP: –56° ± 10°, RVSP: 45 ± 15°, LBAP: 60 ± 11 ms (p < 0,04). Ventricular lead impedance: RVAP: 603 ± 20Ω, RVSP: 560 ± 25Ω, LBBAP: 805 ± 25Ω (p < 0,03). Threshold: RVAP: 0,75 ± 0.50V. RVSP: 0,75 ± 0.25V. LBBAP: 0.5 ± 0.25V (p < 0,05). R wave: RVAP: 10,1 ± 2 mV RVSP: 9,9 ± 3 mV LBBAP: 10,9 ± 6 mV(p: 0,06) There were no adverse events during the 6-month follow-up. Conclusion: The unipolar LBBAP achieved lower QRSd and LAVT, normal QRS axis, lower pacing thresholds, slight increase in ventricular lead impedance, there were no significant difference in the R wave sense compared to RVAP and RVSP. LBBAP is a safe technique that preserves ventricular synchrony, with better electrophysiological parameters compared to RVAP and RVSP, and could be considered as the first choice of ventricular pacing for patients. 111120 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING JOAO PEDRO DA HORA SILVA BARROS1, Júlya de Araujo Silva Monteiro1, Andressa Teoli Nunciaroni2, Renata Flávia Abreu da Silva2 (1) Universidade do Estado do Rio de Janeiro; (2) Universidade Federal do Estado do Rio de Janeiro Introduction: Active methodologies, such as case studies, are teaching strategies in nursing education, making the student an active agent in learning and the teacher a facilitator. Objective: To validate two evaluation instruments for case study discussion in cardiology nursing. Methods: Methodological, prospective, quantitative study, conducted remotely for content validation of measurement instrument. Two instruments were evaluated: Quantitative, with twenty-seven items and three aspects (Psychosocial, Intellectual and Human/Sensitive) and; Qualitative, with six questions addressing the learner’s interest about case studies. Nurses with experience in cardiology for over two years were recruited and the instruments were structured in Google Forms®, being available by link, with questionnaires in Likert Scale with an area for suggestions from experts. Data analysis was performed using Microsoft Excel® and validation was based on the calculation of the Content Validity Index (CVI). Items with at least 80% approval if more than six experts and 100% approval if less than six were valid. The study was approved by the institution’s Research Ethics Committee under Opinion #4,126,925 on July 1, 2020. Results: The data collection took place in October 2020, obtaining 7 responses from jurors with experience ranging from 4 to 30 years; 3 specialists, 2 masters, and 2 PhDs. In instrument 1, 7 items were not validated, namely: 2 on the Intellectual Aspect and 5 on the Human/Sensitive Aspect; in instrument 2, only 1 item on Pertinence. After changes, a new consultation was made, and 7 responses were obtained, validating all items of the instruments. When calculating the CVI of the instrument (CVI-I), the comprehensiveness, clarity and pertinence were: instrument 1 = 0.99, 1 and 0.97 and; instrument 2 = 1, 0.97 and 1. The validation occurred in Portuguese, not being translated so that there would be no reduction in its characteristics. The simultaneous use of the instruments is mandatory. Some limitations: reduced access to hospitals/universities by COVID-19 and no testing with the public. Conclusion: The validated instruments can be used to evaluate learning through case studies in cardiology. With the interpretation of the results of the instruments, the teachers will be able to recognize the weaknesses and form strategies to overcome them, besides being used in actions of permanent education. 110895 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS VISAKHA REVANA IRAWAN1, Frans Wantania2, Janry Pangemanan2 (1) Department of Internal Medicine, Faculty of Medicine, Sam Ratulangi University/Prof. dr. R. D. Kandou General Hospital, Manado, Indonesia; (2) Division of Cardiology, Department of Internal Medicine, Faculty of Medicine, Sam Ratulangi University/Prof. dr. R. D. Kandou General Hospital, Manado, Indonesia Background: The neutrophil-to-lymphocyte ratio (NLR) in peripheral blood has recently been able to act as a biomarker that can predict worse clinical conditions ranging from infectious diseases to cardiovascular diseases. Objective: The study aimed to determine the optimal cutoff level of NLR level to predict in-hospital mortality in acute myocardial infarction (AMI) patients. Methods: We analyzed the data characteristics of 332 consecutive cases of AMI patients admitted in Prof. dr. R. D. Kandou General Hospital, retrospectively, from September 2020 to December 2021. Patients with acute infections, HIV/AIDS, who used corticosteroids in the last three months, and antibiotics in the last 24 hours were excluded. NLR was computed from the absolute values of neutrophils and lymphocytes from the complete blood count (CBC) on hospital admission. Furthermore, the optimal cutoff level of NLR was analyzed using ROC curves to discriminate survivors versus non-survivors during hospitalization. Results: The value of NLR in predicting in-hospital death (AUC = 0.88, p = 0.0001) was higher than the platelet-lymphocyte ratio (PLR) (AUC = 0.79, p = 0.0001), the neutrophil-monocyte ratio (NMR) (AUC = 0.61, p = 0.075), and the lymphocyte-monocyte ratio (LMR) (AUC = 0.17, p = 0.0001). The optimum cutoff NLR level to predict in-hospital mortality was 7.36, with a sensitivity 72% and a specificity of 90.55%. Patients with NLR levels ≥7.36 had a higher probability of mortality when comparing with those who with NLR levels <7.36 (OR = 24.65, 95% CI (9.5–63.94), p = 0.0001). Conclusions: NLR could be an early and important predictor of in-hospital mortality in AMI patients. 111347 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GAUTAM SATHEESH1, Rupasvi Dhurjati1, Abdul Salam2 (1) The George Institute for Global Health, Hyderabad, India; (2) The George Institute for Global Health, University of New South Wales, Sydney, New South Wales, Australia Introduction: Globally, there are large gaps in the diagnosis, awareness, treatment, and control of hypertension. Using evidence-based standard treatment protocols (STPs) may improve hypertension treatment and control. Objective: 1. To summarize major guidelines recommendations for the pharmacological management of hypertension. 2. To identify, characterize and compare available hypertension STPs with the major guidelines. Methods: We summarized recommendations on the pharmacological management of hypertension from four major hypertension guidelines: American Heart Association, European Society of Cardiology, World Health Organization (WHO), and International Society for Hypertension. STPs were identified from websites of WHO, Resolve to Save Lives, Pan American Health Organization and through expert consultations. We defined hypertension STP as series of steps recommended for treating hypertension with information on target patient group (age, comorbidities, race etc.), blood pressure (BP) thresholds for treatment initiation and intensification, BP targets, drugs/classes for each step, follow-up frequency, target time for BP control, and combination therapy/single-pill combinations. Results: All guidelines, except American, presented a stepwise treatment approach and recommended initiation with single-pill combinations in most patients. All guidelines recommended renin-angiotensin-system inhibitors (RASi), thiazide-type diuretics (TZD), or calcium channel blockers as first-line therapy. Except WHO, no guidelines specified individual drugs or doses. Further, WHO and American guidelines did not mention a target time for BP control. All identified STPs (n = 36) presented a stepwise approach, and 86% (n = 31) consisted of 5 or more steps. Majority (86%) recommended calcium channel blockers as initial therapy, and RASi were the second choice in 69%. TZD was introduced as a third line agent in 72% of STPs, second line in 14%, and first line in 11%. Only eight STPs recommended initiation with combination therapy, and only two recommended single-pill combinations. Contradictory to guidelines, 39% of STPs recommended dose-increase if desired BP control is not achieved. No STPs mentioned a target time for BP control. Conclusion: Several deviations from the major guideline recommendations were observed in the identified STPs. Developing optimal, evidence-based, and context-specific STPs is vital to control hypertension, especially in countries with limited resources. 111165 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FERNANDO SARAIVA CONEGLIAN1, Fernando Saraiva Coneglian1, Flávio Henrique Valicelli1, Fernanda Fernandes Souza1, Ajith Kumar Sankarankutty1, Wilson Marques Júnior1, Caroline Lavigne Moreira1, Minna Moreira Dias Romano1, Pedro Manoel Marques Garibaldi1, Rodrigo Tocantins Calado de Saloma Rodrigues1, Marcus Vinicius Simões1 (1) Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto Introduction: Liver transplantation (LT) is an alternative treatment for patients with familial amyloidotic polyneuropathy (FAP), correctly renamed hereditary transthyretin (hATTR) amyloidosis, and is associated with a significant improvement in survival. However, some patients may exhibit progression of the disease latelly, with eventual cardiac involvement. There are few reports on the most common cardiac manifestation in these cases. Objective: To describe the clinical evolution and cardiac involvement in patients who underwent liver transplantation for hATTR in a single interdisciplinary Amyloidosis center. Methods: Observational study of cohort with 8 patients undergoing LT after the diagnosis of hATTR, considering cardiovascular symptoms, ECG, Echocardiogram and therapeutic management. Results: All 8 patients had a diagnosis of hATTR with early-onset V30M mutation, 6 were male (75%). The mean follow-up after LT was 13.5 + 3.9 years and the age at the moment of evaluation was 49.3 + 6.2 years. Cardiovascular manifestations occurred in 4 patients (50%), corresponding in all cases to symptomatic bradycardia with Sinus Node Disease (SND) and need for permanent pacemaker (PP) implantation, with the mean period of cardiovascular manifestation being 14 years. All patients with DNS exhibited symptoms of severe autonomic neuropathy with symptomatic postural hypotension. Only 2 patients(25%) developed echocardiographic alterations compatible with myocardial infiltration by CA and significant increase in the thickness of the interventricular septum (13–15 mm), 1 patient have developed manifestations of heart failure (HF) that started 16 years after LT. This same patient had DNS and PP implant 4 years before the onset of HF. Conclusion: Our results indicate a high prevalence of late development of cardiac organic involvement after liver transplantation in patients with hATTR due to V30M mutation. The preferred form of manifestation of heart disease is symptomatic bradycardia by SND requiring PP implantation. There seems to be an association between the intensity of autonomic denervation and SND, suggesting a pathophysiological link between these abnormalities. 110900 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT SHEILA TATSUMI KIMURA MEDORIMA1, Daniela Camargo Oliveira1, Íkaro Soares Santos Breder1, Vaneza Lira Waldow Wolf1, Luiz Sérgio Fernandes de Carvalho1, Alexandre Anderson de Sousa Munhoz Soares1, Joaquim de Paula Barreto Fonseca Antunes de Oliveira1, Daniel Batista Munhoz1, Jessica da Silva Cunha Breder1, Wilson Nadruz Junior1, Thiago Quinaglia Araújo Costa Silva1, Andrei Carvalho Sposito1 (1) Atherosclerosis and Vascular Biology Laboratory (Atherolab), Cardiology Department, State University of Campinas (Unicamp), Campinas, SP, Brazil. Dapagliflozin significantly reduced cardiovascular death and heart failure endpoints, however sulfonylureas and metformin are the only oral antidiabetics available in Brazilian Primary Care Public Health System. This trial was planned to evaluate their effect on diastolic function and to investigate possible mechanisms underlying this change. Methods: The Assessment of Dapagliflozin effect on Diabetic Endothelial Dysfunction of brachial Artery – Brazilian Heart Study 2 trial is an investigator-initiated prospective clinical trial, single-center, active-controlled, open-label, phase-4 randomized trial. High cardiovascular risk patients with type 2 diabetes were randomized to 12-week treatment with Dapagliflozin (DAPA) or Glibenclamide (GLIB) on top of Metformin, in glucose-lowering equivalent regimens. Echocardiogram was performed at randomization and at 12 weeks. Serum nitrate was analyzed by NO chemiluminescence analyzer. Results: 97 patients completed the study, including 61% males, median age 59(10) years, mean LVEF 62.9% ± 8. After 12weeks, E/e’ratio difference significantly reduced in DAPA group, while increased in GLIB arm [-0.17(1.9) vs 0.87(2.3), p = 0.001]. Tissue Doppler e’ velocities did not change significantly, but E velocity increased with GLIB [0(19.7) vs 4.0(16.0), p = 0.016]. LV mass and LV septum reduced in DAPA group, although without statistical significance. Exploratory secondary analysis was performed with clinical variables tested in multivariable binary logistic regression analysis, with E/e’ ratio improvement as dependent variable. Nitrate raising was an independent predictor of diastolic function improvement (B = 1.098; p = 0.024; Exp(B) = 2.999), adjusted for age, sex, body surface area and randomization group. This multivariable model classifies correctly 70,1% of the patients (model R-squared = 0,27; p-value < 0,001). Conclusion: Dapagliflozin significantly improves diastolic function by reducing E/e’ ratio, while Glibenclamide worsens. In our study, E/e’ improvement was significantly predicted by serum nitrate raising after 12-weeks, offering important clues on the pathophysiology evolved in SGLT2 inhibitors myocardium protective effect. 110947 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION ANABEL LIMA VIEIRA1, Renan Figueiredo de Freitas1, Maria Beatriz Siqueira de Araújo4, Sérgio José Siqueira de Araújo3, Ândrea Virginia Ferreira Chaves2 (1) Hospital Agamenon Magalhães, Recife, PE; (2) Serviço de Referência em Doenças Raras – RARUS, Recife, PE; (3) UniNassau, Recife, PE; (4) Faculdade Pernambucana de Saude, Recife, PE Introduction: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease (1:200). The development of genetic panels for cardiomyopathies has allowed for the diagnosis of phenocopies, the receipt of adequate genetic counseling and, most importantly, the identification of diseases with specific treatment. Objective: To present the genetic characterization of a group of patients (pts) with an echocardiographic diagnosis of HCM. Methods: This was a cross-sectional observational study that looked at pts who had been diagnosed with HCM and had a molecular panel for HCM. The genes analyzed were cardiac α-actin, desmin, filamin C, alpha galactosidase A, lysosomal-associated membrane protein 2, myosin-binding protein C, cardiac β-myosin heavy chain, myosin light chain 2, myosin 3, myopaladin, phospholamban, Protein kinase A γ subunit, non-receptor tyrosine-protein phosphatase type 11, troponin C1, troponin I3, troponin T2, α-tropomyosin 1 and transthyretin. Results: The panel was performed on 25 pts with HCM. The age ranged from 13 to 91 years, mean 39.3 years. Regarding gender, 52% were women. Variants were found in cardiac β-myosin heavy chain genes (sarcomeric, chromosome 14, autosomal dominant and recessive) in ten pts (40%), filamin C in four pts (16%), myosin-binding protein C in three pts (12%), desmin in two pts (8%), myosin light chain 2 in one pts (4%), α-tropomyosin 1 (TPM1) in one pts (4%), troponin T2 in one pts (4%), non-receptor tyrosine-protein phosphatase type 11 (Noonan syndrome – phenocopy, chromosome 12, autosomal dominant) in a pts (4%), lysosome-associated membrane protein 2 (Danon’s disease – phenocopy, X chromosome) in a pts (4%), alpha galactosidase A (Fabry’s disease – phenocopy, X chromosome) in one pts (4%). In one pts two mutations were found and in another three concomitant mutations. In one pts, no mutation was identified. Conclusion: The molecular panel was used to provide adequate genetic counseling to the pts. Three pts with HCM related phenocopies (Noonan syndrome, Danon’s disease, and Fabry’s disease) were identified. The diagnosis of Fabry disease allowed for the recommendation of specific treatment, which had a direct impact on the natural history of this pts and their families. Because the panel used was limited to 18 genes, no mutations were found in three pts. Expanded panels improve the probability of genotype identification and the possibility of effective therapy. 110957 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOGERIATRICS ANABEL LIMA VIEIRA1, Marina Souto da Cunha Brendel Braga1, Amanda Valério Galindo1, Caio Correia da Silva1, Rafael Buarque de Macedo Gadelha1, Matheus Dantas Soeiro2, Sabrina Barreto Braga Pires2, Jessica Myrian de Amorim Garcia1, Francisco Alfredo Bandeira e Farias1 (1) Hospital Agamenon Magalhães, Recife, PE; (2) Faculdade Pernambucana de Saúde, Recife, PE; (3) Universidade de Pernambuco, Recife, PE Introduction: Thyroid hormones have both direct and indirect effects on cardiac cells, so both excess and deficiency will influence the cardiovascular system. Atrial fibrillation (AF) is one of the cardiac manifestations of thyroid dysfunction (TD), particularly hyperthyroidism, and arrhythmia with clinical significance is more common in the context of thyroid diseases. Objectives: To assess the relationship between thyroid dysfunction and the presence of atrial fibrillation in elderly patients with heart failure. Methodology: An observational cross-sectional study. The medical records of 118 patients 65 years old admitted to the cardiology ward with HF from August 2020 to October 2021 were analyzed. For qualitative variable comparisons, SPSS v.25.0 was used, and the assumed significance level was 5%; for quantitative variables AF × TD, the chi-square test was used. Results: There is a mean age of 72.8 years (ranging from 65 to 96 years), with the majority of patients being male (54.2%). There are 36 patients (30.5%) with TD present. 47.2% of these patients are male, 97% have Systemic Arterial Hypertension, 46.9% have Diabetes Mellitus (DM), and 25% are pre-DM. Nonetheless, 38.9% have HFrEF, 19.4% have HFrEFi, and 41.7% have HFrEFp, with an average LVEF of 48.16% (standard deviation: 16.8); 5.7% have coronary artery disease, and 26.3% have a history of stroke. In the TD group, 32 patients (88.88%) had subclinical hypothyroidism, while 4 (11.11%) had clinical hypothyroidism. In this group, the mean thyroid-stimulating hormone (TSH) level was 11.26, with (standard deviation: 13.69). 12 patients (33.3%) in the TD group had AF. Only 11 (13.4%) of the non-DT group had AF. The mean TSH in AF patients was 8.50, with a standard deviation of +–14.7, compared to 4.03, with a standard deviation of +–6.17 in non-AF patients (t(116) = 2.267; p = 0.013). The comparison of TD and AF revealed a close relationship between the two variables: 10.2% of the elderly with TD had AF, while only 9.3% of the sample (69.5%) did not have TD (Chi-Square = 6.325a; p = 0.012). Conclusions: According to the study sample, there is a statistically significant relationship between thyroid dysfunction and atrial fibrillation in elderly HF patients, corroborating what has previously been established in the literature. Among the possible thyroid dysfunctions, this study found a stronger link between AF and hypothyroidism, both clinical and subclinical. 110972 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY RITA CASSIANA MICHELON2, Caroline Engster da Silva2, Ludimila Silveira Parker Lopes1, Claudia Costamilan Paes2, Fernanda Lucchese1 (1) Santa Casa de Misericórdia de Porto Alegre (ISCMPA)/Hospital da Criança Santo Antônio (HCSA); (2) Instituto de Cardiologia/Fundação Universitária de Cardiologia (IC/FUC) Introduction: Congenital Heart Disease (CHD) has a prevalence of about 1 case per 100 live births nationwide in Brazil. Infants with CHD are at greater risk of having developmental delays. In 2012, the American Heart Association (AHA) proposed guidelines for the evaluation and intervention of neurodevelopmental delays in infants with CHD. Multidisciplinary early evaluation and interventions can significantly improve the course of the development of a child with CHD, preventing neurodevelopmental sequelae and increasing quality of life, adaptive skills, and academic and professional achievements later in life. Objective: To present data on the neurodevelopment of infants with CHD participating in a multidisciplinary research clinic seeking to develop low-cost early intervention protocols in Brazil. Method: Fifty-nine infants with CHD under the age of 42 months were recruited from a convenience sample at the Pediatric Cardiology Outpatient Clinic of a reference hospital in Southern Brazil. The information was gathered through parental questionnaires. Infant development was measured by the Bayley-III Infant and Toddler Development Scale. Results: Of the 59 infants recruited, there were 17 drop-outs due to non adherence, schedule incompatibility, fatigue during the assessment or death. Forty-two infants (28 female, at the average age of 10 months). Forty-three infants who underwent surgery had on average 2 procedures, ranging between 1–5 surgeries. Sixty-seven percent of the infants were cyanotic. The average length of hospital stay was 31 days (X–XX range), and eight were preemies. The developmental findings showed that 41% of infants were borderline or delayed in the Cognitive, 31% in Receptive Language, 29% in Expressive Language 29%, 36% in Fine Motor, and 67% in the Gross Motor domains. Conclusion: This study showed high rates of developmental delay in infants with CHD from a public (SUS) outpatient clinic in Southern Brazil. In particular, almost 70% of the sample was at risk or had a delay in gross motor development. These data are congruent with previous research that have shown developmental delays in infants with CHD, in particular gross motor deficits. This evidence corroborates with AHA’s guidelines, and supports the need for multidisciplinary early evaluation and intervention to prevent neurodevelopmental sequelae in low-income infants with CHD in Brazil. 111366 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING MARÍLIA MIGUEL DA SILVEIRA1, Sérgio Augusto Yukio Hissayassu1, Isabela Ramos Ali Ganem1, Lívia Hirayama1, Raíza Silveira da Costa1, Carlos Alexandre Vieira Gomes1, Tiago Augusto Magalhães1, Adriano Camargo de Castro Carneiro1, Carlos Eduardo Elias dos Prazeres1, Juliana Matsumoto Bello1, Valéria de Melo Moreira2, Carlos Eduardo Rochitte1 (1) Hospital do Coração (Hcor); (2) Instituto do Coração (InCor), Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo Background: Cardiovascular complications in Covid-19 is prognostic. Myocarditis and pericarditis can be precisely detected by cardiovascular magnetic resonance (CMR) and present early and/or later (Covid-19 long). Myocardial and pericardial evaluation by CMR in routine clinical practice is scarce. Therefore, we sought to investigate cardiac abnormalities by advanced CMR including subtle tissue chances in myocardium and pericardium. Methods: We included 303 consecutive patients with confirmed prior Covid-19 diagnosis referred to CMR, performed in a 1.5T scanner (Sola CV edition, Siemens Healthineers) with cine-MR, late gadolinium enhancement (LGE), T1(MOLLI), T2 maps and extracellular volume measurements (ECV) using standard parameters. Early-CMR or late-CMR if CMR was defined as before or after 60 days of the infection, respectively. Results: From the 303 patients, 120 (39.9%) were female, mean age was 47.6 ± 13.8 years and mean body mass index of 27.7 ± 4.9. Symptoms were present in 87 patients (28.7%). Mean interval between Covid-19 infection and CMR study was 155.0 ± 138.6 days for the entire study group, with 78 patients (30.3%) with early-CMR. Myocardial LGE (mLGE) was present in 60 patients (19.9%), with 14.1% in early-CMR and 21.8% in late-CMR (p > 0.05). However, pericardial LGE (pLGE) was more frequent in early-CMR than late-CMR (9.0 vs. 2.8%, p = 0.049). The presence of mLGE was similar between symptomatic vs asymptomatic patients (17.7 vs. 20.8%, p > 0.05). Patients with risk factors for coronary artery disease (including obesity) had significantly more mLGE than those without (26.1 vs. 15.9%, p = 0.039). Native T1 and T2 values were higher in patients with CAD (1041 ± 31 vs. 1005 ± 3.8 and 46.7 ± 2.2 vs 44.3 ± 0.3, p = 0.024 and 0.039, respectively), but ECV was similar. No differences were seen in maps and ECV values between early and late-CMR. Thus, mLGE has similar distribution for symptomatic/asymptomatic and early/late phases after infection. However, pLGE was significantly more frequent in the first 60 days after infection. CAD patients had higher T1/T2 myocardial maps. Conclusions: CMR was able to detect subtle myocardial and pericardial changes in patients after Covid-19. Myocardial involvement was similar regardless of symptoms and time after infection, while pericardial involvement was detected more frequently before 60 days of the infection. This might indicate that pericardial involvement is more transient than myocardial involvement. 110983 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY PEDRO HENRIQUE ALMEIDA WAROL 1, PEDRO HENRIQUE ALMEIDA WAROL1, TALLES AYRES LIMA2, ROMULO RODRIGUES BADINI3, NAJLLA DE SALIM RIBEIRO4, MARIANNA RAMALHO DE SOUSA1, EMILIO PANDELÓ LIMA1, EMILIO CONCEIÇÃO DE SIQUEIRA1 (1) UNIVERSIDADE DE VASSOURAS; (2) UNIVERSIDADE ESTACIO DE SÁ; (3) UNIVERSIDADE UNIGRANRIO; (4) FACULDADE DE MEDICINA DE PETRÓPOLIS Cardiovascular diseases (CVD) are the leading cause of death in Brazil. Among such diseases, there are Conduction Disorders and Cardiac Arrhythmias (CBD), electrical changes in the heart that cause changes in its normal rhythm. Even though it is configured as a public health problem, there is a scarcity in the literature regarding the epidemiological aspects of hospitalizations and deaths of this disease, and its analysis is essential in order to prepare health teams to deal with BED. A systematic review of the literature was carried out together with the descriptive, cross-sectional and observational collection of data available in DATASUS – Hospital Information System of the SUS (SIH/SUS) – from 2017 to 2021, evaluating hospitalizations, deaths and pattern of patients: age group, sex and self-reported color/race. In the analyzed period, there were 316,939 hospitalizations for BED with a total expense of 1,391,918,003.70 reais, with 47.5% of these hospitalizations in the Southeast region. The most affected sex was male, with 52.5% of hospitalized cases. The age group with the most hospitalizations was over 50 years old, with emphasis on the 70 to 79 years old group, which corresponded to 25.8% of the total. When analyzing color, white was the most affected with 45.7% of the total, however 20% of hospitalizations did not have this information. As for deaths, in the analyzed period, there were 40,193, with a predominance of males, which corresponded to 54.3% of the total deaths from BED. The age group with the highest mortality was over 60 years, with emphasis on individuals aged 80 years and over, who accounted for 24.8% of deaths. There was an increase in mortality, which in 2017 was 6,854 and in 2021 it reached 8,664. TCACs represent a serious public health problem with high morbidity and mortality. Because of this, and the increase in mortality expressed in the years analyzed by the research, a new dynamics in health is essential. This dynamic must be guided by prevention and health promotion, with effective monitoring carried out by Primary Health Care in association with the care provided by the cardiologist, in order to avoid the development and worsening of these diseases. 111004 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY DANIEL DE MAGALHÃES FREITAS1, Artur Henrique de Souza1, João Alberto Pansani1, Max Weyler Nery1, Patrícia Ferreira Demuner1, Stanlley de Oliveira Loyola1, Maurício Lopes Prudente1, Débora Rodrigues1, Larissa Xavier Alves de Oliveira1, Fernando Araújo Cintra Canedo1, Giulliano Gardenghi1 (1) Hospital Encore Introduction: The feasibility of minimally invasive mitral valve surgery (MIMVS) has been proven extensively and many centers around the world have been using this technique as a standard approach. Some advantages of the minimally invasive technique, compared to the traditional median sternotomy, are the blood loss reduction, lower morbidity, and shorter intensive care unit and in-hospital stay. Objective: In this study we analyzed the results of MIMVS performed at our institution in the year of 2021 and compare with data from the international literature. Methods: 13 patients were submitted to MIMVS. At our institution, a femoral cannulation is utilized to establish cardiopulmonary bypass; and the incision was made at third or fourth intercostal space starting at the mid-subclavicular line with a length of 5–7 cm. The myocardial protection is done with cold-blood cardioplegia. Data were collected from the patients records and tabulated and analyzed in the Excel software®. We used Mitral Valve Academic Consortium safety definitions for comparison purposes. Results: From the 13 cases, 11 (84,6%) were valve replacement and 2 were of valve repair. Age mean was 59,6 years (±14); 9 (69,2%) were at low risk by STS score, 3 were at medium risk (23,2%) and 1 (7,6%) at high risk. The 30-day complications analysis are shown in table 1. Conclusion: Our results are consistent with the world literature corroborating the feasibility and safety of the technique. 111020 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION NAJAH KHAN1, Bindu Chebrolu1, Nadeen N. Faza1 (1) Houston Methodist Hospital Introduction: Twitter influencers exchange information and stimulate discussions from a breadth of expertise. Cardiovascular imaging with echocardiography (ECHO), cardiac computed tomography (CCTA), single-photon and positron emission tomography (SPECT and PET), and cardiac magnetic resonance imaging (CMR) has gained momentum through social media. Objectives: To analyze demographics of top Twitter influencers and imaging modalities they discuss on Twitter. Methods: Twitter topic scores for “cardiac imaging” (CCTA, CMR, SPECT, and PET) and “echocardiography” were collated for individual accounts on Feb 6, 2022 using the Cronycle monitoring platform (integrated with the Right Relevance application programming interface), generating top 75 influencers. Cardiovascular board certifications were confirmed via country-specific board-certifying organizations. Occupation, gender, location, and imaging modalities discussed by influencers were confirmed via Twitter biographies. Influencers’ academic influence (h-indices) from Scopus were correlated to their topic scores. Results: Majority influencers were male (73%) and board-certified cardiologists (87%), followed by non-cardiovascular physicians (9%), and non-physicians (4%). The most discussed imaging modality was echocardiography by 40% of top influencers, followed by CMR (28%), CCTA (23%), and Nuclear/PET imaging (9%). Most influencers reside in the United States (61%) followed by the United Kingdom (17%), Canada (4%), Italy and Portugal (3%), and other countries (Spain, Romania, Serbia, Peru, Germany, Chile, Columbia, Brazil, and Australia, 1% each). There was a positive correlation between the h-index and Twitter topic score (r = 0.3562, p = 0.0009). Conclusions: While most top influencers in cardiac imaging are board-certified male cardiologists in the United States, other influencers consist of international cardiologists, non-cardiovascular physicians, and non-physicians. There is a vast global representation of cardiac imaging influencers, showcasing the reach and expansive growth of the field. These findings emphasize the diverse representation of cardiac imaging influencers on Twitter, hopefully changing the landscape of this field. 111022 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION DANIEL BATISTA CONCIEÇÃO DOS SANTOS2, Luís Henrique Wolff Gowdak2, José Jayme Galvão de Lima2, Luiz Aparecido Bortolotto2 (1) Universidade de São Paulo; (2) Instituto do Coração Introduction: There is a need of a simple, inexpensive and reliable noninvasive testing to predict coronary artery disease (CAD) in CKD patients, a population with the higher prevalence of CV events and death. Object: This study analyzed the association between peripheral arterial disease (PAD) and CAD in patients with stage 5 CKD on dialysis. Methods: This was a prospective and observational cohort study in 201 CKD patients on hemodialysis. PAD was defined by either the absence of peripheral arterial pulse by palpation or by ≥50% narrowing by ultra sound Doppler (USD) and CAD by angiography (≥70% narrowing). Results: Palpation and USD predicted the occurrence of PAD (P = 0.0001, OR = 27.37). CAD was observed in 45% of patients with PAD (by palpation) and in 60% by USD. Absence of pulse by palpation predicted CAD with a sensitivity of 55% and a specificity of 76%; USD showed a sensitivity of 62% and specificity of 60%. The risk of combined serious CV events and death was significantly higher in individuals with PAD diagnosed by palpation but not by USD. PAD assessed by palpation also correlated with the occurrence of multivessel CAD and with the probability of coronary intervention. Conclusion: There is a high prevalence of CAD in patients with PAD. Palpation may be used instead of USD for the diagnosis of PAD because is inexpensive and easier to use. Both methods presented a moderate utility to predict CAD but the diagnosis of PAD by palpation was a predictor of combined cardiovascular events and death and was also associated with the severity of CAD and the probability of indication of coronary intervention. 111043 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SHEILA TATSUMI KIMURA MEDORIMA1, Eduardo Monteiro Diniz Junqueira1, Ana Cristina Seródio1, Maria Fernanda Murijo Righi Turatti1, Fernanda Melo Gomes1, Carolina Rosa Queiroz Fulas1, Valeria Cristina Jodjahn Figueiredo1 (1) Municipal Health Department (SMS-Campinas), Prefeitura Municipal de Campinas Introduction: The COVID-19 pandemic required special management of health services, once outpatients visits were suspended in most units during the lockdown. In Campinas-SP, since 2006, cardiology matrix support is acknowledged by primary health care as a service to case discussion, EKG evaluation and shared care. Since 2019, our team offers telecardiology through institutional e-mail, which acquired great importance during Pandemics. Objective: Evaluate the content of these e-mails, quantifying diagnostic groups with priority risk classification. Methods: This is a cross-sectional observational study, using data from service registry produced by cardiologists while evaluating cases received from e-mail. This research has been approved by ethics committee. Results: In a total of 2224 cases discussed along 2021, 22.8% had missing data, 10.8% were high risk CAD or unstable CAD, 9.4% had acute HF symptoms, 6.4% intermediate risk chest pain, 5.5% structural chronic heart disease, 4.7% paroxysmal supra ventricular tachycardia and 4.1% had abnormal heart murmur. There were 1089 patients (49% of the cases) classified as priority. More priority cases were in the following diagnostic groups: acute atrial fibrillation (AF) (90.2% priorities), high risk CAD or unstable CAD (90.0% priorities), thromboembolic event (87.5% priorities), syncope (78.0% priorities), post-COVID cardiovascular complications (72.0% priorities), structural chronic heart disease (74.6% priorities), acute HF symptoms (72.7% priorities) and chronic AF (69.2% priorities). Conclusion: Telecardiology in Public Health System of Campinas-SP was a successful experience during the COVID-19 pandemics, allowing to evaluate clinical criteria to guide priority risk classification for cardiology consultations, ensuring equity. 111049 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SHEILA TATSUMI KIMURA MEDORIMA1, Eduardo Monteiro Diniz Junqueira1, Ana Cristina Seródio1, Maria Fernanda Murijo Righi Turatti1, Fernanda Melo Gomes1, Carolina Rosa Queiroz Fulas1, Valeria Cristina Jodjahn Figueiredo1 (1) Municipal Health Department (SMS-Campinas), Prefeitura Municipal de Campinas Cardiology matrix support (CMS) is offered to Primary Care services since 2006 in Campinas-SP, allowing closer relationship with the specialists. In 2019, telecardiology was the method used to expand this proposal, through the institutional e-mail for case discussion, clinical orientation, receiving ECG requiring reports and risk classification for cardiology consultation. This method allowed Specialized Care to meet the needs of Primary Care during the COVID-19 Pandemic, allowed priority access to urgent cases and contributed to continued education of Primary Care in Cardiology. Objective: Characterize the CMS performed in Brazilian Public Health System (SUS), Campinas-SP. Methods: This is a cross-sectional observational study with data registered by cardiologists while evaluating clinical cases discussed via e-mail. This research has been approved by ethics committee. Results: In a total of 2224 cases discussed along 2021, the median age was 61 years, most female (N = 1136,51%). A total of 1089(49%) was classified as priority, 538(24%) as intermediate and 46(2%) as low risk. There were 546(24%) referrals with incomplete data, 381(17%) had insufficient data, 809(36%) proposed shared patient care instead of referring the patient to the specialist, 797(36%) received orientation regarding therapeutic adjustments or complementary investigation. The CSM offered cardiology consultation for 477(21%), echocardiography for 303(14%) and exercise testing for 110(5%). Conclusion: Telecardiology was consolidated in Specialized Care at the SUS in Campinas-SP during the COVID-19 Pandemic, allowing quick access from Primary Care to cardiology support. This data will contribute with the analys of the impact of this format of Specialized Care and for future improvements. 111054 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION EMMA SOKOLOVA1, Aleksandra Jeņičeka2, Ieva Bikava3 (1) Riga Stradins University, University of Latvia, RISEBA, Riga East University Hospital, Pauls Stradins Clinical University Hospital; (2) Riga Stradins University, RISEBA; (3) Riga Stradins University Introduction: Available data on the doctor-patient remote consultations all over the world shows that those are mostly provided by radiologists, psychiatrists and cardiologists. The regulatory framework of each country, the procedure for organizing and payment procedure for the specialists, the legal aspects and the complexity of the medical sector create specific conditions and the need to develop a model and procedure for providing remote consultation appropriate to each country and institution. Objectives: The aim of the study was to find out, summarize and analyze the experience of state paid remote consultation of cardiologists in Latvia during the year 2021, identify problems and develop recommendations for safe and effective remote specialist-patient and specialist-specialist consultation in cardiology. Methods: The study was conducted through semi-structured interviews with cardiologists of PSCUH, data provided by NHS of Latvia and Specialized Medicine Center (unit of State EMS). Results: The total number of state provided teleconsultations was 2 884 632, from them only 2194 (0,8%) patient-cardiologist consultations were conducted, which for 1,9 billion of Latvian population is a very low indicator. Remote patient-cardiologist consultations remained under 0,1% – compared to consultations of doctors of other specialities. The majority of cardiologists indicated that the first consultations could not be performed remotely over the phone. At the specialist-specialist level, the demand is stable, with around 1,100 consultations each year and accounting for about 20–25% of the total number of remote consultations. Conclusions: The main obstacles to remote visits, especially for first-time visits, are related to two problems: 1) the specialist does not have access to all examinations and results of the patient, and 2) it is not possible to make an objective assessment of the patient. A video call is recommended for remote consultations, as it is able to provide more additional information and patient evaluation options. Patient monitoring through remote consultations would be possible if visits are sufficiently regular (every 3 or 6 months), the patient is compliant, the patient’s condition has not significantly deteriorated, and the necessary examinations and analyzes have been performed and are available to the doctor. At present, procedures have not been developed and defined at the national level for the implementation of telephone consultations. 111073 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DOMINIQUE VERVOORT1, Rachel Livergant2, Maryam Shams3, Niraj S Kumar4, Sameer Hirji5, Bobby Yanagawa1 (1) Division of Cardiac Surgery, University of Toronto; (2) Office of Global Surgery, Department of Surgery, University of Alberta; (3) Department of Medicine, Stanford University; (4) Department of Medicine, University College London Medical School; (5) Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School Introduction: Rheumatic heart disease (RHD) affects over 30 million people globally and remains one of the most underfunded conditions in global health relative to its disease burden. The lack of access to primary, secondary, and tertiary prevention and care for the majority of the world’s population contributes to these disparities and is a result of a poor understanding of the socioeconomic value of interventions to prevent and manage RHD. Here, we evaluate the current literature to assess economic evaluations of the prevention, screening, diagnosis, and medical and surgical management of acute rheumatic fever (ARF) and RHD. Objective: The objective of this study is to identify the current knowledge and gaps surrounding the cost-of-illness of ARF/RHD and the cost-effectiveness and cost-utility of prevention or care for ARF/RHD. Methods: A systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines using the PubMed/MEDLINE, EMBASE, Web of Science, World Health Organization Global Index Medicus, and EconLit databases to identify original research on economic evaluations of ARF and RHD interventions between January 2002 and December 2021. Variables extracted included bibliometric data, study country income group (high- versus low- and middle-income countries, LMICs), type of economic evaluation, measures of effectiveness, type of interventions, economic outcomes, and the use of decision analysis. Results: Of 3,980 screened articles, 23 were included in the final analysis. Most studies (56.5%, N = 13) took place in LMICs. Primary prevention and care were most commonly studied (52.2%, N = 12), followed by tertiary care (39.1%, N = 9). Cost-effectiveness and cost-utility analyses (65.2%, N = 15) were the most common type of economic evaluation, followed by cost-of-illness studies (34.8%, N = 8). Effectiveness was most commonly measured as quality-adjusted life-years (30.4%, N = 7) and disability-adjusted life-years (17.4%, N = 4). Most studies (78.3%, N = 18) used decision analysis models. Conclusion: The economic burden due to ARF/RHD is large in LMICs, but economic evaluations in LMICs remain limited. ARF/RHD interventions appear consistently but variably effective and cost-effective to prevent and treat ARF/RHD. Increased research, political prioritization, and public health and healthcare funding are needed to address the global burden of ARF/RHD. 111082 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LISAKOVA ANASTASIIA1, Maria Simonenko1, Petr Fedotov1, Larisa Vasilieva1, Yulia Sidorovskaya2, Olga Tarasova1, Svetlana Bagramian1, Aelita Berezina1, Maria Bortsova1, Oleg Mamontov1, Yulia Levashkevich1, Elena Demchenko1 (1) Almazov National Medical Research Centre; (2) Saint-Petersburg state university (SPbU) Introduction: Cognitive impairment and difficulties in coping with negative conditions slows down the rehabilitation. The post-transplant quality of life affects rehabilitation and social adaptation. Objective: To evaluate the dynamics of cognitive and psycho-emotional characteristics of patients with end-stage heart failure before and after heart transplantation (HTx) as a prerequisite for optimization of psychological correction during cardiac rehabilitation. Methods: Study included 60 end-stage heart failure patients (from 07.2019 to 06.2021), 30 of them (mean age–46.9 ± 13.3 yo; 57.1% – male) were examined on pre-HTx and during 6 mo after HTx (1st period – 1–3 mo and 2nd – 4–6 mo), others were excluded due to the various reasons (i.e. still awaiting HTx, death during early-term posttransplant follow-up, etc.). For patients’ examination we used Short Form-36 (SF-36), Luria Memory Words Test, Simple analogies method (for the cognitive sphere) and Integrative Anxiety Test (A. Bizyuk et al.) – the tests validated in Russia. Statistical analysis was performed using Student’s t-tests and repeated measures ANOVA. Results: At the pre-HTx examination the level of long-term memory was lower than at the 3rd mo after HTx, but decreased by the 6th mo (5.5 ± 1.8; 7.8 ± 1.7; 7.3 ± 1.6, p = 0.013 for trends) Social Functioning was decreasing during 6 mo (50.2 –± 7.6; 41.7 ± 6.1 (3 mo); 38.1 ± 10.9 (6 mo), p = 0.011 for trend). Role-Physical Functioning increased to the 3rd mo and dropped to the 6th mo (18.9 ± 17,4; 47.3 ± 40.5 (3 mo); 39.3 ± 34.9 (6 mo), p = 0.01). Mental Health improved to the 2nd period (73.1 ± 15.8 vs 57.1 ± 14.9 pre-HTx, p = 0.011). Physical Functioning increased to the both periods: 30.0 ± 19.1; 68.6 ± 20.4 (3mo); 69.4 ± 22.3 (6 mo), p = 0.004 for trend. General situational anxiety leveled up to the 3rd mo but decreased to the 6th mo (4.5 ± 1.9; 5.9 ± 2.1; 2.6 ± 2.1, p = 0.013 for trend), whereas asthenic component dropped in both periods (6.1 ± 1.9; 4.1 ± 2.5; 3.4 ± 2.5, p = 0.014), alarming assessment of future decreased to the 3rd mo (6.2 ± 2.4; 4.7 ± 2.8, p = 0.046). There were no statistically significant differences in the Simple analogies method. Conclusion: The results can be used for psychological support within rehabilitation of HTx patients. Situational anxiety increased to the 3rd mo, thus, psychocorrection of psycho-emotional states is crucial in 1–3 mo after HTx. Social functioning was dropping in 6 mo after HTx, so psychological rehabilitation is necessary in the 4–6 months after HTx. 111092 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH RICARDO MENDES CARNEIRO1, Eric Costa de Almeida1, Bruno Reznik Wajsbrot1, Daniel Xavier de Britto Setta1, Marcelo Luiz da Silva Bandeira1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Roberta Siuffo Schneider1, Claudia Lanzillotti Weksler1, Andre Volschan1, Fernando Oswaldo Dias Rangel1, Ricardo Mourilhe-Rocha1 (1) HOSPITAL PRÓ-CARDÍACO Introduction: Atrial fibrillation (AF) is an increasingly prevalent condition, with an estimate of almost 60 million people worldwide and 2.5% of the Brazilian population, according to data from the ELSA-BRASIL study, which was not exclusively composed by patients hospitalized for AF. Knowing the clinical profile, cardioembolic and hemorrhagic risk allows personalizing the therapeutic approach in different scenarios. Objective: To identify the clinical and epidemiological profile of patients hospitalized with AF in a quaternary hospital. Methods: Retrospective cohort study of 819 hospitalized patients with a confirmed diagnosis of AF, from January 2018 to December 2021. Data were analyzed using the SPSS20 software. Results: 57.6% of the patients were male, with a mean age of 71 ± 14.4 years. They had the following risk profiles: CHADS2 ≥2 in 79%; CHA2DS2VASc ≥3 in 63.8%; age >65 years in 72.6%, systemic arterial hypertension in 66.4%, heart failure in 12%, diabetes mellitus in 21.7%, stroke or transient ischemic attack in 8.2%, coronary artery bypass graft surgery previous in 8.6%and previous coronary artery disease in 21.6%. The median of CHA2DS2VASc was 3, and the median of HASBLED was 2. There was reversion to sinus rhythm in 68.3% of the cases. Of these, 52.5% underwent electrical cardioversion (EVC), 22.5% chemical cardioversion and 25% had spontaneous reversion. Regarding oral anticoagulants administered during hospitalization, most used apixaban (25.6%) and rivaroxaban (12%) and only 1.1% had contraindications for anticoagulation. Regarding antiarrhythmics used during hospitalization, amiodarone (29.1%) was the most prescribed one and, of the heart rate control medications, beta-blockers (50.2%) were the most important ones. Complication rates were low, with only 3 embolic (0.4%), 10 (1.2%) hemorrhagic, and 7 (0.9%) deaths. Conclusion: Despite being a population of high-risk AF patients with a high mean age and CHA2DS2VASc ≥3, only a few complications occurred. CVE was the method of choice for reversion to sinus rhythm and the most used oral anticoagulant was apixaban. The knowledge of this profile can allow the development of specific care plans for this population. 111097 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ERIC COSTA DE ALMEIDA1, Ricardo Mendes Carneiro1, Daniel Xavier de Britto Setta1, Julia Paulo Mourilhe Rocha1, Thiago Matos Barcellos1, Flávia Prado Fialho Santos1, Bruno Reznik Wajsbrot1, Roberta Siuffo Schneider1, Vitor Hugo Mussi Campos1, Andre Volschan1, Fernando Oswaldo Dias Rangel1, Ricardo Mourilhe-Rocha1 (1) HOSPITAL PRÓ-CARDÍACO Introduction: Strategies aimed at reversing atrial fibrillation (AF) to sinus rhythm aim at better symptomatic control and quality of life. Knowing the clinical factors associated with greater success in rhythm reversal remains a challenge. Objective: To assess failure related factors to revert to sinus rhythm in patients with AF. Methods: Retrospective cohort study of 819 patients hospitalized with diagnosis and AF from January 2018 to December 2021. The parameters analyzed were age, BNP, left atrial size, creatinine and left ventricular ejection fraction (LVEF). Data were analyzed by SPSS 20 software. Results: 57.6% of the patients included were male, with a mean age of 71 ± 14.4 years. They had the following risk profiles: CHADS2 ≥2 in 79%; CHA2DS2VASc ≥3 in 63.8%; age >65 years in 72.6%, systemic arterial hypertension in 66.4%, heart failure in 12%, diabetes mellitus in 21.7%, stroke or transient ischemic attack in 8.2%, coronary artery bypass graft surgery previous in 8.6%, previous coronary artery disease in 21.6%. The CHA2DS2VASc median was 3, and the HASBLED median was 2. There was reversion to sinus rhythm in 68.3% of the cases. From the comparison between patients discharged in sinus rhythm and those who remained in AF, the profile of patients who were unsuccessful in reversal were older (mean 70.23 ± 14.6 vs 73.95 ± 13.83), had higher BNP (median 216 pg/ml vs 515 pg/ml; p < 0.001), higher creatinine (median 1.0 mg/dl vs 1.1 mg/dl; p < 0.023), median LVEF of 64.0% vs 62.0%, p < 0.332; and higher left atrial volumes (median 41 ml/m2 vs 43 ml/m2; p < 0.013) Conclusion: The parameters associated with failure to revert to sinus rhythm were age, BNP, creatinine and left atrial volume. The understanding of this association may allow the elaboration of failure prediction scores in the reversal of patients with AF. 111099 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FILIPPE BARCELLOS FILIPPINI1, Cauyna Gurgel Moreira1, Tiago Costa Bignoto1, Maurício Felippi de Sá Marchi1, Gabriel Kanhouche1, Antônio Fernando Diniz Freire1, Pedro Felipe Gomes Nicz1, Renata de Sá Cassar1, Henrique Barbosa Ribeiro1, Alexandre Abizaid1, Santiago Raul Arrieta1, Fábio Sandoli de Brito Jr.1 (1) InCor – Instituto do Coração do Hospital das Clínicas da FMUSP Aims: Tricuspid bioprosthesis deterioration is a frequent condition and its treatment (Redo tricuspid valve replacement) is complex and associated with high mortality. Percutaneous tricuspid valve-in-valve implantation (TViV) presents as a safe alternative with favorable outcomes. We aim to describe the pilot experience of TViV procedures. Methods and Results: From June 2015 to August 2022, 12 patients were submitted to TViV procedure in one tertiary-care Brazilian hospital. The median age was 30.0 ± 13.1 years old, 50% of patients with previous infective endocarditis and the number of previous cardiac surgery was 3.2 ± 1.5 per patient. The majority of patients had severe heart failure symptoms (NYHA III or IV in 50%) and the congenital disease involved were tetralogy of fallot or Ebstein anomaly in most cases (66.7%). The mean time since last tricuspid valve procedure was 9.5 years, most of the prosthesis were Braile (33%). The principal mechanism of bioprosthetic valve failure (BVF) was regurgitation in 66% and combined prosthesis dysfunction in 75%. Preprocedural echocardiography identified moderate or severe right ventricular systolic dysfunction in 7 (58%), mean tricuspid gradient of 10.4 mmHg and pulmonary arterial systolic pressure of 34 mmHg. The transcatheter heart valve (THV) prosthesis used was either Inovare (Braile) in 10 (83%) or Sapien 3 (Edwards) in 2 (17%), with sizes 24, 26 or 28 mm equally distributed in 9 (75%) of the cases. Balloon pre-dilatation was used in 7 (58%) and post-dilatation of the THV in 2 (17%). Intraprocedural echocardiographic evaluation of mean tricuspid gradient was 3.5 mmHg with no moderate or severe paravalvular leak. Overall technical success was obtained in 11/12 (92%) of the procedures, due to change in access site from jugular vein to transapical approach in one single case. Mean length of stay was 12 ± 5.4 days, with 67% of patients in NYHA I at discharge. No patient died during the median long term follow-up of 29 months, the antithrombotic regimen was vitamin K antagonist in 10 (83%) and late bioprosthesis thrombosis was observed in 1 (8.3%) case during the follow up, in the context of concomitant covid infection. One year echocardiographic evaluation of mean tricuspid gradient was 7.7 ± 2.4 mmHg and 1 case with moderate paravalvular tricuspid leak. Conclusion: The pilot experience of tricuspid valve-in-valve implantation demonstrated safety, effectiveness and marked improvement of symptoms at mid-term follow-up. 111112 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MASHAAL IKRAM1, Mashaal Ikram1, Mina Kerolos2, Mingxi Yu3, Kim Allan Williams2, Max Liebo3 (1) University of Chicago (Northshore University Health System); (2) Rush University Medical Center; (3) Loyola University Medical Center Background: Food choices have been identified as the leading cause of mortality in the U.S., underpinning many chronic diseases, including cardiovascular disease, diabetes mellitus and cancers. During the SARS-CoV-2 pandemic, nutrition quality, specifically plant-based and Mediterranean diets, have been found to be highly protective from moderate to severe COVID-19 illness. In addition, these diets have been promoted by the 2019 ACC/AHA Primary Prevention Guidelines to reduce cardiovascular disease, which remains the leading cause of death of physicians. Objective: We sought to identify the nutritional habits of medical students and practitioners in two Chicago area academic medical centers. The primary aim of the study was to correlate dietary habits that could correlate with the symptomatic response and infection rates of both SARS-CoV-2 virus and its vaccine. Methods: Medical students, residents, fellows and faculty from Loyola and Rush University Medical Centers with exposure to SARS-CoV-2 patients, and who had received two doses of vaccinations were asked to complete a web-based survey between September 2021 to February 2022. Participants provided their dietary habits, any history of SARS-CoV-2 infection, and occurrence of symptoms after either of their two COVID-19 vaccinations. Results: Of the 274 respondents, 14% indicated that they had contracted COVID-19 prior to the survey. Only 18.% had moderate or severe symptoms with the vaccine. The dietary habits, however, indicated that 72% were eating an unrestricted diet (omnivore), 15% were categorized as semi-vegetarian (intentionally reducing, but not eliminating, animal products), 5% consumed seafood, eggs and dairy, but no red meat, 4% ate eggs and dairy without seafood, 4% ate seafood without eggs or dairy, and 0.3% ate no animal products (figure). Given the relative paucity of categorical dietary diversity, there was no analyzable correlation between viral or vaccine symptoms and nutritional habits. Conclusions: This data indicates that very few (11%) of the surveyed physicians follow ACC/AHA Prevention nutritional recommendations, which would lower both cardiovascular and COVID-19 morbidity and mortality. 111121 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION LEILA DAL POGGETTO MOREIRA1, Tuany Mageste Limongi Zamperlim2, Giulia Micali1, Camila Martins1, Higor Quintas Gonçalves1, Thamyres Oliva Bueno1, Pricila Helena de Souza1, Rodrigo Moreno de Oliveira1, Giovanna Dolder1, Luciana Gonzalez Viscardi Auad1, Adriana Sarmento de Oliveira1 (1) Universidade Anhembi Morumbi UAM; (2) Universidade Federal de Juiz de Fora Background: Impairment of cardiac autonomic modulation during the aging process contributes to the development of cardiovascular events, the leading cause of death in the elderly population, and may indicate that the elderly with frailty syndrome have a poor outcome when compared to other elderly. Objectives: To evaluate the cardiac autonomic response to isometric exercise in comparison to baseline and recovery. Methods: After screening, 54 frail elderly (F) and 12 non-frail elderly (NF). Briefly, using Fried’s criteria subject frailty was defined using: handgrip strength, walking speed, unintentional weight loss, report of exhaustion and level of physical activity. The Polar V800 was used to record the RR interval, which was later evaluated by the Kubois HRV Standard. The isometric exercise protocol was performed at 30% of the maximum handgrip strength for 3 minutes. Where appropriate, the Student’s t-test or the Mann Whitney U test was used to compare differences between two independent groups. Statistical significance was set at p < 0.05. Results: The F group has a greater value in the LFun index that is associated with cardiac sympathetic and parasympathetic activity at baseline than the NF group [F:55.3 ± 9.7 vs NF:48.8 ± 7.4; p < 0.02], greater LF/HF value [F:1.2 (0.9–1.5) vs NF:0,9 (0,7–1,2); p < 0.02] and lower HFun index value that is related to cardiac parasympathetic activity [F:44.6 ± 9.7 vs NF:50.9 ± 7.2; p < 0.03]. In the recovery measure, the NF group presented higher values in HFun (REC:40.4 ± 6.0 vs HAND:50.6 ± 12.98; p < 0.05). Additionally, it was observed that at the handgrip moment, group F presented a higher LFun value (HAND:59.7 ± 10.8 vs BAS:55.3 ± 9.7); p < 0.02] and a lower HFun value (HAND:40.1 ± 10.8 vs BAS: 44.60 ± 9.77; p < 0.02). On the other hand, still in the F group, a low HFms index was noted during handgrip [REC:153.5 (61–339) vs HAND:93.9 (57–197); p = 0.02]. For the NF group, the handgrip measurement had higher LFun (HAND:59.4 ± 6.1 vs BAS:48.8 ± 7.4; p < 0.00) and LF/HF [(HAND: 1.73 (1.37–1.98) vs. BAS: 0.99 (0.77–1.20); p < 0.01] and lower for the HFnu index (HAND:40.4 ± 6.0 vs BAS: 50.9 ± 7.2; p < 0.00). There were differences between groups for the HFun index [F:44.6 ± 9.7 vs NF:50.9 ± 7.2); p < 0.03], and the LF index (F:55.3 ± 9.7 vs NF:48.8 ± 7.4) p < 0.03] that were low and high in the F group, respectively. Conclusion: Sympathetic activity is increased and cardiac parasympathetic activity is reduced in the frail elderly at baseline, implying greater cardiovascular risk. 111429 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DANIELI CRISTINA PIGOZZO1, Edson Lessa2, José Rocha Faria Neto2, Ana Caroline Dariva Chula2, Marcia Olandoski2 (1) Hospital Santa Casa de Curitiba; (2) Pontifícia Universidade Católica do Paraná Background: Many countries have shown a decrease in the demand for medical care and an increase in out-of-hospital deaths (EH) caused by cardiovascular diseases (CVD) during the COVID-19 pandemic period. Objectives: To analyze possible differences in the temporal trend of EH deaths and hospital admissions for CVD in the city of Curitiba during the period of the COVID-19 pandemic. Methods: Retrospective, observational, time series study. Individuals admitted in Brazil’s Unified Health System network in Curitiba with a diagnosis coded by ICDs I20–I24 and I60–I64 were included. The number of admissions was correlated with the number of EH deaths from CVD and with the number of new COVID-19 cases. The period analyzed was from January 2019 to August 2021. The descriptive analysis was made through graphics. Results: There were 289941 new cases of COVID-19, 13446 hospital admissions and 2336 EH deaths due to CVD. There was a decrease in admissions in the early period of the pandemic (4352 versus 3736 March-December 2019 and 2020) at the expense of fewer admissions for AMI (3307 versus 2435), while those for stroke increased (1044 versus 1301). EH deaths increased mainly from unspecific cardiovascular causes (UCVC). From March to December 2020 this increase was 71% (185 versus 318) compared to the same period in 2019. Conclusion: This study suggests that the COVID-19 pandemic had a negative impact on the evolution of cardiovascular diseases, through a transient reduction in the number of hospital admissions and an increase in the number of EH deaths from CVD. These at the expense of higher mortality from UCVC. 111129 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ALINE GEHLEN FERRARI1, Raul Cavalcante Maranhão1, Rocío Salsoso1, Vanessa Maria Gomes Taques Fonseca Baldo1, Gabriela Liberato1, Remo Holanda de Mendonça Furtado1, Talia Falcão Dalçóquio1, Luciano Moreira Baracioli1, Aleksandra Tiemi Morikawa1, Cesar Higa Nomura1, Thauany Martins Tavoni1, José Carlos Nicolau1 (1) Instituto do Coração – Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (InCor-HCFMUSP) Background: Inflammation is very important in the pathophysiology of ST-elevation acute myocardial infarction (STEMI), with special role in left ventricular (LV) remodeling. Methotrexate (MTX) is a potent anti-inflammatory drug with a potential benefit in the treatment of STEMI. A formulation of MTX incorporated into lipid nanoparticles (LDE), LDE-MTX, tested in rats with induced myocardial infarction, reduced by 50% the infarct size and improved LV function, without observable toxicity. Purpose: To evaluate effects and safety of LDE-MTX treatment on LV remodeling and infarct size in patients with STEMI. Methods: Randomized, double-blinded, placebo-controlled, proof of concept study. Patients were randomized within 4 ± 2 days after STEMI to receive LDE-MTX (40 mg/m2, intravenous) or LDE-placebo weekly for 6 weeks. The efficacy endpoints were change between 90 ± 7 days and baseline, in LV end-diastolic and end-systolic volumes, LV ejection fraction, LV mass and infarct size, measured by cardiac magnetic resonance. The main safety endpoints were serious adverse events and incidence of hematological, renal, and liver dysfunction. Results: Thirty-five patients were randomized (18 to LDE-MTX, 17 to LDE-placebo). Two patients in the LDE-placebo group and one in the LDE-MTX group refused to continue the protocol and were excluded from the final analysis. The main efficacy results are shown in Table 1. There were no significant differences between the groups regarding safety parameters. Conclusions: In patients with STEMI, LDE-MTX resulted in higher reduction in infarct size and lower LV mass loss, despite no differences in LV volumes. No safety issues were observed. These results encourage further clinical studies on this novel Nanomedicine approach for the treatment of patients with STEMI. 111130 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY EMMA SOKOLOVA1, Ainārs Rudzītis3, Oskars Kalējs2 (1) Riga Stradins University, University of Latvia, Riga East University Hospital, Pauls Stradins Clinical University Hospital; (2) Riga Stradins University, Pauls Stradins Clinical University Hospital; (3) University of Latvia, Pauls Stradins Clinical University Hospital Introduction: The improvement in cardiac inotropic function after restoration of sinus rhythm in patients with persistent atrial fibrillation (AF) is very important. Previous studies shows that antiarrhythmic class Ic medications are decreasing inotropic function of the heart. Objectives: To investigate safety and efficacy parameters of widely used antiarrhythmic Ic class medication in patients with atrial fibrillation. Methods: 70 patients with persistent AF underwent bicycle ergometer stress test, rest ECG, 24-hours Holter monitoring, echocardiography and blood tests within 1 day and 4 weeks (±7 days) following electrical cardioversion. We gathered also information considering the quality of life. The investigation protocol included testing with the baseline 100 mg/day aethacizinum treatment. Results: We observed increase in bicycle stress test time from a mean 5,42 minutes to 6,05 minutes (p = 0,005). During Holter monitoring in n = 4 patients (5,7%) atrial fibrillation was observed. Echocardiography showed improve of systolic function from a mean 50% to 55% (p = 0,005), no worsening of diastolic function was observed. There was no significant changes in ECG parameters. Conclusions: No proarrhythmic events were observed during the study. Medication was well tolerated, proved to have high antiarrhythmic efficacy and safety in patients with persistent atrial fibrillation, having no negative inotropic effect on myocardium. 111163 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MARIA GORDEEVA1, Irina Serdiukova2, Alexander Krasichkov2, Elena Parmon1 (1) Federal State Budgetary Institution “Almazov National Medical Research Centre” of the Ministry of Health of the Russian Federation; (2) Saint Petersburg Electrotechnical University The key investigation for the assessment of the left ventricular ejection fraction (EF) is echocardiography, however, this method is not a screening method, especially in individuals with mildly reduced ejection fraction (mrEF). At the same time, an ECG is performed in almost all patients with suspected cardiovascular disease and as part of a preventive examination. It is known that traditional ECG-pattern associated with a decrease in EF have a low diagnostic value. Recently, new ECG-patterns associated with depolarization abnormality (fragmentation of the QRS complex (fQRS) and early repolarization pattern (ERP)) have been actively studied. The aim of study is investigated ECG-pattern of depolarization abnormalities (fQRS and ERP) in patients with a mrEF. Materials and methods: The study included 148 patients with ischemic and non-ischemic cardiomyopathy. According to the level of EF, patients were divided into three groups: patients with low EF (lEF) (less than 40%): 31 (25 men, mean age 52.0 +/–15.6); patients with mrEF (49%–40%): 29 (23 men, mean age 54.7 +/– 12.4); patients with preserved EF (pEF) (more than 50%): 88 (57 men, mean age 58.2 +/–12.0) – control group. We used the criteria by Das M. et al, 2006 to identify fQRS and the criteria by Macfarlane P.W. et al., 2015 to identify ERP. Results: In the 1st group (lEF), fQRS was registered in 16 (51.6%) patients, in the 2nd group (mrEF) – in 13 (44.8%), in the 3d (pEF) in 2 (13.6%), p < 0.001. ERP in the 1st group (lEF) was registered in 2 (6.5%), in the 2nd group (mrEF) – in 2 (6.9%), in the 3d group (pEF) ERP – in 11 (12.5%), p = 0,5. As a result of the ROC analysis, a relationship was found between fQRS and an intermediate decrease EF (40–49%). Conclusions: The greatest difficulty for the early detection of heart failure is the group of patients with a mrEF. FQRS has shown its predictive value in identifying these patients. This ECG-pattern must be analyzed to assess the risk of heart failure. 111179 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY AMANDA VANESSA DEMARCHI1, Luciana Vidal Armaganijan1, Dalmo Antonio Ribeiro Moreira1, Mariane Higa Shinzato1, Kelvin Henrique Vilalva1, Pablo Santos Graffiti1, Rodrigo Augusto de Miranda Bertin1, Murilo Amato David1, Mathias Antonio Haruno de Vilhena1, Guilherme Dagostin de Carvalho1 (1) Instituto Dante Pazzanese de Cardiologia (IDPC) Introduction: The CHA2DS2-VASc score has been used as a predictor of cardiovascular outcomes even in the absence of atrial fibrillation (AF). Several P wave indexes and echocardiographic parameters are associated with a higher risk of developing AF and thromboembolism; however, there are few studies in patients without AF. Objectives: Primary: Evaluate the association between P wave indexes [P wave duration, dispersion and variability, maximum and minimum P wave duration, P wave voltage in lead I, Morris index, PR interval (PRI), P/PRI ratio and P wave peak time] and CHA2DS2-VASc SCORE in patients without AF and valve disease. Secondary: To assess the relation between echocardiographic parameters [left atrium (LA) and left ventricle (LV) size, LV ejection fraction (LVEF), LV mass and LV indexed mass] and CHA2DS2-VASC score in the same population. Methods: A cross-sectional, descriptive and analytical study in which clinical, electrocardiographic and echocardiographic data from 272 patients without AF and valve disease were collected and analyzed. For statistical analysis, the Chi-Square Test, Mann-Whitney U-Test and Spearman Correlation were used with the significance level of 5%. Results: PRI, LA and LV diameter, LV mass and LV indexed mass were positively associated with CHA2DS2-VASc SCORE, while P wave amplitude, P wave voltage in lead I and LVEF were negatively associated to the same score (Table). The presence of the Morris index was related with high CHA2DS2-VASc. Conclusion: The study of the association between P wave indexes, echocardiographic parameters and CHA2DS2-VASc score may be useful in clinical practice to evaluate patients who trend to have a higher cardiovascular risk using clinical parameters and non-invasive methods in patients without AF. 111181 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM YURI CAVALCANTI ALBUQUERQUE TENORIO1, Claudia Patricia da Silva Gois2, Lamark Melo Silva Moreira2, Stephanny Isabelly Pessôa Neri de Araujo2, Igor Vieira Lima Alexandre2, Priscila Alves da Silva2, Larine Ferreira Lira3, Edecio Galindo de Albuquerque1, Antônio Everaldo Vitoriano de Araújo Filho2, Francisco de Assis Costa2 (1) Hospital Veredas; (2) Centro Universitário Tiradentes; (3) Centro Universitário Cesmac Introduction: Coronavirus disease (Covid-19) is a pandemic disease caused by SARS-CoV-2 virus first described in Wuhan (China) in 2019. This condition causes general inflammation leading to acute respiratory distress syndrome (ARDS) in severe cases, sometimes requiring mechanical ventilation. The aim of this study is to identify the impact of cardiac, renal and hepatic markers in mortality, hospital staying, development of ARDS, and necessity of mechanical ventilation (MV) in Covid-19 patients. Methods: Case-control study in a philanthropic hospital in the Brazilian state of Alagoas, selecting 103 hospitalized patients from 2020 to 2021 aged more than 18 years old and diagnosed with SARS-CoV- 2 infection. Results: Descriptive of the population analysed was 60.2% of male, 47.6% had hypertension, 26.2% had diabetes, 4.9% had cardiac heart failure, 34.9% had history of smoke, 19% had history of alcohol consumption and 2.5% had history of cancer. There was a positive risk between acute renal failure and death (OR = 2.97), similarly there was a positive risk of requiring MV (OR = 1.74), but there was no relation to hospital staying (p = .456). There was no relationship between hepatic injury and death (OR = .862) and hospital staying (p = .429). On the other hand, there was association to the necessity of MV (OR = 2.86). Finally, there was a relation between cardiac injury and death (OR 19.3) and MV (OR 1.38), but no relation in hospital staying (p = .801). Discussion and Conclusion: The study concluded that cardiac, renal and hepatic markers are related to severe Covid-19 requiring MV in the population analysed. There was association between renal and cardiac markers to death, but not hepatic markers to death. 111193 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES ISABELA GALIZZI FAÉ1, Pedro Henrique Oliveira Murta Pinto MD1, Fernando Crespo Torres2, Sofia Caporalli Barbosa2, Gustavo Brandão de Oliveira MD1, Rhuan Braga Oliveira2, Lucas Bretas de Pádua2, Teresa Cristina Abreu Ferrari, MD, PhD1, Maria do Carmo Pereira Nunes, MD, PhD1 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais; (2) Universidade Federal de Minas Gerais Introduction: Infective endocarditis (IE) is a rare disease associated with substantial mortality. In recent decades, the epidemiology of IE has been changing with an increased age of patients and a crescent number of health-care-acquired IE. However, few studies have examined the contemporary outcomes of IE in this growing population at risk of complications. Objective: The present study aimed to describe the incidence of in-hospital mortality over the past 2 decades and to identify the risk factors for early mortality in a cohort of patients with IE admitted to a Brazilian quaternary hospital. Methods: A total of 334 consecutive patients diagnosed with IE based on modified Duke criteria were prospectively included from 2001 to 2021. Data regarding predisposing baseline condition, laboratory findings, etiologic agents, treatment and in-hospital outcomes were analyzed. The primary endpoint was in-hospital death due to any complication related to IE. Results: The median age was 54 years, 60% men. Cardiac device and rheumatic heart disease were the most frequent predisposing conditions. During the treatment, 78% patients presented adverse events, including worsening of heart failure (HF) (34%), embolic events (13%) and the need for cardiac surgery (43%). The overall in-hospital mortality rate was 34.9% with no changing over this time. In multivariable analysis, the characteristics independently associated with death were embolic events, HF development, increasing age and high CRP levels (C statistic of the model 0.84). Conclusion: Despite recent advances, IE continues to be associated with high in-hospital mortality, without changes in the last 2 decades. Early identification of patients who are at high risk of death may offer an opportunity to improve outcomes in IE. 111215 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISABELLE MENDES RODRIGUES SALOMÃO1, Marye dos Santos Xavier Dias1, Luanna Damasceno Amaral de Sousa1, Alessandra Arnez Pacheco1, Larissa Guerra Cunha de Sousa1, Daniel Xavier de Brito Setta1, Julia Paulo Mourilhe Rocha1, Fernando Oswaldo Dias Rangel1, Ana Amaral Ferreira Dutra1, Louise Freire Luiz1, Claudia Lanzillotti Weksler1, Ricardo Mourilhe-Rocha1 (1) Hospital Pró-Cardíaco Introduction: Spontaneous coronary artery dissection (SCAD) is considered an uncommon cause of acute coronary syndrome (ACS), in which typical chest pain is the main manifestation, being more common in women ≤50 years old. It can be triggered by physical or emotional stress, female sex hormones, inflammatory disorders, fibromuscular dysplasia, and connective tissue disease. Objective: To assess the epidemiological profile of patients with acute myocardial infarction (AMI) secondary to SCAD. Methods: This is a retrospective case series study developed at a Quaternary Hospital in the city of Rio de Janeiro. The database and electronic medical records of 1.200 patients with ACS, admitted between July 2013 and February 2022, were analyzed, in which were selected 0,7% diagnosed with SCAD. Results: Of the 9 patients evaluated, all were female, with a median age of 58 years, 67% had arterial hypertension, 78% obesity, 78% anxiety, 44% smokers, 22% diabetes mellitus, 22% previous AMI and 22% with a positive family history for coronary artery disease. Most (78%) had typical chest pain on admission, with 89% Killip I, 78% non-ST segment elevation AMI, 67% with preserved global left ventricle (LV) systolic function. 11% with severe LV dysfunction and 56% with segmental alteration. Median ejection fraction of LV was 59%. All patients underwent coronary angiography (CAT) within 24 hours of admission, with 67% single-vessel involvement and 44% TIMI III. Conservative treatment, with ASA, clopidogrel, high-potency statin, ACEI and bisoprolol was chosen in 78% of the patients. 22% were treated with angioplasty with drug-eluting stent implantation due to unfavorable coronary anatomy. One patient with extensive and severe lesion of the anterior descending artery and another patient with severe lesion of the marginal artery. 33% of patients had pain recurrence and 22% of whom had recent readmissions due to angina. The length of hospital stay was 5 days. There were no in-hospital deaths. Conclusion: SCAD is more prevalent in females and may be associated with stress factors, smoking and obesity. The approach to patients follows the usual ACS protocol, but it must be considered that the dissection can be aggravated by CAT. Conservative treatment is recommended in most low to moderate risk cases. It is important to perform the differential diagnosis of chest pain in order to avoid underdiagnosis of SCAD. 111760 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES GUSTAVO NEVES DE ARAUJO2, Gustavo Neves de Araujo1, Fernando Luis Scolari3, Guilherme Pinheiro Machado3, Anderson Donelli Silveira3, Felipe Pereira Lima Marques3, Rafael Beltrame3, Andre Theobald3, Alan Pagnoncelli3, Rodrigo Vugman Wainstein3, Marco Vugman Wainstein3 (1) Imperial Hospital de Caridade; (2) Instituto de Cardiologia de Santa Catarina; (3) Hospital de Clinicas de Porto Alegre Background: The new SCAI Shock Classification encompasses patients with progressive severity from stage A (at risk) to stage E (Extremis), and early identification of stepping stages is essential to scaling therapy. Lung ultrasound (LUS) evaluates pulmonary congestion, which may be present even in the absence of other signs of overt shock. Our aim was to evaluate prognosis in patients with and without lung congestion evaluated by LUS among SCAI Shock stages. Methods: Cohort of STEMI patients treated with primary PCI in a tertiary center. LUS protocol consisted of 8 scanning zones performed at admission. SCAI shock classification was evaluated within 24h of admission. Primary outcome was in-hospital mortality. Results: We included 582 patients with mean age of 61 ± 12 years and 373 (64.1%) male. SCAI shock stage A was present in 361 (62%) patients, while 115 (19.8%) were class B, 44 (7.6%) class C, 58 (10%) class D, and 4 (0.7%) class E. In-hospital mortality in patients with SCAI Shock classification A-E was 1.4%, 14.4%, 48.9%, 63.8% and 50%, respectively. Among SCAI B patients, mortality in mild (0–3 positive sites) and severe congestion pattern (4–8 positive sites) were 8.8% and 22.2%, respectively. Mortality in SCAI C patients without of LUS congestion (14.3%) was similar to patients in SCAI B stage (p = 0.583). Mortality in SCAI D patients without of LUS congestion (50%) was similar to patients in SCAI C stage (p = 0.631). Conclusion: Among STEMI patients at risk for cardiogenishock, LUS reclassifies SCAI Shock Classification regarding mortality prediction. Absence of lung congestion was associated with one-step decrease of mortality rates in SCAI C and D patients. While this must be tested in larger cohorts, LUS should potentially be included in SCAI Classification. 111288 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ANDRÉ PINHEIRO ZYLBERMAN1, Lara Teles Alencar Duarte1, Allexa Gabriele Teixeira Feitosa1, Cláudia Bispo Martins-Santos1, Yasmin Juliany de Souza Figueiredo1, Cleovaldo Ribeiro Ferreira Júnior1, Edvaldo Victor Gois Oliveira1, Arthur Leite Lessa1, Octavio Morais Veloso1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe Introduction: The left bundle branch block (LBBB) is a degenerative condition of the cardiac conduction system that is easily diagnosed by an electrocardiogram. Its prevalence ranges from 0.2 to 1.1%, increasing with age. As it is a chronic illness, patients may also develop several associated pathologies that require careful investigation, including coronary disease. As a result, the determination of heart ischemia predictors takes its place as an important evaluation, especially considering the exercise test’s diagnostic difficulties in LBBB subjects. Objective: To determine the predictors of heart ischemia in LBBB patients undergoing physical stress. Methods: This is an observational, cross-sectional and analytical study. It was used a dataset of LBBB patients from a private institution, all of which underwent an exercise stress echocardiography. The selection process excludedindividuals with previous coronary events. A total of 15 variables were studied in relation to the test’s result, including: sex, age, body mass index, family background, diabetes mellitus, dyslipidemia, hypertension, obesity, smoking, aortic diameter (AO), left atrium diameter (LA), left atrium volume, left ventricular mass index, ejection fraction and diastolic function. Statistical analysis was performed by the chi-square test (X²) and by the student’s t-test, both considering p < 0,05 as significant. The software SPSS Statistics version 22.0 was used. Results: From the 252 patients included in the study, 115 (45.63%) were men and 137 (54.37%) were women. Ages ranged from 30 to 92 years old, with a mean of 64.07 (±10.93). From the results of the echocardiography, two groups can be clearly identified: positive (n = 64; 25.40%) and negative for ischemia (n = 188; 74.60%). Among the qualitative variables, analysis showed that the male sex was the only associated with heart ischemia (p = 0.01) in LBBB patients. However, among the quantitative variables, AO (p = 0.00), LA (p = 0.02) e left ventricular index (p = 0.02) had significant higher means in the ‘positive for ischemia’ group. Conclusion: Only four of the 15 possible predictors were significant in the present study. Sex was deemed the only important personal factor for the echocardiography’s positive result. However, considering heart measurements as possible predictors, LBBB patients with either a larger atrium or aort, or with a greater ventricular mass were more susceptible to ischemia while facing stress. 111299 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT TARCIO SADRAQUE GOMES AMORAS1, Giovana Salomão Melo2, João Simão de Melo Neto2, Christelaine Venzel Zaninotto1, Vitor Bruno Teixeira de Holanda1, Sheila Santos de Oliveira1, Luana da Silva Freitas1, Luana Santos Nunes1 (1) Division of Cardiology, Gaspar Vianna Clinical Hospital Foundation; (2) UPCEURG, Institute of Health Sciences, Federal University of Pará Introduction: Currently, one of the most common causes of aggravation in hospitalized patients with heart failure (HF) is the worsening of renal function. Approximately one-third of patients with acute HF may develop acute renal injury (AKI); this condition has recently been termed cardiorenal syndrome type 1 (CRS-1). Objectives: To identify predictors of CRS-1 and mortality in patients with acute HF in the Eastern Amazon. Methodology: This was an observational, case-control study in which descriptive and inferential analyses were performed. We included 183 patients with acute HF aged older than 18 years admitted from January 2017 to May 2021 at a public referral hospital in the department of nephrology and cardiology. Patients were categorized into two groups: G1, patients with SCR-1 (n = 72), and G2, patients without SCR-1 (n = 111). The prevalence of sociodemographic and clinical factors was determined for both the groups, and independent predictors of SCR-1 and mortality were identified using the logistic regression model. All statistical analyses were performed using SPSS Statistics for Windows, version 21.0. Results: Among 183 patients, 72 (39.3%) developed SCR-1 and 33 (16.6%) evolved with dialysis AKI. Prevalence of chronic kidney failure (CRF) (p = 0.003), uncontrolled hypertension (p = 0.005), non-adherence to food/water (p = 0.043), advanced age (p = 0.004) and elevation of systolic BP (p = 0.021) was observed in the group with SCR-1. Among the factors that contributed to the worsening of acute HF, only non-adherence to dietary and water intake guidelines was considered a predictor for the incidence of SCR-1 (p = 0.003; OR = 81.45; 95% CI = 4.38–1513.13). For in-hospital mortality, only AKI dependent on renal replacement therapy was considered a predictor for SCR-1 incidence (p = 0.010; OR = 15.5; 95% CI = 1.93–124.35). Conclusion: Non-adherence to dietary and water intake guidelines was considered a risk factor for SCR-1. As it is a modifiable factor, preventive actions in public health become relevant for minimizing the occurrence of SCR-1. 111335 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KHOLIKOVA AZIZA OYBEK1, Abdullaev Timur Atanazarovich1, Kholikova Aziza Oybek1, Tsoy Igor Arsenevich1 (1) Republican Specialized Scientific and Practical Medical Center for Cardiology Purpose of the study: Evaluation of the effect of sacubitril/valsartan on central hemodynamic parameters in patients with chronic kidney disease (CKD), chronic heart failure (CHF) and type 2 diabetes. Material and methods: Randomization included 42 patients with CHF FC class II–III according to NYHA, EF less than 40% with concomitant type 2 diabetes and CKD stages 3a–3b, mean age 50.4 ± 16.6 years. Every 3 months, all patients were determined the content of urea, serum creatinine, the content of potassium, sodium, magnesium and calcium in the blood, GFR, protein in the urine and blood pressure. CKD in history occurred in 100% of patients, arterial hypertension 90%, diabetes mellitus 100%. Previously, patients received standard baseline cholesterol therapy. After 3–4 days of observation, patients were switched to sacubitril/valsartan by standard dose titration. The follow-up period was 18 months. Results: Patients included in the observation who used sacubitril/valsartan were divided into 2 groups according to CKD stages: CKD group 1 GFR up to 45 ml/min/1.73 m2, GFR group 2 above 46 ml/min/1.73 m2 Analysis of integral hemodynamic parameters showed the following. The severity of the decrease in SBP, DBP and HR was greater and had statistical power in patients with eGFR ≤45 compared with the group of patients with eGFR >46 ml/min/1.73 m² (∆ SBP 16.9 ± 6.24 VS 10 ± 3.18 mm Hg, ∆ DBP 10.76 ± 3.48 VS 1.72 ± 2.1 mm Hg, ∆ HR 10.38 ± 4.02.24 VS 1.151 ± 3.99 bpm), respectively with an average intergroup t = 2.25; P = 0.03. Those. hemodynamic response was the best in the general group with eGFR ≤45 and was almost completely determined by patients with type 2 diabetes. DBP – 14 ± 4 (t = 2.298; P = 0.03); HR 8.4 ± 4.48 was significantly higher in the group with eGFR ≤45 than in the group with eGFR >46 SBP – 3.34 ± 4.32 (t = 0.701; P = 0.03); DBP – 2.5 ± 2.17 (t = 0.045; P = 0.8), heart rate 6.25 ± 4.47 (t = 1.423; P = 0.105). The content of urea, creatinine in blood serum did not change significantly in both groups (t1 = 0.439408; t2 = 0.220387). Conclusion: In patients with CHF FC II–III according to NYHA, with an EF less than 40% with concomitant type 2 diabetes, therapy with a neprilysin inhibitor/RAAS blockade was not accompanied by a noticeable decrease in hemodynamic parameters in patients with CKD C3a and C3b. 111350 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS THALES CARDOSO WHATELY1, Joao Gabriel Monteiro Junqueira1, Ana Salomé Eurico1, Ana Luiza Iannarella Lacerda1, Barbara Bezerra de Almeida1, Karen Sanae Takehara Vieira1, Mayara Bastos Souza1, Constantino Gonzales Salgado1, João Addison Pessoa1, Roberto Pozan1, Esmeralci Ferreira1 (1) Hospital Universitário Pedro Ernesto; (2) Universidade do Estado do Rio de Janeiro Introduction: COVID-19 is a predictor of mortality in acute coronary syndrome (ACS). In a university hospital, in patients with ACS treated by percutaneous coronary intervention (PCI), an increase in mortality and in-hospital complications was observed. In view of these observations and because there are no data related to PCIs in COVID-19, we developed this work. Objectives: The main objective was to assess in-hospital mortality in patients with and without COVID-19 infection and ACS undergoing urgent PCI. The secondary objective was to evaluate cause of mortality and the association with clinical presentations, risk factors and angiographic findings. Methods: It’s a comparative, retrospective and consecutive study. A total of 598 patients undergoing PCI in ACS were evaluated. Patients were divided into groups: GI with COVID-19 (N = 76) and GII without COVID-19 (N = 522). Results: The mean age was 62 years. The rate of diabetes, obesity, dyslipidemia, smoking, previous coronary artery disease and systemic arterial hypertension (SAH) were similar. SAH was the most frequent factor (GI = 68.8%; GII = 69.2%, p = 0.99). Renal failure and previous coronary artery bypass graft surgery were more prevalent in GI (11.7%, p = 0.005). ST-segment elevation myocardial infartcion (STEMI) was more frequent (GI = 57.1%; GII = 61.5%, p = 0.53), followed by non-STEMI (GI = 35.1%; GII = 25 .1%, p = 0.07) and unstable angina (GI = 6.5%; GII = 13.4%, p = 0.09). We observed a higher frequency of interventions in anterior descending artery (ADA) (GI = 42.9%; GII = 58.8%, p = 0.01), followed by right coronary artery (GI = 13.3%; GII = 34 .9%, p < 0.001) and circumflex artery (GI = 6.5%; GII = 16.9%, p = 0.02). Mortality was higher in GI (GI = 22.3%; GII = 3.6%, p < 0.001) related to severe acute respiratory syndrome (SARS) (GI = 47.0%; GII = 5.3%, p < 0.001). Cardiogenic shock was greater in GII (GI = 41.2%; GII = 78.9%, p < 0.001). Septic shock was similar in the groups (GI = 11.8%; GII = 10.6%, p = 0.9). In multivariate analysis, the presence of COVID, age, renal failure and the involvement of the ADA showed a positive and significant association with in-hospital death. Conclusion: Emergency care and hemodynamic assessment of ACS took on great importance in pandemic. This study proved that presence of COVID-19 was the main predictor of ACS mortality due to SARS and shock. In addition to COVID-19, mortality is related to age, ADA involvement, and renal dysfunction. 111351 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH THALES CARDOSO WHATELY1, João Gabriel Monteiro Junqueira1, Luciana Ludwig Nigri1, Cláudia da Silva Lunardi1, Ivana Picone Borges Aragão1, Sara Cristina Marques dos Santos1, Ana Salomé Eurico1, Esmeralci Ferreira1 (1) Hospital Universitário Pedro Ernesto; (2) Universidade do Estado do Rio de Janeiro Introduction: The hospital scenario in the state of Rio de Janeiro (ERJ) was overloaded before SARS-Cov-2. Associated with the collapse of the pandemic, there was a reduction in demand for medical services, especially in patients with cardiovascular disease (CVD). COVID-19 represents an additional risk to these individuals. Previous studies have shown lower rates of hospitalization and death from CVD, and an increase in the fatality rate in the pandemic. The lack of epidemiological data related to COVID-19 and mortality in the ERJ led to this study. Objective: Analyze hospital CVD mortality in the health regions of the ERJ in the pre- and per-pandemic period of COVID-19 in patients of the brazilian unified health system (UHS). Methods: Descriptive, population-based, retrospective epidemiological study, using the hospital information system database of the UHS informatics department. The periods evaluated were divided in pre-pandemic (2018 and 2019) and per-pandemic (2020 and 1st quarter of 2021). Mortality data were obtained from the International Code of Diseases, which took place in the ERJ, divided into: ischemic diseases (G1), heart failure and cardiogenic shock (G2), cardiac arrhythmia (G3) and myocarditis (G4). Hospitalizations decreased in the regions of Baixada Litorânea (21.21%), Center-South (14.50%), Middle Paraíba (20.86%), Metropolitan I (18.37%), Metropolitan II (5.32%), North (19.44%) and Serrana (16.38%), and increased in Ilha Grande Bay (7.02%) and Northwest (1.72%). There was a drop in mortality in the Center-South (5.07%), Middle Paraíba (25.38%), Metropolitan I (18.09%), Metropolitan II (2.40%), North (11.02%) and Serrana (11.75%) and increase in Ilha Grande Bay (11.32%) and Northwest (9.28%). Conclusion: Hospitalizations in the CVD groups were reduced, especially in G2 patients. There was a significant increase in mortality caused by myocarditis, probably related to the virus. The health regions with an increase in hospitalizations and mortality were Ilha Grande Bay and Northwest Rio de Janeiro. 111352 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY JOÃO LINS DE ARAÚJO NETO1, João Lins de Araújo Neto1, Eduardo Arrais Rocha1 (1) Universidade Federal do Ceará Background: Postoperative atrial fibrillation is the most common sustained arrhythmia after cardiac surgery and occurs in approximately 30% of postoperative patients. Because of the large amount of evidence recommending prophylactic treatment and lack of data indicating who should receive such treatment, this study aimed to develop a new predictive score for atrial fibrillation after cardiac surgery. Methods: This retrospective cohort study included 989 adult patients who underwent cardiac surgery, except for heart transplantation and implantation of a ventricular assist device. Patients with previous atrial fibrillation or those using amiodarone were excluded. The analyzed variables were subjected to a univariate analysis of the occurrence of postoperative atrial fibrillation and a multivariate analysis using logistic regression. Results: Statistically significant variables in the multivariate analysis were age ≥60 years (P < .001), left atrial enlargement according to echocardiography (P = .025), inotrope use within 24 hours after surgery (P = .002), and the need for reoperation within 24 hours after surgery (P = .016). The score comprises these four variables and has an accuracy of 77% for predicting outcomes. Scores ≥3 were related to a 34% risk of postoperative atrial fibrillation. Conclusions: The proposed score represents the disease pathophysiology well and will be useful in clinical practice. 111354 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE ALBERTO BARRETO GRIMALDI1, Gustavo Monnerat1, Dahienne Ferreira de Oliveira1, Fabio C S Nogueira3, Gilberto B Domont3, Helena Cramer Veiga Rey2, Jose Hamilton Matheus Nascimento1, Antonio Carlos Campos de Carvalho1, Leonardo Maciel1 (1) Instituto de Biofísica Carlos Chagas Filho (Federal University of Rio de Janeiro);; (2) Instituto Nacional de Cardiologia (INC/RJ); (3) Instituto de Química (Federal University of Rio de Janeiro) Remote ischemic preconditioning (RIPC) provides myocardial resistance to ischemia/reperfusion (I/R) injuries. The protection mechanism by RIPC is suggested to be mediated by humoral factors. However, the identity of these humoral factors remains enigmatic. We aimed to characterize and identify the humoral factors responsible for the cardioprotection induced by RIPC. Human volunteers signed a consent form and answered a health survey. The volunteers were submitted to RIPC protocol (3 cycles of 5 minutes of ischemia alternated with 5 minutes of reperfusion in the arms). The venous blood was collected before (Placebo plasma) and after RIPC (RIPC plasma). The human plasmas were fractionated in different molecular weight ranges and the cardioprotection was evaluated in isolated hearts of rats submitted to 30 minutes of ischemia and 120 minutes of reperfusion in an isolated heart apparatus. Mass spectrometry (MS) was performed in placebo and RIPC plasma. The fraction less than 10kDa from RIPC plasma reduced infarct size in 50% and induced hemodynamic recovery of hearts submitted to I/R compared to control. The fraction less than 10kDa from placebo plasma did not induce protection. Hearts perfused with the fraction greater than 10kDa or total RIPC plasma also had no cardioprotection. MS showed differences in the protein content, including higher content of adenosine and kininogen in quantitative analysis and the presence of 15 putative cardioprotective proteins in qualitative analysis in the RIPC plasma compared with placebo. The cardioprotective humoral factors are in the less than 10kDa fraction of RIPC plasma. Moreover, the cardioprotection by RIPC can be transferred between species. Adenosine, kininogen, and other 15 proteins could be responsible for the RIPC cardioprotection. 111362 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KHOLIKOVA AZIZA OYBEK1, Xudoyberganov Otabek1, Abdullaev Timur Atanazarovich1, Tsoy Igor Arsenevich1 (1) Republican Specialized Scientific and Practical Medical Center for Cardiology Objective: To study the features of electrocardiographic changes in patients with peripartum cardiomyopathy in comparison with female patients with dilated cardiomyopathy. Material and methods: ECG and HMECG data of 68 women with peripartum cardiomyopathy and 43 patients with dilated cardiomyopathy were examined. The frequency of occurrence of complete bundle branch block (PBBB) was analyzed. QS phenomenon, atrial fibrillation (AF), ventricular extrasystoles according to Lown’s gradation (PV), AV bdokady. Results: ECG analysis showed that complete blockade of the left bundle branch block (LBBB) occurred in patients with DCMP in 12 (27.9%) (χ2 = 7.061; p = 0.008) cases, and in group 1 in 6 (8.8%) cases, respectively. Blockade of the right bundle branch of His (RBBB) was detected in 1 (1.4%) (χ2 = 0.638; p = 0.008) patients from group 1 and was not detected in patients of group 2. A permanent form of atrial fibrillation was in 4 (9.3%) patients with DCMP, and in 1 (1.4%) (χ2 = 3.756; p = 0.053) cases in patients with PCMP (χ2 = 3.756; p = 0.053). Absence of R wave growth or QS complex in chest leads in 8 (12.7%) and 5 (11.6%) (χ2 = 0.000; p = 0.983) patients in groups 1 and 2. AV blockade of the 1st degree was recorded in 4 (5.9%) cases in group 1, in patients from group 2 AV blockade of the 1st degree in 14 (32.5%) cases (χ2 = 13.797; p = 0.001). Negative T wave in 24 (35.3%), group 2 DCMP in 8 (18.6%) (χ2 = 3.576; p = 0.059) cases. HMECG found that ventricular arrhythmias of grades 1 and 2 were 60 (88%) in PPCM, and high grades: paired VA – in 25 (36.8%) and 23 (53.4%) cases (χ2 = 3.002; p = 0.084), respectively, in groups 1 and 2; group VA – 11 (16.1%) and 14 (32.5%) (χ2 = 4.051; p = 0.045); unstable forms of VT – 6 (8.8%) and 9 (20.9%) (χ2 = 3.304; p = 0.070). A stable form of VT was recorded in one case in both groups. Conclusion: In DCM, PBLBBB and 1st degree AV block are significantly more common. At the same time, high grade ventricular extrasystoles and a negative T wave in the chest and standard leads are significantly more common in patients with PPCM. 111367 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KHOLIKOVA AZIZA OYBEK1, Abdullaev Timur Atanazarovich1, Tsoy Igor Arsenevich1, Mirzarakhimova Saodat Temurovna1, Bekbulatova Regina Shavkatovna1 (1) Republican Specialized Scientific and Practical Medical Center for Cardiology The aim: To study the clinical and functional features at patients with familial form of dilated cardiomyopathy. Material and Methods: We examined 47 females with the familial form of dilated cardiomyopathy compared with the same of idiopathic dilated cardiomyopathy (n = 50). Examination included: ECG Holter monitoring, echocardiography, test 6-minute walk distance (6MWT). Results: Disease duration was on average 9,4 ± 1,4 and 12,4 ± 1,4 months. (p > 0.05). The length of distance traveled during 6MWT was 264,5 ± 17,3 and 198,4 ± 11,4 m (p > 0.05). Patients with familial form of DCM disease has developed in a relatively early age 36,9 ± 1,6 years, which was significantly lower than in the comparison gr (42,4 ± 1,1 years, p < 0.01). It was shown that AV-blockade I degree was observed with the same frequency in the two grps (12.7% and 14.2%, p > 0.05), however, in the gr with the familial form of DCM in 5 (10.6%) were cases of complete atrioventricular block. Among patients with the familial form of DCM, the LBBB met less frequently (14.8% vs. 21.2%), and AF – more often (19.1% vs. 17.5%, both p > 0.05). The analysis of echocardiographic parameters showed that patients with familial DCM had lower values of ESD (5,6 ± 0, 1 and 6,2 ± 0,1 cm, p < 0.01) and EDD (6,9 ± 0,1 cm and 7,4 ± 0,1 cm, p < 0.01) and EF (37,8 ± 1,8 vs 31,1 ± 1,1%; p = 0.03) compared with patients with idiopathic dilated cardiomyopathy. Conclusions: This form of the disease is associated with younger age, the development of complete atrioventricular block and high rates of left ventricular ejection fraction. 111368 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES JULIO LEANDRO BOBADILLA1, Cristhian Emmanuel Scatularo1, Melisa Antoniolli1, Ezequiel Lerech1, Ignacio Manuel Cigalini1, Ezequiel José Zaidel1 (1) CONAREC, Consejo Argentino de Residentes de Cardiología Background: Pulmonary embolism (PE) current stratification includes intermediate-risk patients, in which the specific treatment is controversial yet. The aim of this study was to evaluate short term outcomes of intermediate-high risk patients that received different treatment strategies. Methods: We performed an analysis of a multicenter PE registry. Patients with intermediate-high risk (classification of the European Society of Cardiology) were selected, and clinical variables were stratified by type of treatment received (only antocoagulation or reperfusion with systemic trhombolysis, local thrombolysis, thrombus fragmentation-aspiration or surgical thrombectomy). Results: From 684 consecutive acute PE patients from 75 centres, 178 (26%) were classified as intermediate-high risk. Sixteen percent (n = 28) of this cohort received reperfusion treatment, either systemic thrombolysis (89%) or endovascular treatments, while the rest received only anticoagulation. On multivariable analysis, we found that patients receiving reperfusion strategies were younger (57 ± 17 years vs 68 ± 14; p < 0.001) and had more frequent bilateral PE (78% vs 43.7%, respectively; OR 4.72; 95% CI 1.8–12.3; p < 0.001). No significant differences were observed in total bleeding and major bleeding. 30 days absolute mortality was 3.6% in the reperfusion group and 14% in the non-reperfusion group (OR 0.22, 95% CI 0.02–1.76; p = 0.1). Conclusions: Comparing with anticoagulation only, the use of different reperfusion strategies for intermediate-high risk was safe and showed a trend to improved short-term outcomes, highlighting the need to develop a randomized clinical trial for this specific group of PE patients. 111370 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION JOSIANE APARECIDA DE ALMEIDA1, Ana Paula Delgado Bomtempo Batalha2, Isabela Coelho Ponciano1, Mariana Balbi Seixas2, Tiago Peçanha1, Patrícia Fernandes Trevizan3, Raquel Rodrigues Britto3, Lilian Pinto da Silva2 (1) Graduate Program in in Rehabilitation Sciences and Physical-Functional Performance Faculty of Physical Therapy, Federal University of Juiz de Fora, Juiz de Fora, Brazil; (2) Graduate Program in Physical Education, Faculty of Physical Education and Sports, Federal University of Juiz de Fora, Juiz de Fora, Brazil; (3) Graduate Program in Rehabilitation Sciences, Federal University of Minas Gerais, Department of Physical Therapy, Belo Horizonte, Brazil Background: The COVID-19 pandemic imposed critical social restrictions that affected people’s behaviors and lifestyles as decreased physical activity. At the same time, healthcare systems recommend home-based exercises as a safe and effective alternative to maintain the population’s physical activity levels. Therefore, this study aimed to evaluate motives to maintain physically active and adherence to exercise, as recommended, by patients with prediabetes and type 1 (T1D) and type 2 (T2D) diabetes that participated in a structured exercise intervention delivered partially on-site and partially remote. Methods: This is a cross-sectional study including participants of a pilot randomized trial (NCT03914924) who had completed a 12-week exercise intervention. The exercise intervention was switched from an on-site to a remote delivery in March 2020 due to the social restrictions imposed by the COVID-19 pandemic. All pilot randomized trial participants were instructed to maintain at least 150 minutes of moderate- or vigorous-intensity aerobic physical activity, as measured by the Borg scale, and 2 to 3 sessions/week of resistance exercise. The motives for maintaining physically active were evaluated by the Motives for Physical Activity Measure-Revised scale (MPAM-R), a twenty-six self-administered questionnaire encompassing five motives (enjoyment, competence, appearance, fitness, and social). Exercise adherence, as recommended, was evaluated by the Brazilian Portuguese version of the Exercise Adherence Rating scale (EARS-Br), a six-items self-administered questionnaire containing six items scored from 0 to 24. A score of seventeen is a cutoff point that demarks adequate adherence to the recommended exercise. Results: Fifteen patients (58 ± 11 yrs; 53% female; 13% prediabetes, 27% T1D, 60% T2D) participated in the study. Thirteen participants (87%) reported maintaining physical activity three months after the exercise intervention conclusion. The descending order of median scores obtained in each motive for physical activity was fitness, enjoyment, competence, social, and appearance. The median (interquartile range) of the EARS-Br total score was 17 (13–23), revealing over half, 53% (n = 8), of the participants adhered to physical exercise as recommended at the end of the exercise intervention. Conclusion: Health concerns are the main motive for patients with prediabetes and diabetes to maintain physical activity during the pandemic COVID 19. 111371 Modality: E-Poster Young Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES WHESLEY TANOR SILVA1, Matheus Ribeiro Ávila1, Pedro Henrique Scheidt Figueiredo1, Lucas Frois Fernandes de Oliveira1, Vanessa Amaral Mendonça1, Ana Cristina Rodrigues Lacerda1, Vanessa Pereira Lima1, Henrique Silveira Costa1 (1) Universidade Federal dos Vales do Jequitinhonha e Mucuri UFVJM Introduction: Physical training seems to promote important clinical, functional, echocardiographic and quality of life changes in patients with Chagas cardiomyopathy. Thus, it is important to verify the differences in the parameters mentioned in the different levels of physical activity presented by the patients. Objective: To verify the functional, echocardiographic and quality of life differences in different levels of physical activity in patients with Chagas‘ heart disease. Methods: Seventy-eight patients with Chagas cardiomyopathy (53 ± 10 years, 55% male, NYHA I–III, LVEF 53 ± 16%) were selected and evaluated by echocardiography, Cardiopulmonary Stress Test and by Minnesota Living with Heart Failure Questionnaire. Patients‘ physical activity level was assessed by the International Physical Activity Questionnaire (IPAQ), classifying patients as sedentary, insufficiently active, active and very active. Resulted: Insufficiently active Chagas patients (n = 13, 17%) presented younger age (p = 0.019), greater curve of ventilatory carbon dioxide equivalent (VE/VCO2 slope) (p = 0.003) and greater left ventricular diastolic diameter (p = 0.020) in relation to active (n = 38.49%) and younger age (p = 0.023), lower left ventricular ejection fraction (p = 0.017) and greater left ventricular diastolic diameter (p = 0.012) in relation to the very active (n = 27, 34%). There was no difference in quality of life, resting heart rate, blood pressure and VO2peak between the groups. When comparing the active with the very active, there was no difference between any of the variables analyzed. In the total sample, the level of physical activity correlated with the VE/VCO2 slope (r = –0.447; p < 0.001), with age (r = 0.249; p = 0.028), with left ventricular ejection fraction (r = 0.276; p = 0.016) and left ventricular diastolic diameter (r = –0.300; p = 0.009). Conclusion: Insufficiently active chagasic patients had worse values of the main functional and echocardiographic parameters. In addition, the level of physical activity correlated with well-established prognostic markers for this population, however, without influencing the quality of life of patients with Chagas cardiomyopathy. 111372 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KHOLIKOVA AZIZA OYBEK1, Abdullaev Timur Atanazarovich1, Gulomov Khumoyun1, Tsoi Igor Arsenevich1, Akhmatov Yashin Ravshanovich1 (1) Republican Specialized Scientific and Practical Medical Center for Cardiology The aim of the study: To assess the relationship between the parameters of cardiac dysfunction according to echocardiography and the level of the ST2 biomarker in patients with dilated cardiomyopathy. Materials and Methods of the trial: The study included 38 patients (28 men and 10 women) with dilated cardiomyopathy, with an average age of 49 years. Depending on the ST2 level at admission, patients were divided into 2 groups: a group with an ST2 level ≥35 ng/ml and a group with an ST2 level <35 ng/ml. In addition to the general clinical examination of patients, an echocardiographic assessment of the state of intracardiac hemodynamics was carried out. The ST2 content in blood plasma was determined by the enzyme immunoassay. Results of the study: The 1st group included 20 people with an increased level of expression ST2 ≥35 ng/ml, the 2nd one – 18 patients with ST2 expression level <35 ng/ml. The concentration of soluble ST2 isoform in blood serum in the 1st group was 77.3 ± 24.7 ng/ml, and in the 2nd – 20.6 ± 7 ng/ml (p = 0.000). According to the results of a comparative analysis of structural of the LV functional state in participants at the time of inclusion in the study, it was found that in patients from the 1 group LVEF was 29.9 ± 4.3% and was less by 2.2% (p = 0.035) in comparison with patients in the 2group (LVEF = 32.1 ± 10.9%). EDD – 72.8 ± 3 mm in the 1group 67.6 ± 9.5 mm, in contrast to the 2 group ESD- 62.1 ± 4 mm, 57.7 ± 10.8 mm, LA- 45.6 ± 3.2 mm, 41.5 ± 4.3 mm, RV- 41.2 ± 6.1 mm, 40 ± 9.2 mm, LVMM – 355.4 ± 118.4 g, 293.3 ± 74 g, respectively, for the 1st and 2nd groups. Analysis of the relationship between the parameters of the structural and functional state of the LV according to echocardiography revealed a weak positive correlation between LVEF (r = 0.193, p < 0.05) with the concentration of sST2, as well as a positive correlation between the concentration of soluble sST2 in serum and the size of the LV cavities (end- diastolic dimensions (EDD), r = 0.467, p < 0.05; end systolic (ESD), r = 0.376, p < 0.05; LA size r = 0.273, p < 0.05), and LV myocardial mass (r = 0.375, p < 0.05). Conclusion: In patients with dilated cardiomyopathy, an increased level of soluble ST2 isoform correlates the left ventricular function; the presence of an increased ST2 level in patients with dilated cardiomyopathy is a significant marker of a worse course of CHF. 111376 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES VANESSA SANCHES CORCIOLI BELLINI1, Gustavo André Boeing Boros1, Claudia Bernoche1, Milena Frota Macatrão Costa1, Silvia Helena Gelas Lage Pasqualucci1 (1) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da USP -INCOR-FMUSP Background: Intra-aortic balloon pump (IABP) is a short-term device that is widely used in patients with advanced heart failure (HF) awaiting heart transplantation (HTx). Complications can be more frequent with prolonged use of the support until HTx. Purpose: Evaluate IABP complications in patients with advanced HF awaiting HTx. Methods: We retrospectively analyzed from 2009 to 2020 in a single center intensive care unit, patients with advanced HF who underwent HTx and received IABP therapy pre-transplant. Patients whose IABP was removed before the transplant were excluded. Prolonged use of IABP was considered over 20 days. We considered IABP relevant complications bleeding, induced thrombocytopenia, infection, and leg ischemia. They were classified as mild or severe. Transfusion or removal for blood complications, replacement and removal for infection and leg ischemia were considered severe. Results: We included 195 advanced HF patients with IABP therapy before HTx. Mean time of IABP onset to HTx was 26 ± 23 days and 107 days was the longest used time. The main cardiomyopathy etiology was Chagas Disease, followed by idiopathic. Left ventricle ejection fraction (LVEF) was 24 ± 6% (TABLE 1). Fifty-two patients had IABP complications. The most frequent complications were induced mild thrombocytopenia and severe infection (TABLE 2). Bleeding occurred in 12 patients and leg ischemia in 5. The IABP replacement occurred in 43 patients and the main reason for replacement was infection. Infection was the only complication that correlated with prolonged use (p = 0.0004). No deaths were attributable to IABP complications. Conclusion: IABP is a safe therapy with low complication rates, most of them minor. Except for infection, complications analyzed were independent of IABP duration. 111384 Modality: E-Poster Young Researcher – Non-case Report Category: DIGITAL HEALTH/INNOVATION MIGUEL BARELLA NETO1, Daniel Fontes1, Sávio Batista3, Jadhe Maillard4, Heitor Olegário6, Ana Amaral Ferreira Dutra2, Louise Freire Luiz2, Gabriel Paes5 (1) Daktus; (2) Hospital Pró Cardíaco; (3) Universidade Federal do Rio de Janeiro; (4) Universidade Estácio de Sá; (5) Universidade Federal de São Paulo; (6) Universidade Federal de Juiz de Fora Introduction: Virtual training is a tool already used for medical education, adaptation of good practices and training of health professionals to new care flows. Goals: In this work, we report a retrospective observational study demonstrating the results of a Virtual Simulation Training (VST) used as a tool to measure medical knowledge, which allowed a focused face-to-face training intervention during the COVID-19 pandemic in a hospital specializing in cardiology. Materials and methods: During the 3-month period, a VST was performed by the COVID-19 team of the hospital specialized in cardiology, with delivery of a pre and post-test form to evaluate the results. The questions on the form were divided into 5 main criteria: A) Appropriate indication of diagnostic tests for COVID-19 and rational use of resources; B) Recognition of signs of severity and adequate follow-up of patients with suspected COVID-19; C) Use of antibiotic therapy and opening of a sepsis protocol in patients with suspected COVID-19; D) Management of oxygen therapy; E) Airway Management; and F) Cardiovascular emergencies concomitant with COVID-19. The answers to these forms were evaluated according to the percentage of correct answers in good (>90%), medium (between 70–90%) and low (<70%) at the end of the training and, after the results, a face-to-face intervention focused on low performance criteria. Results: The results of the post-test criteria showed Criterion C presenting a good result, Criteria A, B, D and F an average result and Criterion E a low result after the test. VST Criterion E (airway management), due to its low result, was selected for a new face-to-face intervention for airway management. The training consisted of training the entire medical team of the hospital in orotracheal intubation (OTI) through dummies, in airway management. After training, when analyzing the number of complications associated with OTI in the period prior to VST, there was an end to complications of airway management in COVID-19 patients. Conclusions: In addition to being an ally in online medical training, the VST can be a possible tool to show medical training deficits in teams, which may result in choices efficient face-to-face training in specialized teams. 111390 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH WILLIAM YANG ZHAO1, Lili Song2 (1) The George Institute for Global Health, China; (2) The George Institute for Global Health, Faculty of Medicine, University of New South Wales, Sydney, Australia Background: Cardiometabolic disease has been a clinical challenge worldwide and a major public health issue. We aim to examine the epidemiology of cardiometabolic multimorbidity and the association of cardiometabolic multimorbidity with all-cause mortality and premature mortality. Methods: This cohort study utilizes data from the China Health and Retirement Longitudinal Study between 2011 and 2018, which includes 9,712 adults aged 45 years and older. Poisson-distributed Generalized Linear Models were conducted to determine the association of cardiometabolic multimorbidity with all-cause mortality and premature mortality. Results: Overall, the prevalence of cardiometabolic multimorbidity was 32.3% among 9,712 Chinese adults, and increased with age. Compared with the group of none and single disease, cardiometabolic multimorbidity was associated with a higher risk of all-cause mortality (Relative risk ratio [RR] = 1.355, 95% CI = 1.140, 1.611) and all-cause death (RR = 1.601, 95% CI = 1.166, 2.199), after adjusting socio-demographic and lifestyle behavioral covariates. Stratified analyses revealed stronger relationships between cardiometabolic multimorbidity and all-cause mortality (RR = 1.486, 95% CI = 1.126, 1.960) as well as premature mortality (RR = 1.994, 95% CI = 1.137, 3.497) among the females compared to the males. The associations of cardiometabolic multimorbidity with all-cause death (RR = 1.784, 95% CI = 1.317, 2.417) and premature death (RR = 1.781, 95% CI = 1.098, 2.890) were only statistically significant in urban residents. Conclusions: Healthcare systems need to shift from single-disease models to new clinical and public health service delivery models to effectively manage cardiovascular and metabolic diseases as well as multimorbidity. 111413 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MONIZZE VICTÓRIA ROCHA SENTALIN1, Wilson Nadruz Júnior1, Andrei C. Sposito1, Fabiana G. A. M. Feitosa2, Audes D. M. Feitosa2, Marco A. Mota-Gomes3, Weimar S. Barroso4, Roberto D. Miranda5, Andréa A. Brandão6, Eduardo C. D. Barbosa7 (1) Universidade Estadual de Campinas – UNICAMP; (2) Universidade Federal de Pernambuco – UFPE; (3) Centro Universitário CESMAC; (4) Universidade Federal de Goiás – UFG; (5) Universidade Federal de São Paulo – UNIFESP; (6) Universidade Estadual do Rio de Janeiro – UERJ; (7) Hospítal São Francisco – Santa Casa de Porto Alegre Introduction: Has been hypothesized pandemic of COVID-19 may have led to inadequate control of cardiovascular risk, like arterial hypertension (HT), due to social isolation and changes in lifestyle habits. However, the impact of pandemic on blood pressure (BP) phenotypes derived from office and out-of-office measures (true normotension, AH sustained, AH white apron, AH masked) is still poorly known. Objective: To compare BP phenotypes before and during the pandemic, and their relation to pandemic’s severity in patients São Paulo city. Methodology: Retrospective study was conducted analyzing clinical characteristics and measures of BP in office and residential measure of BP (RMBP) in 1529 patients who weren’t using anti-hypertensive drugs, from TELEMRPA platform between 2017 and September 2021. Obtained information regarding number of cases and deaths from COVID-19 (SIVEP and SIM/SMS-SP) and medium temperature of São Paulo’s city in same period. Through analysis of multivariate logistic regression – such as adjustment of BMI, age, sex and ambiental temperature – was avaliated relations between SAH phenotypes, pandemic and its number of cases and deaths from COVID-19. Results: There was an increase in sustained HT (28,2% to 42,2%) and decrease in true normotension (44,5% to 31,8%) in individuals evaluated at the pandemic compared to those evaluated before the pandemic. In addition, BMI and age were greater among patients during pandemic (Table). The relation between pandemic and sustained HT persisted (p < 0,001) and the relation between sustained HT and COVID-19 cases (p < 0,001) and deaths (p = 0,015) every two weeks of COVID-19, even after adjustment for confounding factors. Conclusion: There’s a relation between BP phenotypes and COVID-19 pandemic and its severity in São Paulo. 111422 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JEFERSON DOS SANTOS1, Suelen Maiara dos Santos1, Luiz Fernando Souza Santos1, Anisia Vieira Souza Fontes3, Aline Barreto Hora2, Adrielly Meneses dos Santos2, Beatriz Silva Ferreira Dantas2, Caio Nemuel Nascimento Santos2, Luiz Ricardo Gois Fontes2, Maria Luíza Leal Paranhos de Oliveira2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe; (3) Centro Universitário Uninassau Introduction: During to the acute ST-segment elevation myocardial infarction (STEMI), the occurrence of clinically relevant events is directly proportional to the time of initiation of reperfusion therapy. Objective: To perform a comparative analysis regarding to the reperfusion therapies used in patients treated in the Unified Health System (SUS) and in the private network. Method: Through the ACCEPT registry database, a project to document the care of acute coronary syndrome in Brazil, patients treated at participating hospitals were selected, whose data were collected from admission to discharge. This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: The procedures performed in 1550 patients (Public = 66.4%; Private = 33.6%) with STEMI were evaluated. Coronary angiography was predominant (Public = 89.3%; Private = 94.8%; p < 0.001) and angioplasty (Public = 73.6%; Private = 81.2%; p = 0.001) in both services. Furthermore, the administration of thrombolytics was performed in 21.8% of patients from the SUS and 10% from the private network (p < 0.001). Only 1.8% of the total number of patients underwent coronary artery bypass graft surgery and 16.9% did not undergo any reperfusion therapy. Conclusion: There was a statistically significant difference in the performance of coronary angiography, angioplasty and use of thrombolytics. 111432 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH WILLIAM YANG ZHAO1, Siqi Zhao3 (1) The George Institute for Global Health, China; (2) The George Institute for Global Health, Faculty of Medicine, University of New South Wales, Sydney, Australia; (3) Yantaishan Hospital of Yantai, Yantai, Shandong, China; (4) Yantai Sino-French Friendship Hospital, Yantai, Shandong, China Background: Cardiometabolic disease has been a clinical and major public health challenge worldwide. We aim to examine the epidemiology of cardiometabolic multimorbidity and the association of cardiometabolic multimorbidity with the onset of functional disability. Methods: This cohort study utilizes data from the China Health and Retirement Longitudinal Study between 2011 and 2018, which includes 9,016 adults aged 45 years and older. Poisson-distributed Generalized Linear Models were conducted to determine the association of cardiometabolic multimorbidity with the activities of daily living (ADL) limitation and the instrumental activities of daily living (IADL) limitation. Results: The prevalence of cardiometabolic multimorbidity was 31.9% among 9,016 Chinese adults, and increased with age. Compared with the group of none and single disease, cardiometabolic multimorbidity was associated with a higher risk of ADL limitation (Relative risk ratio [RR] = 1.297, 95% CI = 1.160, 1.450) and of ADL limitation (RR = 1.165, 95% CI = 1.054, 1.288), after adjusting socio-demographic and lifestyle behavioral covariates. Stratified analyses revealed stronger relationships between cardiometabolic multimorbidity and the onset of ADL limitation (RR = 1.309, 95% CI = 1.138, 1.505) among the females compared to the males. The association of cardiometabolic multimorbidity with IADL limitation was only statistically significant (RR = 1.473, 95% CI = 1.158, 1.676) in urban residents. Conclusions: Cardiometabolic multimorbidity is associated with the greater onset of functional impairment. The Health system needs to shift from single-disease models to new clinical and healthcare delivery models to effectively manage cardiometabolic multimorbidity. 111460 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY JULIA LEAO BATISTA SIMOES1, Júlia Leão Batista Simões1, Margarete Dulce Bagatini1 (1) Universidade Federal da Fronteira Sul Heart failure (I/R) injury is an important cause of myocardial infarction and heart failure after cardiovascular surgery. Among the therapeutic possibilities, the modulation of the cholinergic system has been highlighted as a prevention of cardiac dysfunction after a cardiac event. The central mechanism of the cholinergic system is related to the stimulation of inflammatory agents in the afferent branch of the vagus nerve. From this perspective, the literature suggests that the greater availability of ACh during and after ischemic deficiencies is associated with a preservation of cardiac function. Thus, the present study was designed to investigate the enzymatic activity of cholinesterases in patients with RHI, given the relationship between Ach and protection against heart problems. Seventy patients with IHD from the Hospital of the Federal University of Santa Maria were selected for the study. Ten milliliters of blood were obtained from each patient and used for biochemical determinations. The protocol was approved by the Human Ethics Committee of the Health Sciences Center of the Federal University of Santa Maria. Thus, the present study demonstrated that the enzymatic activity of AchE and BuChE is increased in patients with IHD, directly responsible for the reduction of ACh in the middle and triggering a worsening in the outcome of the disease. 111444 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT IVNA GIRARD CUNHA VIEIRA LIMA1, Ivna Girard Cunha Vieira Lima1, Jairo Tavares Nunes2, Igor Henrique de Oliveira2, Fernanda Ronco4, Edimar Alcides Bocchi2 (1) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; (2) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; (3) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; (4) Therapeutic Area Leader – CVRM, AstraZeneca, Brazil Background: Previous observational trials suggested a U-shaped association between potassium disorders and clinical outcomes in HF. However, the mechanism and modes of death, as well as the factors influencing this association are not yet fully understood. Aim: Evaluate the association of potassium disorders with all-cause mortality, as well as to describe the association of potassium disorders with the mode of death in patients with coexisting HF. Methods: A retrospective cohort study conducted in a tertiary hospital from January 2013 to December 2020 including 10390 patients. In-hospital deaths were reported by cause and outside of the hospital deaths were considered sudden deaths. Results: Among the causes of in-hospital death HF was reported in 41.7%, cardiogenic shock in 31.2%, and other causes of death in 25.3%. Death outside of the hospital was reported in 12% of patients. The association between serum potassium disorders and mortality was stronger for severe hyperkalemia, adjusted HR (aHR) of 2.06 (1.36–3.12, p < 0.01), and hypokalemia, aHR of 1.93 (1.42–2.63, p < 0.01). Mortality outside of the hospital was more frequent in patients with hyperkalemia (8.7%) in comparison with hypokalemia (4%). Conclusion: Concurrent potassium disorders were associated with mortality in HF. The potassium disorders may have potential to be a new frontier in the management of HF. Out-of-hospital deaths, were more frequently reported in patients with hyperkalemia. 111451 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING EMILY JUSTINIANO1, Leticia Orlandin1, Luisa Mendes Silveira1, Simoni Chiarelli Da Silva Pokorski1 (1) Hospital de Clínicas de Porto Alegre Introduction: During the SARS-CoV-2 pandemic, the population also suffers from the damage associated with the reduction in the number of outpatient visits and elective exams. In this sense, telemedicine was promoted and expanded, and it served as a strategy in the follow-up and control of patients with chronic diseases, such as heart failure (HF). Objectives: To report the experience of a care protocol, including nursing teleconsultation, during the covid-19 pandemic in patients with heart failure. Method: Experience report of descriptive character constructed from the experience of teleconsultations with patients from a university hospital in southern Brazil during the Covid-19 pandemic. Experience report: In HF, multiprofessional programs focused on care transition, patient education, medication regimen simplification and outpatient follow-up are effective in reducing costs and readmissions. However, during the pandemic period, there was need to reduce outpatient care, which caused a greater team distancing of the patient to the care team which may have contributed to HF decompensation. Faced with this challenge, the nursing team implemented teleconsultation for patients with HF. The team composed of nurses and academic students, physiotherapists and dietitians was trained to use a telephone care protocol based on the application of the European scale, validated for use in Brazil, which evaluates self-care – European Heart Failure Self Care Behavior Scale – EHFScBS. The process begins during hospitalization for decompensated HF, where the patient is evaluated for his/her self-care, receives health education by a multidisciplinary cardiology team, establishes bonding; and has post-discharge follow-up by the team through teleconsultations. In the immediate post-discharge period (5th to 7th day) a telephone call is made with the same questions applied during the hospital evaluation and educational guidelines for self-care are resumed. For the patients who were not hospitalized during the pandemic, teleconsultations were also carried out. Conclusions: The team observed that teleconsultation proved to be efficient for the control of adherence to therapies and it also served as an instrument to reconnect the patient to the team. However, it is worth mentioning that the inability of some patients to use electronic devices, as well as the absence of physical examination during the consultation are some of the difficulties encountered in the use of such strategy. 111487 Modality: E-Poster Young Researcher – Non-case Report Category: NUTRITION FERNANDA SILVA MOTTA1, Gabriela Godinho Rezende1, Rodrigo Máximo Silveira1, Samuel Barud Massensine1, Rafael Matoso de Oliveira Figueiredo1, Amanda Gonçalves Vieira Martins1, Bianca de Fátima Pereira1, Moisés de Toledo Vilela1, Paula Gouvea Abrantes1, Marcela Rabelo1, Eliane Ferreira de Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Internship Department, Medical School of the Federal University of Juiz de Fora (UFJF), Juiz de Fora, Minas Gerais, Brazil Introduction: Abdominal obesity represents an important additional risk factor for both the development of chronic non-communicable diseases, cardiovascular diseases, cancer and diabetes. There is evidence in the literature of the association between smoking and abdominal obesity, in which nicotine acts by increasing insulin resistance and, consequently, is related to the deposition of fat in this region. Objectives: To analyze characteristics associated with abdominal obesity in smokers with multimorbidities. Methods: Longitudinal study, from the “Free Tobacco” group, attended between August/2021 to April/2022, referring to treatment groups consecutive sessions for smoking cessation (with mixed intervention, face-to-face and by telemedicine), composed of a multidisciplinary team, including sessions of cognitive behavioral approach, drug treatment and follow-up. Abdominal obesity was defined as waist circumference (WC) >80 cm for women and >94 cm for men. Light smokers, for use ≤10 cigarettes/day. Abusive use of alcohol, for Audit-C ≥5 points. High risk for Obstructive Sleep Apnea Syndrome (OSAS), Stop-Bang questionnaire ≥5 points. Abnormal systolic blood pressure (SBP), >130 mmHg. Results: 36 users were evaluated, referring to 6 intervention groups. When comparing those with abdominal obesity (60% of the sample) with those who did not, it was observed that these users were predominantly male (p > 0.031), light smokers (p > 0.057) and with alcohol abuse (p > 0.002). As for chronic conditions, they had a higher prevalence of obesity (p > 0.001), OSAS (p > 0.049), diabetes mellitus (p > 0.001), previous acute myocardial infarction (p > 0.001) and peripheral vascular disease (p > 0.001). Allied, higher abnormal SBP (p > 0.021) and increased levels of total cholesterol and LDL-cholesterol (p > 0.021 and p > 0.021, respectively). Conclusions: Among users undergoing treatment for smoking cessation, abdominal obesity was a frequent condition, with the majority of men in abusive alcohol use, but light smokers. Comorbidities associated with vascular damage, as well as lack of lipid and blood pressure control, in addition to OSAS, made up the profile of this population. 111488 Modality: E-Poster Young Researcher – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CARLOS ROCA GUERRERO1, Carlos Roca-Guerrero1, Joana Brito2, Beatriz Silva2, Pedro Carrilho-Ferreira2, Claudia Jorge2, Pedro Cardoso2, Hugo Corte-Real2, Marcelo Rizzo1, Pedro Canas-Silva2, Fausto Pinto2, Luís Brás-Rosário2 (1) Hospital Universitario Parc Taulí de Sabadell (Barcelona); (2) Hospital Santa Maria de Lisboa – Centro Hospitalar Universitário Lisboa Norte Introduction: Severe aortic stenosis patients frequently suffer other conditions that require antithrombotic treatment. Features regarding different antithrombotic therapy (AT) and their clinical impact on patients admitted for elective transcatheter aortic valve implantation (TAVI) are not well known. Our aim was to describe AT’s diversity and explore its clinical impact in patients admitted for TAVI. Methods: We analyzed retrospectively clinical, laboratory, echocardiographic, angiographic, drug therapy and procedure-related variables from 202 consecutive patients with TAVI between January 2019 and October 2021. Results: This cohort had a mean age of 82,3 ± 5,9 years, 56% male, 83% hypertensive, 62% dyslipidemia, 35% diabetic, 20% chronic kidney disease, 32% had coronary artery disease, 20% previous percutaneous coronary intervention (PICP), 38% Atrial Fibrillation, 18% peripheral arterial disease, 12% cerebrovascular disease. 50% of TAVIs were balloon-expandable and 50% self-expandable. The mean follow up was 11 ± 8 months. 128 (63,4%) patients were under any antithrombotic treatment before admision. 44 (21,5%) under single antiplatelet treatment (SAPT), 11 (5%) under double antiplatelet treatment (DAPT), 9 (9%) under SAPT plus oral anticoagulation (OAC), 2 (1%) under DAPT plus OAC; 62 (30,5%) were under isolated OAC (2 of them with warfarin and the rest with direct OAC). 73 patients (36%) were receiving OAC and 84 (41,6%) were under an antithrombotic strategy different than just SAPT. At the univariate analysis receiving OAC, higher mean hemoglobin during hospitalization and previous percutaneous coronary intervention were all statistically associated with survival at the end of follow up; P < 0,05. At the multivariate analysis adjusted by the previous variables, sex, history of coronary artery disease and age, only PICP, and hemoglobin levels were statistically associated with survival at the end of follow up; P < 0,05. Conclusions: In our patient’s population, AT before TAVI-related admission was frequent and diverse. Previous PCI and bleeding were the only variables independently-asociated with mortality. These findings emphasize the importance of balancing bleeding and thrombotic risk when treating these patients, even before the TAVI procedure. 111494 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MARIANA RIBEIRO SILVA1, Gualter Santos Silva1, Pedro Ribeiro Queirós1, Rafael Teixeira1, Sara Fernandes1, João Almeida1, Paulo Fonseca1, Marco Oliveira1, Helena Gonçalves1, Alberto Rodrigues1, João Primo1, Ricardo Fontes-Carvalho1 (1) Centro Hospitalar de Vila Nova de Gaia/Espinho Introduction: Recurrence of atrial fibrillation (AF) after catheter ablation (CA) is estimated to be between 20% and 45% and the prediction of recurrence AF in patients (pts) with paroxysmal AF undergoing CA remains challenging. Objectives: To determine the clinical and procedural predictors of recurrence AF after CA in pts with paroxysmal AF. Methods: Single-centre retrospective study that included all pts with paroxysmal AF who underwent AF CA between 2017 and 2019. Ablation procedures included radiofrequency and second-generation cryoballoon CA. All pts underwent cardiac computed tomography prior the procedure. AF recurrence was defined as any recurrence of AF, atrial flutter or atrial tachycardia >30 seconds (recorded in 12-lead electrocardiogram or Holter) after 90 days of CA. The independent association between clinical and procedural variables and AF recurrence was evaluated with Cox regression analysis. Results: We included 351 pts, 63,5% male and with a mean age of 57,1 ± 11,4 years. CHADSVASC score ≥2 points were present in 43,7% of the pts, median duration of AF prior the intervention was 3 years (IQR 1–6) and most pts were treated with some antiarrhythmic drug at the time of CA (56,9%). Overall, median follow-up was 27 months (IQR 19–39). AF recurrence was present in 96 pts (27,4%), on average, 15,2 ± 10,4 months after CA. Univariate logistic regression identified female gender, thyroid disease, left atrium (LA) enlargement (defined as LA indexed volume >34 mL/m2 or LA diameter >38 mm for female or >40 mm for male), coronary calcium score >100, epicardial adipose tissue volume, number of previous electric cardioversions, treatment with antiarrhythmic drugs prior CA and the extent of CA (only pulmonary vein isolation (PVI) or PVI and ablation of other lesions) as predictors of recurrence AF after CA in pts with paroxysmal AF (p < 0,05 for all). Cox regression analysis identified female gender (OR 1,615, 95% CI 1,005–2,597; p = 0,008), LA enlargement (OR 2,084, 95% CI 1,207–3,596; p = 0,008) and coronary calcium score >100 (OR 1,958, 95% CI 1,133–3,385; p = 0,016) as independent predictors of AF recurrence. Conclusions: In our cohort, AF recurrence was significantly higher in pts with LA enlargement, with coronary calcium score >100 and female gender pts. 111499 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARIANA RIBEIRO SILVA1, Gualter Santos Silva1, Mariana Brandão1, Cláudio Espada Guerreiro1, Marco Oliveira1, Helena Gonçalves1, Gustavo Pires Morais1, Bruno Melica1, Lino Santos1, Alberto Rodrigues1, Pedro Braga1, Ricardo Fontes-Carvalho1 (1) Centro Hospitalar de Vila Nova de Gaia/Espinho Introduction: Conduction disturbances after TAVI remains one of the most frequent complications of the procedure. Right bundle brunch block (RBBB) is the strongest risk factor, leading to an increased risk of permanent pacemaker (PM) implantation and is associated with an increased risk of early and late mortality following TAVI. Objectives: To evaluate whether pre-existing RBBB is associated to higher risk of permanent PM implantation and short and mid-term all-cause mortality in patients (pts) undergoing TAVI. Methods: Retrospective study of all pts submitted to TAVI between 2016 and 2018. ECG data before, immediately after the procedure, at day 3 post-TAVI and at discharge were collected, and continuous telemetry monitoring was recorded. We evaluated the rates of temporary and permanent PM implantation during hospital stay and at 1-year follow-up (FUP), ventricular pacing rates in the first visit after permanent PM implantation and all-cause mortality at 30-days, 3 and 6 months and 1-year after TAVI. Results: 220 pts were included, 57,3% female, mean age of 80,7 ± 7,0 years; RBBB prior TAVI was present in 18 pts (8,2%). No significant differences were found regarding baseline characteristics between pts with or without RBBB prior TAVI procedure. Pts with RBBB presented higher baseline QRS duration compared with pts without RBBB (140,0 ± 16,9 ms vs 107,9 ± 26,6 ms; p = 0,002), without differences in QRS duration immediately or at day-3 after TAVI (p > 0,05). High-degree atrioventricular block and complete atrioventricular block immediately after the procedure were more frequent in pts with RBBB (44,4% vs 14,5%, p = 0,004). We found no significant differences in the rates of temporary PM in pts with or without RBBB. Pts with baseline RBBB presented significantly higher rates of permanent PM implantation during hospital stay (55,6% vs 20,0%; p = 0,002) – without differences regarding the timing of PM implantation – and higher rates of PM implantation at 1-year FUP (58,8% vs 21,4%; p = 0,002). The rates of ventricular pacing at the first visit after PM implantation in pts with RBBB was 75,0% (vs 47,2% in pts without RBBB; p = 0,139). No differences were found regarding 30-days, 3 and 6 months and 1-year FUP regarding all-cause mortality, between patients with and without RBBB prior TAVI. Conclusion: Pre-existing RBBB significantly increased the risk of permanent PM implantation but was not associated with a higher risk of short- and mid-term all-cause mortality. 111509 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BEATRIZ STEPHAN FARHAT JORGE1, Pedro Drumond Maia1, Rodrigo Máximo Silveira1, Samuel Barud Massensine1, Rafael Matoso de Oliveira Figueiredo1, Amanda Gonçalves Vieira Martins1, Bianca de Fátima Pereira1, Fernanda Silva Mota1, Gabriela Godinho Rezende1, Moisés de Toledo Vilela1, Eliane Ferreira Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Cardiology Department, Medical School, Federal University of Juiz de Fora – UFJF Introduction: Women exhibit peculiar characteristics that influence the smoking habit. The identification of the female profile is an important tool to be used in the approach to smoking, identifying triggers and factors that prevent cessation or favor relapse. Objectives: To analyze biopsychosocial characteristics among women smokers treated in groups for smoking cessation. Methods: Study with a mixed strategy, cross-sectional and longitudinal follow-up regarding smoking cessation, evaluating smokers of the “Livres do Tabaco” Project, intended for smoking cessation, between 09/2021 and 03/2022. Abdominal obesity was defined as abdominal circumference >80 cm in women and >94 cm in men; neck circumference was abnormal if >40 cm. Normal systolic blood pressure <130 mmHg. Depression, score Patient Health Questionnaire (PHQ-9) ≥9 points. Cognitive deficit, Montreal Cognitive Assessment <26 points. Results: Thirty-six patients were evaluated, 66.7% of whom were women, 56.63 ± 10.80 years old. Comparing women to men, it was noted that women had statistically significant associations regarding sedentary lifestyle (p < 0.030), abnormal neck circumference (p < 0.002), abdominal obesity (p < 0.031), arterial hypertension (p < 0.007), arrhythmias (p < 0.022), cancer (p < 0.057), depression (p < 0.052) and greater cognitive deficit (p < 0.001). Regarding the use of other drugs, marijuana (p < 0.004) and crack (p < 0.002) had negative associations for females. Regarding smoking history, women had a higher frequency of triggers in terms of habits (p < 0.027), while dependence (p < 0.001) and stress (p < 0.001) were higher among men. In addition, they also presented higher nicotine dependence (p < 0.007). Regarding adherence to meetings, it was observed that the first (face-to-face) and the second (remote) were the most frequent among women (p < 0.052 and p < 0.012, respectively). It was women who exhibited the lowest prevalence of cessation, both in the 4th week (p < 0.052) and in the 12th week of treatment (p < 0.012). Conclusion: The present study revealed that female smokers showed cardiovascular risk factors and tobacco-dependent comorbidities more frequently than men. In addition, high nicotine dependence and greater trigger related to habits were evidenced. Greater adherence to meetings was not enough to give women the highest rate of smoking cessation. 111519 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION MARIANA RIBEIRO SILVA1, Eduardo Vilela1, Ana Mosalina Manuel1, Ana Raquel Barbosa1, João Almeida1, Ana Tavares1, Daniel Caeiro1, Olga Sousa1, Alberto Rodrigues1, Pedro Braga1, Madalena Teixeira1, Ricardo Fontes-Carvalho1 (1) Centro Hospitalar de Vila Nova de Gaia/Espinho Introduction: Severe aortic stenosis (AS) and mitral regurgitation (MR) often coexist. However, conflicting data reside concerning the impact of MR on outcomes after TAVI. Also, very few data exist regarding the benefits of a cardiac rehabilitation program (CRP) following TAVI in pts with MR. Objectives: To evaluate the effect of a CRP in functional parameters after TAVI, particularly in pts with MR grade ≥ II. Methods: Retrospective study which included all pts submitted to TAVI between 2014 and 2020 that completed a CRP following the procedure. Cardiopulmonary exercise tests (CPET) were performed after TAVI at baseline (pre-CRP) and post-CRP. We evaluated pre- and post-CRP peak oxygen consumption (pVO2), pVO2 at the anaerobic threshold (AT), respiratory exchange ratio (RER), VE/VCO2 and CPET duration. MR grading severity was assessed with transthoracic echocardiography performed after TAVI and was divided into 2 groups (grade < II vs grade ≥ II). Results: 52 pts were included, 59,6% were male, mean age of 78,6 ± 8,6 years-old. Mean STS risk score was 4,9%. Twenty-seven (51,9%) pts had MR grade ≥ II. Baseline characteristics were similar between pts with MR grade < II vs MR grade ≥ II, with the exception of the prevalence of coronary artery disease which was higher in MR grade ≥ II (p = 0,036). Pts with MR grade < II had higher maximum and median aortic gradients before TAVI (p < 0,05 for all). The mean number of cardiac rehabilitation sessions was 21 ± 7, without differences between both groups. In pts with MR grade ≥ II, there was an improvement in CPET duration after CRP (HF protocol from 03:57 min to 05:02 min; p = 0,017 and modified Bruce protocol from 06:03 min to 06:41 min; p = 0,049) but without significant changes in pVO2 (14,7 mL/kg/min to 14,9 mL/kg/min; p = 0,990), RER or VEVCO2/VO2 ratio. Patients with MR grade < II significantly improved pVO2 (13,8 mL/kg/min to 14,7 mL/kg/min; p = 0,015), and CPET duration with HF protocol from 05:04 min to 06:23 min; p = 0,006 after CRP. There was also an improvement in VEVCO2/VO2 ratio, although not statically significant. Conclusions: Patients with MR grade < II after TAVI who underwent a CRP significantly improved pVO2 and CPET duration. Although pts with MR grade ≥ II did not improved pVO2 after a CRP, an improvement in CPET duration may translate into a clinical benefit in these pts. These results highlight the importance of further research and personalization among this potentially higher risk subset of pts. 111521 Modality: E-Poster Young Researcher – Non-case Report Category: DYSLIPIDEMIA DANNYELLY HYLNARA DE SOUSA CAVALCANTE MAIA1, Antônia Gabriela de Araújo1, Mariana Roberta Santos de Melo1, Maria Eduarda Oliveira Amorim1, Josianne Alves de Freitas Maia1 (1) Faculdade Nova Esperança de Mossoró (FACENE/RN) Introduction: The worldwide prevalence of obesity is increasing, both in adults and children. When it occurs during childhood, there is an increased risk of developing cardiovascular disease (CVD) in adulthood. Thus, early interventions become necessary to reduce the risk of morbidity and mortality during adult life. Objectives: This study aims to assess whether vitamin D supplementation is efficient to treat childhood obesity. Methods: The present study is a qualitative systematic review, in which we used the prism method for making, we also emphasize that the guiding question was based on the pico strategy. Two databases were used: PubMed and VHL and the descriptors were: “vitamin D” and “Pediatric Obesity”, combined with the Boolean operator “AND”. We obtained as an initial result a total of 354 articles, however, after adding filters, we obtained a total of 49 articles, which went on to remove duplicates and read the title together with abstract in pairs, according to the inclusion and exclusion factors.; secondary and primary studies, with the exception of clinical trials, a study sample not compatible with the pediatric age group and studies in which the relationship between vitamin D dose and obesity was not detailed, resulted in a total of 09 articles. The selected articles went on to read the full texts which resulted in a total of 04 articles which had their data extracted. Results: Most studies did not statistically perceive the reduction in BMI or change in body mass in the supplemented group. In all studies, patients in the supplemented group had an increase in the serum concentration of 25OHD. According to Mazzochi and colleagues, the association of DHA with vitamin D supplements could help improve vitamin D status, body composition and metabolic markers. Most articles also highlight that the dose used in studies may have been insufficient to reach concentrations in obese patients, which would act actively to reduce their lipid profile. However, Alves et al. highlight that supplementation with 1,000 IU/day for 90 days reduced serum TC, LDL-c and LDL-c/HDL-c ratio without altering body composition. 111531 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY RICARDO CZARNOBAI SOCCOL1, Denise Pellegrini1, Ricardo Lasevitch1, Natalia Lamas Bueno1, Carlos Alberto S Mattos2, Augusto Lima Filho3, Noriaki Takeshita4, Silvio Sergio Pontes5, Leandro Zacarias6, Marcelo Sabedotti7, Pablo Teixeirense Tomé8, Paulo Caramori1 (1) Hospital São Lucas da PUCRS; (2) Hospital da Cidade de Passo Fundo; (3) Santa Casa de Belo Horizonte; (4) Sociedade Hospitalar Angelina Caron; (5) Hospital São Marcos; (6) Santa Casa de Goiânia; (7) Hospital Geral de Caxias do Sul; (8) Instituto dos Fornecedores de Cana Aims: The goal of this study was to evaluate the performance of a Sirolimus-eluting coronary stent, composed of a metallic chromium-cobalt platform with thin struts (75 µm), which releases sirolimus towards a bioresorbable polymer applied only to the abluminal surfaces, in a real-world scenario as a post-marketing clinical follow-up evaluation. Methods: This is a prospective, multicenter, single arm registry of patients treated with the stent in 18 participant sites. All patients with lesions in native coronary arteries treated with the coronary stent were included. Exclusion criteria were target lesion located at a saphenous vein or arterial graft, and use of another stent than study stent during the index procedure. The primary endpoint was the rate of MACE (cardiac death, myocardial infarction and target lesion revascularization) at 12 months of clinical follow-up. Secondary endpoints were target lesion revascularization and stent thrombosis rates at 12 and 24 months. Results: Between June 2017 and January 2022, 2506 patients were included (2991 lesions). The mean age was 63 ± 11 years and 64.2% were male. Clinical presentation was acute coronary syndrome in 53.2%, diabetes mellitus was present in 35.5%, previous myocardial infarction in 27.4% and previous percutaneous coronary intervention in 18.2% of patients. Most of procedures were successful (99.9%) with only one failure related to a vessel perforation followed by cardiac tamponade. To date, 1688 patients have completed the one-year follow-up. The incidence of MACE at 1 year was 3.5%. Cardiac death was 2.2% and non-fatal myocardial infarction was 1.3%. Target lesion revascularization was only 1.1%. Definitive stent thrombosis was 0.3%. Conclusion: In this interim report, the 1-year MACE rate, as well as the individual components of this endpoint were excellent and consistent with previous results available for this stent and others 3rd generation drug eluting stent. The result of this study demonstrates the safety and efficacy of this stent in daily clinical practice. 111542 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIORESPIRATORY PHYSIOLOGY/BASIC SCIENCE OLÍVIA FAGUNDES BRUNO1, RODOLFO PASSOS ALMEIDA1, JULIA DA COSTA ANCIÃES2, VICTOR EMANUEL FAGUNDES BRUNO3, JOSÉ JARJURA JORGE JUNIOR1 (1) PONTIFÍCIA UNIVERSIDADE CATÓLICA DE SÃO PAULO; (2) UNIVERSIDADE FEDERAL DE ALFENAS; (3) PONTIFÍCIA UNIVERSIDADE CATÓLICA DE MINAS Objectives: To evaluate the repercussions of the respiratory pattern of Obstructive Sleep Apnea Syndrome (OSAS) in relation to the occurrence and prevalence of cardiovascular pathologies. Methodology: Systematic review of articles in the databases: Scielo, PubMed, Lilac‘s, NHR and Cochrane. The following search terms are used: Cardiovascular Disease, Apnea and OSAS, selected without restrictions for the language of the articles and for the gender of the observed population, excluding works from the population under the age of 18 years. The specified outcomes were: hypertension, resistant hypertension, Incident coronary heart disease, heart failure in men, cardiovascular death in mild or moderate apnea, cardiovascular death in severe apnea. Results: A joint analysis of stratified disease data revealed 95% confidence, a positive and significant relationship between apnea and cardiovascular diseases, with the treatment of these diseases being a variation that reduces this correlation. 111549 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION NATÁLIA ALBERTIN DOS SANTOS1, Luiza do Nascimento Ghizoni Pereira1, Natália Albertin dos Santos1, Simone Rolim Fernandes Fontes Pedra1, Carlos Augusto Cardoso Pedra1, Oswaldo Passarelli Junior1, Márcio Gonçalves de Sousa1, Fernanda Marciano1, Antonio Gabriele Laurinavicius1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Hypertension (HTN) diagnosis depends on the accuracy and representativeness of blood pressure (BP) obtained by different methods. In pediatric obese patients, office BP can present great variability and does not detect nighttime changes. Ambulatory Blood Pressure Monitoring (ABPM) allows recognition of HTN phenotypes and predicts HTN severity in an earlier and assertive way. Objectives: To compare HTN stages defined by office BP with ABPM, and describe prevalence of masked HTM in pediatric sample from a reference service. Methods: Retrospective cohort of pediatric patients with primary HTN. Patients underwent a detailed clinical history and examination. BP was measured by auscultatory method with technique adequacy. BP was checked at 2 other visits (2 week interval) and the mean BP of these 3 visits was used to classify BP according recommendations. ABPM was performed with pediatric validated device, with a revised report for this analysis, according to guidelines. Mean 24-hours awake and sleep BP and load were considered to identify HTN phenotypes according to 95th percentile for sex, age and height. Diagnosis and stages of HTN based on office BP were compared with ABPM. Patients with sustained HTN had secondary causes discharged after investigation and target organ damage (TOD) was also evaluated. Results: Were included 16 patients with primary HTN, mean age 13 ± 3.3 years, 62% male, 87% obese or overweight (mean weight 89 ± 28.9 kg) and 75% with first degree family history of HTN. Of these, as in Figure 1, masked hypertension was detected in 37.5% (6/16), white coat HTN in 12.5% (2/16), and in 68% of the sample (11/16) ABPM classified HTN at higher stage compared to office BP. Nocturnal HTN was present in 81% (13/16). None patient had TOD and at follow-up, 12 required antihypertensive drugs, with 68% of BP control. Conclusion: For this obese primary hypertensive pediatric sample, ABPM seems to be essential for HTN diagnosis and stratification, evidencing frequent nocturnal changes in BP. Complementary tests to investigate obstructive sleep apnea weren’t done but this could be an associated factor. 111552 Modality: E-Poster Young Researcher – Non-case Report Category: PSYCHOLOGY PEDRO DRUMOND MAIA1, Beatriz Stephan Farhat Jorge1, Bianca de Fátima Pereira1, Ramon José Moreira Silva1, Fernanda Silva Mota1, Gabriela Godinho Rezende1, Moisés de Toledo Vilela1, Paula Gouveia Abrantes1, Rafael Matoso de Oliveira Figueiredo1, Maria Fernanda Monteiro Lamas1, Eliane Ferreira Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Cardiology Department, Medical School, Federal University of Juiz de Fora, Juiz de Fora – Minas Gerais. Introduction: Smoking is a serious public health problem. Among the several factors that hinder smoking cessation and the maintenance of abstinence, there is the high rate of psychiatric comorbidities, such as depression. Mood disorders are important factors to be considered in the evaluation of smokers, aiming at a better approach to the habit and a greater reduction in it. Objectives: To evaluate the impact of depression on biopsychosocial characteristics among smokers with multimorbidities in the smoking cessation process. Methods: Observational cross-sectional study evaluating medical charts of a public service for smoking cessation (“Livres do Tabaco” Project), between 09/2021 and 03/2022. Depression defined by the score Patient Health Questionnaire (PHQ-9) ≥9 points. Cognitive deficit evaluated by the Montreal Cognitive Assessment <26 points. Abdominal obesity was defined as abdominal circumference >80 cm in women and >94 cm in men. Normal systolic blood pressure (SBP) <130 mmHg. Results: Thirty-six patients were evaluated, aged 56.63 ± 10.80 years, and depression was present in 55.9%. Comparing smokers with and without depressive symptoms, it was observed that those with depression had statistically significant associations regarding obesity (p = 0.007); arterial hypertension (p = 0.035) and uncontrolled blood pressure (p < 0.035); greater cognitive deficit (p = 0.022); COVID-19 infection (p < 0.003). In addition, the occurrence of cardiovascular events also had a positive association in these patients: acute myocardial infarction (p = 0.002); heart failure (p < 0.001); peripheral vascular accident (p < 001); greater hospitalization secondary to COVID-19 (p < 0.001). Regarding smoking history, those with depression had a higher frequency of triggers for stress (p < 0001). Regarding motivation, depressed patients had lower rates (p < 0.056), as well as adherence to meetings and smoking cessation in the 12th week of treatment (p < 0.022 and p < 0.001, respectively). Conclusion: Smokers with depression showed significantly more frequent cardiovascular outcomes. Low motivation reflected in lower adherence to cognitive-behavioral approach meetings and smoking cessation. The association between smoking and depression reveals the need for a multidisciplinary approach to the habit to develop more effective interventions, and to remember that depression is also a chronic disease that should be tracked and treated simultaneously to the process for the success of smoking cessation. 111562 Modality: E-Poster Young Researcher – Non-case Report Category: PERIOPERATIVE EVALUATION RODRIGO PINHEIRO AMANTÉA1, Virgílio Olsen2, Laura Hastenteufel1, Julia Bueno1, Santiago Tobar1, Lívia Goldraich1, Roberto C. Manfro1, Flavia K. Borges3, Nadine Clausell1 (1) Hospital de Clínicas de Porto Alegre (HCPA); (2) Santa Casa de Misericórdia de Porto Alegre; (3) Faculty of Health Sciences, McMaster University Introduction: Cardiovascular risk assessment is important among renal transplant recipients. Biomarkers, such as cardiac troponin and BNP, have been used as adjunctive tools in cardiovascular risk stratification in non cardiac surgeries, but their use in the context of kidney transplantation is not well studied. Objectives: To describe the perioperative profile of cardiac biomarkers in kidney transplant recipients and its association with delayed graft function (DGF). Methods: Prospective cohort study including deceased donor kidney transplant recipients between September 2018 and March 2020 at Hospital de Clínicas de Porto Alegre. We evaluated BNP at hospital admission and 24 hours after kidney transplantation. High-sensitivity cardiac troponin (hs-cTn) was evaluated at admission, 24 and 48 hours after kidney transplantation. Only patients who presented with no major adverse cardiovascular events (MACE) during the first month of follow-up were included in the analysis. Results: We prospectively included 102 patients, of which 52.9% were male, 82.4% were hypertensive and 19.6% had diabetes mellitus. Admission’s BNP median value was 225 pg/ml (IQR 98.6–626 pg/ml), and 24 hours after transplantation was 288 pg/ml (IQR 180–597 pg/ml). Admission’s Hs-cTn above the 99th percentile was observed in 35.3% of patients, and was associated with age at transplantation (OR 1.099/CI 1.043–1.173) and male sex (OR 4.667/CI 1.644–14.838). Hs-cTn’s median change from admission to 24 hours after transplantation was –13.5% (IQR –32–9%) and from 24 hours to 48 hours after transplantation was –1.2% (IQR –17–5%). After transplantation, 52% of the patients presented with DGF, which was associated with concomitant preoperative cardiac troponin above the 99th percentile and BNP above 100 pg/ml (OR 3.989/CI 1.217–15.765). Conclusions: Baseline cardiac troponin and BNP are elevated in a large proportion of kidney transplant recipients. While BNP has an ascending trend after renal transplantation, troponin tends to decrease following surgery. Both biomarkers might be predictive of non-cardiac endpoints such as DGF. 111640 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIANE LOPES DA SILVA1, Débora Gomes da Rocha2, Nathalia Bottega Banaletti2, Fernanda Lucchese-Lobato2 (1) institute of Cardiology, University Foundation of Porto Alegre; (2) Hospital da Criança Santo Antônio, Irmandade Santa Casa de Misericórdia Children with congenital heart disease (CHD) are exposed to multiple sources of risk for delayed neurodevelopment. Early intervention (EI) studies show positive results in preventing delays. To evaluate the effectiveness of a home-based parent-mediated EI protocol, on the neurodevelopment of children with CHD, in a 6–9-month cohort. Methods: A single-blind randomized controlled trial of a parent-mediated early intervention for infants with CHD. The 6-week cost-effective protocol of activities for early home stimulation was performed by the parents with the support of a specialist. The protocol was remotely guided by a combination of booklet/toy kit/videos, and WhatsApp check-ins. The Bayley-III was used to assess developmental outcomes. Results: Twenty dyads participated in the final evaluation, 10 in the intervention group (IG), and 10 in the control group (CG), with a mean age in months of 6 ± 2, in both groups. Both had greater female participation (58%). The IG performed better than the CG, at T2, in four of the five developmental domains: cognitive (p < 0.05), receptive language (p < 0.001), expressive language (p < 0.05), and fine motor (p < 0.05). The gross motor subscale was not significantly different between IG and CG at T2 (p = 0.58). The CG group did not differ in scores at T1 and T2 in all domains: Cog, Rec lang, exp lang, fine motor, and gross motor (p = 0.9, p = 0.7, p = 0.3, p = 0.3, p = 0.9, p = 0.6, respectively). The IG significantly differed between T1 and T2 scores for four domains: Cognitive, Rec. Lang., Exp. lang. (p < 0.001), and fine motor (p < 0.05). Gross motor performance in the IG did not differ significantly between T1 and T2 scores (p = 0.1). Conclusion: The intervention was effective in improving the developmental scores of the IG in four out of five areas of development at T2. This study showed that a low-cost remote parent-mediated program can be successful in reducing the risks of significant delays in child development associated with CHD, which can also promote long-term academic and professional success in this population. Trial registry: Protocol registered at register.clinicaltrials.gov: NCT04152330. 111602 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION STEFAN TOTH1 (1) SLOVACRIN, Slovak Clinical Research Infrastructure Network, Pavol Jozef Šafárik University, Trieda SNP 1, 040 11 Kosice, Slovakia Introduction: Short-time cold-water immersion (CWI) is associated with significant acute cardiovascular, metabolic, and endocrinological responses. There is, however, no available study following the long-term effect of repeated CWI on atherogenesis, lipid parameters, and fat distribution. This study aimed to explore the suggested protective effect. Investigations and methods: 35 healthy volunteers were followed during the 5-month CWI exposition under standard conditions (three times per week 7–10 min). Neoprene equipment was not allowed; volunteers with followed weight or muscle mass changes over 5% were excluded. Equivalent sham control (N = 30) was included. In the beginning and at the end of the study, blood collection and clinical examinations were made. Lipid and non-lipid parameters, including PCSK9 and hsCRP levels, were quantified. Vascular changes were detected by carotid ultrasound (cIMT) and by echo-tracking for detection of arterial stiffness parameters (PWV; AI; Beta). Liver steatosis quantification was based on the calculation of hepatorenal index (HRI), fat distribution was measured by the quantification of subcutaneous and visceral fat thickness changes. Results: 28 volunteers have completed the given protocol. Significant decrease of cIMT (p = 0.0001); AI (p = 0.0002); Beta (p = 0.0001) and PWV (p = 0.0001) was detected after the long-term repeated CWI. In comparison with the entry values, a significant decrease of hsCRP (p = 0.01) and PCSK9 (p = 0.01) was observed. Liver fat accumulation decreased by an average of 11% in comparison with the entry values (HRI p < 0.001). A significant decrease in LDL; TC; TG and VLDL was followed as well. Conclusion: We suggest a possible beneficial effect of repeated-CWI on atherogenesis, liver fat accumulation, lipid, and non-lipid parameters. 111636 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIO-ONCOLOGY ANA FLÁVIA OLIVEIRA DE SOUZA1, Afonso Moraes Melo Júnior2, Davi Gabriel Barbosa3, Daniel Oliveira da Costa4, João Lucas Watrin Braga5, Juliana Ayumi Azevedo Kurosawa6, Lucas Guimarães Junqueira7, Paola Bitar de Mesquita Abinader8, Rafaela Oliveira Cardoso9, Rafael Silva Lemos10, Wanda Maria de França Pires11, Luis Eduardo Werneck de Carvalho12 (1) Centro Universitário do Estado do Pará (CESUPA); (2) Centro Universitário do Estado do Pará (CESUPA); (3) Universidade Estadual do Pará (UEPA); (4) Universidade Estadual do Pará (UEPA); (5) Centro Universitário do Estado do Pará (CESUPA); (6) Centro Universitário do Estado do Pará (CESUPA); (7) Universidade Estadual do Pará (UEPA); (8) Centro Universitário do Estado do Pará (CESUPA); (9) Centro Universitário do Estado do Pará (CESUPA); (10) Universidade Estadual do Pará (UEPA); (11) Universidade Federal do Pará (UFPA); (12) Oncológica do Brasil Introduction: Intracardiac tumours can be divided into intramural thrombus, primary cardiac neoplasms and metastatic tumours. The management of these pathologies is generally determined by obtaining the tissue pathology, the type of tumour and its degree of involvement. Among the types of treatment, chemotherapy is the main strategy, as well as radiotherapy, immunotherapy and surgery. These neoplasms, when untreated, present a poor prognosis, which highlights the need for epidemiological studies that highlight the therapeutic modalities available today. Objectives: To establish an overview of the treatment of malignant neoplasms of the heart, mediastinum and pleura in Brazil, between 2013 and 2020. Methodology: This is a descriptive, quantitative and retrospective study, whose data came from the DATASUS database. The data were analysed according to the division by federative units and the variables used were the therapeutic modality, cancer staging, year of treatment, age group, gender and total number of cases per year in the selected period. Results: When evaluating the treatment of these neoplasms in Brazil in the period 2013 to 2020, it was observed the prevalence of treatment lasting up to 30 days (82.2%), with the main therapeutic modality being surgery (66.4%), followed by chemotherapy (20.2%), and radiotherapy (13.2%). Among the cases analysed for treatment, staging 3 prevailed (32.7%), followed by 4 (31.4%), and the year 2019 (29.5%) was the one in which the beginning of treatment was recorded the most. Regarding the age range, the ages 55 to 59 years represented 11.3% of cases, followed by 60 to 64 years with 10.9% and the predominance in the male gender (50.9%). Conclusion: It is observed that primary malignant neoplasms of the heart have a poor prognosis and are submitted to palliative treatment, while in metastatic heart tumours the therapy depends on the tumour of origin. Moreover, there is a slight predominance among males and between the ages of 55 and 64 years. Moreover, the TNM stages 3 and 4 were the most prevalent, as well as surgical and chemotherapeutic therapy. Therefore, it is evident the need for continuous scientific and technological advances employed both in early diagnosis and in therapeutic modalities in order to provide a positive prognosis for these patients. 111635 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS EDVALDO VICTOR GOIS OLIVEIRA1, Enaldo Vieira de Melo1, Cláudia Bispo Martins Santos1, Lara Teles Alencar Duarte1, Antônio Carlos Sobral Sousa1, Eduardo José Pereira Ferreira1, Allexa Gabriele Teixeira Feitosa1, Cleovaldo Ribeiro Ferreira Júnior1, Irlaneide da Silva Tavares1, José Icaro Nunes Cruz1, Gabriela de Oliveira Salazar1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe Introduction: Left ventricular (LV) diastolic dysfunction is one of the important factors for survival and long-term prognosis in patients with Chronic Coronary Syndrome (CCS). However, the connection between diastolic dysfunction during exertion, Chronic Coronary Syndrome, Myocardial Ischemia (MI) and the profile of increased cardiovascular risk has not been demonstrated over the years. Therefore, there is a need to predict the factors that can aggravate this connection, in order to enable the creation of secondary prevention strategies and decrease the occurrence of unfavorable outcomes. Objectives: To evaluate LV diastolic function and factors associated with the presence of Myocardial Ischemia (MI) in patients with Chronic Coronary Syndrome (CCS). Methodology: A cross-sectional study between January 2000 and January 2022 with individuals with CCS who underwent Physical Stress Echocardiography (FES) in a cardiology referral service. A total of 2000 patients (62 ± 19 years) were categorized according to the presence or absence of MI. Statistical analysis included the chi-square test and t ’s Student using SPSS Statistics version 22.0. Results: We found 937 (46.9%) patients without MI and 1059 (53.0%) with MI – of the latter, 22.3%, 60.7% and 17.1% had induced ischemia, fixed ischemia and ischemia fixed and induced, respectively. There was no association between MI and diabetes mellitus, obesity, physical inactivity (p > 0.05). Myocardial ischemia was associated with: hypertension (77.9% vs. 69.8%), dyslipidemia (77.7% vs. 67.9%), positive family history of coronary heart disease (74.1% vs. 63.8%) and previous myocardial infarction (51.8% vs. 22.3%) – p < 0.001. MI was associated with diastolic dysfunction (p < 0.001): most patients with normal diastolic function belonged to the group without MI (17.6% vs. 6.5%, p < 0.00001), the impaired relaxation was not significant between groups (p = 0.4839), pseudonormal function was more prevalent among patients with MI (28.9% vs. 20.9%; p = 0.0002), as was the pattern restrictive (1.8% vs. 0.1%; p = 0.0007). Left ventricular ejection fraction did not differ between the groups with and without MI (p > 0.05). Conclusion: Arterial hypertension, dyslipidemia, family history and previous myocardial infarction have been shown to be predictors of myocardial ischemia. On the other hand, LV diastolic dysfunction was more prevalent in patients with myocardial ischemia. 112114 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANA CAROLINA PEDROSO 1, Andreá Ferreira Haddad1, Lara Camporez Menezes Trindade1, Antonio Vitor Martins Amaral1, Priscila Oliveira Diaz1, Bernardo Cleto Teles e Silva1, Joao Felipe Tamiozzo Reis1, Thaisa Rodrigues Garcia1, Alessandra godomiczer1, Flávia de Moraes Pedro Moisés1, Monica Amorim de Oliveira1, Luiz Fernando Simvoulidis1 (1) Unimed Rio Hospital Venous thromboembolism (VTE) is the most common preventable cause of death in hospitalized patients. Hospitalization increases the risk of VTE by 8x, generating important clinical consequences such as deep vein thrombosis (DVT) and pulmonary embolism (PE) that can leave severe sequelae and even being the main cause of death that can be prevented in hospitalized patients. Covid19 is associated with a concomitant prothrombotic state, increasing risk of VTE This is a retrospective cohort that evaluated clinical and surgical patients admitted for at least 48 hours at a private quaternary hospital, where we compared data on thromboembolic events in the pre-pandemic period, from January 1, 2018 to November 30, 2019 with patients hospitalized in the period pandemic from January 1, 2020 to March 14, 2021. All patients were evaluated according to risk stratification for VTE by the physician and prophylaxis was instituted according to the risk found, classified as low, moderate and high risk. Patients with COVID19 were classified as high risk. Patients admitted with DVT and/or PE, arterial thrombosis events and those in palliative care were excluded from the analysis. The events of DVT (distal and proximal), device thrombosis (PICC and AVP) and PE were recorded and all results were confirmed with imaging exams through the EcoDoppler study. Results: In the historical cohort Jan 18 to Nov 19, 114 VTE events were reported in a universe of 81,445 patients, corresponding to 0.14% of hospitalizations in this period. Of these, DVT corresponded to 81.5%, PE 15% and 3.5% central venous catheter thrombosis. There were 23 deaths during hospitalization, corresponding to 23.96% of patients who presented VTE during this period. These events account for 0.03% of in-hospital mortality. In the period from January 1, 2020 to March 14, 2021, 126 events occurred among hospitalized patient ina a universe de 15,000 patients. Of these, 120 (95.2%) were due to DVT and 06 (4.8%) to PE, causing 48 deaths, which corresponds to 38.1% of patients who presented VTE within the in-hospital mortality. VTE is a frequent condition in hospitalized patients and is related to longer hospital stay and in-hospital mortality. It is the most common preventable cause of death in hospitalized patients. We have to consider that SARS-COV2, COVID-19 disease, has been associated with a concomitant prothrombotic state and an increased risk for VTE frequency, therefore, an increase in mortality. 111653 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY AYA MUTAZ ZEIDAN1, Aya Mutaz Zeidan1, Yangyang Dai1, Zhouyang Xu1, Jonathan Behar1, Steven Williams1, Richard Housden1, Aruna Arujuna1, Kawal Rhode1 (1) King’s College London KCL Robotically assisted catheter ablation procedures have been shown to be safe, effective and confer both better catheter stability and tissue contact. Despite promising results from robotic cardiac CA systems, all systems require manual navigation for the transseptal puncture access, thus motivating the need to develop a fully robotic TP system. This would potentially improve patient safety, further reduce radiation dose to cardiologists, and pave the way to more complete robotic solutions for CA. The research aim is to present a preliminary design and prototype for a robotic TP system, and demonstrate real-time operation via simulation. By employing computer-aided design (Fusion360, Autodesk, USA) and additive manufacturing methods (Ender 3, Creality, China), a first prototype for a robotic TP system has been designed to achieve the cooperation principle of the transseptal needle and dilator sheath. The prototype implements four degrees-of-freedom to accomplish translation and rotation of the needle and sheath, individually. Safety features include speed and position constraints. The open-source Robot Operating System (ROS 2, Open Source Robotics Foundation, USA) was used for the control architecture. Additionally, ros2_control was used to employ Forward Command Controller for efficient and reliable actuator control. We demonstrated constant positional relationship between the virtual dilator-sheath and the BRK-1 needle, achieved within the ROS 2 graphical interface. In coupling the needle and sheath, the realised system was able to successfully drive to a maximum length of 144.5 mm, with an positioning accuracy of 0.10 mm. The deterministic performance of the system and its real-time functionality exemplified the benefits of leveraging ROS 2. Movement commands were executed within the order of a few microseconds, providing real-time, low-latency operation. This research confirms proof-of-concept for a first prototype real-time control of a Robotic TP system implemented based on the ROS 2 framework. Our next step is to develop a joystick interface and evaluate the system by using an anthropomorphic cardiac phantom. 111655 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION PIETRA PETRICA NEVES1, Maycon Junior Ferreira2, Tânia Plens Shecaira2, Michel Pablo dos Santos Ferreira Silva1, Débora Conte Kimura2, Maria Claudia Irigoyen3, Guiomar Nascimento Gomes2, Kátia De Angelis1 (1) Nove de Julho University (UNINOVE), São Paulo, Brazil; (2) Federal University of São Paulo (UNIFESP), São Paulo, Brazil; (3) Heart Institute (InCor), University of São Paulo (USP), São Paulo, Brazil Cardiovascular diseases are currently the main causes of mortality and morbidity worldwide. Systemic arterial hypertension(SAH)is one of the main risk factors for cardiovascular outcome. Pharmacotherapy associated with exercise training is clinically recommended for the control of blood pressure(BP)to manage SAH. To investigate the effects of concurrent exercise training(CET)and pharmacological treatment with hydrochlorothiazide on hemodynamic and renal parameters in an experimental model of SAH. Female spontaneously hypertensive rats(SHR) were allocated into 4 groups:sedentary ovariectomized(OS, n = 8), trained ovariectomized (OT, n = 8), sedentary ovariectomized treated with hydrochlorothiazide(OSH, n = 8) and trained ovariectomized treated with hydrochlorothiazide(OTH, n = 8). Ovariectomy was performed on the first day of the study. Hydrochlorothiazide(30 mg/kg) was dissolved in the drinking water. The OT and OTH groups were submitted to CET with intensity of 40–60% of maximum capacity,3 days per week, during 8 weeks. In the seventh week, all rats were kept in metabolic cages during 24 hours for urine collection. In the sequence, the rats underwent cannulation of the carotid artery for direct BP recording. Both trained groups showed an increase in their performances when compared to their initial tests, and to the sedentary groups at the end of the protocol(p < 0.0001). Furthermore, OTH group showed an additional increasing when compared to the OT group at the end of the protocol in the maximal load test (p < 0.0001). The OSH, OT and OTH groups presented lower systolic(OSH:189.0 ± 13; OT:179.3 ± 5; OTH:174.1 ± 15), diastolic(OSH:138.9 ± 7; OT:131.3 ± 11; OTH:127.6.1 ± 14) and mean BP(mmHg) (OSH:163.0 ± 9; OT:154.3 ± 13; OTH:149.8 ± 14) when compared to the OS group (SBP:207.6 ± 15; DBP:155.4 ± 18; MBP:180.2 ± 8). The OT group(332 ± 20) presented resting bradycardia (bpm) compared to the sedentary groups(OS:380 ± 25; OSH:374 ± 28). The OTH group showed reduced plasma creatinine levels in relation to the OS group(p < 0.0001). In addition, hydrochlorothiazide combined or not with CET induced higher clearance of creatinine when compared to the OS group(p = 0.0029). Plasma urea levels were similar between groups. The results suggest that the association of CET and pharmacological treatment can promote additional cardiorenal benefits in post menopaused rats, suggesting an important role of the combination of these therapies, probably impacting on quality of life and SAH-related risk factors and morbimortality. 111664 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MATHEUS SOUZA DA SILVA1, Jéssica Haline de Souza dos Reis1, Thalles Franklin Souza Santos1, Daniel Ferreira Cunha1, Lucca Dal Moro1, Carlos Lutian da Silva Andrade1, Bruno Artur de Almeida Santos1, Lucas Gama Pacheco1, Kamylla Batista Brito1, Raul Antônio Lopes Silva Campos1, João Marcos Rodrigues Silva1, Luciano Moura de Assunção2 (1) Universidade Federal do Pará; (2) Fundação Santa Casa de Misericórdia do Pará Introduction: Pre-hospital care aims to reduce the time between the onset of an ischemic event in the myocardium and effective treatment. It is at this point that mortality in the first few hours can be reduced (1). Therefore, a good quality of care and adequate management of cases of Acute Myocardial Infarction (AMI) must have a good infrastructure, which is closely related to the number of deaths. Knowing the complexity and differences between the different care contexts by regions, the present study compares the number of deaths from AMI and the number of hospitalizations by regions in Brazil. Objectives: To establish a quantitative analysis on the ratio of the number of deaths to the number of hospitalizations due to AMI and to compare the results by regions of Brazil. Methods: The study is a cross-sectional, retrospective, descriptive and quantitative analysis carried out based on data obtained from the Department of Informatics of the Unified Health System (DATASUS) for Brazil, with a temporal analysis between the period 2015 and 2019. Results: Between 2015 and 2019, there were a total number of 571,473 hospitalizations for AMI in Brazil; being 24,158 in the North Region; 111,422 in the Northeast Region; 283,438 in the Southeast Region; 113,733 in the South Region and 38,722 in the Midwest Region. Of which, 61,499 died in Brazil; 2,892 in the North Region; 13,476 in the Northeast Region; 29,699 in the Southeast Region; 11,392 in the South Region and 4,040 in the Midwest Region. Of the total number of hospitalized patients by region, 10.76% died in Brazil, 11.9% in the North, 12% in the Northeast, 10.47% in the Southeast, 8.79% in the South and 10.43% in the Midwest Region. Thus, there were 9.29 hospitalized for each death in Brazil; 8.3 hospitalized for each death in the North Region; 8.2 hospitalized for each death in the Northeast Region; 9.5 hospitalized for each death in the Southeast Region; 9.98 hospitalized for each death in the South Region and 9.58 hospitalized for each death in the Midwest Region. Conclusions: In view of the results, it can be concluded that the North and Northeast regions have a higher ratio of deaths due to hospitalization due to AMI and do not follow the national average for the same statistics. As a limitation of the study, data referring to out-of-hospital deaths from AMI are highlighted. 1.Myerburg RJ, Castellanos A. Cardiac arrest and sudden death. In: Braunwald E. Textbook of cardiovascular medicine. Philadelphia. 111679 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MARIA FERNANDA MONTEIRO LAMAS1, Beatriz Stephan Faraht Jorge1, Paula Gouvea Abrantes1, Pedro Drumond Maia1, Ramon José Moreira Silva1, Rodrigo Máximo Silveira1, Samuel Barud Massensine1, Rafael Matoso de Oliveira Figueiredo1, Amanda Gonçalves Vieira Martins1, Bianca de Fátima Pereira1, Arise Garcia de Siqueira Galil1, Eliane Ferreira Carvalho Banhato2 (1) Universidade Federal de Juiz de Fora – UFJF; (2) Centro Universitário UniAcademia Introduction: Tobacco is responsible for 4 milion adults death per year. The tobacco problem seems to be concerned with changing the cognitive system as well as the use of cognitive function Impairments skills are designed by psychometrically valid brief screening instruments. Montreal Cognitive Assessment (MoCA), Clock Drawing Test (CDT) and Verbal Fluency (VF) are validated tests that provide information particularly on cognition such as proven activities, planning, decision making and impulse control. Goal: Identify the cognitive profile of smokers participating in a smoking cessation program, using neuropsychological screening tests. Methods: Longitudinal study, “Livres do Tabaco” group, Cardiology Service, Federal University of Juiz de Fora, Juiz de Fora/Minas Gerais. Users with multimorbidities were treated between September/2021 and March/2022, in consecutive treatment groups, mixed intervention (in person and telemedicine), multidisciplinary team, cognitive behavioral approach sessions, drug treatment and follow-up. Definitions: Cognitive deficit, Montreal Cognitive Assessment <26 points (CDT and VF, inserted in MoCA) was used. Low level of education <8 years. Results: Sample of 36 smokers, equivalent to 6 consecutive treatment groups. Women, 66.7%; 8 years or more of schooling, 57.1%; 65.7% were not in a stable relationship. Age, 56.64 ± 10.61 years. Number of cigarettes smoked daily, 22.77 ± 14.64 cigarettes; high nicotine dependence, 68.6%. As for the cognitive profile, there was low performance on the MoCA (79.4%). In the VF, 55.9%; in the CDT, 19.4%. Age was correlated negatively with MoCa (r = –0.508, p = 0.002) and positively with years of addiction (r = 0.791, p = 0.000) and pack years (r = 0.426, p = 0.011). The CDT was negatively correlated with age (r = –0.479, p = 0.008) and years of addiction (r = –0.602, p = 0.000). Verbal Fluency was correlated negatively with years of addiction (r = –0.353, p = 0.040) and pack years (r = –0.409, p = 0.016). Conclusion: The negative and significant association among variables related to smoking history and cognitive tests showed the presence of cognitive impairment in the studied sample. It is possible that the impairment in planning and decision-making capacity is an important barrier to the difficulty of smoking cessation among smokers with the characteristics described. 111678 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JEFERSON DOS SANTOS1, Suelen Maiara dos Santos1, Luiz Fernando Souza Santos1, Anisia Vieira Souza Fontes3, Aline Barreto Hora2, Thiago Vaz de Andrade2, Matheus Jhonnata Santos Mota2, Victória Rafaela Nunes dos Santos2, Carlos Felipe Amado Abud2, Jullia beatriz araujo souza2, Karenn santos souza cruz2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe; (3) Centro Universitário Uninassau Introduction: Due to the efforts and efforts of the Brazilian Society of Cardiology (BSC), with national records, it is possible to ensure detailed data that allow us to analyze the differences between public and private hospitals in the care provided and outcomes of patients with Acute Coronary Syndrome (ACS). Objectives: To determine differences in patient characteristics, access to well-equipped services, treatment offered, and intra-hospital and 12-month outcomes between public and private hospitals in Brazil for patients with acute coronary syndrome. Method: This is a prospective observational study that included inpatients diagnosed with ACS in 47 Brazilian hospitals. Patients were followed from admission to discharge and then follow-up was performed by telephone contact at 30 days after discharge, 6 months after discharge and 12 months after discharge. Clinical, personal (previous history) and index event data were collected, such as medical prescription and occurrence of major cardiovascular events (cardiovascular mortality, reinfarction and stroke). The current series compared the results obtained by the population using the Public Health System and the population using the private network. Values of p < 0.001 were considered statistically significant. The ACCEPT registry included, from August 2010 to April 2014, a total of 5,047 patients, making it the largest prospective registry ever published with ACS in Brazil. For the present analysis, all patients who completed the 12-month follow-up (n = 4375) were considered. This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: Patients treated in the Public hospitals had less direct access to a specialized service, with a greater number having to be transferred (38.9%; p < 0.001), in addition to spending more days in the hospital (after 7 days, 40.7% were still hospitalized p < 0.001) and death in this subgroup also showed a statistical difference, both in the period of hospitalization and at 12 months after discharge. Conclusion: In Brazil the management of patients with Acute Coronary Syndrome is influenced by the type of health service, public or private. Patients were more likely to have worse outcomes in public hospitals. 111682 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SILVIO CÉSAR ALVES DO NASCIMENTO JUNIOR1, Karimi Mohamed El Bacha1, Karine Corcione Turke2 (1) Universidade Nove de Julho – São Bernardo do Campo; (2) Faculdade de Medicina do ABC Introduction: Rheumatic fever is a prevalent disease in our country, with an incidence of 30,000 people/year in Brazil. This disease is directly related to environmental and economic factors, and it is known that it is a disease with a higher prevalence in regions with less financial conditions. The disease can be responsible for several long-term consequences. There are few studies that link expenditures of the Sistema Único de Saúde (SUS) with deaths from rheumatic fever in adults. Objectives: Correlate spending on rheumatic fever in SUS and late mortality associated with the disease in different administrative regiões od Brazil. Methods: Ecological study. The data collected through the DATASUS platform. SUS spending on rheumatic fever and deaths from this disease were evaluated through the Sistema de Informações Hospitalares for each administrative region of the country in adults over 20 year old. Obits were adjusted by age group and were stratified by sex, age group and region. For correlations, the Pearson or Spearman tests were used depending on the normality of the data, assessed by Shapiro-Wilk. Results: Throughout the period of 2008 to 2016, were reported 6254 deaths from rheumatic fever in Brazil in adults over 20 years and the central-west region had the highest mortality rate (0,63:100.000). No correlation was found between the overall mortality rate and SUS spending with rheumatic fever (cor: 0.374, p = 0.32). A strong and positive correlation was observed between mortality and outgoing in the northeast region (cor: 0.853, p = 0.003) and central-west (cor: 0.757, p = 0.018). In addition, no correlation was observed between mortality and HDI (cor: 0.552, p = 0.333). No correlation was found by age group or gender. Conclusion: In this study, it was possible to observe that there was no correlation between regional costs and mortality from rheumatic fever in the country, however, central-west and northeast a correlation was found. In view of the above, this correlations show a significant cost associated with higher mortality. In this way, we can assume that the investment these regions receive, there is a right mortality rate from rheumatic fever in adults, what can reflect an ineffective distribution of resources in these places. 111692 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS SUELEN MAIARA DOS SANTOS1, Jeferson dos Santos1, Luiz Fernando Souza Santos1, Aline Barreto Hora2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe Introduction: In the last decade many scholars have been concerned about the differences in treatment (acute and intermediate) offered by the public and private health systems, particularly for patients with Acute Myocardial Infarction (AMI), who belong to the group cardiovascular disease – the leading cause of death in the world. Objectives: The present study aimed to compare population subgroups in order to evaluate the access to health care based on clinical recommendations and the difference in clinical outcomes of patients affected by Acute Coronary Syndrome (ACS), who participated in the ACCEPT Registry, comparing them with regard to the type of service (Public Network versus Private Network). Method: The present study compared the results obtained by the public system user population and the private network user population. Values of p < 0.001 were considered statistically significant. The ACCEPT Registry included, from August 2010 to April 2014, a total of 5,047 patients, making it the largest prospective registry ever published with ACS in Brazil. This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: In both subgroups, there was a predominance of males (Private Network: 65.8%; Public Network: 69.9%). Regarding risk factors, smoking (current or previous) showed a significant difference in patients from the Public Network (63.5%; p < 0.001), as well as dyslipidemia (59.2%; p < 0.001) and sedentary lifestyle. (62.6%; p < 0.001) in patients from the Private Network. Patients treated in the Public Hospitals had less direct access to a specialized service, with a greater number having to be transferred (38.9%; p < 0.001), in addition to spending more days in the hospital (after 7 days, 40.7% were still hospitalized; p < 0.001) and death in this subgroup also showed a statistical difference, both in the period of hospitalization and at 12 months. Patients affected by the group of diseases that constitute the Acute Coronary Syndrome who need to use the Public Health System are more likely to spend more time in hospital and have worse in-hospital outcomes and after 12 months. Conclusion: The need to be transferred to a specialized service seems to contribute to this difference. Thus, in the face of this unequal access to health care, the need for robust, effective and effectively health policies for all is evident. 111694 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM SILVIO CÉSAR ALVES DO NASCIMENTO JUNIOR1, Natalia Gil Prado1, Uelra Rita Lourenço1 (1) Universidade Nove de Julho – São Bernardo do Campo Introduction: On March 11, 2020, the World Health Organization declared a COVID-19 (Coronavirus Disease – 2019) pandemic. Data from April 21, 2021 record 14.12.,795 confirmed cases and 381.475 deaths across the country. It is known that there is a correlation between cases of patients infected with COVID-19 and heart disease. However, there are few studies that show an increase in numbers of cases of heart failure (HF) and arrhythmias in adults in the state of São Paulo (SP) after the beginning of the pandemic in Brazil. Objectives: To analyze the incidence of deaths from HF, conduction disorders and arrhythmias registered in the Health Unic System (SUS) from Jan/2010 to Dec/2019, and 2020 when the COVID-19 pandemic was declared. Methods: Ecological study. Data were collected through the DATASUS platform. Deaths from HF, conduction disorders and arrhythmias were evaluated in the SUS through the Hospital Information System (SIH/SUS) for the state of São Paulo in adults over 20 years of age. Deaths were adjusted by year of care and were stratified by sex, age group and administrative region. For the analysis, hypotheses were used, associated with a t-Student test, when deriving the normality of the data. Results: From 2010 to 2019, 283.834 deaths from HF, conduction disorder and arrhythmias were reported in Brazil in adults over 20 years of age, with 76.190 cases registered in the state of SP, mortality rate (16:100.000). There was no correlation between deaths in 2010 to 2019, and 2020, when the pandemic was declared, recorded in the SUS. However, a probability associated with a t-Student test was observed, with a two-tailed distribution (p: 0.00793), with no significant incidence. When stratified by sex, the mean was 7567.63 (M: 49.90%; F: 50.1%), 80% among the population over 50 years old. Conclusion: It was observed that there was no higher incidence of deaths from HF, conduction disorder and arrhythmias in the pre-pandemic period and the total number of deaths in 2020. However, a slight increase was found when compared to the average mortality in the years 2010 to 2020. 2020 between men and women, and also in older populations. Therefore, it is known that more women die from these diseases in SP. In view of this, cross-sectional studies can be carried out to prove whether there is a relationship between the number of deaths from these pathologies in the pre- and post-pandemic. 111695 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RAPHAELA PAULA PINHEIRO1, Carlos Renato de Oliveira2, Allice de Souza Rodrigues1, Maria Julia Montebeller Meneses1, Renato Jorge Alves3 (1) Santa Casa de Sao Paulo School of Medical Sciences; (2) Santa Casa de Misericordia de Sao Paulo Hospital, Cardiology; (3) Santa Casa de Sao Paulo School of Medical Sciences; Santa Casa de Misericordia de Sao Paulo Hospital, Cardiology Background: Hyperlipidemia is an important cardiovascular risk factor. Pulse wave velocity (PWV), the gold standard to assess arterial stiffness (AS), can detect subclinical atherosclerosis with accuracy, however, it’s a high-cost tool. Neck circumference (NC) and neck-to-height ratio (NHR), inexpensive and easily obtainable anthropometric measurements, may be associated with AS. Aims: To evaluate the relationship between NC, NHR, and AS assessed by PWV in treated hyperlipidemic patients. Methods: We performed a cross-sectional study with 47 hyperlipidemic patients, over 18 years. Excluded those with cervical anatomical abnormalities. Brachial PWV (bPWV) was obtained by a non-invasive oscillometric method. NC and NHR were assessed. Data were present as frequency (%) or mean ± S.D., used Pearson correlation for analyses, P-value:5%. Results: Sample: 53,2% woman, mean age of 63,6 ± 13,2 years, 63,8% Fredrickson’s phenotype IIa, 63,8% used high-potency statin, 87,2% hipertensives, 14,9% current smokers. On average, patients were overweight (BMI 28 ± 3,5). Mean NHR of 23,5 ± 1,8 cm/m, NC of 37,6 ± 3,8 cm, bPWV of 9,3 ± 2,2 m/s; • bPWV was positively associated with NHR (figure 1), but not with NC (r = 0.083; p = 0.580); • Age (years), a well-established risk factor for increased AS, was strongly correlated with PWV (r = 0.962, p = 0.000) and was also associated with NHR (r = 0.386, p = 0.007). Conclusion: Our findings suggest that NHR, but not NC, was positively associated with bPWV in hyperlipidemic patients and might be an inexpensive potential predictor of arterial stiffness, contributing to the clinical follow-up, and preventing cardiovascular events in this population. 111700 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUIZ FERNANDO SOUZA SANTOS1, Jeferson dos Santos1, Suelen Maiara dos Santos1, Aline Barreto Hora2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe Introduction: Although we have records that observe patients with Acute Coronary Syndrome (ACS), there is still a lack of studies that show the differences between the public and private service in the care of patients with ACS, in particular the presentation of STEMI (ST-Elevation Myocardial Infarction). The present study aimed to investigate the differences in acute treatment and the presence of outcomes after 12 months of patients affected by STEMI, who participated in the ACCEPT Registry, comparing them with regard to access: Public versus Private Network. Method: The ACCEPT Registry is a prospective observational study that included inpatients diagnosed with ACS in 47 Brazilian hospitals. Patients were observed from admission to discharge and then followed up for 12 months. Clinical data, previous history and index event, such as medical prescription and occurrence of major cardiovascular events (cardiovascular mortality, reinfarction and stroke) were collected. The current study compared the results obtained by the public system user population and the private network user population, specifically affected by the STEMI. Values of p ≤ 0.01 were considered statistically significant. The ACCEPT Registry included a total of 5,047 patients from August 2010 to April 2014, making it the largest prospective registry ever published with ACS in Brazil. For the present article, only patients with STEMI were considered (n = 1550), which corresponds to the most common diagnosis of ACS (35.8%). This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: Patients treated in the public health service spent more days hospitalized (after 7 days, 40.7% were still hospitalized) and the death rate in this group was 12.3% in 1 year. Age and diabetes were independently associated with the occurrence of major cardiovascular events. Patients with STEMI who need to use the Public hospitals are more likely to spend more time in hospital and have worse outcomes 12 months after the event. Conclusion: The need to be transferred to a better equipped service seems to contribute to this difference. Thus, there is a need to understand in a better way these variables that impact the dichotomy between services, as well as the implementation of strategies that ensure adequate patient management. 111702 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT HENRIQUE IAHNKE GARBIN1, Fernando Luis Scolari2, Felipe Costa Fuchs2, Edileide de Barros Correia3, Beatriz Piva e Mattos2 (1) Post-Graduation Program in Cardiology and Cardiovascular Sciences, Faculty of Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; (2) Division of Cardiology, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; (3) Division of Cardiomyopathy, Instituto Dante Pazzanese de Cardiologia, São Paulo, Brazil Background: Atrial fibrillation (AF) is a common arrhythmia in hypertrophic cardiomyopathy (HCM), frequently related to adverse outcomes. The AF-HCM point-score is a recently validated predictive model for the assessment of AF risk. Objectives: We sought to independently evaluate the novel AF-HCM score in a tertiary HCM center cohort in Brazil. Methods: A longitudinal HCM cohort followed between 2007–2022 was retrospectively stratified for new onset AF, according to the presence of left atrial dimension (+2 points per 6 mm increase), age at clinical evaluation (+3 points per 10-year increase), age at HCM diagnosis (–2 points per 10-year increase) and heart failure symptoms (+3 points if symptomatic). The AF-HCM score was classified as low (<1,0%/year; score ≤17), intermediate (1,0–2,0%/year; score 18 to 21), and high risk (>2,0%/year; score ≥22) for AF development. Cox regression model and Kaplan-Meier survival free from AF were analyzed, P < 0,05. Results: A total of 116 patients, aged 56 ± 13 years, 64 (55%) females, were followed for 7.0 ± 5.5 years. The left ventricular maximal wall thickness was 18 ± 3 mm, the left atrial diameter 46 ± 6 mm, 46 (40%) subjects were in NYHA class I, 49 (42%) in class II and 21 (18%) in class III. The AF-HCM score stratified risk as low in 13 (11%) individuals, as intermediate in 49 (42%) and as high in 60 (52%). Over the follow-up, 37 (32%) were diagnosed with AF. Among those with newly diagnosed AF, none was classified as low risk, 14 (38%) as intermediate and 23 (62%) as high, P = 0.027. The area under the ROC curve to detect AF was 0.671 (95% CI 0.565–0.777, P = 0.003). The Kaplan-Meier curve showed that patients in intermediate and high-risk were more likely to develop AF [P (Log-Rank) <0.001]. The intermediate and high-risk groups were associated with developing arrhythmia with a hazard ratio of 56.8 (95% CI 3.4–944.6), P = 0.005. The specificity and the negative predictive value were 100%. On the contrary of the previous North-America study population, most patients of the Brazilian cohort were in the high-risk category (37% vs. 52%). Conclusion: The AF-HCM score is a reasonable tool for recognizing patients not prone to develop AF among those stratified as low risk in a Brazilian cohort. However, the model was limited to identify intermediate and high risk individuals in contrast to the previously stratified North-American population. 111708 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BÁRBARA BRANDÃO LOPES1, Bárbara Brandão Lopes1, João Joadson Duarte Teixeira1, Maria das Graças da Silva Guerreiro2, Nádya dos Santos Moura5, Ivana Rios Rodrigues1, Rebeca Silveira Rocha3, Maria Luziene de Sousa Gomes1, Weslley Tiago Sousa Alves4, Bárbara Gomes Santos Silva1, Mônica Oliveira Batista Oriá1 (1) Universidade Federal do Ceará – UFC; (2) Universidade Estadual do Ceará – UECE; (3) Maternidade Escola Assis Chateaubriand – MEAC/UFC; (4) Prefeitura Municipal de Fortaleza – PMF; (5) Universidade Federal do Piauí – UFPI Introduction: Hypertensive disorders occupy the first place in the ranking of causes of maternal deaths in developing countries, as well as being responsible for about 14.0% of all maternal deaths in the world, especially preeclampsia (PE). Despite the lack of knowledge about the etiology of PE and the complexity of its pathophysiology, some factors are related to the development of the disease. Therefore, the early identification of risk factors and the correct management of pregnant women at risk of preeclampsia is a decisive factor in the maternal-fetal clinical outcome. Objective: To investigate the risk factors and maternal characteristics for the development of preeclampsia in pregnant women. Method: Prospective cohort study carried out in 18 Primary Health Care Units in the city of Fortaleza, Ceará, Brazil between March 2018 and February 2020. The study population consisted of pregnant women captured in the 1st gestational trimester, being followed up until postpartum. Sociodemographic, clinical and obstetric data were collected using a form and processed using the Statistical Package for Social Sciences (SPSS) version 24.0 and R software version 3.5.5. Study approved by the Research Ethics Committee of the Federal University of Ceará, according to opinion number 2.448.308. Results: Final sample consisted of 146 patients, of which 39 developed preeclampsia (26.71%). No sociodemographic and clinical aspects were associated with preeclampsia. However, obstetric data such as previous hypertensive disorder (p = 0.03), family history of preeclampsia (p = 0.007) and body mass index (p = 0.01), with a mean of 28.52 (SD ± 7,32) had a strong relation with preeclampsia. Conclusion: In prenatal care, the identification of risk factors related to the development of PE is essential, in order to early identify pregnant women at greater risk for the disease and offer them specialized follow-up, with actions aimed at reducing maternal and perinatal morbidity and mortality. 111847 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LARA CABRITA1, Ana João Taveira2, André Gomes Roque3, Estela Cabral4, Pedro Damião5 (1) USF Fénix de Aveiro; (2) UCSP Mira; (3) USF Santa Joana; (4) USF Atlântico Norte; (5) USF Fénix de Aveiro Introduction: Cerebrovascular disease (CVD) is a major morbidity and mortality cause. Systolic blood pressure (BP) is classically referred to as a predictor of cerebrovascular events (CVE), and clinical attention is generally given to the mean values of SBP. However, several studies have shown that blood pressure variability (BPV) is a predictor of hypertension mediated organ damage, CVE, and all-cause mortality. Objective: Our study aims at describing long term systolic BPV distribution based on different combinations of modifiable cardiovascular risk factors, before and after a CVE. Methods: We performed an exploratory data analysis on a convenience sample of patients with previous CVE (stroke and transient ischemic accident (TIA)), from a single primary care center. Data was obtained from clinical registries in the software in use on our center. The variables included were age, sex, tobacco use, hypertension, dyslipidemia, obesity, overweight, type 2 diabetes and systolic blood pressure. BPV was calculated as the variability coefficient from the closer 3 systolic BP measures pre and post event, to a maximum of 5 years pre and post event. Statistical analysis was performed with R® software and presented in cardinality boxplots. We compared before and after BPV with the Wilcoxon test. Results: Our initial sample included 255 patients with cerebrovascular events, from which only 80 had a minimum of 3 office BP measurements pre and post CVE, and as such were eligible for systolic BPV analysis. Blood pressure target met (defined as systolic BP below 140 mmHg) proportion of patients, pre and post CVE, were 0.475 and 0.508, respectively. No significant differences were found between pre and post event BPV (p = 0.78 global events; p = 0.99 for stroke; p = 0.38 for TIA). No trend was identified for post event reduced BPV for a single or combinations of risk factors. Conclusion: Our study shows that office long term BPV does not vary before and after a CVE. However, in our sample, systolic BP mean proportion of patients that meet the recommended target also remains the same after a CVE, probably meaning a lack of control of this important predictor. This study has major limitations, being observational with a small sample of patients, which renders definite conclusions for the relevance and eventual change of BPV before and after cerebrovascular events. However, it clearly shows that there is a need to improve risk factor goal attainment after a CVE. 111716 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY LUIS HENRIQUE OLIVEIRA PEREIRA1, Kelvin Câmara1, Tamires Santos Pinheiro1, Matheus Mônaco Lemos1, Ana Laísa Andrada Oliveira2, Maria Eduarda Pereira de Oliveira3, Gabrielly Machado Trindade4, Marjorie Francisca Raksa1, Manoel Flávio Silva Kanisky1, Gerlânio César da Silva1, Valdano Manuel5 (1) Universidade Centro de Ensino de Maringá (UNICESUMAR); (2) Faculdade Pernambucana de Saúde (FPS); (3) Universidade Vila Velha (UVV); (4) Centro Universitário Max Planck (UniMax); (5) Clínica Girassol, Complexo hospitalar de doenças cardio-pulmonares Cardeal Dom Alexandre do Nascimento, Instituto do coração (InCor) do Hospital das Clínicas da Universidade de São Paulo Introduction: Rheumatic heart disease (RHD) remains a public health problem, especially in developing countries. The mitral valve is the main affected cardiac structure, requiring intervention in many cases. The discussion of the best intervention is still a controversy; repair or replacement. Objectives: This systematic review aims to evaluate the survival of patients with RHD submitted to valve replacement or repair. Method: We systematically reviewed the English literature through PubMed, LILACS, SciELO and Google Scholar between January 1, 2021, and February 28, 2022, based on PRISMA methodology. Articles with a sample of at least 30 patients under 66 years-old who underwent mitral valve (MV) replacement or repair were included. The articles were classified by Newcastle-Ottawa Scale. Results: Six studies including 2874 patients were analyzed. Most of the patients were female (2001; 69.6%) with a ratio of 2.30:1 for female:male. The age ranged from 11 to 66 years old, being 10 to 65 years old in the repair group and 12 to 66 years old in the replacement group. The mean follow-up time varied from six to 106 months. In MV repair group, the mortality was 3.2% and reoperation was 6.2%, while in the MV replacement the mortality was 7.5% and 3.2% required reoperation. Patient’s survival was found to be similar (85% for repair and 87% for replacement). About the main complications post-MV repair and MV replacement, respectively, were: stroke (1.8%, 2.5%) and endocarditis (0.5%, 1.3%). One article, composed by 148 patients, compared datas between mechanical and bioprosthetic surgical replacement: mortality (2%, 17%), reoperation (5.6%, 5.2%) and survival in four years (98% ± 2%, 70% ± 10%), in that order. The article also presented that the main general complications were stroke (0%, 5,2%) and endocarditis (1%, 4%). Conclusion: MV repair demonstrated lower mortality in patients compared to MV replacement, even though it had a higher rate of reoperation. These facts support the idea of shared decision with the heart team and the patient, considering their clinical status, life expectancy, measuring risks and benefits of the surgical decision. 111731 Modality: E-Poster Young Researcher – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY HENRIETTE MÉSZÁROS1, Pál Ábrahám1, Andrea Ágnes Molnár1, Dorottya Pásztor1, Sándor Nardai1, Béla Merkely1, Béla Merkely1 (1) Heart and Vascular Centre of Semmelweis University Introduction: Half of the cryptogenic strokes are attributed to persistent foramen ovale (PFO), their transcatheter closure significantly reduces the risk of recurrent stroke. Aims: We aimed to do a comparative analysis of the baseline clinical and follow up data from our PFO closure patients with the data from the largest randomized trial on PFO(441 patients). Methods: We prospectively enrolled 105 patients in our single-centre registry who had PFO-closure between January 2018 and March 2021. We analysed retrospectively their baseline clinical data, PFO anatomy, procedural outcome and prospectively arrhythmias, device-related complications, and recurrent stroke events during a median follow-up time of 585 days. Data was compared to the published data from the PFO-closure arm of the REDUCE. Results: The mean age of the patients(45 ± 10.6 vs.44.4 ± 9.3(p = 0.56); and the distribution of genders was similar(males 64% 67/105 vs.59.2% 261/441; p = 0.44). More of our patients had hypertension(42.9% 45/105vs.25.4% 112/441p = 0.0007), and were smokers(36.2% 38/105vs.14.3% 63/441 p = 0.001), the prevalence of diabetes (4.7% 5/105 vs.4.1% 18/441(p = 0.80) and the proportion of complex PFO-s was similar(57.2% 57/105 vs.42.8% 268/441 p = 0.23). The success rate of closures was similarly high:91.4% 96/105 vs.93.7% 413/441 p = 0.40. Device dislocation occured in 1 patient vs.in 3 patients(0.9% 1/105 vs.0.6% in 3/441 patients p = 0.58). No recurrent stroke occurred in our cohort, while 5 patients (1.2%) had stroke in REDUCE in the first 2 year follow-up period, this difference is non-significant. 1 of our patients(0.9%) had postoperative paroxysmal atrial fibrillation, while 29 patients(6.6%) of the REDUCE cohort, however their follow-up time for arrythmias was longer, therefore this data is not comparable. Conclusion: The short and midterm outcome of our PFO program yielded similar results to REDUCE, despite treating patients of higher cardiovascular risk. Further follow-up is needed to determine the long-term prevalence of atrial fibrillation, therefore we need imaging markers in the future for to predict the developement of this arrhytmia. 111759 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GHABRIELA SILVA RABELO1, Ghabriela Silva Rabelo1, Júlia Souza Siqueira1, Lorena Oliveira Carneiro2 (1) Universidade Católica de Brasília; (2) Universidade Federal do Goiás Introduction: One of the most prevalent acquired heart diseases in children and young adults in Brazil is the Rheumatic Heart Disease. When the patient has already been exposed to the patogen and wasn‘t treated, the use of antibiotic prophylaxis (AP) prevents further complications. Goal: Given the social/economical difficulties and the high incidence of rheumatic disease in Brazil associated with the prolonged period of antibiotic administration, this study aims to understand the impact of the AP in Brazil in preventing heart damage. Methods: A systematic review carried out in accordance with the recommendations of the PRISMA. The databases used in the study were: SciELO, Lilacs and PubMed. Those search descriptors were used: Rheumatic Heart Disease, Brazil and antibiotic prophylaxis. The inclusion criteria consisted of original articles, of free access, published in English and/or Portuguese and that met the objective of the study. Exclusion criteria included reviews and monographs. Results: The total of 5 articles were screened, 1 of which were duplicate; however, only 3 were selected and included in the review, since the study not selected is a narrative review. A cross-sectional study shows a growth of 215% from 1998 to 2016 of the mortality rate from acute rheumatic fever. A randomized clinical trial with children and adolescents with latent rheumatic heart disease were given intramuscular benzathine penicillin G (BPG) every 4 weeks for 2 years or no prophylaxis (control). Among the participants who completed the study, only 0.8% had echocardiographic progression, compared with 8.2% in the control group. A prospective cohort study followed 593 patients with rheumatic fever for 32 years, starting AP with BPG every 3 weeks, in which 10.4% of patients changed treatment to every 4 weeks after turning 21 years of age. This study was divided into two groups, both with AP, the first with patients without cardiac lesions (41%) and the second with cardiac lesions (59%), among these, 17 patients, in irregular AP, were affected by the worsening cardiovascular; however, no patient on regular AP experienced progression of cardiac damage. Conclusions: This study demonstrates the efficiency of regular use of AP to reduce the recurrence of heart damage and the high rates of rheumatic heart disease in Brazil. However, there are few studies comparing the accessibility and effectiveness of the Brazilian prophylactic scheme in relation to international protocols. 111762 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUIZ FERNANDO SOUZA SANTOS1, Jeferson dos Santos1, Suelen Maiara dos Santos1, Aline Barreto Hora2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe Introduction: Cardiovascular diseases represent the main cause of death in Brazil and in the world. Acute Coronary Syndrome (ACS) is one of the presentations of these diseases, with ST elevation acute myocardial infarction (STEMI) being the type with the highest morbidity and mortality. Objective: To assess the correlation between patient outcomes in STEMI episodes between public and private health care networks. Method: The present study used the ACCEPT registry database. Patients with ACS treated in selected Brazilian hospitals were compiled. Only patients with STEMI were considered (Public Network = 1029, Private Network = 521). P-values (Fisher’s exact) were reported to three decimal places. This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: Regarding to the clinical outcomes in the first 7 days, there was no significant difference between episodes of reinfarction, stroke and severe bleeding (Table 1). However, there was a difference in death in the first 7 days (Public Network – 3.8%; Private Network – 1%; p = 0.001). Conclusion: It was concluded that there are statistically significant differences between patients who use the Public Network suffering worse outcomes, death showed differences in the two studied moments, while at hospital discharge there was a worse prognosis for SUS users, the other variables were not significant. 111766 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARIANA DA SILVEIRA CASTRO1, Leonardo Sandrini Costa1, Rafaella Quirino Alcântara1, Geovana Oliveira de Paula1, Felilpe Fonseca dos Reis1 (1) Pontifícia Universidade Católica de Goiás (PUC-Goiás) Introduction: Chagas disease is an infectious tropical disease, caused by the flagellate protozoan Trypanosoma cruzi, transmitted by contact with the feces of vector insects, called “barbeiros” in Brazil, and it reamins still quite neglected. It has a greater distribution in Brazil, reaching about 40% of the territory, and it is estimated that 30% evolve to cardiac and digestive form, the main responsible for the high disease morbidity and mortality. Objective: To analyze the number of deaths from Chagas disease reported in the Brazilian population, between 2010 and 2020. Methodology: Epidemiological, retrospective, descriptive and quantitative study, carried out by collecting data from the Mortality Information System (SIM) on the platform of the Department of Informatics of the Unified Health System (DATASUS). Mortality data from Chagas disease in the Brazilian population were analyzed from January 2010 to December 2020. The sociodemographic and clinical variables selected were: region and Federative Unit of residence, year of death, sex and age group. Results: In the analyzed period, the total of 49.574 deaths from Chagas disease in the entire national territory was recorded, with 26.886 being men and 22.684 women. Most deaths occurred in age group 70–79 years, corresponding to 28,2% of the total. The variation in mortality in this 10-year interval followed a specific pattern, with a decreasing rate of 14,6%, with the year 2020 having the lowest rate with 4.165 deaths, while the year 2010 represented the highest number of deaths, with 4.876. According to the Federative Unit residing, it was observed that Southern remained as the largest holder of deaths from Chagas disease in the country, with emphasis on the state of São Paulo. On the other hand, region North presented the lowest number with 2% in relation to the total. Conclusions: It was possible to identify a high concentration of deaths from Chagas cardiomyopathy in the the most populated region of the country, even with a drop in the number of cases in a ten year period. In view of the national perspective, primary care should be further promoted in terms of the access of the Brazilian population to effective diagnosis and treatment of the disease, since they are crucial for improving the quality of life of patients with this disease, as well as for the prevention of its chronic, and even fatal, condition. 111781 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS SUELEN MAIARA DOS SANTOS1, Jeferson dos Santos1, Luiz Fernando Souza Santos1, Aline Barreto Hora2, Andreza Oliveira Almeida2 (1) Universidade Tiradentes; (2) Universidade Federal de Sergipe Introduction: Acute ST elevation myocardial infarction (STEMI) is one of the main causes of death caused by cardiovascular diseases. Combined events such as reinfarction, respiratory arrest, severe bleeding and stroke are factors that affect the prognosis of STEMI. Objective: To evaluate events that may contribute negatively to the prognosis of STEMI in the population studied. Method: The present study used the ACCEPT registry database, which selected patients with Acute Coronary Syndrome treated in Brazilian hospitals in all regions. Only patients with STEMI were considered (Public Network = 1029; Private Network = 521). P-values (Fisher’s exact) were reported to three decimal places with p-values less than 0.001 reported as p < 0.001. This study was approved by the Research Ethics Committee of the Federal University of Sergipe, under registration number 302.544. Results: In this present study, the variables: attendance at the Unified Health System (SUS), region, age, systemic arterial hypertension (SAH), diabetes, Congestive heart failure (CHF), current smoking and dyslipidemia were the factors that most contributed to the negative prognosis. (Fig. 1). The variables of female sex, reperfusion with primary and non-primary angioplasty only, and thrombolytic-only reperfusion reduce the likelihood of a negative prognosis. Conclusion: Care in the SUS, SAH, CHF, diabetes and current smoking were factors that most contributed to the negative prognosis. 111856 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MIRIAM ALLEIN ZAGO MARCOLINO1, Miriam Allein Zago Marcolino1, Rodrigo Antonini Ribeiro2, Luis E. Rohde3, Carisi Anne Polanczyk4 (1) Graduate Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; (2) National Institute of Science and Technology for Health Technology Assessment – INCT/IATS (CNPq 465518/2014-1), Porto Alegre, Brazil; (3) Cardiology Center, Hospital Moinhos de Vento, Porto Alegre, Brazil; (4) Graduate Program in Cardiology and Cardiovascular Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction: Cardiac implantable electronic devices (CIED), including pacemakers (PM), implantable cardioverter-defibrillator (ICD), and cardiac resynchronization therapy (CRT), are interventions long established in the cardiology practice, but restricted for selected patients. They include simple devices to highly complex and costly procedures. Objective(s): To describe the profile of patients that received a CIED covered by the Brazilian public health system (SUS) over a 12-year period. Methods: Retrospective epidemiological study including open data of all hospital admissions with a CIED implantation as primary procedure reimbursed by the SUS between 2008 and 2019, obtained from the Hospital Information System of SUS Informatics Department (DATASUS). Mean annual rate per million population was age and sex standardized by the OMS standard population. All analysis were performed using R. Results: SUS reimbursed 216,440 CIED implant procedures from 2008 to 2019 (mean annual rate of 87.1 per million population), mainly PM (89.3%). Patients mean age was 68.8y (15.3), and were mostly male (54%), residents in the Southwest (45%), South (22%), or Northeast (21%) regions. Arrhythmias diagnoses were present in 87% of the patients, mostly in PM (91%) and ICD (83%). Heart failure was present in 61% of CRT and 38% of CRT-D patients. The mean length of stay was of 4.1 (6.3) days, 1.5% died during hospitalization, and mean hospitalization reimbursement values varied by CIED type (R$8,309 to R$ 56,791). (Table 1) Conclusions: Twelve-year cumulative data indicates that CIED implantation in Brazil was performed mostly on aged patients, males, residents in the Southwest, South or Northeast, PM predominantly. These data might help guide health policy actions on access to CIED therapies. 111871 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RAPHAELA PAULA PINHEIRO1, Carlos Renato de Oliveira2, Allice de Souza Rodrigues1, Maria Julia Montebeller Meneses1, Renato Jorge Alves3 (1) Santa Casa de Sao Paulo School of Medical Sciences; (2) Santa Casa de Misericordia de Sao Paulo Hospital, Cardiology; (3) Santa Casa de Sao Paulo School of Medical Sciences; Santa Casa de Misericordia de Sao Paulo Hospital, Cardiology Background: Hyperlipidemia is a well-established cardiovascular risk factor. The overweight is associated with low-grade chronic inflammation and is correlated to cardiometabolic risk factors. Pulse wave velocity (PWV), the gold standard for arterial stiffness assessment, can detect subclinical atherosclerosis with accuracy. Studies suggest an association between PWV and nutritional status. AIM To describe the overweight prevalence, and correlate with arterial stiffness assessed by PWV and cardiometabolic risk factors in treated hyperlipidemic patients. Methods: We performed a cross-sectional study. There were included hyperlipidemic patients, over 18 years. Sociodemographic data and clinical background were collected by a standardized questionnaire and medical records. Nutritional status was determined by body mass index (BMI), considering “overweight” BMI ≥25 kg/m² to adults <60 years and BMI ≥28 kg/m² to elderly ≥60 years. The brachial PWV was assessed by the non-invasive oscillometric method and the patient’s laboratory tests were recently collected by medical records. Data were presented as frequency (%), prevalence(%), or mean ± S.D. Results: Sample: 62 patients. Mean age of 63,9 ± 12,5 years, 53,23% female, 43,5% diabetics, 88,7% hypertensives, 64,5% Fredrickson’s phenotype IIa, 67,7% used high potency statin and 64,5% overweight prevalence, mean BMI of 28,8 ± 3,7 kg/m². Overweight patients presented a lower mean of PWV (8,95 m/s), although higher use of high potency statin (55,65%) when compared to those without overweight (10,06 m/s; 38,46%). The patients with overweight presented higher means of LDL-c (113,7 ± 61,3 mg/dl), total cholesterol (190,3 ± 74,6 mg/dL), triglycerides (202,3 ± 157,7 mg/dL), fasting blood glucose (113,2 ± 40,4 mg/dL), glycated hemoglobin (6,5 ± 1,4%), uric acid (6,2 ± 2,4 mg/dL) and lower means of HDL-c (42,9 ± 11,6 mg/dL), when compared with individuals without overweight (99 ± 30,5; 169,1 ± 47,1; 165,2 ± 100; 108,4 ± 30,3 mg/dL; 6,2 ± 1,0%; 5,7 ± 1,1; 43,1 ± 17,3 mg/dL respectively). Conclusion: The sample had a 64,5% prevalence of overweight, and these individuals presented a worse cardiometabolic profile when compared with individuals without overweight. Contrary to most of the publications, overweight patients presented lower PWV, although they used more high potency statin when compared to those without overweight. Our results may be related to reverse endothelial remodeling, but longitudinal studies can be used to elucidate these correlations. 111884 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JHESSICA MACIEIRA PEREIRA1, Thayrine Rosa Damasceno1, Rafael Dias de Brito Oliveira1, Enrico De Francisco Magnani1, Danielle Aparecida Gomes Pereira1, Denise Mayumi Tanaka2, Eduardo Elias Vieira De Carvalho2, Júlio César Crescêncio2, Eduardo Rubio Azevedo2, Marcus Vinicius Simões2, Luciano Fonseca Lemos De Oliveira1 (1) Universidade Federal de Minas Gerais – UFMG; (2) Faculdade de Medicina de Ribeirão Preto – FMRP/USP Introduction: Increased pulmonary artery systolic pressure (PASP) and reduced left ventricle ejection fraction (LVEF) in individuals with heart failure (HF) is related to disease progression, morbidity and mortality. On the other hand, booth PASP and LVEF are not related to functional capacity (FC). Patients with Chagas HF are known to present worst prognosis and may develop myocardial dysfunction and pulmonary hypertension (PH). However, the association of FC with cardiac function and PH in Chagas HF is still unclear. Objective: To evaluate the association of FC with cardiac function and PH in individuals with Chagas and non-Chagas HF. Methods: Cross-sectional observational study with 178 patients with HF who underwent clinical examination, cardiopulmonary exercise test (CPET) and 2-dimensional echocardiography. The sample was divided between Chagas (CH, n = 101) and non-Chagas (NCH, n = 77) HF. Data normality was analyzed using the Kolmogorov-Smirnov test. Data comparison was verified with Student’s t test or Mann-Whitney test. The association was performed by using the Pearson or Spearman correlation coefficients. Results: Compared to NCH group, CH group presented higher VO2peak (16.8 ± 6.1vs 13.8 ± 4.3 ml.kg.min–1, p < 0.001) and LVEF (42.6 ± 18 vs 28.9 ± 9.7%, p < 0.001), lower left atrial diameter (LAD; 43.1 ± 8.4 vs 47.7 ± 7.2 mm, p < 0.001), left ventricle diastolic diameter (LVDD; 59.5 ± 9.1 vs 66.7 ± 9.9 mm, p < 0.001) and PASP (37.3 ± 13.4 vs 51 ± 15 mmHg, p < 0.001), and similar ventilatory efficiency given by the VE/VCO2slope (35.5 ± 10.6 vs 35.8 ± 8.2, p = 0.83). In the CH group, VO2peak correlated significantly with PASP (r = –0.60, p < 0.001), LVEF (r = 0.51; p < 0.001), LVDD (r = –0.36, p < 0.001), LV mass index (r = –0.36, p < 0.001), LAD (r = –0.34; p < 0.001), E wave (r = –0.32; p = 0.01) and TAPSE (r = 0.27, p = 0.04). The VE/VCO2slope was correlated with LVEF (r = 0.53; p < 0.001), PASP (r = 0.46; p = 0.001), LVDD (r = 0.42; p < 0.001), LAD (r = 0.33; p = 0.001), TAPSE (r = 0.3, p = 0.03) and LV mass index (r = 0.21, p = 0.047). In the NCH group, VO2peak was only correlated with TAPSE (r = 0.60, p = 0.013) and LAD (r = –0.25, p = 0.04). Moreover, VE/VCO2slope was not correlated with echocardiography variables. Conclusion: In patients with Chagas HF, PASP as well as myocardial function and morphology impacts the functional capacity of the subjects when compared to patients with different etiologies. In addition, the findings suggest that PH may be related to the disease severity and progression. 111891 Modality: E-Poster Young Researcher – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES SAMUEL BARUD MASSENSINE1, Amanda Gonçalves Vieira Martins1, Beatriz Stephan Farhat Jorge1, Bianca de Fatima Pereira1, Fernanda Silva Mota1, Gabriela Godinho Rezende1, Moisés de Toledo Vilela1, Paula Gouvea Abrantes1, Pedro Drumond Maia1, Ramon Jose Moreira Silva1, Eliane Ferreira Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Cardiology Department, Medical School, Federal University of Juiz de Fora – UFJF Introduction: Smoking and alcohol abuse usually coexist and have synergistic effects, leading those with this duo addiction to worse outcomes than those who only smoke. This culminates in personal and family suffering and high social cost. Then it’s important to evaluate the effects of alcohol consumption in smokers. Objectives: To evaluate alcohol abuse (UAA) with clinical characteristics and smoking history, among smokers during the smoking cessation process. Methodology: Longitudinal study, group “Free Tobacco”, Juiz de Fora/Minas Gerais. Users with multi-morbidity were treated between September/2021 and March/2022, in consecutive treatment groups, mixed intervention (face-to-face and telemedicine), multidisciplinary team, cognitive behavioral approach (ACC) sessions, drug treatment and follow-up. Definitions: UAA, Audit-C ≥5 points. Low level of education, <8 years. Normal systolic blood pressure (SBP) <130 mmHg. Depression, Patient Health Questionnaire (PHQ-9) score ≥9 points. Cognitive deficit, Montreal Cognitive Assessment <26 points. High nicotine dependence, Fagerstrom test ≥5 points. Results: The sample consisted of 36 smokers with multi-morbidity who participated in a group with a multidisciplinary team, intended for treatment for smoking cessation. UAA was found in 20% of the sample. Comparing these patients with those without this use, they were younger individuals (p < 0.049), with a higher prevalence of abdominal obesity (p < 0.002), arterial hypertension (p < 0.002), of cognitive deficit (p < 0.001) and with a tendency to greater use of crack together (p < 0.063). Allied, they had a lower prevalence of depression (p < 0.031), anxiety (p < 0.021), and curiously, sedentary lifestyle (p < 0.049). As for smoking history, they had greater nicotine dependence (0.001), greater triggers for alcohol itself (p < 0.008), for habits (p < 0.003), but not for the use of coffee (p < 0.019). Allied, greater adherence to meetings for ACC, (p < 0.001). Conclusion: The association between smoking and alcohol consumption is associated with the perpetuation of this addiction and increased consumption of illicit drugs. Screening for alcohol use should be encouraged for all smokers, and interventions aimed at encouraging the cessation of both harmful habits should be the target audience. As it is also associated with other unfavorable outcomes, it is necessary to track and discourage alcohol consumption by smokers. 111922 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSIOTHERAPY THAYRINE ROSA DAMASCENO1, Enrico de Francisco Magnani1, Rafael Dias de Brito Oliveira1, Jhessica Macieira Pereira1, Denise Mayumi Tanaka2, Mariana Duarte de Souza1, Jorge Mejia Cabeza3, Camila Godoy Fabricio2, Alessandra Arantes Resende2, Dawit Albieiro Pinheiro Gonçalves1, Marcus Vinicius Simões2, Luciano Fonseca Lemos de Oliveira1 (1) Universidade Federal de Minas Gerais (UFMG); (2) Faculdade de Medicina de Ribeirão Preto (FMRP/USP); (3) Hospital Israelita Albert Einstein Introduction: Chronic Chagas cardiomyopathy (CCC) may present with morphological, functional and myocardial perfusion alterations. However, the influence of these changes in the reduction of functional capacity and the effects of aerobic physical training (APT) on them are still unknown. Objective: To evaluate the association of aerobic capacity with morphology, function and myocardial perfusion as well as to evaluate the effects of APT on such variables in a model of CCC in Syrian hamsters. Methods: Female Syrian hamsters infected with 3.5 × 10000 trypomastigote forms of the Y strain of T. Cruzi (n = 37) and their respective controls (n = 14) were used. Seven months after infection, the surviving animals underwent two-dimensional echocardiography, myocardial perfusion scintigraphy to assess myocardial perfusion defects (PD) and cardiopulmonary testing (CPT). The animals were then divided into 4 groups: Sedentary Chagas (CH-SED, n = 14), APT Chagas (CH-APT, n = 9), Sedentary Control (CT-SED, n = 6) and APT Control (CT-APT, n = 8). The APT was performed for 8 weeks, 5 times a week, for 50 minutes at an intensity equivalent to 50% of the peak velocity evaluated in the TCP. All animals repeated the examinations at the end of the experiment. Results: At baseline, 9 (24%) of the 37 infected animals showed myocardial involvement with reduced left ventricular ejection fraction (LVEF, pANNOVA <0.01), VO2peak (pANNOVA = 0.01) and higher PD (pANNOVA = 0.01) when compared to control and infected animals without cardiac involvement. VO2peak correlated with PD (r = –0.66, p < 0.01), LVEF (r = 0.37, p = 0.02), left ventricular diastolic diameter (LVDD, r = –0.53, p < 0.01) and LV systolic diameter (LVSD, r = –0.49, p < 0.01). In the multiple regression analysis, only PD remained independently associated with VO2peak (r² = 0.42). After the follow-up period, the mixed analysis of variance showed that the groups submitted to APT and the CH-SED group showed eccentric LV remodeling with an increase in LVDD. However, an increase in LVSD and PD, as well as a decrease in LVEF were only observed in the CH-SED group. In addition, the CH-APT group showed an increase in VO2peak and an increase in the maximum distance covered after the intervention. Conclusion: APT was able to prevent the progression of perfusion defects and LV dysfunction, in addition to improving functional capacity, which is determined by morphology, function and myocardial perfusion in an experimental model of CCC. 111952 Modality: E-Poster Young Researcher – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MICAELE FARIAS NASCIMENTO1, Beatriz Souza de Albuquerque Cacique New York1, Milton Antonio Gonçalves de Oliveira1, Kátia Elizabete Galdino1, Ana Tereza do Nascimento Sales Figueiredo Fernandes1 (1) State University of Paraíba – UEPB Introduction: Cardiac surgery (CS) may be associated with several organic repercussions responsible for the appearance of cardiac risk factors during the postoperative period. These, associated with prolonged hospital length of stay (LoS), may trigger critical manifestations in individuals undergoing this surgical procedure. Objective: To investigate the relationships between postoperative cardiac risk factors, LoS, and changes in functioning state. Methods: Patients undergoing reconstructive, substitutive, or corrective cardiac surgeries were evaluated. The presence of postoperative cardiovascular risks was assessed using the InsCor score, while LoS and functionality were collected from medical records. Results: One-hundred patients with a mean age of 59.2 ± 12.3 years were included. Significant correlations between functionality and both the hospital and Intensive Care Unit (ICU) LoS (p < 0.0001, ρ = 0.56; p = 0.002, ρ = 0.29, respectively), as well as between hospital LoS and the number of comorbidities (p = 0.003, ρ = 0.28) were found. No significant relationships were observed between the number of postoperative risk factors and LoS. Conclusion: Functionality and comorbidities are associated with increased hospital and ICU LoS in patients undergoing cardiac surgery. 111976 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MANUELLA DE AMORIM SILVA4, Marcelo Antônio Oliveira Santos Veloso3, Gabriela Cisneiros Arcoverde4, Andrea Virgínia Chaves Ferreira3, Eveline Barros Calado1, Manuel Markman5, Lucas Soares Bezerra3, Brivaldo Markman Júnior1, Dinaldo Cavalcanti de Oliveira3 (1) Hospital das Clínicas da Universidade Federal de Pernambuco HC-UFPE; (2) Serviço de doenças raras RARUS; (3) Programa de Inovação Terapêutica da Universidade Federal de Pernambuco – UFPE; (4) Hospital Alfa de Referência ao COVID-19 – HRE; (5) Hospital Agamenon Magalhães – HAM Background: Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death (SCD) in youth. The recommendations for prophylactic implantable cardioverter-defibrillators (ICDs) are divergent. Objective: To evaluate the agreement in the indication of ICD in patients with HCM, as per recommendations of the European Society of Cardiology 2014 and American Heart Association 2020, and evaluate fragmented QRS (fQRS) as a predictor of cardiovascular outcome. Methodology: Retrospective cohort with 81 patients evaluated between 2019 and 2021. Patients with HCM ≥16 years old were included. Exclusion criteria: secondary myocardiopathy, follow-up <1 year. Kappa coefficient was used to determine the agreement. Survival and incidence curves were determined by Kaplan-Meier method. A p-value <0.05 was considered significant. Results: The fQRS was identified in 44.4% of patients. There were no differences between patients with and without fQRS regarding clinical parameters, echocardiography, fibrosis and SCD risk. During follow-up of 4.8 ± 3.4 years, there was no SCD, but 20.6% patients with ICD had at least one appropriate shock. Three of the seven appropriate shocks occurred in European Society of Cardiology low-to-moderate risk patients. Three shocks occurred in moderate-risk patients and four in American Heart Association high-risk patients. Overall recommendations agreement was 64% with a Kappa of 0.270 (p = 0.007). C-statistic showed no differences regarding the incidence of appropriate shock (p = 0.644). Conclusion: SCD risk stratification algorithms present discrepancies in ICD indication, both with low accuracy. 111977 Modality: E-Poster Young Researcher – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MAICON BORGES EUZEBIO2, Maicon Borges Euzébio2, Priscila Valverde de Oliveira Vitorino3, Sayuri Inuzuka1, Weimar Kunz Sebba Barroso4 (1) UNIVERSIDADE FEDERAL DE GOIÁS UFG; (2) CENTRO UNIVERSITÁRIO DE MINEIROS UNIFIMES; (3) PONTIFÍCIA UNIVERSIDADE CATÓLICA DE GOIÁS PUC GOIÁS; (4) LIGA DE HIPERTENSÃO ARTERIAL LHA HC UFG Introduction: Several studies have demonstrated a higher prevalence and severity of arterial hypertension (AH) in blacks than in whites. However, this is an aspect related to the epidemiology of AH that has been little studied in Brazil. Objectives: To characterize the Brazilian black population of the first Brazilian Hypertension Registry in relation to anthropometric, clinical and pharmacotherapeutic characteristics. Methods: This is a cross-sectional, multicenter descriptive study of the population subgroup of self-declared black from the First Brazilian Registry of Arterial Hypertension, whose collection was carried out from June 2013 to October 2015. A descriptive analysis was performed and the Shapiro- Wilk test was used to verify the data distribution of the variables and adopted as significant p < 0.05. Anthropometric variables were considered: Age, in years; Body mass index (BMI) in kg/m2; Waist circumference (WC) in cm. In the clinical variables, the presence of: Diabetes, Dyslipidemia, Cerebrovascular disease, Cardiac disease, Renal disease, Peripheral artery disease, Smoking, Physical Activity, Alcohol dependence, Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP) in mmHg, Blood Pressure (BP) on target (cut-point for BP control <140 × 90 mmHg), Glucose, mg/dL, Total cholesterol, mg/dL, HDL-Cholesterol, mg/dL, LDL-Cholesterol, mg/dL and Triglycerides, mg/dL. As a pharmacotherapeutic conduct, the use of: Acetylsalicylic acid, Clopidogrel, Metformin, Insulin and Statins. Results: Of a total of 2,646 participants (Whites, Blacks, Browns and Yellows) from 45 locations in Brazilian regions, 452 (17.8%) declared themselves to be black. The median age was 62.5 years (54.2–69.7), 57.5% were female; BMI 29.8 ± 5.36 and WC 98 (91–106). Dyslipidemia was the main disease in 182 (40.3%) individuals, followed by Diabetes Mellitus 128 (28.3%) and other heart diseases with 93 (20.3%). Smoking was found in 31 (6.9%) participants and 273 (60.4%) reported performing some type of physical activity. The median of SBP was 139 mmHg (128–151), of BPD was 85 mmHg (80–92.6) and the frequency of controlled casual blood pressure in 233 (51.54%) participants. Statins 167 (36.9%), acetylsalicylic acid 148 (32.7%) and metformin hydrochloride 82 (18.1%). Conclusions: The black population evaluated was predominantly female. On average, the body mass index was above the recommended, most had controlled blood pressure and performed some type of physical activity. 111988 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LETICIA DE SOUSA PERES1, Thiago Moreira Bastos da Silva2, Iliana Regina Ribeiro Menezes1, Nathalia Duarte Camisão1, Mariana Moreno Canário da Silva1, Renata Mexias Abdala Felix1, Giovanni Possamai Dutra1, Anna Butter1, Henrique Custódio Goudar1, Gláucia Maria Moraes de Oliveira2, Bruno Ferraz de Oliveira Gomes1, João Luiz Fernandes Petriz1 (1) Hospital Barra Dor; (2) UFRJ Background: During the COVID-19 pandemic, cardiovascular comorbidities were associated with greater severity during hospital stay. However, little is known about its association with other long-term outcomes and death. Goals: To assess the association of cardiovascular comorbidities with myocardial injury, lung parenchyma involvement, need for mechanical ventilation, and long-term death in patients hospitalized for COVID-19. Methods: Retrospective cohort study with patients who were hospitalized with a confirmed diagnosis of COVID-19. We evaluated the association of comorbidities diabetes mellitus (DM), arterial hypertension (SAH), chronic kidney disease (CKD) and obesity with the outcomes long-term mortality, in-hospital mortality, myocardial injury, lung parenchymal involvement and use of mechanical ventilation. The variables were evaluated using the chi-square method (categorical variables) and Student’s t test (continuous). Results: 1454 patients were included, mean age 59.8 ± 17.0, 62.6% men. There were 269 deaths (18.5%) during the study period (mean follow-up = 338 ± 209 days). 44.7% of patients had myocardial injury. Obese subjects had greater involvement of the lung parenchyma (>50%) than non-obese subjects (18.5 × 12.1%, p = 0.013). However, they had a lower risk of long-term mortality (OR 0.70; 95%CI 0.51–0.96) as well as a lower prevalence of myocardial injury (OR 0.74; 95%CI 0.59–0.94). We observed an association of DM with long-term mortality (OR 2.30; 95%CI 1.75–3.03), in-hospital mortality (OR 2.21; 95%CI 1.65–2.96), myocardial injury (OR 1.49; 95%CI 1.18–1.87) and need of mechanical ventilation (OR 1.92; 95%CI 1.47–2.50). SAH was also associated with long-term mortality (OR 2.87; 95%CI 2.15–3.84), in-hospital mortality (OR 3.25; 95%CI 2.37–4.44), myocardial injury (OR 2.66; 95%CI 2.15–3.30), need of mechanical ventilation (OR 2.42; 95%CI 1.85–3.17) and pulmonary involvement >50% (17.1 × 11.0%, p = 0.005). Finally, CKD was associated with long-term mortality (OR 3.93; 95%CI 2.39–6.44), in-hospital mortality (OR 3.73; 95%CI 2.24–6.20), myocardial injury (OR 5.73; 95%CI 3.10–10.58) and need of mechanical ventilation (OR 2.61; 95%CI 1.58–4.31). Conclusion: In patients hospitalized for COVID-19, SAH, DM and CKD were associated with long-term mortality, as well as myocardial injury and need of mechanical ventilation. On the other hand, obese individuals had lower mortality and less myocardial injury, despite the greater involvement of the lung parench. 112019 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MANOEL FLÁVIO SILVA KANISKY1, Gerlânio César da Silva1, Kelvin Câmara1, Luís Henrique Oliveira Pereira1, Marjorie Francisca Raksa1, Matheus Mônaco Lemos1, Tamires Santos Pinheiro1, Ana Laísa Andrada Oliveira2, Gabrielly Machado Trindade3, Maria Eduarda Pereira de Oliveira4, Valdano Manuel5 (1) Universidade Cesumar (Unicesumar); (2) Faculdade Pernambucana de Saúde (FPS); (3) Centro Universitário Max Planck (UniMax); (4) Universidade Vila Velha (UVV); (5) Clínica Girassol; Complexo Hospitalar de Doenças Cardiopulmonares Cardeal Dom Alexandre do Nascimento; Instituto do Coração (InCor) do Hospital das Clínicas da Universidade de São Paulo Introduction: COVID-19 can affect the myocardium causing myocarditis. Some patients need ventricular support with Extracorporeal Membrane Oxygenation (ECMO), however, its therapeutic efficacy is not clear yet. Objectives: To know the role of ECMO in patients with myocarditis due to COVID-19. Methods: We systematically reviewed the English literature, searching for patients over 18 years-old on Google Scholar, LILACS and PubMed, using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), between March 2020 to February 2022. The keywords used were “myocarditis”, “COVID-19” and “extracorporeal membrane oxygenation”. The quality of the articles was assessed by Newcastle-Ottawa Scale. Results: Ten studies including 609 patients were analysed. Most of them were male (70,6%) and 29,4% female. Age range was 18 to 80 years. Mean follow-up was 103 days (10–180), the length of stay at the intensive care unit was 26 days (0,9–51,9) and at the hospital was 33 days (21–56,6). The average ECMO duration was 15 days (6,5–38,5). Its most used type was venovenous, which was applied to 469 patients; only 19 received venoarterial, in accordance with eight studies. Although three studies did not report weaning rate, 214 patients (35,1%) weaned off successfully, whereas it is estimated that approximately 165 (27%) did not wean. The most relevant ECMO-related complications, evidenced in eight articles, were: bacterial infection (207), acute kidney (156), liver injury (69) and multiorgan failure (11). The global mortality was 216 deaths (35,4%) which varied from three to 63 deaths. Conclusion: Patients with severe COVID-related ventricular dysfunction submitted to ECMO achieved considerable results of successful weaning off, even though the death rate had a significant result. In addition, venovenous intervention demonstrated an effective strategy in selected patients. However, ECMO application still needs further studies to validate the benefits above its negative outcomes in these patients. 112022 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH CELSON JÚNIO DO NASCIMENTO COSTA1, Paula Simplicio da Silva2, Roberto Magalhães Saraiva2, Andrea Rodrigues da Costa2, Alejandro Marcel Hasslocher-Moreno2, Luiz Henrique Conde Sangenis2, Marcelo Teixeira de Holanda2, Henrique Horta Veloso2, Gilberto Marcelo Sperandio da Silva2, Fernanda Martins Carneiro2, Daniel Arthur Barata Kasal1, Mauro Felippe Felix Mediano1 (1) Departamento de Pesquisa e Ensino, Instituto Nacional de Cardiologia; (2) Instituto Nacional de infectologia Evandro Chagas Introduction: Chagas disease (CD) is a neglected disease that infected 6 to 7 million people worldwide. Individuals with CD are usually from low socioeconomic status and, therefore, more prone to food insecurity (FI). Objective: To assess the prevalence of FI in CD and its association with clinical forms, nutritional status (NS), comorbidities, and biomarkers. Methods: This is a cross-sectional study including patients diagnosed with CD (confirmed by two serological tests), from both sexes. Patients diagnosed with diseases that affect the immune system, other infectious diseases during the data collection period, those using corticosteroids or anti-inflammatory drugs, cancer patients, pregnant women, and cognitive alterations were excluded. The FI was evaluated according to the Brazilian scale of FI (EBIA 2003). The classification of the clinical form of Chagas disease was obtained following the determinations of the Brazilian Consensus on CD (2016). Parameters established by the Brazilian Institute of Geography and Statistics were used to collect the socioeconomic variables. Anthropometric measurements (weight, height, waist circumference) were collected to assess NS. Biomarkers included lipid profile and plasma glucose. Descriptive statistics included mean (standard deviation for numerical) and percentage (frequency) for categorical variables. Comparisons between participants without and with FI were performed using t-test for numerical and chi-squared test for categorical variables. Linear and logistic regression models adjusted by age, sex, education level, and race were fitted to evaluate the association between FI status and clinical forms, NS comorbidities, and biomarkers. Results: Three hundred sixty individuals (56.1% of women) were included in the study, with a mean age of 60.7 (10.8) years. Of those, 30.8% had FI. Participants with FI were more likely to be women, had a lower per capita income, a lower height, a greater percentage of illiterate/incomplete elementary education, and greater frequency of obesity (Table). No significant association was observed between FI and clinical forms of CD, NS, comorbidities, and biomarkers after adjustments for age, sex, education level, and race. Conclusion: Despite the elevated prevalence of FI among patients with CD, no association was observed for health outcomes. Longitudinal studies examining the impact of FI on health parameters and mortality of individuals with CD are warranted. 112038 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH CAMILA DOS SANTOS MOREIRA DE SOUZA1, Dalmo Valério Machado de Lima1, Christianne Bretas Vieira Scaramello1 (1) UNIVERSIDADE FEDERAL FLUMINENSE UFF Introduction: Cardiovascular diseases (CVD) are the main cause of death in Brazil and worldwide. Socioeconomic determinants of health are considered independent risk factors for its occurrence. The Human Development Index (HDI) summarizes local human development encompassing health, knowledge and standard of living, being important to investigate if it can be reflected in CVD premature mortality. Objective: Considering the relevance of socioeconomic determinants on CVD deaths, the aim of this work was to evaluate premature cardiovascular mortality (30–69 years of age) in two Brazilian cities with similar populations and different HDI. Methods: The ecological observational study evaluated proportionate CVD premature mortality in two cities of Rio de Janeiro State – Niterói and Belford Roxo – from 2008 to 2019. Populations of these cities encompass about 500,000 habitants and while Niterói presents a very high HDI (0.837), Belford Roxo presents a medium one (0.684), occupying the 1st and the 71st position in the state HDI ranking, respectively. An Excel spreadsheet was used for data analysis whose significance was accepted if p < 0.05. The local Human Ethics Committee approved the work. Results: Proportionate CVD premature mortality was lower in Niterói compared to Belford-Roxo throughout the period studied (27.6 ± 1,3% × 33.5 ± 2.1%; p < 0.01). Pearson coefficient indicates a strong correlation between proportionate CVD premature mortality in these two cities despite the absence of statistical significance (r = 0.4072; p = 0.1888). According to HDI, the mean porportionate CVD premature mortality seems to vary between genders, with Belford Roxo presenting a higher proportion in females (34.2%) while Niterói shows a higher proportion in males (28.3%). Conclusions: Data suggest that socioeconomic determinants may impact CVD premature mortality. It is important to investigate deeply considering ethnicity and gender aspects to allow better discussions of health policies. 112050 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RHANNIEL THEODORUS HELHYAS OLIVEIRA SHILVA GOMES VILLAR1, José Victor de Sá Santos3, Murilo Jorge da Silva3, Victor Luis Peixoto Pereira Botelho3, Bianca Aparecida Colognese3, Márcio Andrade Barreto Filho4, Marcelo Vincenzo Sarno Filho3, Pollianna de Souza Roriz1 (1) Protocolo IAM – SAMU Salvador; (2) Hospital Ana Nery; (3) Universidade Federal da Bahia; (4) Escola Bahiana de Medicina e Saúde Pública Introduction: The pandemic caused by the coronavirus that started in 2020 changed the pre-established dynamics in health systems and also in the user’s itinerary in the search for medical care. Before, larger general hospitals shared the entrance of patients in the emergency network with pre-hospital units in a similar way. Objective: Identify possible changes in the itinerary of patients with Acute Myocardial Infarction with ST segment elevation (STEMI) in the urgency and emergency care network in the city of Salvador-BA. Methods: An ambispective study with patients with STEMI treated by the Acute Myocardial Infarction Protocol (AMIP) in Salvador during the pre-pandemic (Jan/2019 to Feb/2020) and pandemic (March/2020 to Dec/2021) period. The AMIP is a group designed to assist the entire process of care for patients with suspected STEMI, from diagnosis to reperfusion, in addition to training health professionals for management. Data were collected regarding the unit of origin: fixed pre-hospital (FPH) and general hospital (GH); assistance times; and mortality. Statistical significance was considered for p < 0.05. Results: In the pre-pandemic period, AMIP performed 542 attendings to STEMI, with 25.3% of access through GH. During the pandemic period, 935 visits to STEMI were performed, with the GH being the gateway in 12.0% of the calls (p < 0.001). During the pandemic, there was a higher frequency of activations through the FPH compared to the pre-pandemic period (76.3% × 61.4%; respectively; p < 0.001). Comparing the pandemic to pre-pandemic period, there was a shorter door-to-ECG time [33 (15–77) min × 38 (19–95) min, respectively; p = 0,04] and a shorter door-to-needle time [115 (80–180,5) min × 149 (101,8–206) min, respectively; p < 0,001] in the FPH access. Regarding GH entrance, comparing pandemic to pre-pandemic period, there was a lesser door-to-ECG time [28 (13,5–67) min × 44 (17–149) min, respectively; p = 0,01] and a lesser door-to-ballon time [221 (158,3–294,5) min × 297,5 (203,5–415,8) min, respectively; p = 0,049]. There was no difference in mortality between the groups (15.8% GH × 16.5% FPH; p = 0.92). Conclusion: After closure of general hospitals for non-COVID emergencies during the pandemic, there was a shift in the demand for medical care of patients with STEMI to the pre-hospital units, with shorter care times and without a major impact on mortality in our sample. 112061 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MIRIAM ALLEIN ZAGO MARCOLINO1, Miriam Allein Zago Marcolino1, Rodrigo Antonini Ribeiro2, Luis E. Rohde3, Carisi Anne Polanczyk4 (1) Graduate Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; (2) National Institute of Science and Technology for Health Technology Assessment – INCT/IATS (CNPq 465518/2014-1), Porto Alegre, Brazil; (3) Cardiology Center, Hospital Moinhos de Vento, Porto Alegre, Brazil; (4) Graduate Program in Cardiology and Cardiovascular Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction: Cardiac implantable electronic devices (CIED) implantation and other high-complexity procedures in the Brazilian Public Health System (SUS) are restricted to tertiary care centers located in strategic geographic regions that should provide integral access to care with equity to all population. Objective(s): To describe geographic variations in the access to CIED implantation in the SUS over 12 years. Methods: A retrospective epidemiological study including open data of all hospital admissions with CIED implantation as primary procedure reimbursed by SUS from 2008 to 2019, obtained from the Hospital Information System of SUS Informatics Department (DATASUS). The mean annual rate per million (MM) population per region and state of residency was age and sex standardized by the OMS standard population. All analyses were performed using R. Results: SUS reimbursed 216,440 CIED implantations in the period (87.1/MM). Implantation rates varied according to residency region (South 124.9, Central-West 124.6, Southeast 86.7, Northeast 75.4, North 64.6/MM). The national distribution of different types of CIED implants showed distinct patterns of access by the residents of each state. (Figure 1) Conclusions: Although the regionalization principle of SUS should ascertain equity in the access to health care, 12-year data reveal disparities in the access to different types of CIED in Brazil, showing the need of management actions on this field. 112098 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH JOSÉ PEDRO DA SILVA SOUSA1, José Pedro da Silva Sousa1, Evaldo da Costa Sá Borges de Rezende2, Maria Eliza Alves Teixeira3, Juliana Leite de Oliveira2, Daniel Sorna Labeca Guerra2, Anna Luiza Alves de Oliveira Miranda1, José Wilker Gomes de Castro Júnior1, Beatriz Siems Tholius1, Maria Eduarda dos Santos Lopes Vasconcelos1, Matheus Rocha Maia1 (1) Centro Universitário do Estado do Pará; (2) Universidade Federal do Pará; (3) Universidade de Gurupi Introduction: Heart failure (HF) is a syndrome that the main characteristic is the inability of heart to pump enough blood that the body’s needs, due to damages in contraction and/or relaxation. Objective: Trace the epidemiological profile of HF patients in the Northern Region of Brazil between 2017 and 2021. Methods: Epidemiological, quantitative and observational study, with a retrospective design based on Health Unic System Informatical Department (DATASUS) data, through access to information on Epidemiology and Morbidity (SUS Hospital Morbidity) on HF from January 2017 to December 2021. Results: The age group with the highest number of hospitalizations, corresponding to 248,343 patients and 27.1% of hospitalizations in Brazil for HF, involved patients between 70 and 79 years of age. In relation to the region, there were 46,403 hospitalizations in the North of the country, with of these, 12,272 (26.4%) were patients between 70 and 79 years. In the analysis between sexes in the same region, the number of hospitalizations among men is higher (58.9% of hospitalized patients). When it comes to the mortality rate, the prevalence is among patients over 80 years old (17.64%) and the North region takes the second position with 12.30% of the total. In the analysis related to sex, in the North, the mortality rate is higher in females (12.97%). Conclusion: Data analysis corroborates the existence of an intrinsic relationship between advanced age and the hospitalization rate of patients with HF in the North region. This scenario is the result of the natural aging of individuals and the bad prognosis of the disease, which the survival rate is 35% after 5 years of diagnosis. In addition, it was observed that the North region ranks second among the Brazilian regions that have the highest mortality rate in patients over 80 years of age, which denotes inefficiency in the hospital management of this group. Furthermore, the study indicated a higher rate of hospitalization associated with men, due to their more negligent lifestyle in relation to health, but it identified higher mortality rates in females, which indicates less efficient therapeutic approaches for women in the management of HF. 112105 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING EDMAR GERALDO RIBEIRO1, Lilian Cristina Rezende1, Júlia Bicas Buback1, Tulio Batista Franco2, Deborah Carvalho Malta1, Luisa Campos Caldeira Brant1 (1) Universidade Federal de Minas Gerais UFMG; (2) Universidade Federal Fluminense UFF Introduction: Heart failure (HF) is the main cause of clinical hospitalizations for cardiovascular diseases in Brazil. Hospitalization for HF is related to adverse outcomes, including loss of quality of life, rehospitalizations and death. Digital health interventions (DHI) to improve cardiovascular care is an evolving field, with some studies showing a reduction in adverse outcomes in high-income countries. However, social disparities may be a barrier to the implementation of DHI in low resource settings, particularly due to lower access and education. Purpose: To compare the sociodemographic characteristics of patients discharged from hospital due to HF who refused or accepted to participate in a clinical trial that aims to evaluate the effectiveness of a DHI to improve HF care. Methods: In a randomized clinical trial with patients discharged from hospitalization for HF in a Brazilian capital city, 527 patients were screened in 6 months in 5 public hospitals, which accounted for 73% of hospitalizations for HF in the city. From these, 448 were not eligible, 54 refused to participate, and 79 participants were randomized in the study (43 in the intervention group and 36 in the control group). In a preliminary analysis, sex, age, race, marital status, education, and living in or outside the city were compared between those who refused or accepted to participate in the trial using Fischer’s exact test. A p-value <0.05 was considered statistically significant. Results: Individuals who refused to participate in the study were older (69 vs. 62y, p < 0,001), less educated (53 vs. 30% with less than 4y of education, p = 0.007), with a greater proportion of individuals who self-reported their race as black/pardo compared to white (88 vs. 73%, p < 0.05), and lived more frequently outside the capital city (36 vs. 20%, p < 0.05) than those who accepted. There was no difference between the proportion of females or marital status between the groups. Conclusions: DHI have the potential to overcome major barriers in HF care, such as: access to GDMT care, treatment adherence, and health education. However, our results show that among the eligible population, the most socially vulnerable individuals refused to participate in the trial. As such, DHI studies must evaluate and address the barriers for the acceptance of the intervention to guarantee that DHI in HF care do not increase inequalities. 112108 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MIRIAM ALLEIN ZAGO MARCOLINO1, Rodrigo Antonini Ribeiro2, Luis E. Rohde3, Carisi Anne Polanczyk4 (1) Graduate Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; (2) National Institute of Science and Technology for Health Technology Assessment – INCT/IATS (CNPq 465518/2014-1), Porto Alegre, Brazil; (3) Cardiology Center, Hospital Moinhos de Vento, Porto Alegre, Brazil; (4) Graduate Program in Cardiology and Cardiovascular Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction: Cardiac implantable electronic devices (CIED) are technologies long available in the Brazilian Public Health System (SUS). The aging population and increased incidence and prevalence of cardiovascular diseases can impact the rates of CIED implantation, which are essential information for health policy strategies. Objective(s): To describe the temporal trend of CIED implantation in the SUS before the pandemic, according to devices and patient characteristics. Methods: A retrospective epidemiological study including open data of all hospital admissions with CIED implantation as a primary procedure reimbursed by the SUS between 2008 and 2019, obtained from the Hospital Information System of the SUS Informatics Department (DATASUS). The annual rate per million (MM) population per CIED type was age and sex standardized by the OMS standard population. All analyses were performed using R. Results: In the period, 216,440 CIED implants were reimbursed by SUS, with a mean annual rate of 87.1/MM, mostly pacemakers (78.1/MM). Despite the growing number of implants per year, from 14.403 to 20.635 (+43.2%), CIED implantation rate reduced from 92.8 in 2008, to 83.7/MM in 2019 (–9.7%), mainly due to the reduction of pacemaker implants (–12.3%). Cardiac resynchronization therapy (CRT) decreased 57% from 2008 to 2019 (3.55 to 1.53/MM), while CRT with defibrillator implantation increased 3.5 times (0.82 to 2.87/million) and implantable cardioverter-defibrillator (ICD) increased 30%. (Figure 1). Conclusions: There was a decrease in the implantation rates of CIED in SUS, mainly derived from a decrease in the implantation of pacemakers and CRT. The decrease in the implantation rates may be due to an unbalance between the populational growth and the increase of these technologies‘ access in the period. Moreover, implantation of CRT with a defibrillator and ICD increased in the observed period, in accordance with contemporary guidelines. 112119 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR SURGERY MICAELE FARIAS NASCIMENTO1, Beatriz Souza de Albuquerque Cacique New York1, Milton Antônio Gonçalves de Oliveira1, Kátia Elizabete Galdino1, Ana Tereza do Nascimento Sales Figueiredo Fernandes1 (1) State University of Paraíba – UEPB Introduction: Functional changes can now be assessed in a more complete and reliable way through the International Classification of Functioning, Disability and Health (ICF), created by the World Health Organization (WHO), improving communication between interested people and health professionals and bringing an important facility for international comparisons. Objective: To investigate the prevalence of functional changes in the postoperative period of cardiac surgeries based on the ICF codes. Methods: Cross-sectional study, where 100 patients undergoing reconstructive cardiac surgery (coronary artery bypass grafting and valve repair), replacement (valve replacement) and correction (correction of congenital heart diseases) in a public referral hospital in Cardiology were evaluated, with prior permission from the patient regarding the use of their postoperative data. The functional alterations present were evaluated through the ICF, according to the 8 chapters of the Body Functions Component. The data computed through the form used were collected, quantified and presented through means, standard deviation and percentages. Results: The mean age was 59.2 ± 12.3 years. Changes in sensory and pain functions (96%) were the most present postoperatively, followed by functional changes in the cardiovascular system, hematological and immune systems, and respiratory system (31%), mental functions (17%), functions of the digestive system and metabolic and endocrine systems (5%), neuromusculoskeletal and movement-related functions (4%), and changes in genitourinary and reproductive functions (3%). No alteration of voice and speech functions (Chapter 3) was presented in the study patients. Chest pain after the surgical procedure was reported by 98% of the patients, being justified mainly by the sternotomy process performed in cardiac surgeries. Conclusion: Cardiac surgeries have several organic repercussions that can alter physiological mechanisms and may lead to a critical postoperative state, implying the need to reinforce evaluation methods and intensive care in order to establish a good recovery for patients. 112148 Modality: E-Poster Young Researcher – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS PAULA SANTIAGO TEIXEIRA2, Julio Paiva2, Alessandra Oliveira2, Carlos Campos3, J. Ribamar Costa3, Henrique B Ribeiro2, Milena Fonseca1, Bárbara Freitas1, Jamil Cade1, Breno O. Almeida1, Adriano Caixeta1 (1) HOSPITAL ISRAELITA ALBERT EINSTEIN; (2) UNIVERSIDADE FEDERAL DE SAO PAULO; (3) INSTITUTO DO CORAÇÃO DO HCFMUSP Background: Spontaneous coronary artery dissection (SCAD) is a non-atherosclerotic, traumatic or iatrogenic epicardial dissection. The pathophysiology is not yet fully elucidated, but is related to triggering factors such as emotional stress, physical stress, or pregnancy. It occurs by intramural hematoma formation or intimal rupture that obstructs the coronary artery, causing acute myocardial infarction, especially in young women (<50 years). Demographic, clinical and angiographic description of SCAD in a Brazilian cohort is poorly explored. Objective: To evaluate the demographic, clinical, angiographic profile and triggers of SCAD in a Brazilian population. Methods: The SCALIBUR registry is a retrospective and prospective study of patients with SCAD involving 22 Hospitals in Brazil. REDcap database review in the period 2010–2022. Results: There were 183 patients with SCAD, with a mean age of 50.15 ± 10.56 years (29 to 84 years) with prevalent incidence in female gender (85%). Patients had none or few risk factors for coronary artery disease, including hypertension (6%), family history of early coronary artery disease (22.5%), mixed dyslipidemia (5%), and active smoking (18%). Ten percent of SCAD cases occurred in the gravid-puerperal cycle. Most cases of SCAD manifested as acute myocardial infarction without supra-ST, NSTEMI (45%), acute myocardial infarction with supra-ST, STEMI (34%), and unstable angina (9%). Among the triggering factors, present in 57.8% of the cases, emotional stress (21%) and menopause (16%) stand out. Fibromuscular dysplasia, not systematically investigated, was observed in a few cases (7%), pregnancy (0.5%), puerperium (9%), physical stressor (5.4%), use of hormone therapy (3%) and psychiatric diseases (2.7%). The anterior descending artery was the most affected vessel, followed by the right coronary and circumflex arteries. SCAD type II occurred in (48.82%). Most frequent serious complications were cardiogenic shock (3.27%) and cardiac arrest (1%). Conclusion: In this large Brazilian cohort, SCAD affected mostly young women with no or few classical risk factors for coronary heart disease and the predominant trigger was emotional stress. It is an underdiagnosed condition, but should be considered in the differential diagnosis of acute myocardial infarction in young women. Demographic and angiographic findings seems to be similar to other ethnicities including in the United States, Canada and Europe. 112149 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JULIANA DA ROCHA FERREIRA1, Juliana da Rocha Ferreira1, Julia Passarelli Pereira1, Anna Paula Arpini Botelho1, Marcelo Machado Melo1, Glauber Monteiro Dias2 (1) Instituto Nacional de Cardiologia – INC; (2) Universidade Estadual do Norte Fluminense Darcy Ribeiro – UENF Aortic diseases arising in Marfan Syndrome (MFS), such as in aneurysms and dissections of the thoracic aorta, are related to genetic alterations in the FBN1 gene. Databases, such as Universal Mutations-FBN1, ClinVar and The Human Gene Mutation, contain more than a thousand FBN1 mutations associated with MFS. The FBN1 gene, which encodes fibrillin-1, is responsible for the integral production of different protein domains. Possible genetic changes may lead to a weakening of blood vessels, leading to the development of aortopathies. In this study, we present the association of a novel FBN1 variant with MFS. The proband is a man who presented ascending aortic aneurysm and dissection (TAAD) at 42-yr-old, which was surgically treated. Clinical investigations were performed in all family members enrolled in the study. Marfan signs were observed in the proband, daughters and granddaughter. Direct sequencing of the FBN1 gene in the proband identified a novel truncation variant p.(Glu2019Ter) and a cascade screening were done. The variant was classified as pathogenic and causal for MFS according to the American College of Medical Genetics and Genomics (ACMG) criteria and revised Ghent nosology for MFS diagnosis, respectively. Proband’s daughter and granddaughter harbor the variant, however without aortic alteration. This work reports for the first time a patient with the FBN1-p.(Glu2019Ter) variant and its association with MFS/TAAD. 112121 Modality: E-Poster Young Researcher – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JOÃO GABRIEL GUEDES DA CUNHA MELLO1, João Gabriel Guedes da Cunha Mello1, Santiago Raúl Arrieta1, Pedro Alves Lemos Neto1, Marcelo Biscegli Jatene1, Cauyna Gurgel Moreira1, Estela Azeka1 (1) Instituto do Coração/InCor Introduction: Heart transplantation is an effective option for the treatment of end-stage heart failure. Cardiac allograft vasculopathy (CAV) is considered one of the leading causes of long-term mortality in these patients. The objective of this study is to evaluate the type of angiographic lesions and the impact of CAV on the survival of heart transplant recipients at a referral center. Methods: Retrospective cohort of cineangiography between 2013 and 2022 in patients that underwent heart transplantation. Demographic, clinical and examination data were obtained from medical records as well as the institutional database. Results: The number of transplanted patients during the period of the study was 196, and coronary angiography was performed in 130 (66.3%) of them. Of these (n = 130), 65 (50%) were males, the median age at transplantation was 9.6 ± 7.8 years, and the median age at diagnosis of the first lesion was 6.2 years. CAV was observed in 29 of them (22.3%). The affected arteries followed the given profile: left anterior descending (LAD) 30.9%, right coronary 16.9%, marginal 14.1%, circumflex 12.7%, posterior descending and posterior ventricular 12.7%, diagonal 8.5% and left main coronary artery 4.2%. The CAV classified as moderate and severe were the most common (37.9% each). The event-free survival curve in transplant patients with CAV shows significant sequential drops in 10 years of transplantation, when compared to the curve for patients without CAV (log-rank p = 0.0001). Other coronary angiographic findings correspond to 11.6% of the total of exams, which were, coronary-cavitary fistulas, pulmonary artery fistulas, mediastinal venous plexus fistulas, myocardial bridges, single ostium coronary artery, anomalous origin of coronary artery and thrombi. Conclusions: A prevalence of 22.3% of CAV was observed in transplanted patients that underwent coronary angiography. Moderate and severe lesions were the most common, and the LAD artery was the most affected. CAV has a major negative impact on event-free survival in transplant recipients. 112131 Modality: E-Poster Young Researcher – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARIA EDUARDA KOSTECKI1, Wilson Nadruz Junior2, Odilson Marcos Silvestre3, Ramon Conde4, Daniel Macedo4, Ana Carolina de Franca5, Giulia Caldeira Gaelzer5, Mariana De Nadai Andreoli4, Carlos Cunha6, Rodrigo Julio Cerci6, João Vicente Vitola6, Miguel Morita Fernandes-Silva6 (1) Hospital Santa Casa de Misericórdia de Curitiba (HSC); (2) Universidade Estadual de Campinas (Unicamp); (3) Universidade Federal do Acre (UFAC); (4) Universidade Federal do Paraná (UFPR); (5) Pontifícia Universidade Católica do Paraná (PUCPR); (6) Quanta Diagnóstico por Imagem (QDI) Background: Cardiovascular risk factors (RF) can lead to left ventricular (LV) remodeling, regardless of the presence of coronary artery disease (CAD). The contribution of each RF to non-ischemic LV dysfunction in the population is uncertain. Aim: To estimate the proportion of non-ischemic LV systolic dysfunction attributable to each CV risk factor in patients undergoing myocardial perfusion imaging (MPI). Methods: This is a cross-sectional study of patients undergoing MPI in an imaging center in Curitiba, Brazil, from 2010 to 2021. We excluded those with known previous coronary artery disease or any perfusion myocardial defects at MPI. Data on traditional CV risk factor, including hypertension, diabetes mellitus, and smoking status, were obtained during medical history before MPI exam. Obesity was defined as body mass index ≥30 Kg/m2. LV dysfunction was defined as LV ejection fraction below 50% from rest gated-SPECT. The population attributable fraction (PAF) for LV dysfunction was estimated for each CV risk factor. Results: We evaluated 28,156 patients (60 ± 12 years old, 51% women), 632 (2%) having VL dysfunction. After adjusting for age, sex and body mass index, hypertension (adjusted Odds Ratio [95% Confidence Interval]: 1.34 [1.12, 1.60]), diabetes mellitus (adj OR: 1.43; 95%CI 1.18, 1.74) and smoking (adj OR: 1.69; 95%CI 1.35, 2.13) were independently associated with LV dysfunction, but obesity was not (adj OR: 0.89; 95%CI 0.68, 1.17). Hypertension had the highest PAF for LV dysfunction, followed by diabetes mellitus and smoking. Conclusion: In this large study of patients referred to MPI, hypertension and diabetes mellitus were the CV risk factors that mostly contributed to non-ischemic LV dysfunction in the population, followed by smoking status. 112158 Modality: E-Poster Young Researcher – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIELE CARRA FORTE1, Cristina Carra Forte1, Rubens Gabriel Feijó Andrade1, Luis Miguel Millan Diaz1, Bruno Hochhegger1 (1) Pontifícia Universidade Católica do Rio Grande do Sul – PUCRS Background: Severe acute respiratory syndrome coronavirus 2 is recognized as a multisystemic disease. Clinical studies have reported a complex interplay between cardiovascular disease and worse outcomes in COVID-19 patients. Purpose: To evaluate the association between coronary artery calcification (CAC) and intensive care unit admission among COVID-19 patients. Methods: This is a retrospective cohort study design. All eligible patients underwent a chest CT scan, who were admitted in tertiary university hospital, at the first wave from April 01 until December 31, 2020, were included. A positive COVID-19 case was defined as one polymerase chain reaction test positive for COVID-19. Clinical outcomes were collected from electronic medical records. CAC was graded with a semiquantitative grading system and classified as 0 = absent, 1 = mild, 2 = moderate, and 3 = severe. All data were rated in consensus by 2 readers. The readers were blinded for patient outcomes. Results: 441 consecutively admitted COVID-19 patients were included. Among them, 159 (36.4%) had coronary calcification: 80 (18.3%) patients classified as mild degree, 73 (16.7%), as moderate and six (1.4%) as severe. Sixty-four (14.5%) required admission to an intensive care unit; and 54.7% of these required invasive mechanical ventilation. The presence of CAC was associated with intensive care unit admission (OR 4.37, 95%CI 2.46–7.74, p < 0.001), adjusted for multiple confounders. Conclusion: Estimated risks for intensive care unit admission were consistently higher for patients with COVID-19 and coronary artery calcification. In addition, including CAC standard chest CT for these patients may help guide clinical decision and prognosis. 112161 Modality: E-Poster Young Researcher – Non-case Report Category: NURSING NICHOLLAS COSTA ROSA1, Brenda Gonçalves Donay Alves1, Melissa Schiwe1 (1) Centro Univeristário Ritter dos Reis Introduction: Heart failure (HF) is the final pathway of most heart diseases, it has a great impact on the quality of life of patients with this condition, due to progressive symptoms. Telemonitoring of patients with HF aims to reduce hospitalizations, mortality and improve quality of life. Objective: To identify the impact of telemonitoring as a care tool in patients with HF. Method: This study is an integrative review. The bibliographic search was carried out from August to October 2021. The combination of the following descriptors in health sciences (DeCS) was used as a search strategy: “Telemonitoring” and “Heart failure” crossed using the Boolean connector AND or OR. Randomized clinical trials articles were included, indexed in the aforementioned databases in the last 5 years (2016 to 2021). Results: The search resulted in 285 articles, from the reading of the title and abstract, 60 publications were selected, after reading in full, the final sample consisted of 12 articles. The study by Feijó et al. 2021, had 206 randomized patients and demonstrated that the use of a diuretic adjustment algorithm improves the combined outcome in these outpatients, demonstrating a reduction in HF admissions and clinical instability. The studies by Koehler et al. 2021 and Kalter Leibovici et al. (2017), randomized 710 and 1360 patients respectively, demonstrated that care improved depressive symptoms and had a positive influence on quality of life in patients with HF and moderate depression. The study by Winkler et al. 2021, obtained 1119 randomized patients, as a result obtained a reduction in unplanned hospitalizations and emergency calls could be considered a marker of higher morbidity and mortality. Galinier et al. (2020) and Frederix et al. (2018), randomized 937 and 160 patients respectively, telemonitoring did not result in a significantly lower rate of all-cause deaths or unplanned hospitalizations in patients with HF, but in the study of there was a significant reduction in the number of days of hospitalization for HF. Conclusion: Telemonitoring is a tool that has great potential to improve self-care regardless of the patient’s clinic, however, impacts on readmissions and mortality were not a consensus in all studies. This can be attributed to the different ways of applying telemonitoring and different audiences addressed in each study. 112173 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION BERNARDO CLETO TELES E SILVA1, Lara Camporez Menezes Trindade1, Priscila Diaz1, Antonio Amaral1, Ana Pedroso1, Joao Reis1, Flavia Moises1, Alessandra Godomiczer1, Monica Amorim1, Claudio Carvalho1, Thaisa Garcia1, Andrea Haddad1 (1) Unimed Rio Goals: To evaluate data from patients who had thromboembolic events during hospital stay from Jan 2020 to Mar 2021, correlating length of stay and age with in-hospital mortality in a private quaternary hospital. Methods: Retrospective cohort where consecutive patients admitted to a private quaternary hospital from January 2020 to March 2021 were evaluated, for clinical and surgical hospitalization for at least 48 h. All were evaluated according to risk stratification for VTE by the physician and prophylaxis instituted according to the risk found. Patients admitted with DVT and/or PE, arterial thrombosis events and those in palliative care were excluded from the analysis. DVT (distal and proximal), device thrombosis (PICC and AVP) and PE events were recorded. These outcomes were confirmed with EchoDoppler imaging. Results: We correlated data on thromboembolic events with length of stay and age with an increased risk of in-hospital mortality. In the described period, there were 126 events, of which 120 (92.5%) were DVT and 6 (4.8%). The mean age was 70.8 ± 12.3 years. The age group distribution is shown in table 1. 74 of the patients (58.7%) were male and the mean BMI was 29.6 ± 6.4 kg/m2. The most common risk factors were age >65 years (89.7%), immobility or bedridden (49.2%), neoplasia (13.6%) and surgery or trauma (11.1%). Among patients who had VTE during hospitalization, 63.5% were hospitalized due to COVID-19 infection (figure 1), 35% remained hospitalized for 7–29 days (Graph 1) and 49 patients (38.9%) died. A total of 78.5% were hospitalized for >7 days. 48 patients died, of which 60.3% had more than 7 days of hospitalization among patients who had VTE. There is a correlation between length of hospital stay and higher risk of VTE and mortality, especially in patients with COVID19. Conclusão: VTE is a frequent condition in hospitalized patients and is related to longer hospital stay, as well as advanced age with in-hospital mortality. COVID-19 caused patients to stay in hospital longer, which increased the frequency of VTE. 112193 Modality: E-Poster Young Researcher – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GIOVANNA LÍDIA GONDIM OLIVEIRA DIAS1, Paulo Carvalho Ximenes de Aragão Filho1, Joaquim Francisco Cavalcante Neto1, Mateus de Sousa Cavalcante1, Leandro Cordeiro Portela1, Lucas Almeida Magalhães1 (1) Federal University of Ceará Introduction: Cardiovascular diseases are the most prevalent comorbidities worldwide and one of the main causes of hospital admissions and mortality, including in Brazil. It is essential to identify the most common conditions in this morbidity group and to guide populations on how to prevent the development of these diseases and their respective negative outcomes. Methods: This is a descriptive, cross-sectional, prevalence study retrospective in nature. The objective is to compare the number of hospitalizations and deaths in Brazil among the main diseases of the cardiovascular system, between 2015–2021. The data used for this work came from DATASUS, in a public electronic data platform of the Brazilian government named “TabNet”. The following informations were consecutively selected: “Epidemiology and Morbidity”, “Hospital Morbidity on SUS (SIS/SUS)”, “General, by hospitalization site – from 2008”, and “Brazil by regions”. The line for table it is classified by “ICD-10 Morb List”, in the column has been selected “Year of attendance” and in the content has been placed “Admissions”, “Deaths” and “Mortality Rate”. The period settled was between “January 2015” and “December 2021”. Results: In Brazil, between 2015–2021, the number of hospitalizations due to circulatory system diseases was 7,714,326, of which 667,815 patients died. Among these hospitalizations, 1,374,104 were due to heart failure – with 156,484 deaths; 1,082,705 due to stroke (ischemic or hemorrhagic) – with 169,224 deaths; 841,559 due to acute myocardial infarction – with 87,432 deaths; 1,017,709 from other ischemic heart diseases – with 27,162 deaths; 457,861 due to varicose veins of the lower limbs – with 1,707 deaths; 440,936 due to conduction disorders and cardiac arrhythmias – with 52,139 deaths; and other morbidities with fewer hospitalizations. Discussion: Analyzing the results presented, the impact of cardiovascular diseases on the public health system is undeniable. Annually, it corresponds to a large number of hospital admissions in Brazil. The comorbidity that caused the highest number of hospitalizations in the last six years was heart failure, followed by stroke. However, stroke has the highest gross number of deaths and the highest mortality rate. In addition, not all diseases with high hospitalization evolve with a significant number of deaths, such as varicose veins of the lower extremities and other ischemic heart diseases (except acute myocardial infarct). 112220 Modality: E-Poster Young Researcher – Non-case Report Category: PHYSIOTHERAPY KAMYLLA MARIA ALCANTARA SILVA ALVES1, Kamylla Maria Alcantara Silva Alves1, Ana Eugênia Vasconcelos do Rêgo Barros1, Bruna Thays Santana de Araújo1, Talyta Oliveira de Almeida1, Daniella Cunha Brandão1 (1) UFPE The pandemic caused by the SARS-Cov-2 virus brought several new challenges in the health area, among them, in the lives of patients, generating a decrease in functional capacity and an increase in the fatigue of patients who contracted the disease, having a direct impact However, as in the context of the current pandemic, the recommendation is to maintain as much social isolation as possible, telerehabilitation has proved to be an alternative resource to help improve the functional capacity of these patients and improve their quality of life. Objective: To compare the effectiveness of face-to-face rehabilitation (PR) and telerehabilitation in relation to quality of life, intensity and impact of fatigue in post-COVID-19 patients. Methods: Patients with a history of hospitalization for COVID-19 were allocated to the face-to-face rehabilitation group, whereas those who were not hospitalized were allocated to the telerehabilitation group. the activities of daily life and, consequently, in the quality of life. Thus, cardiopulmonary rehabilitation has been shown to be of great relevance and efficiency in the recovery of such patients. All participants answered the Medical Outcomes Study Short – Form 36 questionnaire and the Fatigue Pictogram. PR was performed and telerehabilitation was performed via the online platform google meet, during 12 sessions, twice a week. The intervention protocol consisted of flexibility, aerobic, strengthening and breathing exercises. This work is an experimental study with a non-probabilistic sample. Results: 12 patients in each group completed rehabilitation, totaling 24 patients. The mean age was 42.83 ± 14.45 years for the telerehabilitation group and 47.67 ± 10.83 years for the PR group. There was an improvement in quality of life in all domains of the SF-36, both in the telerehabilitation group, with a total score of 267.08 ± 125.50 vs 453 ± 186.03 (p < 0.01), and in the RP group. 298.38 ± 205.61 vs 470.31 ± 142.09 (p < 0.01) with no difference between the groups. Regarding fatigue, a reduction of 66.66% and 100% in the “very tired” category was observed for the telerehabilitation and RP groups, respectively. And the reduction in the impact of fatigue was observed by the greater number of individuals who reported being able to “do almost everything”. Conclusion: It was observed that both face-to-face rehabilitation and telerehabilitation are capable of promoting an improvement in quality of of life, with no differences between them. 112221 Modality: E-Poster Young Researcher – Non-case Report Category: CARDIOVASCULAR IMAGING GABRIELE CARRA FORTE1, Cristina Carra Forte1, Rubens Gabriel Feijó Andrade1, Luis Miguel Millan Diaz1, Bruno Hochhegger1 (1) Pontifícia Universidade Católica do Rio Grande do Sul – PUCRS Introduction: Coronavirus disease 2019 had a great impact worldwide, being an important cause of morbidity and mortality in several countries. Although the typical clinical presentation is acute respiratory syndrome, cardiovascular disease is frequent and reported as an important determinant of poor clinical outcome. Purpose: To evaluate the association of thoracic aorta calcification on in-hospital mortality in COVID-19 patients. Methods: All patients with SARS-CoV-2 infection, who underwent a chest computed tomography (CT) in a tertiary university hospital in southern Brazil, between April and December 2020 were prospectively included. Clinical outcomes were collected from electronic medical records. Thoracic aorta calcification, body composition and bone mineral density was evaluated by low-dose chest computed tomography imaging (chest CT scan), widely performed in moderate to severe SARS-CoV-2 patients. Patients were classified as without calcification (0), having mild calcification (1), moderate (2) or having severe (3) calcification. The readers were blinded for patient outcomes. Results: In all, 441 patients were enrolled in the study. There was a predominance of females (54%), and the mean age was 55 ± 19.2 years. Visceral and subcutaneous fat area was 116.9 m² (55.4–174.0) and 115.3 m² (67.0–181.1), respectively, and muscular area was 102.5 m² (84.2–126.8). The bone mineral density was 171.5 (120.0–219.0)HU. After adjustment for covariates, thoracic aorta calcification [OR 11.45 (95%CI 1.745–75.194); p < 0.001], mechanical ventilation [OR 25.45 (95%CI 3.542–182.972); p < 0.001), visceral fat area [OR 1.01 (95%CI 1.003–1.023); p = 0.014] resulted to be independent predictors of in-hospital mortality. Conclusion: Mechanical ventilation, thoracic aortic calcification and visceral fat area permits to stratify the COVID-19 patients in-hospital mortality risk. In addition, to include the thoracic aorta calcification evaluation on standard chest CT for these patients may help guide prognosis and clinical decision. 112228 Modality: E-Poster Young Researcher – Non-case Report Category: ANTICOAGULATION ANA CAROLINA PEDROSO1, Ana Carolina Pedroso1, Monica Amorim1 (1) Unimed rio hospital Patients with femoral neck fractures have an increased mortality rate. Femoral neck fracture is frequently found in women over 60 years of age and its incidence has been increasing in many parts of the world due to the increase in life expectancy in this age group. Total hip arthroplasty (THA) is one of the most successful surgical procedures in the field of orthopedic surgery. Oral anticoagulants are indicated in the postoperative period (PO) of hip arthroplasty to prevent thromboembolic events. Direct oral anticoagulants (DOACs) were considered by studies to be effective in preventing thromboembolic events, as was enoxaparin. goal Ensure the use of anticoagulant in patients discharged from hospital in the PO of femur fracture, as prophylaxis for venous thromboembolism. Methods: Retrospective cohort from Jan 2019 to Dec 2021 in hospitalized patients with a diagnosis of femur fracture undergoing hip arthroplasty surgery. In a total of 34,167 patients, 373 patients were admitted with a femoral fracture, 28 patients died during hospitalization, and were considered to be patients aged >60 years. All patients were evaluated according to risk stratification for VTE by the physician and prophylaxis was instituted according to the risk found. All patients in the PO period of hip arthroplasty are considered high-risk patients. Results: Of the 34,167 hospitalized patients, 1.1% were due to femoral fractures, of which 7% died during the hospitalization period. Follow-up was performed for 30 days. Of the 345 patients who were discharged from hospital, 100% received anticoagulation with enoxaparin or rivaroxaban or apixaban. Conclusion: Anticoagulation is safe and effective in prophylaxis of thromboembolic events in the postoperative period of femur fracture, showing no risk of increased bleeding with the use of DOACs. Despite the reduced follow-up time of these patients, there was no readmission for VTE. 108565 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES JOÃO MIGUEL MARTINS DE CARVALHO1, Tânia Proença1, Ricardo Alves Pinto1, João Carlos Silva1, Filipe Macedo1 (1) Centro Hospitalar Universitário São João (CHUSJ), Porto, Portugal Severe aortic stenosis patients who are critically ill are in high risk for mortality. Aortic balloon valvuloplasty (ABV) can be done on emergency, improving hemodynamic status on short-term and can be life-saving. A 59 years old male, with known medical history of hypertension, diabetes, renal cell carcinoma treated with parcial right nephrectomy, had severe aortic stenosis, severe left ventricle dysfunction (left ventricle ejection fraction [LVEF] 15%) and 3 vessel coronary artery disease (50% stenosis on the medium segment of the left anterior descending [LAD], 90% stenosis on the proximal segment of the circumflex, 90% stenosis in the proximal right coronary artery, followed by a occlusion on the middle segment; the distal vessels were in good condition) was awaiting surgery when he was admitted to the Internal Medicine ward on January 2019 with a respiratory infection with hypoxemia and severe systemic inflammatory response syndrome. On the same day, he presented with an inferior ST elevation myocardial infarct and cardiogenic shock. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) and aminergic support were initiated and he was submitted to emergent coronary angiography that revealed progression of the LAD disease, with 90% stenosis on the medium segment. It was decided in Heart Team to perform an emergent ABV; the mean left ventricle-aortic gradient reduced to 10 mmHg. It was decided not to perform LAD angioplasty due to the high procedure risk and the probable need for surgical treatment soon. A week later, the patient was submited to surgery with VA-ECMO support, having performed a coronary artery bypass graft with the left internal mammary artery to the LAD; he didn’t perform aortic valve replacement surgery due to highly calcified aortic root. On the 8th day pos-op, the patient presented with a hemopericardium and tamponade, needing to be submitted to a new surgery. He was off VA-ECMO and aminergic support by day 12 pos-op. On the 21st day pos-op, the patient was submitted to transcatheter aortic valve implantation (TAVI). The pos-procedure echocardiogram showed a normofunctional aortic prothesis and an improvement in LVEF (36%). The patient was discharged, remaining during follow-up in NYHA functional class I. ABV can be a bridge to TAVI or surgery in highly unstable patients, contributing to patient stabilization. It can be done on an emergency basis, can alter the patient prognosis and should be considered in these patients. 108006 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ANDRÉ FILIPE MACEDO ALEXANDRE1, Dias de Frias1, Andreia Campinas1, João Silveira1, Severo Torres1 (1) Centro Hospitalar Universitário do Porto, Porto, Portugal Disulfiram is used for the treatment of chronic alcoholism. Yet, it is not a safe drug in patients who still drink alcohol due to disulfiram-ethanol reaction (DER). Acute myocardial infarction due to DER has been rarely reported, and most reports suggest coronary vasospasm as the causative mechanism. We present a case of myocardial infarction probably due to DER, in which “white thrombus” formation was found instead of vasospasm. This 65-year-old man with history of schizophrenia and chronic alcoholism was admitted for out-of-hospital cardiac arrest. After 10 minutes of advanced cardiac life support with four defibrillation shocks, the patient returned to spontaneous circulation and the ECG revealed an anterior “shark fin” STEMI. The patient was referred for emergent coronary angiography, which revealed acute thrombotic occlusion of the left anterior descending artery. Aspiration thrombectomy was performed with recovery of a large “white thrombus”, and a drug-eluting stent was implanted. This patient has been prescribed disulfiram the week before STEMI presentation, yet he was still drinking alcohol while taking the drug. We suspect that DER may have been responsible for plaque instability due to close temporal relationship. This clinical case highlights that DER can not only cause myocardial ischemia by coronary vasospasm, but can also predispose to thrombus formation, leading to extensive myocardial infarction. 108133 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FRANCIELLY DOS SANTOS VIEIRA1, Marina Vitória Silva Costa1, Nathália Abdo Zuliani1, Jéssica Evangelista de Queiroz1, Leonardo Teixeira de Melo1 (1) Universidade Federal de Uberlândia – UFU Preface: Cardiovascular diseases are an important cause of gestational and puerperal morbidity and mortality. Because it is rare, aortic dissection is difficult to diagnose during pregnancy and the puerperal period, contributing significantly to maternal mortality due to its high fatality rate, comprising 4% of the causes of maternal death. Case Report: A 38-year-old primiparous patient, on the fifth day of the puerperium (cesarean section), with left renal agenesis, without other gestational comorbidities or complications, presented with severe chest pain with irradiation to the back, upper limbs, neck and jaw. She immediately sought tertiary care, where she was admitted for dyspnea, sweating, bradycardia, and hypotensiveness. Electrocardiogram suggestive of acute myocardial infarction with inferior wall ST elevation. She was referred for coronary angiography, which showed type A aorta dissection associated with acute occlusion of the right coronary artery and right renal artery, without occlusion of supraaortic or mesenteric vessels. She underwent stent implantation in the right coronary artery, followed by emergency surgery with replacement of the ascending aorta using a number 28 Dacron tube and replacement of the aortic valve with a Carpentier Edwards biological prosthesis number 23, replantation of the left coronary artery (Bentall de Bono surgery), associated with implantation of a saphenous vein bypass for the right coronary artery. She remained in the intensive care unit for six days for hemodynamic reestablishment, being discharged on the 11th postoperative day without major complications. Conclusion: Aortic dissection is a rare, life-threatening condition for two main reasons: increased risk of aortic rupture and compromised blood flow from the systemic circulation to major organs by occlusion of the arterial lumen. Pregnancy is one of the main risk factors for arterial dissection among young women. Approximately 60% of them occur related to pregnancy and puerperium, being the third cause of maternal death related to cardiovascular diseases. The therapeutic approach does not differ much from that proposed for other patients, and emergency surgery to replace the ascending aorta with a Dacron tube is the best option in cases of type A acute dissection. 110102 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING LAÍS ANTONUCCI FERREIRA1, Katia Gleicielly Frigotto1, Washington Luiz Batista da Costa1, Daniela Roberta Alves Silva1, Luciano de Figueiredo Aguiar1 (1) Hospital São Lucas Copacabana Introduction: Kinking artery is an angular alteration that forms a sharp angle in an artery. Embryogenic and acquired causes can lead to this alteration. Congenital factors are clinically significant in older age, aggravated by arterial hypertension (AH), diabetes mellitus (DM), dyslipidemia, smoking, rheumatological and heart diseases. This alteration is more common in the internal carotid artery, but is rare in subclavian and common carotid arteries. That condition is often asymptomatic, but can present neurologic symptoms, such as mimicking a transient ischemic attack (TIA) due artery stenosis. Diagnostic investigations can be made by anamnestic and clinical data, doppler ultrasonography, computed tomography angiography (CTA), and magnetic resonance angiography (MRA). The therapeutic includes anti-aggregation and anticoagulation therapy, antihypertensives, antilipidemic, cerebral vasodilatation therapy, and surgical treatment if indicated. Case description: The 77-year-old female patient was admitted to the Emergency Room, due dysarthria and loss of upper body strength, with spontaneous improvement in approximately 15 minutes. She has been reported to have diagnosis of HAS, DM, dyslipidemia, parkinson disease, and previous TIA. In the neurological examination she was conscious and disoriented, presenting miosis and ptosis on the right side, without strength deficit. CTA indicated kinking in the right subclavian and common carotid arteries. The patient was treated with acetylsalic acid, clopidogrel, atorvastatin, antihypertensive and antidiabetic therapy. Conclusion: A severe degree of kinking can cause neurological symptomatology and even simulate TIA. The kinking artery should be considered as a differential diagnosis, especially in older patients and/or with risk factors. 108467 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CYBELLE LUZA COSTA1, Cybelle Luza Costa1, Paulo Lucas Moraes Pimenta2, Fábio Queiroga1 (1) Hospital Agamenon Magalhães; (2) Faculdade Pernambucana de Saúde Introduction: Infectious endocarditis (IE) is a serious pathology caused by colonization of the cardiac endocardium, presenting itself in an insidious way, with few specific symptoms, but with catastrophic consequences if not properly treated. Due to its high morbidity and mortality, early recognition and targeted treatment can prevent irreversible sequelae. Objective: Case report of a difficult-to-diagnose infectious endocarditis in a patient assisted at Agamenon Magalhães Hospital in Recife, Brazil. Methodology: Retrospective, qualitative, observational case report through medical chart and literature reviews. Case Report: MSJ, 79 years-old, female, arrives at the Internal Medicine service referred from a reference hospital in ophthalmology for venous antibiotic therapy after a surgical procedure in the left eye, performed for the treatment of Endogenous Endophthalmitis. She went to the hospital due to bilateral eye pain and a drop in visual acuity, evolving with only light perception in the day she sought medical care. She denied previous eye surgery or trauma. She reported being admitted to the ICU for 5 months due to sepsis and bloodstream infection, evolving after discharge with bilateral eye pain. On admission, she was clinically and hemodynamically stable, with bilateral amaurosis, with hospitalization scheduled for completion of broad-spectrum antibiotic therapy for the endophthalmitis. There was no report of fever. During hospitalization, a new systolic murmur in the mitral focus was detected in the physical examination, and a transesophageal echocardiogram (TE-ECHO) and blood cultures were performed to rule out the possibility of infectious endocarditis (IE) as the cause of endophthalmitis, since the diagnosis of this pathology would imply in extension of antibiotic therapy time. Cultures were negative, but this was not valued because of the use of intravenous Vancomycin in progress. The result of the TE-ECHO found vegetation adhered to the mitral valve, confirming the main suspicion of IE, and empirical treatment with Vancomycin and Gentamicin was performed for 6 weeks. Conclusion: IE is a disease of difficult differential diagnosis due to its symptomathological non-specificity. Therefore, it is important to know the guidelines of ESC and AHA, since the non-confirmation of the disease through one does not exclude confirmation by the other, reducing the number of underdiagnosed patients and, consequently, avoiding severe consequences. 108477 Modality: E-Poster Young Researcher – Case Report Category: CARDIO-ONCOLOGY VICTOR BAROUKI KORMANN1, Carolina Limongi de Oliveira1, Marcela Kondo Sato1, Claudinei Collatusso1, Taiana Emílio Checchia de Lima1 (1) Hospital Santa Casa de Curitiba Introduction: Based upon the data of 22 large autopsy series, the frequency of primary cardiac tumors is approximately 0.02%. Primary heart tumors are stated to occur at all ages and to have an equal sex incidence. Three quarters of the primary cardiac tumors are benign and one quarter are malignant. Myxomas and sarcomas were the most common types. Description: Patient, 34 years old, reports that in May 2021 she started with pain in the left scapular region, associated with progressive dyspnea, which became limiting for light physical activities after 3 months. Evolved with edema of the face and upper limbs, severe dyspnea, cyanosis, poor peripheral perfusion, requiring emergency pericardiocentesis. Magnetic resonance showed a tumor measuring 8.3 × 5 × 4.8 cm in the right atrium, compressing the superior vena cava and signs of cardiac tamponade. The patient underwent surgical resection of the tumor and cavity reconstruction with bovine pericardium. Biopsy confirmed the diagnosis of Angiosarcoma, with surgical margins free of neoplastic involvement. Conclusion: Cardiac tumors are rare entities, but they must be diagnosed early due to the high potential for serious complications. In this report, we present a case of right atrial sarcoma, coursing with superior vena cava syndrome and cardiac tamponade, successfully treated surgically. The patient remained asymptomatic, and no metastasis was evidenced on the tomographic study. 108544 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING DANIEL GAMA DAS NEVES1, Mario Ricardo Amar1, Rafael Vilanova Lima1, Alair Augusto Sarmet Moreira Damas dos Santos1 (1) DASA – Complexo Hospitalar de Niterói Aortic intramural haematoma (IMH) in a type of acute aortic syndrome, in which a haematoma is formed inside the aortic wall. In this presentation we aim to report a case of acute aortic IMH diagnosed and treated in our institution, that had findings corroborating one of the etiological theories A 58-year-old female patient is admitted at the hospital after being initially evaluated in another institution of lower complexity. The patient arrived at our institution still complaining of crushing chest pain nonrelated to physical exertion, associated with dull pain in the right upper quadrant of the abdomen. Troponin and CKMB where normal but D-dimer was elevated. The medical team decides to investigate the pain with a computed tomography angiography (CTA) of the thoracic and abdominal aorta. That shows an eccentric dense thickening of the aortic wall dislocating the parietal calcifications inwards in the nonenhanced images. Contrast images showed a small irregularity in the luminal surface about 6 cm from the aortic valve plane, suggestive of a small intimal flap. The medical team then decided to admit the patient to the ICU for close surveillance and strict control the double product. After seven days of hospital care, the chest pain returned. New aortic CTA showed increase of wall thickness, formation of ulcer like projections in the ascending aortic wall covering the same location as the previously described luminal irregularity. The images also demonstrated the formation of pericardial effusion. The attending team then decides that surgery was the best course of action. The surgeons description of the procedure confirmed the location of the intimal flap described in the first CTA. Control CTA performed 10 days after the surgery showed no complications. A new CTA performed a year after the procedure demonstrated complete regression of the haematoma. This case aggregates to the body of evidence that intimal flap is a cause of not only classical aortic dissection, but also of aortic IMH. As better CT scanners becomes more available, and cardiologists, cardiovascular surgeons as well as radiologists become more aware of aortic IMHs, the understanding of this pathology will likely improve, and the diagnosis and treatment criteria may become more defined. 108541 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES MARCELO VIAL FELIX DE SOUSA1, Maicon Felipe Ribeiro da Cruz1, Renata Muller Couto1, Silvério Albano Fernandes Júnior1, Walter Emanoel Magalhães Rocha1 (1) Universidade Estadual de Campinas – UNICAMP We’ve reported the case of a patient who has presented with symptoms of neurological alterations and, under investigation, was diagnosed with endocarditis by Staphylococcus aureus (SA) involving the mitral, aortic and pulmonary valves. A 53-year-old male sought medical attention due presenting with right arm paresis, right eyelid ptosis and diplopia. During the investigation, he developed fever, lack of appetite, weight loss and diastolic murmur in the pulmonary focus. His exams had shown positive blood culture for SA. In a transthoracic echocardiogram (TTE), vegetations were identified in the pulmonary and mitral valves. After the changes were observed, it was requested a transfer to a tertiary level hospital. In a new TTE, performed in the tertiary service, vegetations were visualized in the pulmonary valve, in the anterior leaflet of a mitral valve and in the right coronary leaflet of aortic valve. After 2 months of vancomycin, the vegetations in the aortic and mitral valves disappeared. Afterwards was performed valvuloplasty in the pulmonary valve with success. Patient maintained in outpatient follow-up without complaints so far. A review of the literature was carried out and it is not uncommon to have 2 valves involved, but the involvement of 3 valves is rare. Multivalvular involvement is related to more severe infections and has higher mortality. Despite the poor prognosis in these cases, the patient in question developed uneventfully. 109943 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES PEDRO GUIMARÃES SILVA1, BRUNA MITIE IKARIMOTO1, GUILHERME RAPOSO DE MEDEIROS1, MARILIA TAILY SOLIANI1, PAULO ROGERIO SOARES1 (1) INSTITUTO DO CORAÇÃO DO HCFMUSP Introduction: Vasculitis is a rare cause for coronary stenosis. However, it can still be responsible for life-threatening conditions. This group of diseases usually present with nonspecific symptoms and involvement of various systems. Here, we expose a case in which its initial manifestation was the development of ischemic cardiomyopathy due to a severe stenotic lesion of the left main coronary artery (LMA). Case Report: A 63-year-old woman without any previously known comorbidities began an outpatient investigation due to a 1-year history of dyspnea and angina under minimal efforts. Dilated cardiomyopathy with reduced ejection fraction (37%) was diagnosed on transthoracic echocardiography and an elective coronary angiography was requested for assessment of coronary disease A 70% focal stenosis in the distal segment of the left main coronary artery was detected, without any other significant narrowing in the other vascular beds. The patient was the admitted to the hospital for an emergent surgical revascularization. A CT angiography of the aorta and its branches was ordered. It showed a circumferential parietal thickening in the transition between the subclavian and axillary arteries to the right, determining significant segmental luminal reduction (>50%); circumferential parietal thickening of the aorta along its entire length, reaching a thickness of approximately 0.3 cm, suggestive of vasculitis. A PET-CT was ordered, which corroborated the diagnosis with the detection of diffuse and heterogeneous increase in glycolytic metabolism, of mild/moderate intensity, suggestive of an inflammatory process compatible with large-vessel vasculitis. Blood tests rendered negative for Chagas, syphilis, ANCA and antinuclear antibody serology. Pulse therapy with methylprednisolone 1 mg/kg/day was initiated and angioplasty of the LMA was performed with the use of 2 drug-eluting stents in the anterior descending and circumflex arteries, following the mini-crush and post-dilation with kissing-balloon techniques. An intra-aortic balloon was used for coronary protection during the procedure. Aspirin and Ticagrelor (60 mg in loading dose followed by maintenance 10 mg per day) were administered as antiplatelet therapy prior to the intervention. A good angiographic result and resolution of the anginal was achieved and the patient was discharged from the ward after 7 days for out-patient follow-up with a rheumatologist. 108609 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ALICE MIRANE MALTA CARRIJO1, Veronica Perius de Brito1, Gabriel Alvarenga Santos1, Samuel Gomes Tomaz da Silva1, João Lucas O’Connell1 (1) Hospital de Clínicas da Universidade Federal de Uberlândia – HC/UFU Introduction: Patent foramen ovale associated with a thrombus in transit represents a rare clinical entity difficult to manage. Its association with other thrombotic events and the hyperinflammatory state of novel coronavirus disease (COVID-19) is the subject of discussion in this study. Case description: A 69-year-old woman with grade III obesity, a history of systemic arterial hypertension, hypothyroidism and chronic venous insufficiency, was hospitalized for severe respiratory condition of COVID-19. On the 16th day of symptoms of COVID-19, the patient was diagnosed with Acute Myocardial Infarction with ST-segment elevation of the inferior wall after presenting with typical anginal pain, radiating to the left upper limb, associated with worsening dyspnea. Treatment was carried out with primary coronary angioplasty with drug-eluting stent implantation in the right coronary artery. One day after angioplasty, a routine transthoracic echocardiogram was performed, and identified preserved global systolic function of the left ventricle and the presence of a thrombus straddling the patent foramen ovale, with mobile segments in the right and left atria (Figure 1A). Full anticoagulation with enoxaparin was initiated, acetylsalicylic acid (ASA) was maintained and clopidogrel was changed to tirofiban to schedule an embolectomy. The patient evolved with clinical stability, improvement in the respiratory condition and resolution of the atrial thrombus, documented on preoperative echocardiography (Figure 1B), without the need for surgical embolectomy. Pulmonary artery CT angiography was performed, and confirmed a pulmonary thromboembolism (PTE). The patient remained without hemodynamic instability and ASA, clopidogrel and oral anticoagulation were maintained after hospital discharge. Conclusion: The identification of thrombus between the atria associated with the presence of patent foramen ovale is a rare event with high mortality rates, especially in the context of severe COVID-19, associated with PTE and Acute Coronary Syndrome. Management in this context is complex and there is still no consensus on the best therapy. 108633 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY ANA PAULA DALPICOLLI CORSO1, Alessandra Bossardi1, Bruna Valduga Dutra2, Bibiana Maggi3, Leandro Gazziero Rech4 (1) HGCS – Hospital Geral Caxias do Sul; (2) Federal University of Rio Grande do Sul UFRGS Introduction: Alcapa Syndrome (anomalous left coronary artery from the pulmonary artery) is a rare congenital disease that presents as a coronary anomaly, consisting of the origin of the left coronary artery (LCA) in the pulmonary trunk. The objective of the report is to emphasize the importance of early diagnosis of cardiac alterations to obtain better prognosis results in surgery. Case Description: A 28 years-old female sought care at the hospital due to tight retrosternal pain radiating to the left upper limb associated with dyspnea worsen on exertion in 2021. Patient reports that she had a typical chest pain episode 3 months ago, but with ECG and normal troponins. Previous history of Alcapa Syndrome, with correction of the anomalous origin of the left coronary artery in 2002. Initial laboratory assessment showed no alteration, with a troponin value of 0.005 (reference value >0.0012), and the patient was in use of furosemide. Transesophageal Echocardiography demonstrated a preserved ejection fraction with mitral valve thickening with a non-dysplastic appearance with mild regurgitation and minimal aortic valve regurgitation. At this point coronary angiography was performed, demonstrating severe stenosis in the anastomosis of the aortic tunneling. After this procedure, they underwent coronary bypass graft without complications with release to the ICU, then to the ward. After 4 days of the intervention, the patient was discharged with instruction. Conclusions: Alcapa Syndrome is a rare disease with an anomalous origin of the LCA from the pulmonary artery. There are two forms of the syndrome, infantile and adult. The adult form is characterized by the formation of a collateral system between the Right Coronary Arteries (RCA) and LCA, with a compensation usually keeping the patient asymptomatic. The patient in the case presented a complication of the tunneling previously performed. Corrected with coronary bypass graft surgery in order to restore coronary flow. 108635 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GIOVANNA MARIN LESSE1, Larissa Ventura Bruscky1, Edileide Correa de Barros1, Jéssika Mayhara Souza Tolentino1, Fabiano Castro Albrecht1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Cardiac amyloidosis is a underdiagnosed cause of heart failure. Hereditary transthyretin amyloidosis can occur in more than 130pathogenic variants with variable clinical presentation depending on the type of mutation. They two main phenotypes are peripheral polyneuropathy and amyloidotic heart disease. The variant Val50t is the most prevalent in the world and most important representative of the polyneur phenotype. Val142Ile is the second most prevalent and is mostly expressed with heart disease. We describe a patient who presents a rare association of these two pathogenic variants in composed heterozygosity. Case report: A 66-year-old black male patient from Juazeiro-BA, previously healthy, with a family history of neuropathy and heart disease in two brothers who died with 64 and 73 years old. She had altered sensitivity in the tips of her fingers, hands and feet, burning pain and reduced strength in her left leg and both hands for tree years. She also reported weight loss of 9 kg in two years, dizziness when assuming an orthostatic position and early satiety. Cardiological complaints were just heart palpitations during exercise and lower limb edema. In physical examination she presented with postural hypotension, rediced grip strength and pinching movement in the fingers and atrophy of the intrinsic muscles of the hands. Electrocardiogram: left anterior fascicular block and inactive zone of inferior and anterior walls. Echocardiogram: preserved left ventricle ejection fraction (global longitudinal strain of –8.7%), grade III diastolic dysfunction and enlargement of the left ventricle wall (20 mm) and interatrial septum (9 mm). Pyrophosphate scintigraphy showed pergini grade 3 uptake with a 2.22 rate. Genetic sequencing demonstrated the presence of two pathogenic variants on chromosome 18, both in heterozygosity: Val50Met and VAL142Ile. Conclusion: An association of two pathogenic variants is rare and is associated with a more severe expression of the disease and worse prognosis. In this case the apresentation was mixed: heart disease, polyneuropathy and dysautonomia, with a predominance of the last one. Heart disease symptoms were not exuberant, perhaps because of the physical limitation of the patient. However, the uptake of pyrophosphate on scintigraphy was much more intense than usual, suggesting high degree of deposit and worse prognosis. 110326 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING RAFAEL RODRIGUES COUTINHO1, Paulo Roberto Dutra da Silva1, Arnaldo Rabischoffsky1, Nilton Lavatori Correa1, Claudio Tinoco Mesquita1 (1) Hospital Pró-Cardíaco/RJ Introduction: Prosthetic valve endocarditis (PVE) has an increasing prevalence. In this case report, we sought to highlight the importance of early diagnosis in cases of previous valve-in-valve intervention. We highlight the need for integrated multidisciplinary action in an endocarditis team for the proper diagnosis. Case: Patient 86 years old, male. Hospitalized due to cough and dyspnea associated with low-grade fever and reports that he had a dental procedure two weeks ago. Previous history of heart failure functional class II, TAVI (2004 and 2021) valve-in-valve, Hypertension, dyslipidemia, smoking and alcohol consumption. He performed 2 blood cultures in the current hospitalization, positive for Streptococcus mitis. TTE and TEE without visualization of vegetations or thrombi and preserved ventricular function. CT angiography with absence of aortic vascular occlusion and shows enlarged heart and small bilateral pleural effusion. Normal lower limb venous Doppler. Scintigraphy with labelled leukocytes showing an infectious/inflammatory process in aortic valve prosthesis topography. Initiated antibiogram-guided ceftriaxone with frame resolution. Discussion: The valve-in-valve transcatheter aortic valve implantation (ViV-TAVI) is already a well-established procedure for degenerated prosthetic valves, being also used for patients who would not tolerate open valve replacement surgery. ViV-TAVI is an alternative for high-risk patients, but there is no evidence of lower mortality of patients using this therapeutic approach compared to valve replacement surgery. Nuclear medicine emerges as a useful tool in the diagnosis of infective endocarditis, with two main evaluation modalities in this scenario, PET-CT using the 18-F-FDG agent and SPECT-CT with labelled leukocytes. Leukocytes are deposited in sites where there an active infection, being an interesting tool for suspected PVE. For the diagnosis of PVE, sensitivity and specificity of labelled leukocytes are above 90%, being then included as one of the modified DUKE criteria for infective endocarditis. Therefore, the use of SPECT-CT with labeled leukocytes has proved to be useful in the identification of PVE and should be included in the diagnostic flow of patients with suspected infective endocarditis. 108686 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING JANINE DAIANA STÜRMER1, Raphael dos Santos Silva1, Tiago Hansel Basile Vigil1, Willer Cesar Bica1, Gabriel Soder1 (1) Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia – ICFUC Introduction: Left ventricular (LV) pseudoaneurysm is a rare complication of myocardial infarction (MI) with high mortality rates. It consists of an intramyocardial dissecting hematoma communicating with the ventricular cavity when an injured myocardial wall rupture. The definitive diagnosis can be made with transthoracic echocardiogram (TTE) in only 26 percent of patients. Case report: A 60-year-old male with a history of smoking habit was admitted with typical chest pain lasting 20 hours. The electrocardiogram showed ST-elevation in I, aVL, V5 and V6 leads (Panel A), suggestive of lateral MI. He received dual antiplatelet therapy and was immediately transferred for coronary angiography, which displayed occluded mid-circumflex coronary artery. During the exam, he presented acute deterioration of hemodynamic state and a TTE was performed. Pericardial effusion was ruled out in subcostal-view (Panel B). At the apical 4 and 5-chamber-view the mitral valve was not visualized and in its topography a muscular band-like image that could suggest rupture of left ventricular free wall (Panel C). Finally, a long-axis-parasternal-view demonstrating a pericardial hematoma compressing the left atrium referring to a LV pseudoaneurysm (Panel D,E) which was confirmed on left ventriculography (Panel F). We theorize that as the pseudoaneurysm progressed he obstructed the LV inflow, as seen by the limited mitral valve diastolic flow at color Doppler (Panel E). The patient collapsed and died few minutes later. Conclusion: LV pseudoaneurysms can present as congestive heart failure, chest pain, arrhythmia or sudden death. Our case is a highly unusual instance of pseudoaneurysm producing severe extrinsic compression obliterating the LV inflow diagnosed with TTE in a sudden cardiac death presentation. 108710 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MATHEUS HENRIQUE MENEZES1, Diogo França Souza Camargo1, José Alaor da Veiga Junior1, Deborah Christina Nercolini1, Wilton Francisco Gomes1 (1) Instituto de Neurologia e Cardiologia de Curitiba Introduction: Central venous catheters are important devices for medical care. Although they improve life-quality for patients with chronic conditions, complications are not negligible. We report a rare case of embolization of a catheter fragment into the coronary sinus. Case: A 43-years-old woman with a history of breast cancer undergoing chemotherapy through a totally implantable catheter was referred to CT scan due to a chest x-ray suggestive of a catheter fracture and embolization. CT scan images showed an embolized catheter fragment, with one extremity in the coronary sinus and the other in the right ventricle. The patient was referred for percutaneous removal of the fragment. The right femoral vein was punctured and a 10F sheath was installed. We approach the case using the “pigtail through snare” technique, using a 5F pigtail catheter placed into a 20mm loop snare before its insertion. With a 0.035 hydrophilic guidewire, we then positioned the pigtail catheter into the pulmonary artery and, with a pull-back maneuver, we successfully entangled the extremity located into the right ventricle. With coordinated movement, the snare loop was positioned right above the pigtail catheter and the guidewire inside the catheter was pushed, as an extension, allowing for the grasping of the fragment and the pigtail catheter, simultaneously. The fragment was then extracted, pulling out the assembly and sheath as a unit. Discussion: Embolization of central venous catheter fragments is uncommon, occurring in 1.1%–2.1%. The pulmonary artery and right chambers are the most frequent sites of embolization. The coronary sinus is 15–65 mm long structure and the Thebesian valve protects the ostium in almost 88% of people. This demonstrates the complexity and rarity of the case reported, with fragments in the coronary sinus and right ventricle. To our knowledge, only 6 similar cases have been reported before. The “pigtail through snare” technique seems to be a useful tool in these challenging situations. 108738 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT SILVÉRIO ALBANO FERNANDES JÚNIOR1, Maicon Felipe Ribeiro da Cruz1, Marcelo Vial Felix de Sousa1, Otávio Rizzi Coelho Filho1, Wilson Nadruz Junior1 (1) Hospital de Clínicas da Universidade Estadual de Campinas – UNICAMP Introduction: Phaeohyphomycosis is a chronic fungal disease located mainly in the skin, with Fonsecaea pedrosoi being the most frequent fungus. The infection develops after a traumatic inoculation into the skin, and later, as a small papule. They may ulcerate, evolving with lymphatic or hematogenous dissemination. Infections are the main cause of death in heart transplant patients, with fungi being the third predominant etiologic agent. Case report: Male, 44 years old, vigilant, heart transplanted in 2017, using Azathioprine and Tacrolimus. He started with ulcerated lesions began, with exposure of the deep plane in the temporal region and right upper limb. About 2 months later, he developed aphasia and drowsiness. Seen on cranial tomography, frontal expansive lesion on the left, submitted to surgical resection. Diagnosed with Cerebral Chromomycosis, through the result of culture demonstrating the fungus of the genus Fonsecaea sp. Complementary treatment was performed with prolonged use of Amphotericin B during hospitalization and after discharge, replaced by Voriconazole. He was discharged from the hospital with an improvement in his neurological and skin condition. Discussion: Cerebral phaeohyphomycosis corresponds to brain infections caused by dematiaceous fungi. The predominant symptoms are produced by mass effect on the brain parenchyma. The risk of heart transplant patients presenting infection is directly related to the use of immunosuppressive drugs, such as mycophenolate and tacrolimus, which severely damage all components of immunity. Management usually combines surgical and drug treatment, the main antifungals being itraconazole, voriconazole and posaconazole. Conclusion: Phaeohyphomycosis of the central nervous system is a rare pathology in heart transplant patients. The mortality rate among patients with these infections is at least 50%, the high mortality rate highlights the need for aggressive therapy. The case described stands out for the severity of the condition, but with good resolution with combined surgical and antifungal treatment, even in the case of an immunocompromised patient. 108768 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES FERNANDA MARIA FRANCO CASTRO1, FÁBIO MORATO DE CASTILHO1, EDUARDO BELISÁRIO FALCHETTO1, THIAGO PINHEIRO JUNQUEIRA1, NATHÁLIA SIMÕES FERNANDES CAMPOS1 (1) Hospital Felicio Rocho – HFR Intimal sarcoma of the pulmonar artery is a rare and potentially lethal tumor. Its presentation is similar to pulmonary thromboembolism, what makes the diagnosis between these two diseases a clinical challenge. A 50-year-old woman was diagnosed with pulmonary embolism by Computed tomography angiography (CTA) and anticoagulated with warfarin. Despite 8 months of warfarin therapy she remained symptomatic. She complained of dyspnea and fatigue upon mild exertion. An echocardiogram demonstrated pulmonary hypertension and right ventricular dysfunction and the second CTA exam had demonstrated no change in the pulmonary artery filling defects. In view of the clinical worsening a diagnostic hypothesis of chronic pulmonary thromboembolism associated with acute thromboembolism was raised. She was referred for arteriography which showed the appearance of bilateral acute thrombi, unsuccessful mechanical thrombectomy attempt and subsequent local infusion of thrombolytic. With the findings leading towards a diagnosis of acute pulmonary embolism in a patient who was already on anticoagulant therapy, the implantation of vena cava filter was placed. Subsequently, a pulmonary artery endarterectomy was performed, which did not identify a thrombus. The material sent to anatomopathological and immunohistochemical study, has confirmed the diagnosis of intimal pulmonary artery sarcoma. The lesion was partially removed and the treatment with docetaxel and gemcitabine started with a good initial tumor response. After the surgery to remove the tumor, there was an improvement in the right ventricular function and patient’s symptoms. Pulmonary artery sarcomas have no gender preference and generally occur in the fifth decade of life. Histologically, they are mostly undifferentiated or poorly differentiated tumors. The case presented here catch the attention to the need to raise the differential diagnosis in patients who do not respond to the current treatment of pulmonary thromboembolism because the treatments differ markedly and prognosis depends on the early diagnosis and prompt surgical resection of the tumor. 108837 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING ELISAMA PIMENTEL ZAMIAN COTIAS1, Fabrício Thebit Bortolon1, Patrick Ventorim Costa1, Herbert Felipe Heimbeck1, Berilurdes Wallacy Garcia1 (1) Hospital Universitário Cassiano Antonio Moraes (HUCAM) Unicuspid aortic valve is a rare congenital malformation associated with aortic stenosis and/or regurgitation. This condition can be categorized into two types: unicommissural and acommissural type. This case report presents a 25-year-old man, with a history of heart murmurs, dyspnea only with unusual exertion. His physical examination demonstrated an ejection systolic murmur heard loudest over the aortic valve. Transthoracic echocardiography showed concentric left ventricular hypertrophy with preserved systolic function and dome-shaped aortic valve during systole on a paraesternal long-axis view (Figure 1). Unicuspid aortic valve had nodule calcifications with a lateral zone of attachment to the aorta and an eccentric orifice consistent with unicommissural type on transthoracic and tridimensional transesophageal echocardiography on a short-axis view (Figure 3 and 4). Severe regurgitation at color doppler (Figure 2) and severe aortic stenosis with a maximum velocity of 3.95 m/s, a mean gradient of 38 mmHg and calculated aortic valve of 0.58 cm²/m². In conclusion, echocardiography is a solid imaging method that enables diagnosing morphological anomalies of heart valves, as well as evaluating their functioning. 108874 Modality: E-Poster Young Researcher – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY PATRICIA MATTOS VIERA DO PAÇO1, Olga Ferreira de Souza1, Nilson Araujo de Oliveira Junior1, João Pantoja1, Antônio José Carneiro1 (1) Hospital Copastar(HCS) Introduction: Ablation(ABL) is a safe treatment for atrial fibrillation(AF). However, injury of the anterior vagal plexus can occur when energy is applied to the posterior wall of the left atrium, resulting in gastric hypomotility, mostly oligosymptomatic and reversible. The reported case presents a patient with severe gastroparesis after cryoablation (CB). Case description: A 58 years old male patient with recent paroxysmal AF, was submitted to CB (MEDTRONIC ARCTIC FRONT II). The day after, he complained of indigestion and nausea. The symptoms were ameliorated with oral prokinetic drugs. After 10 days, he was readmitted with diffuse abdominal pain and not tolerating food and liquid ingestion. The patient was afebrile, had stable vital signs, marked abdominal distention and tenderness. CT scan showed significant gastric distention and mild diverticulitis(DI). Ciprofloxacin and metronidazole were initiated. Despite the clinical and radiologic improvement of the DI, the gastroparesis(GP) persisted. So, we ruled out any role of the DI in the GP genesis and diagnosed vagal damage due to CB. We started clarithromycin 250 mg BID to enhance gastric motility with excellent response. A new CT showed the resolution of gastric distention and a liquid diet was initiated. After a couple of days, he could eat regularly and get discharged. The figure shows the evolution of the CTs, from right to left side. Conclusions: This case helps us understand the GP related to AF ABL. Besides, he presented a second abdominal pathology, making the diagnosis difficult. The resolution was obtained from an unusual drug for this condition. Despite the protective measures there was an injury to the vagus fibers. After this case, we observed the need for further studies to seek effective protective measures. 108899 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FELIPE REZENDE GIACOMELLI1, Marcelo Bettega1, Lara do Norte Garcia1, Júlia Guidi1 (1) Hospital Israelita Albert Einstein Introduction: Acute coronary syndrome (ACS) associated with hypersensitivity represent predominantly eosinophilic and mast cell endothelial and microvascular dysfunction. This spectrum encompasses spontaneous coronary artery dissection (SCAD), non-atherosclerotic cause of ACS, and Kounis Syndrome (KS), defined by the concomitance between allergic reactions, cardiac symptoms and electrocardiographic changes after the triggering event. Case report: Male, 41 years old, with refractory asthma, chronic sinus disease, nasal polyposis, urticaria, IgG immunodeficiency and hypereosinophilic syndrome. It started in August/2021 typical anginal chest pain in the absence of ischemia on electrocardiogram, elevation of markers of myocardial necrosis (MMN) or atherosclerotic lesions on coronary angiotomography. After 2 weeks, he presented with pain, ST and MMN elevation, DACS and vasospasm in coronary angiography (CA). Angioplasty was performed, but it evolved with 2 recurrences of vasospasm refractory to intracoronary nitrate and the need for new angioplasties, totaling 6 stents. Simultaneously, there were allergic skin and respiratory manifestations, intense headache and abdominal pain. Transferred to this service after angina associated with purulent rhinosinusitis. Documented on CA nitrate-responsive vasospasm and patent stents, absence of vasculitis, myocarditis or arteritis on myocardial magnetic resonance imaging and PET-CT. Satisfactory clinical response was observed after corticosteroid therapy and optimization of antianginal agents. Conclusion: ACS and hypersensitivity are correlated with eosinophilic endothelial infiltrates, mast cell activation, and release of cytotoxic agents such as histamine and thromboxane. DACS results in disruption of the intimal layer and expansive intramural hemorrhage. In KS, cerebral and mesenteric territories are also affected and 3 coronary variables are observed:type I includes vasospasm without previous coronary artery disease; type II vasospasm with quiescent atheromatosis and type III in-stent thrombosis. Drugs, food, hives, bronchial asthma and the presence of an intracoronary stent are triggers and treatment consists of corticosteroids, antihistamines and vasodilators. Therefore, allergic angina is an underdiagnosed condition, with high morbidity, mortality and iatrogenic risk, whose treatment remains unspecific. Eosinophilstabilizing therapies and monoclonal antibodies are promising in attenuating inflammatorydependent myocardial injury. 108929 Modality: E-Poster Young Researcher – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES LEONCIO BEM SIDRIM1, Maria das Neves Dantas da Silveira Barros2, Rodrigo Brito de Barros Correia e Silva1, Fiamma Ferreira Nogueira1, Taciana Queiroz Medeiros Gomes1 (1) Pronto Socorro Cardiológico de Pernambuco (PROCAPE); (2) Universidade de Pernambuco Introduction: Thromboembolic phenomena are frequent in patients with Chagas heart disease.1,2 Its incidence is 36% in patients with heart failure with this etiology.3 Mortality due to these complications is related, in most cases, to pulmonary and brain embolisms. 4–6 Coronary artery embolisms have been rarely described and their prevalence has not been estimated by clinical studies.2,7 Several anatomopathological and clinical studies have shown a subgroup with higher risk: presence of myocardial dysfunction, presence of intracavitary thrombi, and previous thromboembolic events. 8–10. Case description: Female, 54 years old, history of megaesophagus at 11 years of age and serological diagnosis of Chagas disease 3 years ago. No history of heart disease and no regular use of medication. Transthoracic echocardiogram (TTE) showed preserved left ventricular (LV) diastolic and systolic functions and an ejection fraction (EF) of 71%. For 30 days, she presented chest pain and tightness, in the left hemithorax and radiating to the left upper limb and mandible. Electrocardiogram (ECG) showed ST-segment elevation, measuring 2.0 mm from V2 to V4. Despite looking for the local health service, she was not submitted to emergency coronary angiography or chemical thrombolysis, with spontaneous improvement of chest pain in about 48 hours. After two weeks, the patient sought an outpatient clinic specialized in Chagas disease, reporting what had happened. A new TTE was performed, which showed: dilated LV, with hypokinesia in the anterior (middle), inferolateral (middle) and apical (septal and inferior) septum region, LV EF: 65%. The clinical history and compatible echocardiographic findings allowed the retrospective diagnosis of Acute Myocardial Infarction (AMI). Coronary angiography showed coronary arteries free from atheroma. Ventriculography showed aneurysm at the LV apex. Such findings corroborate the hypothesis of coronary thromboembolism as the etiology for AMI. Conclusion: Chagas disease is always a hypothesis to be raised in thromboembolic phenomena, given its high prevalence. Ventricular aneurysms may represent an important cardioembolic source and may be present even in patients with normal EF. The etiological definition of AMI in this case has an important practical implication for its management, given the need to use anticoagulants as opposed to antiaggregants and statins. 108961 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING UANDY DIAS TERRAS1, Arthur Gabriel Soares Costa Monteiro1, Larissa Amaral de Abreu1, Larissa Rosa de Rezende1, Solange de Sousa Andrade2 (1) UNIVERSIDADE DE RIBEIRÃO PRETO – UNAERP; (2) INSTITUTO DO CORAÇÃO DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO Introduction: ALCAPA syndrome (known as Bland-Altman-Garland syndrome) is a rare congenital anomaly in which it occurs in the coronary arteries. We present a case report of ALCAPA syndrome in a 37-year-old female patient, who presented with syncope followed by seizures and cardiorespiratory arrest, remaining in the intensive care unit for 7 days. Case report: Brazilian citizen, female, 37 years old, residing in Santos – São Paulo, presented syncope followed by convulsive crisis and cardiorespiratory arrest during intense physical activity at the gym. Cardiopulmonary resuscitation maneuvers were performed for 15 minutes by the personal trainer until the Mobile and Emergency Care Service arrived, which in the initial evaluation verified a Ventricular Fibrillation (VF) rhythm and performed defibrillation with a return to sinus rhythm, palpable pulse and hemodynamically stable. She was intubated and taken to the Emergency Care Unit. Upon physical examination, it was observed that the patient was in poor general condition, Glasgow 4 and mitotic pupils. After 24 hours, the patient presented with spontaneous breathing, Glasgow 15. The Echocardiogram that showed diffuse hypokinesia of the left ventricle, more accentuated in the apical region, Ejection fraction of 52%. Coronary catheterization was requested, which showed filling of the distal third of the left coronary artery and it was not possible to catheterize the origin of this vessel. Angiotomography of the coronary arteries was then indicated, which showed the origin of the left coronary artery from the trunk of the pulmonary artery. The case was referred to the cardiac surgery team with a diagnosis of Alcapa syndrome and surgical treatment was indicated. Patient underwent cardiac surgery for reimplantation of the left coronary artery in the left coronary sinus with stable postoperative evolution and clinical improvement. One year later, the patient is asymptomatic and performs regular moderate-intensity physical activity. Conclusion: Alcapa syndrome is a rare disease among the group of anomalous coronary arteries, with a high risk of sudden death, as was presented in the case that presented a satisfactory result with the performance of cardiopulmonary resuscitation maneuvers and the correction of this anomaly was fundamental for the evolution and patient prognosis. 108985 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS VALÉRIA BRAGA SANTIAGO DE SÁ1, VALÉRIA BRAGA SANTIAGO DE SÁ1, GLAUCIENE FERREIRA DE SOUZA XAVIER2, DANIEL MEDEIROS MOREIRA1, GILVAN MAGALHÃES PINTO1 (1) INSTITUTO DE CARDIOLOGIA DE SANTA CATARINA; (2) UNACON RR Introduction: Coronary artery anomalies are a diverse group of cardiac anatomical congenital disorders presenting a wide variety of clinical manifestations. Patients may be asymptomatic or have varying degrees of symptoms correlating with the anatomical presentation of the anomalous coronary path. In some cases, they may lead to sudden death. Case report: A 43-year-old Venezuelan male patient sought care presenting short-term chest pain episodes for a week. Previous medical history reported systemic hypertension treated with losartan and suffering a transient ischemic stroke 5 years ago. Electrocardiogram showed no ischemic alteration, negative myocardial necrosis markers. Coronary angiotomography presented anomalous origin of the middle and distal segments of the anterior descending artery in a way that left coronary trunk originates the circumflex artery and the proximal segment of the left anterior descending artery (LAD), which originates the 1st septal and 1st diagonal, as usual. However, the medial and distal segments of the LAD originates from the right coronary ostium with its path anterior to the pulmonary trunk until reaching the anterior interventricular groove in the middle segment, providing 2 more diagonal branches. Conclusion: This finding encourages further study and discussion regarding its potential role in the symptomatic report of this case and other similar clinical situations, helping to evaluate the need for some intervention. 108990 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY RAFAEL MASSUTI1, Bibiana Guimarães Maggi1, Marcelo Sabedotti1, Rafaela Oliveira Leite2, Leandro Gazziero Rech1 (1) Hospital Geral de Caxias do Sul; (2) Fundação Universidade de Caxias do Sul Introduction: Pulmonary arteriovenous malformations (PAVMs) are abnormal pulmonary vessels in which an artery is directly connected to a vein, forming a right-to-left shunt and resulting in clinical manifestations such as hypoxemia, predisposition to infections, and ischaemic cerebrovascular accident (CVA). Case report: Patient with a history of right hemiplegia at fifteen years old, normal hematological investigation and magnetic resonance of the encephalon at that time, and transesophageal echocardiogram demonstrating a patent foramen ovale (PFO) with a small passage of air bubbles under Valsalva maneuver, without septum aneurysm. The assistant physician discussed PFO closure, but decided for clinical management with acetylsalicylic acid, 100 mg per day. At 21-year-old, this patient seeks medical care presenting aphasia and right hemiplegia. Brain computed tomography revealed hypodense areas on the right parietal cortex region, and in left white matter, as well as encephalomalacia areas on the left brain hemisphere, suggesting vascular injury as aetiology of the stroke. Transcranial Doppler (TCD) ultrasound revealed more than 100 high-intensity transient signals (HITS), considering the Spencer Logarithmic Scale, a large shunt. Despite the lesion’s anatomy, the patient improved symptoms to the absence of neurologic deficits. PFO closure was discussed once more by the assistant physician team, which opted to investigate differential embolic sources, since a PFO with such anatomical characteristics in a young patient was not considered a high risk for CVA. A pulmonary angiography revealed a massive aneurysmatic PAVM (with 3,3 cm of diameter) on the right lower lobe, nurtured by an interlobar artery branch and drained to the pulmonary vein. We proceeded with the percutaneous embolization of the PAVM through puncture of the right femoral vein and the selective catheterization of the lesion with a Progrear® 2.7 microcatheter. The PAVM was embolized with 8 Microplex® 18 coils, achieving successful occlusion of the defect. A control chest computed tomography angiography 30 days after the procedure revealed the absence of flow through the PAVM, and new TCD ultrasound demonstrated the absence of HITS. Conclusion: Even though PFO is associated with ischaemic events, ischaemic stroke in young patients must be thoroughly investigated, especially when the clinical picture is not compatible with the anatomical alterations. PAVM, although rare, must be remembered. 109221 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY JORDANA PIRES MENDONÇA1, Flavio Rosa Vieira1, Rogério Las Casas1, Tannas Jatene1, Vinicius Daher Vaz1 (1) Hospital do coração Anis Rassi Introduction: Transcatheter aortic valve implantation (TAVI) for aortic stenosis has been well documented over the last years. Nevertheless, TAVI for pure aortic regurgitation (AR) it’s stilll challenge due to the absence of calcification of the aortic valve and the high flow state. This features make difficult to anchor the prosthesis in the right spot, that can evolve to acute or late migration with tragic consequences. Case Description: Female 79 y/o fragile lady, at NYHA III on optimal medical therapy. At Physical examination she was tachydyspneic, with severe AR on Doppler, within 100% of the LVOT, the final velocity in the descending aorta was 2.1 m/s, mean gradient (MG) was of 21 mmHg, secondary to hyper flow, PASP of 66 mmHg. Euroscore II 8.39% and STS of 8,5%. showed aortic annulus perimeter of 76.3 mm and mean diameter of 24.3, mean sinus of Valsalva of 34 mm, left main coronary artery height of 12.8 mm and right coronary artery of 11.5 mm with zero aortic valve calcium score. After discussion with the heart team the decision to perform TAVI was made. The procedure was exceptionally performed under general anesthesia and guided by a transesophageal ECO. To secure the pacing during all the deployment a transvenous pacing was chosen. An EVOLUT Pro (Medtronic) #29 was chosen to achieve 19% of oversize. Taking into account the difficulties already anticipates, we did a full paced release. The deployment was done with pacing of 140–160 bpm in order to achieve less than 50 mm/hg of mean arterial pressure until point of no return. After careful checking with angiogram and ECO we did slow full release with 180 bpm and after we kept pacing 120 bpm for 10 minutes. The procedure duration was 69 minutes, with 60cc of contrast and without any major complications. The final MG was of only 4 mmHg and there were no paravalar leak whatsoever. The paciente was discharge from the hospital within 48 hours. At the 30-day follow-up patient has clinically improved markedly, without any further complication. Conclusion: TAVI using self-expanding prosthesis and specific deployments tips may be an alternative for patients with pure aortic regurgitation and high surgical risk or fragile profile. 108998 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY DOUGLAS VINÍCIUS DE OLIVEIRA SANTOS1, Iara dos Santos Ferreira2, Ana Caroline Mendonça Cardoso3, Ana Flávia Reis Costa4, Daniela Passos Garcia Campos5 (1) Hospital Santa Casa de Misericórdia de Belo Horizonte; (2) Hospital Santa Casa de Misericórdia de Belo Horizonte; (3) Hospital Santa Casa de Misericórdia de Belo Horizonte; (4) Hospital Santa Casa de Misericórdia de Belo Horizonte; (5) Hospital Santa Casa de Misericórdia de Belo Horizonte Introduction: Arterial Coronary-cameral fistulas, congenital or acquired, are rare coronary anomalies, capable of generating serious repercussions in their carriers. We report a case of a 10-year-old child with a coronary-cameral fistula, between the right coronary artery (RCA) and right ventricle (RV). Case report: A 10-year-old female patient with asthma sought medical care denouncing recurrent fevers, associated with prostration and hyporexia, with a month of evolution. On physical examination, a heart murmur was detected, and an echocardiogram was performed suggesting Interventricular Communication (IVC). The patient was fowarded to a high complexity hospital to complete clinical, laboratory and imaging exams, in which evidenced the following: presence of lower dental arch cavities, echocardiographic findings compatible with mobile vegetations, significant regurgitation in the tricuspid valve and coronary-cameral fistula between RCA and RV. The diagnosis of infective endocarditis (IE), ACD-VD coronary-cameral arterial fistula were established, and VSD was ruled out. Antibiotic therapy was instituted (Oxacillin 200 mg/kg for 35 days, Amikacin 15 mg/kg for 22 days) and tooth extraction was performed. The patient evolved with clinical, laboratory and imaging improvement, being discharged with Amoxicillin/Clavulanate for another 7 days, along with Furosemide 40 mg/day, Carvedilol 6,25 mg/day and Beclomethasone 4 g/day. Three months later IE treatment, corrective surgery for a coronary-cameral fistula was successfully performed and all medications were discontinued. Conclusion: Coronary artery fistula with cardiac chambers are rare, being present in 0.002% of the population, representing 0.4% of all cardiac malformations. They can be congenital or acquired, the first being apparently associated with persistence of embryonic sinusoids in the myocardium. Manifestation is very variable, being capable of generating serious repercussions in approximately 19% of patients under 20 years of age, such as infarction, heart failure, cardiomyopathy, arrhythmias and IE. Heart murmur is the main semiological finding, associated with the specific symptomatology of complications. The case exemplifies the challenges of early diagnosis and treatment of coronary-camera fistulas, essential for good outcome, as well as the importance of accurate clinical and echo-Doppler evaluations, given the spectrum of manifestations and differential diagnoses, such as VSD and IE. 109000 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LEANDRO GAZZIERO RECH1, Rafaela Oliveira Leite2, Marcelo Sabedotti1, Rafael Massuti1, Bibiana Guimarães Maggi1 (1) Hospital Geral de Caxias do Sul; (2) Fundação Universidade de Caxias do Sul Introduction: Treating patients diagnosed with atrial fibrillation (AF) during the use of direct oral anticoagulants (DOACs) and submitted to elective surgeries is a common daily situation, but its management remains a challenge. The correct suspension period of DOACs before interventions varies according to several factors, and recent studies are committed to adopting the best way of managing these patients to minimize risks. Clinical picture: A 72-years-old female was referred to cervical arthrodesis. She had a history of surgical correction of the atrial septal defect (ASD) 20 years before, chronic AF, diabetes, arterial hypertension, and permanent pacemaker due to sinus node dysfunction. The patient was on rivaroxaban 20 mg per day, suspended 72 hours before the procedure. In the transoperative, the patient evolved to cardiac arrest in pulseless electrical activity, which returned to spontaneous circulation after two minutes of resuscitation by following the advanced cardiac life support (ACLS) protocol. In cardiogenic shock, she was referred to urgent coronary angiography, performed 20 minutes after the cardiac arrest, and the exam demonstrated coronary embolization, with a large quantity of thrombus burden, which were aspirated with a dedicated instrument. The coronary was infused with tirofiban. Hemodynamic instability was corrected with norepinephrine and crystalloids replacement, resulting in a favorable and satisfactory evolution of the instability, allowing the extubation on the same day. A transesophageal echocardiogram was performed and demonstrated spontaneous echo contrast in the left atrium and thrombus in the left atrial appendage. Despite the clinical picture, the patient did not present any degree of ventricular dysfunction. She was on enoxaparin (full dosage) during the hospitalization, and after 48 hours, we introduced Dabigatran 150 mg twice a day. The patient was discharged seven days after the occurrence, asymptomatic. Conclusion: Perioperative management of AF patients during the use of DOACs remains a challenge. Recent studies suggest individualizing treatments, which can be discontinued one to five days before the procedure, according to the surgical port, risk of bleeding, drug half-life, and renal function. Individualization remains the best strategy in addition to the possibility of non-invasive procedures for thrombosis evaluation that can corroborate clinical decisions in problematic situations. 109041 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY DAVID EMMANUEL BEDOYA GOYES1, Rodolfo Vaz1, Luis Antônio Machado1, Bruno Mahler Mioto1 (1) Instituto do coração – INCOR Introduction: Coronary artery anomalies are congenital alterations that include an extensive group of malformations with a wide variety in origin, course, and distribution. Coronary artery fistula is defined as a very rare anomalous connection between a coronary artery and a cardiac chamber or a main vessel, being present in 0.002% of the population and representing 0.4% of all cardiac malformations, being asymptomatic and benign or could present with symptoms such as chest pain and cardiac dysfunction in young adults, depending on the location. Fistulas are usually diagnosed by computed tomography angiography, coronary angiography, transesophageal echocardiography, or cardiac magnetic resonance imaging. Case report A previously hypertensive, dyslipidemic 45-year-old male patient with symptoms of severe grip-related chest pain with irradiation to the mandible and left arm without other associated symptoms. Attended in the emergency room, electrocardiogram was performed without dynamic changes and markers of myocardial necrosis with slight increase; acute coronary syndrome was considered, so coronary angiography was requested, which showed the presence of an important anterior descending artery fistula (AD) for pulmonary artery trunk (AP) with QP/QS of being programmed into a second moment intervention. The patient was successfully submitted to percutaneous closure of the DA-AP fistula by embolization with interlock coil, successfully, control echocardiogram without segmental alterations, and with preserved ejection fraction. Discussion: Coronary fistulas were first described in 1865, being the first successful surgical correction in 1947; coronary artery fistulas (FAC) are congenital anomalies with an estimated incidence of 0.2 to 0.4% and are the result of persistence of primitive myocardial sinusoidal circulation and primordial epicardial vessels and may be associated with other congenital heart diseases such as arterial canal persistence, tetralogy of Fallot and interventricular communication; however, CAS can also be acquired, secondary to complications of procedures such as coronary angioplasty, myocardial revascularization surgery or after heart transplantation and myocardial biopsy. Conclusion: Coronary fistulas, despite having a low incidence among congenital heart diseases, have become increasingly frequent due to technological advances in diagnostic methods, especially in hemodynamic studies offer the possibility of treatment. 109082 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GABRIEL KANHOUCHE1, Filippe Barcellos Filippini1, Pedro Felipe Gomes Nicz,1, Fabio Sandoli de Brito Jr.1, Alexandre Abizaid1 (1) Heart Institute of Sao Paulo – HCFMUSP Background: About 2–5% of patients undergoing transcatheter aortic valve implantation (TAVI) experience stroke, which substantially increases the in-hospital mortality. Atrial fibrillation (AF) prevalence in patients undergoing TAVI ranges from 16–51% and balancing the risk of cardioembolic and bleeding events represents a major challenge. We report a combined procedure strategy for treating a high bleeding risk patient with AF and severe aortic stenosis (AS). Case report: A 81-year-old woman, with past history of permanent AF, diabetes and previous pacemaker implant, evolved with heart failure class NYHA III due to severe AS. The risk-scores were CHA2DS2VASc = 6 and HAS-BLED = 3 and she was on irregular rivaroxaban use, with one prior gastrointestinal bleeding. Transesophageal echocardiogram (TEE) indicated a severe AS with aortic valve area = 0.6 cm2, mean aortic gradient = 56 mmHg, normal left ventricle function and chicken wing morphology in the left atrial appendage (LAA). After careful cardiac computed tomography evaluation, anatomy was deemed suitable for transfemoral TAVI, LAA occlusion and cerebral protection device. During Heart Team discussion, according to the intermediate risk score (STS = 6.8%), frailty, high bleeding risk and suitable anatomy, she was considered to the minimalist combined approach. First, the Sentinel cerebral protection device (Boston Scientific, CA, USA) was positioned in the brachiocephalic trunk and left carotid artery, followed by left transfemoral TAVI with Acurate Neo 2 size 25 mm (Boston Scientific, CA, USA) and right venous transfemoral LAA occlusion with Watchman FLX size 27 mm (Boston Scientific, CA, USA). Intraoperative TEE revealed absence of leak in both devices and no procedural complications. After removal of the Sentinel equipment, many embolic debris were found in the filters. She was discharged 10 days after the procedure, on aspirin use and functional class NYHA I. Clinical follow-up after 30 days was uneventful. Discussion/Conclusion: The combined procedure of TAVI and LAA occlusion with the use of a cerebral protection device followed by single antiplatelet therapy was feasible and safe. Since both TAVR and LAAO are associated with a non-negligible risk of stroke, the use of cerebral protection devices might become the standard of care for this combined approach. 109097 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GABRIEL KANHOUCHE1, Mauricio Felippi de Sá Marchi1, Marcelo Harada Ribeiro1, Carlos Augusto Homem Magalhaes de Campos1, Henrique Barbosa Ribeiro1 (1) Heart Institute of Sao Paulo – HCFMUSP Introduction: Excimer laser catheter atherectomy (ELCA) has improved significantly in recent years, emitting high-energy ultraviolet (UV) and short wavelength with less penetration and heat emission, ultimately leading to less tissue damage and fewer complications. Case report: We described a real-life case of a 66-year-old male patient with hypertension, diabetes mellitus, dyslipidemia and former smoker. He was submitted to percutaneous coronary intervention (PCI) with bare metal stent in left descending anterior (LAD), coronary bypass in 2001 including right mammary internal artery to right coronary artery. In the past 20 years, was performed innumerous PCIs including a LAD drug eluting stent (DES) overlapping. The last LAD stent presents an acute thrombosis and was managed only with thrombolysis. A new cardiac catheterization was performed because the patients developed chest pain in the last couple months. A severe stent under-expansion in the proximal LAD was observed and ELCA was the device of choice due a overlapping underexpanded stent with bulcky calcification that make crossover the lesion with balloons or others devices impossible. A progressive load of 40/40, 60/60 and 80 mJ/mm2/80Hz and saline injection method to prepare the in-stent lesion was performed and subsequent implantation of a DES Firehawk Liberty (MicroPort, Shangai, China) 3.50 × 38 mm from the left main coronary to the LAD. Discussion: ELCA was a precise option in this case because its mechanisms of action allow reaching atherosclerosis beyond the stent without disrupting the stent architecture. The photochemical role makes dissociation of the cell’s molecular bonds, the photothermal effect generated by vibration leads to the softening of collagen and protein fibers of the atheroma and the photomechanical effect generated by the explosion and implosion of the vapor bubbles leads to plaque rupture. In addition to stent under-expansion, ELCA is indicated for in-stent restenosis, calcification, balloon uncrossable lesions, and chronic total occlusion. Similar cases have been shown sporadic in the literature, but it is necessary more publication of successful results with ELCA to remove the past stigma of laser. Thereby more operators could be encouraged to use this useful device. 109141 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MAURICIO FELIPPI DE SÁ MARCHI1, Filippe Barcellos Filippini1, Antonio Fernando Diniz Freire1, Alexandre Abizaid1, Fábio Sândoli de Brito Júnior1 (1) Instituto do Coração do Hospital das Clínicas da FMUSP (InCor) Introduction: Transcatheter Aortic Valve Replacement (TAVR) has emerged as a less invasive procedure for severe aortic stenosis. It’s not rare to observe concomitant Aortic Stenosis (AS) with left ventricular outflow tract (LVOT) obstruction. This condition is mainly due to ventricular septal hypertrophy, which can potentially impact TAVR result, increasing cardiovascular mortality. We report two cases of patients with AS and high values of intraventricular gradient prior to TAVR. In both cases alcohol septal ablation (ASA) was successfully performed before TAVR. Case: 1 A 75-years-old woman with history of AS, coronary artery disease, paroxysmal nocturnal hemoglobinuria and anemia was admitted in the emergency room (ER) for sudden loss of consciousness. Her initial exams demonstrated aortic stenosis, and a 59-mmHg intraventricular gradient measured at cath lab. Considering this, ablation of the first septal artery branch was executed, with a OTW balloon and 2 mL alcohol. Residual post intervention value of the intraventricular gradient was 7 mmHg. TAVR was indicated after discussion with the Heart Team. After the procedures the patient remained assymptomatic and was referred for out-of-hospital follow-up. Case 2 An 82-years-old woman was referred for TAVR for aortic stenosis. Initial 3D echo showed significant aortic stenosis and a hypertrophic septum of 16 mm. Alcohol septal ablation was performed with an OTW balloon and 1.5 mL alcohol. Initial intraventricular gradient was 100 mmHg intervention and became 24 mmHg afterwards. A transfemoral 23 mm Sapien 3® was implanted. Postprocedural echo reported a 65% EF, absence of dynamic intraventricular gradient, a 23-mmHg maximum and 14 mmHg mean gradient. The patient was discharged after three days and remained in out-of-hospital follow-up. Discussion: Outflow-tract gradients of 30 mmHg or more under resting conditions are independent determinants of symptoms of progressive heart failure and death, especially threating in the context of coexistent AS. Percutaneous alcohol septal ablation is a minimally invasive procedure, which can reduce the left-ventricular outflow gradient, therefore reducing symptoms and mortality in many patients. 109151 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MAURICIO FELIPPI DE SÁ MARCHI1, Gabriel Kanhouche1, Filippe Barcellos Filippini2, Luiz Junya Kajita1, Alexandre Abizaid1 (1) Instituto do Coração do Hospital das Clínicas da FMUSP (InCor) Introduction: Endomyocardial biopsy is the gold standard for the evaluation of graft rejection following orthotropic heart transplantation. It is a relatively safe procedure, with <1% chance of serious complications. Case report: A 69-year-old patient with history of heart transplant in 2018 due to Chaga’s disease underwent a 2-years control myocardial perfusion imaging scintigraphy which demonstrated minor stress-induced apical ischemia. He was asymptomatic and his last endomyocardial biopsy was 2 years ago, with no history of graft rejection. After the scintigraphy finding of ischemia a coronary angiography was performed, which displayed a fistula between the left anterior descending artery and right ventricle with subsequent coronary occlusion. This is a rare complication of endomyocardial biopsy. Fistula occlusion was planned after the scintigraphy finding. The procedure was performed with three micro-coils. First, a 5.5 × 5 mm VortX® (Boston Scientific) was deployed, followed by a 6.5 × 6 mm VortX® (Boston Scientific) and finally a 5.5 × 5 mm VortX Diamond® (Boston Scientific), delivered through a Renegade® (Boston Scientific) microcatheter. Final angiography demonstrated closure of the coronary artery fistula. After the procedure patient remained asymptomatic and was later discharged. Conclusion: We report a seldom case of fistula between left anterior descending artery and right ventricle. This patient was later successfully treated with micro-coils and was discharged for out-of-hospital follow-up. 109164 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT OLIVIA SHELLARD JUNQUEIRA FRANCO1, Rodrigo Batista Rocha1, Bruno Normande Colombo1, Rafael Augusto Mendes Domiciano1, Guilherme D‘Andrea Saba Arruda1 (1) Rede D’Or – Hospital São Luiz Anália Franco Introduction: Cardiac sarcoidosis (CS) is considered the second most common cause of death in sarcoidosis patients globally. Isolated cardiac sarcoidosis (ICS) is defined by the absence of systemic involvement, responsible for 9% of total CS and more common in male patients. Clinical manifestations depend on the location, extent, and activity of the disease and may be evidenced as ventricular arrhythmia, high-grade block, sudden death and heart failure (HF). This is a case report of a young female with ICS initially manifested as syncope. Case description: W.C.M, 30 years, female, was admitted at the emergency room reporting palpitations and an episode of syncope during effort. Eletrocardiogram showed right bundle brunch and first-degree atrioventricular block. Due to an ejection fraction (EF) of 28% evidenced in the transthoracic echocardiogram, a cardiac magnetic resonance was indicated and showed a reduction in thickness and transmural thinning, extensive areas of fibrosis (35,9%) and myocardial edema. The endomyocardial biopsy was compatible with granulomatous miocarditis. Screnning for systemic sarcoidosis was negative. She had frequent sustained ventricular tachycardia episodes and antiarrhytmic therapy was prescribed. Treatment for HF followed current guidelines up to the maximum tolerated doses, in addition to corticosteroids and methotrexate. Hence, an implantable cardioverter-defibrillator (ICD) was implanted to prevent sudden death. After 3 months, patient remained in functional class I, there was improvement in the EF by the transthoracic echocardiogram (44% rate) and lower density of arrhythmias were observed. Conclusion: ICS has poorer prognosis when compared to CS with systemic involvement, presenting itself with lower left ventricular EF, more frequent ventricular tachycardia and ICD indication. The diagnosis may be challenging, due to endomyocardial biopsy low sensitivity. Advanced non-invasive cardiac imaging techniques may allow earlier detection of cardiac involvement. The mainstay of medical therapy for ICS is immunosuppression in addition to therapies for HF manifestations. We have reported a case of ICS with an unusual presentation in a woman with syncope. Early diagnosis, initiation of therapy and recognition of patients in risk of sudden cardiac death and demand ICD prove to be crucial. 109171 Modality: E-Poster Young Researcher – Case Report Category: CARDIO-ONCOLOGY NATÁLIA AARÃO BERNARDI1, Marina Sahade Gonçalves1, Fábio Sândoli de Brito Júnior1, Antonio Fernando Diniz Freire1, Luana Alencar Fernandes Sampaio1 (1) Hospital Sírio Libanês Introduction: The use of immune checkpoint inhibitors (ICI), including programmed death protein inhibitors-1 (anti-PD1), brought large increases in survival, representing a paradigm shift. However, the management of immune-mediated side effects is still a challenge. Case report: A 76-year-old male patient with a history of dyslipidemia and coronary artery disease, was diagnosed with acral melanoma in the calcaneus. He started Nivolumab as the first line of systemic treatment, but before initiation, he underwent coronary angioplasty (AD – 2 stents and Mg-1 stent). After the first infusion of Nivolumab, he developed palpebral ptosis and a condition suggestive of immune-mediated myasthenic syndrome. Because symptoms worsened, it was decided to initiate immunoglobulin. During the first infusion, he presented cardiorespiratory arrest with ventricular tachycardia rhythm. Coronary angiography showed previous stents without complications and important progression of coronary artery disease compared to the examination 6 weeks before, being submitted to new angioplasty (AD – 2 stents, CD – 1stent, and balloon in Dg and VP). Cardiac magnetic resonance showed preserved biventricular function and late basal anterolateral epicardial enhancement, suggestive of a previous inflammatory process, and may represent previous inflammatory cardiomyopathy (myocarditis) or even cardiotoxicity. Data from the literature show that ICIs induce an increase in CD8+ T cells in the atherosclerotic plaque, leading to a change in the predominance of macrophages to lymphocytes in its microenvironment. Therefore, activated T cells favor the production of pro-atherogenic cytokines, contributing to the growth and destabilization of the atherosclerotic plaque. Conclusion: The profile of immune-mediated side effects can directly affect the cardiovascular system, culminating in increased morbidity and mortality in previously frail patients due to oncological diagnosis, thus demonstrating the importance of active surveillance of these patients. 109177 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS PATRICIA COSTA DE ALMEIDA1, Vitor Motta Inacio1, Bruno Azevedo da Cruz1, Felipe José Monassa Pitella1, Gabriel Camargo1 (1) Instituto Nacional de Cardiologia Introduction: The objective of this study is to report a rare case of a 15 mm coronary artery aneurysm (CAA) in the left main coronary artery (LMCA) in a patient with trivascular coronary artery disease (CAD), who underwent a surgical approach to CAD and conservative treatment of the coronary artery aneurysm. 68-year-old female patient with arterial hypertension, dyslipidemic and current smoker, with angina and transthoracic echocardiogram showing inferior and apical hypokinesia, with preserved left ventricular systolic function. Case report: The patient underwent stratification for CAD with myocardial perfusion scintigraphy, showing low uptake in the apical and anterior wall and a drop in ejection fraction >10% during exercise, being subsequently referred for coronary angiography with diagnosis of LMCA lesion 95% in the middle and distal third, with the presence of a 15 mm aneurysm, anterior descending artery (ADA) with 70% severe lesion in the middle third, circumflex artery (ACX) of small anatomical importance with 90% ostial lesion, diagonal branch (Dg) with severe lesion at the origin and posterior descending (PD) of the right coronary artery (RCA) with 50% in the middle third. After coronary angiography showed multivascular coronary artery disease, Coronary artery bypass graft surgery (CABG) was indicated and the treatment of the aneurysm was conservative due to technical difficulties. Discussion: Coronary aneurysms are defined as a focal dilatation of a coronary segment at least 1.5 times the size of the adjacent normal segment. The right coronary artery is generally the most affected artery (40%), with the LMCA being the least affected artery (3.5%). In contemporary studies, the incidence of true coronary aneurysms is less than 1%, with a greater predilection for males and for proximal segments of the coronary arteries. The reported clinical case contradicts the epidemiology, showing a rare case of AAC of LMCA in a female patient under investigation for CAD. Most current recommendations on coronary aneurysms are based on small case series or anecdotal reports; AAC involving LMCA, multiple or giant aneurysm. 109190 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY CATARINA MAARCHON DA SILVA2, Rodrigo A. S. Sardenberg2, Catarina Marchon da Silva1, Andrea Galvão2, Pedro Ismael Amaral Silva2 (1) Universidade Municipal de São Caetano do Sul; (2) Hospital Alemão Oswaldo Cruz; (3) União das Faculdades dos Grandes Lagos Cardiac lipomas are rare and benign tumors. We describe a case of a giant lipoma located in the right atrioventricular groove in a 62 year-old female patient who had no related symptoms. The diagnosis was done with an echocardiogram, and confirmed through intra and postoperative histopathology. Chest angio-CT scan was compatible with giant lipoma in the RAV groove or located at the RAV groove, measuring about 9.0 × 4.5 × 5.0 cm, circling the right coronary artery (RCA), ascending aorta and compressing the right atrium, causing dilation. The patient underwent complete tumor resection and was discharged without complications. The combination of an uncommon primary tumor of the heart in a rare location (AV groove) has, according to our researches, has only been reported once in the literature, so far. Cardiac lipomas are so infrequent that no therapeutic guidelines have been established for surgical indications in such cases. They are usually benign, however, tumor embolism, growth capacity, or intracardiac obstruction can cause a dangerous situation for the patient. Consequently, complete surgical resection might be indicated even in asymptomatic patients, varying according to the patient’s or the lipoma’s characteristics. 109199 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY BRUNA SANTI DOS SANTOS1, Eduardo Schlabendorff1, Eduardo Keller Saadi1, Ana Paula Tagliari1, Marcelo Haertel Miglioranza1 (1) Hospital Mãe de Deus Background: Transcatheter heart valve therapies such as mitral valve edge-to-edge repair with the MitraClip device and transcatheter aortic valve implantation (TAVI) have been demonstrated as an effective and valid therapeutic option in high-risk patients with heart failure. One-time double-valve treatment appears to be safe, feasible, and an innovative alternative. We present an exceptional case of concomitant MitraClip implantation for severe mitral regurgitation (MR) and TAVI with valve-in-valve technique in a patient with prior bioprosthetic aortic valve replacement and refractory heart failure. Case: An 80-year-old man with hypertension, diabetes, and prior bioprosthetic aortic valve replacement 6 years ago was referred to our hospital with a history of several hospitalizations for decompensated heart failure in the last 6 weeks. Besides clinical treatment optimization, he persisted with recurrent dyspnea in rest. Transthoracic echocardiography revealed severe MR with effective regurgitant orifice 80 mm2 and biological aortic valve prosthesis dysfunction with severe aortic regurgitation (AR). The left ventricle was dilated (5.8 cm/3.5 cm) and ejection fraction was 69%. This high-surgical-risk patient was accepted for concomitant transcatheter treatment with Mitraclip and TAVI valve-in-valve by the heart team, and it was well succeeded. After the procedure, the patient’s symptoms improved and on discharge, he was ambulating, without dyspnea in ordinary physical activity. Conclusion: Our case illustrates a success case of concomitant MitraClip implantation for severe MR and valve-in-valve TAVI for treatment of refractory heart failure, with significant improvement of symptoms. We conclude that the combination of the two transcatheter therapies would appear to be a viable and safe approach for managing high-risk patients with concomitant severe AR and MR and refractory heart failure, and it is likely to show better results in clinical improvement if performed before severe left ventricle remodeling with severe dilation occurs due to non treated valve disease. 109620 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY SARAH PINI DE SOUZA1, Cíntia Chaves Mattoso1, Maria Carolina Terra Cola1, Fábio Akio Nishijuka1, Thaíssa Santos Monteiro1 (1) Instituto Nacional de Cardiologia Introduction: Atrial Septal Defect (ASD) can remain undiagnosed until adulthood. Sinus venosus atrial septal defects (SVASD) account for approximately 5–10% of ASDs. Associated lesions can be found, including anomalous pulmonary venous connection (APVC), persistent left superior vena cava (SVC), pulmonary valve stenosis and mitral valve prolapse. Case report: A 34 year-old male patient with a history of obesity and systemic arterial hypertension, had a known cardiac condition since he was 13 year-old, but lost follow-up and didn’t know any details about the condition. He reported the dyspnea had worsened gradually in the past 20 years, especially after he was diagnosed with atrial fibrillation, when he sought again for medical follow-up. Echocardiogram (Echo) demonstrated SVASD with bidirectional shunt and pulmonary hypertension. The patient had clinical signs of systemic congestion, suggesting right ventricle dysfunction. Right heart catheterization (RHC) was performed to evaluate if the patient was still eligible for ASD closure, and it confirmed SVASD with severe pulmonary hyperflow, increased pulmonary pressures and resistance, but with satisfactory response to vasodilator. The RHC wasn’t able to exclude the presence of partial APVC. The patient was then submitted to heart surgery for the ASD closure and at the procedure, anomalous drainage of two right pulmonary veins was identified, as they were connected to the SVC. For this reason, Warden repair was performed, with redirection of the APVC in the SVC through the interatrial communication into the left atrium. In the postoperative period, the patient complicated with severe pericardial effusion, requiring drainage, and was discharged 21 days after surgery. Postoperative echo showed improvement of pulmonary pressure and right ventricular function. During follow-up consultations, patient presented as NYHA I. Conclusion: Patients with ASD may remain asymptomatic until adulthood, and diagnosis delay can impact significantly in prognosis, as some complications can become irreversible. ECHO is the first line diagnostic technique and RHC is mandatory in cases of signs of pulmonary hypertension. Patients with pulmonary vascular resistance ≥5 wu should be evaluated with extreme caution, considering not to proceed the ASD closure, or leastwise to maintain an interatrial fenestration. 109211 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MAURICIO FELIPPI DE SÁ MARCHI1, Cauyna Gurgel Moreira1, Gabriel Kanhouche1, Santiago Raul Arrieta1, Pedro Alves Lemos Neto1 (1) Instituto do Coração do Hospital das Clínicas da FMUSP (InCor) Introduction: Kawasaki’s disease is an acute, self-limited febrile vasculitis of unknown cause that predominantly affects children and may lead to coronary dilatation and obstruction. We report the case of a child with extreme coronary aneurysm and stenosis, treated with percutaneous coronary intervention. Case report: A 9-year-old male child was referred to our hospital with chest pain for major efforts. At age of 2, he was diagnosed with Kawasaki’s and received treatment with acetylsalycilic acid (ASA) and Intravenous Immunoglobulin (IVIG). At the time, a computerized tomography angiogram large aneurysmatic dilatations in both the right coronary and the left descending arteries, measuring 11.2 mm and 10.2 mm in diameter, respectively. The patient remained asymptomatic in out-of-hospital care and received ASA and clopidogrel. He remained asymptomatic in clinical follow-up for 6 years. Due to the patient’s complaints, we performed a coronary angiography which revealed a calcified giant coronary aneurysm in the LAD with a 90% focal stenosis before the aneurysm and a giant aneurysm in the RCA with a 60% focal stenosis. After the diagnostic angiography showed signs of coronary stenosis and aneurysm in the left descending artery our Heart Team opted for a new angiographic study with Intravascular Ultrassond. Due to the findings, balloon angioplasty was successfully performed, initially with semicomplacent balloon and finally with a non-complacent balloon. Angiography after the procedure demonstrated reduction of the previously documented stenosis and the patient reported relieved of the symptoms. Discussion: This case demonstrates a rare, albeit reported complication of Kawasaki’s and the treatment option performed. Predisposing factors are not consensual, and an important genetic contribution is observed. Although Kawasaki’s leads to coronary aneurysms in approximately 25% of untreated patients, the timely treatment IVIG has decreased this risk to 3–5% of patients. In our case, despite timely IVIG, the patient still developed two giant aneurysms. 109227 Modality: E-Poster Young Researcher – Case Report Category: ANTICOAGULATION MARIANA INOCÊNCIO MARTINHO1, Rita Calé1, Ana Glória Fonseca2, Melanie Ferreira2, Hélder Pereira1 (1) Cadiology Department, Hospital Garcia de Orta (HGO); (2) Internal Medicine Department, Hospital Garcia de Orta (HGO) Thromboangiitis obliterans (TAO) is a rare vasculitis of young smokers affecting the small and medium sized vessels of the extremities. Although pulmonary embolism (PE) has been described, risk factors for large vessel occlusion and venous thromboembolism (VTE) recurrence are not known and there is no evidence favoring indefinite oral anticoagulation (OAC) for VTE prevention. Our first patient (pt) is an ex-smoker male diagnosed with PE and TAO at 44yo. He was under indefinite warfarin until 64yo, when OAC was switched to clopidogrel due to absence of VTE recurrence. Within 1 month, he had a massive unprovoked PE. Screening for autoimmunity (AI), antiphospholipid syndrome (APLS) and thrombophilia were negative and he was discharged under indefinite warfarin. At 7y follow-up (FUP) he had arterial ischemia but no VTE recurrence. The second pt is a male smoker diagnosed with TAO at 30yo and previous PE treated with warfarin for 6 months. AI and APLS screening were negative and he had hyperhomocysteinemia associated with MTHFR A1298C heterozigoty. At 36yo he had a submassive central PE treated with indefinite rivaroxaban. At 3y FUP despite arterial ischemia there were no VTE events. Thrombophilia and TAO have been associated and this may correlate with vessel inflammation and peripheral disease severity, but no link has been made to atypical presentation such as large vessel involvement. Hyperhomocysteinemia in MTHFR polymorphisms do not seem to increase VTE recurrence risk in the general population and do not determine prolonged OAC after a first VTE event. To our knowledge, this is the first time that this MTHFR mutation is associated with TAO. Although controversial, chronic OAC has been proposed to alleviate peripheral symptoms with some evidence suggesting that warfarin and rivaroxaban are able to reduce inflammatory markers in TAO. In both cases (the first not having any risk factor other that TAO and the second having hyperhomocysteinemia that alone should not determine secondary prevention), OAC did not prevent arterial events but seemed effective for VTE recurrence, leading us to believe that the proinflammatory state in TAO may warrant long-term OAC after a first event – as already happens with other AI diseases. Regardless, the authors acknowledge that we need more evidence to determine the most suitable approach. 109707 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY BRUNO GONÇALVES GARCIA1, Igor Daumas de Souza1, Amanda Dias Bomfim1, Stephan Lachtermacher Pacheco1, Marcelo Machado Melo1 (1) National Institute of Cardiology (INC) – Rio de Janeiro, Brazil Introduction: Ascending Aortic Pseudoaneurysm (AAP) is a rare complication in cardiac surgery. It is a discontinuation of layers of vessel, with extravasated blood being contained by neighboring structures. Understanding of this rare event deserves to be discuss, in view of the potential severity of complications. Case report: Male, 46-year-old, with history of bicuspid aortic valve with aortic insufficiency, who underwent metallic aortic valve replacement in 2005. 15 years later, an increase in diameters of ascending aorta (54 mm) was observed. Submitted to implantation of a non-valved tubular graft, and reimplantation of coronary. In immediate postoperative period, patient develops infective endocarditis of aortic prosthesis, treated only with antibiotic therapy. CT angiography of the aorta 1 year after surgery reveals pseudoaneurysm of ascending aorta measuring 8.4 × 5.9 cm extending up to 9 cm to the arch, with partially thrombosed lumen, and extravasation through anastomosis of the left main coronary artery. Although asymptomatic, by the risk of progression and rupture, was submitted to surgical correction, with closure of the orifice using bovine pericardium patch. On control CT angiography 1 and 2 weeks after surgery, new contrast uptake measuring 9 mm was identified in region adjacent on origin of the coronary arteries, without progression. Re-discussion between Heart Team was made, and conservative approach chosen, with periodic short-term follow-up. Conclusion: The case shows a patient with AAP discovered accidentally, after considerable postoperative period of aortic and aortic valve manipulation. Although asymptomatic, there is a risk of generating compressive symptoms of thoracic organs. It can complicate with rupture and bleeding, in addition to being a site of infection, or thrombosis. Predisposing factors are previous manipulation of the aorta, aortotomy site, cannulation points, coronary graft anastomosis, connective tissue diseases, degenerative changes in the aorta, dissection, local infection. Regardless of size, treatment is indicated. Endovascular therapy is possible, but limited in cases of complex anatomy or active infection. Surgical approach can be performed with patch correction, or Dacron conduit. Cardiopulmonary bypass via femoral vessels is suggested for safe sternotomy. There is a risk of recurrence. Although rare, due to relevant complications, PAA should not be forgotten in the postoperative follow-up of cardiac surgeries. 109300 Modality: E-Poster Young Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION SUELEN DAGOSTIM GISLON1, Roberto Gabriel Salvaro1 (1) Universidade do Extremo Sul Catarinense (UNESC) The Carotid Web is a fibrous tissue projection to the carotid artery into the lumen with a pathological characteristic of intimal fibromuscular dysplasia. It is proposed as a mechanism of ischemic cerebrovascular accident (CVA) in which the hemodynamic alteration provoked can cause blood flow stagnation and clot embolisation. The main objective of this search is showed, clinically, the characteristic findings of carotid web in two different patients as well as their imaging tests. APC, female, 78 years old (patient 1), presenting complaints of headache in the occipital region radiating to the frontal region and dizziness for two months. History of Systemic Arterial Hypertension (SAH), dyslipidemia and ischemic heart disease with coronary artery bypass graft surgery in 2017. Due to the history of atherosclerosis and dizziness, an ultrasound of carotid was requested, which showed significant atheromatosis. The evaluation was complemented with carotid angiotomography (left figure), which demonstrated the presence of a linear image with a vertical orientation based on the posterior wall of the right carotid bulb, extending to the site of the internal carotid, determining stenosis in about 70%, probably related to Carotid Web, in addition to partially caicified plaques in the ostium of origin of the left vertebral column, determining stenosis in about 50%. MMF, male, 68 years old, farmer (patient 2), comes for consultation after having seizures with loss of consciousness for a few seconds. History of four CVA, SAH, venous insufficiency in the left lower limb and use of alcohol and tobacco. In investigation of stroke and embolic sources was performed Cranial Magnetic Resonance and Arterial Angio-AM of the neck (right figure), which showed Web Carotid on the right in addition to significant right vertebral lesion. Web Carotid is related to a higher risk of ischemic stroke, being important their identification to carry out the treatment and secondary prevention since the mechanism is also associated with recurrences of stroke, therefore, in some cases, dual antiplatelet aggregation or even anticoagulation is indicated. 109263 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING JONAS PACHECO DO DESTERRO1, Rafael Rodrigues Coutinho1, Nilton Lavatori Correa1, Izabella Caterine Palazzo1, Cláusio Tinoco Mesquita1 (1) Hospital Pró-Cardíaco Introduction: Infective endocarditis is an infection of the endocardium and is characterized by vegetation, an infected clot of platelets and/or fibrin, containing leukocytes and red blood cells, mainly in valve topography. The diagnosis involves clinical, laboratory and imaging criteria and the prognosis is directly related to the early initiation of treatment. Scintigraphy with MDP-99mTc-labeled leukocytes plays a key role in this pathology. Case report: We report the case of an 85-year-old female patient, hypertensive, dyslipidemic, without history of heart surgery with skin excoriations, fever, poor general condition and tachycardia. COVID19 infection was ruled out and the sepsis protocol started. The patient had leukocytosis with a left shift, elevation of CRP, lactate, massive pyuria and positive blood cultures for Staphylococcus aureus. Cranial, chest and abdomen CT scans showed no signs of infection. The transesophageal echocardiogram showed caseous degeneration in the mitral valve, and it was not possible to rule out the possibility of infective endocarditis (IE). A scintigraphy with labeled leukocytes was requested, which showed anomalous uptake in the mitral-aortic topography, confirming IE in the native valve. Conclusions: In this case report above the scintigraphic method had a crucial role confirming, with high specificity, IE of the native valve and supported the change in the therapeutic management and patient’s prognosis. 109285 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JULIANA JANGELAVICIN BARBOSA1, Fabiana Braga Sanches1, Rafael de Castro Hendges1, Silvio Marques Povoa Junior1, Pedro Silvio Farsky1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Cardiovascular disease has been the leading cause of mortality worldwide since the 1960s, including coronary syndrome and valve disease. Surgical implantation of an aortic valve prosthesis is usually a low-risk procedure, but it can be challenging in selected cases. We describe here a compression of the left main coronary artery (LMCA) by a surgical implanted Aortic Valve Prosthesis. Case report: Female patient,53 years old, dyslipidemic, HIV positive, previous coronary artery disease(CAD)-elective percutaneous coronary intervention of the left anterior descending artery in 2020,and surgical aortic valve replacement on May, 2021. On December 24 this patient presented in the Emergency room with chest pain with 2 hours duration. Initial electrocardiogram demonstrated ST-segment elevation in AvR and persistent ST-segment depression in anterolateral wall of up to 2.0 mm. Cardiac catheterization was indicated, demonstrating severe LMCA ostial lesion (90%), suggestive of extrinsic compression by the aortic bioprosthesis. Twenty four hours after the onset of the chest pain, a significant elevation of troponin was detected (12,810 ng/L – Reference value <11 ng/L). After a Heart Team Decision, emergency coronary artery bypass surgery was indicated. The patient was discharged from hospital in 4 days after a successful procedure. Conclusion: Extrinsic compression of the LMCA as a cause of acute coronary syndrome is uncommon. The literature describes cases of coronary obstruction as a complication in patients undergoing transcatheter aortic valve implantation, in cases of pulmonary hypertension and due to aneurysmal structures. However, extrinsic compression of LMCA by a surgical prosthetic valve is a very rare condition, as in this report, that may guide future therapeutic decisions in atypical cases. 109337 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARCELA GOMES DE SOUZA1, Alice Mirane Malta Carrijo1, Vinicius Ferreira Aratani1, Flávia Bittar Britto Arantes1 (1) Universidade Federal de Uberlândia (UFU) Introduction: The Left Internal Thoracic Artery (LITA) fistula to the pulmonary artery and its branches is a rare clinical condition, but should be suspected in cases of persistent angina after myocardial revascularization surgery (MRS). From a pathophysiological perspective, there is a pressure difference between the vessels, resulting in coronary flow diversion and myocardial ischemia. Diagnosis requires coronary angiography and treatment may include drug therapy, surgical or endovascular intervention. Case Description: A 75-year-old man, with chronic obstructive pulmonary disease, hypertension, former smoker with a 60 pack-year smoking history. In 2014, the patient underwent MRS with LITA grafts to the anterior interventricular artery (AIA) and bypass graft to the right coronary artery, without complications. Five years later, he developed dyspnea on mild exertion, whose investigation revealed lung adenocarcinoma in the left apex, and a lobectomy was performed. After eight months, he presented with dry cough and dyspnea. The propaedeutics indicated a substernal goiter and heterogeneous thyroid, thus requiring a new surgical intervention in the mediastinum. After two months, he presented with complaints of chest pain on moderate and mild exertion associated with dyspnea. Pharmacological stress echocardiogram showed transient hypokinesia of the left ventricular anterior wall. Coronary computed tomography angiography detected an arterial fistula originating from the LITA following the lateral wall of the heart, confirmed by an angiographic study which indicated its destination for the left bronchial artery, besides blood flow diversion from the AIA. The patient underwent percutaneous embolization and trapping, with Onyx polymer and the release of six Axium micro-coils (Medtronic) into the fistular tract, via left radial access with a modified Seldinger technique. The procedure was successfully performed, and flow was immediately restored to the AIA. The patient was discharged remaining asymptomatic in the early follow-up. Conclusions: Due to the number of MRS procedures performed in the modern era and the preference for LITA as a graft, although rare, the occurrence of fistulas should be considered in cases of persistent angina after MRS. Notably, with the advancement of techniques, endovascular therapy, in addition to sparing patients from the risks of a thoracotomy, has been well documented in the treatment of symptomatic patients with a favorable anatom. 109339 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARINA PETSERSEN SAADI1, Anderson Donelli da Silveira1, Guilherme Heiden Teló1, Alan Pagnoncelli1, Felipe Homem Valle1 (1) Hospital de Clínicas de Porto Alegre Background: Utilization of steroids to improve physical performance increase the risk of adverse cardiovascular events by acceleration of atherosclerosis and thrombogenesis. We report a case of acute two-vessel coronary thrombotic subocclusions in an otherwise young healthy testosterone user. Case report: 37 year-old male, bodybuilder, presented with anterolateral myocardial infarction, six hours progression. He reported steroid use since 2013, using 250 mg of intramuscular testosterone decanoate daily at this time. He was taken to primary percutaneous coronary intervention (pPCI), which depicted highly thrombotic proximal left anterior descending coronary artery (LAD) and proximal right coronary artery (RCA) subocclusions, both with TIMI III antegrade flow. Given the high thrombotic burden and the presence of normal flow, pPCI was deferred and intravenous IIb/IIIa inhibitor and unfractionated heparin were administered for the following 24 hours. Repeated angiography with coronary intravascular ultrasound assessment demonstrated an atherosclerotic plaque rupture at proximal LAD, so PCI with a drug-eluting stent strongly discouraged and aggressive secondary prevention of ischemic heart disease was initiated. Conclusion: The potential increased risk of acute coronary syndromes with exogenous testosterone administration needs to be put in perspective and the testosterone to improve physical performance should be avoided. 109365 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES IZABELLA SILVA FIGUEIREDO1, PATRICIA VIEIRA DE SA1, CLEICIANE RAMOS CAPEL1, ALINE SOUZA DE OLIVEIRA1, RAFAEL LUÍS FERREIRA SILVA1 (1) HOSPITAL SANTA CASA DE MISERICÓRDIA DE BELO HORIZONTE – HSCM BH Introduction: Ebstein’s abnormality, a rare congenital heart defect caused by malformation in the posterior and septal leaflets of the tricuspid valve, has an incidence of around 1:20,000 births and a prevalence of about 0.5% among patients with congenital heart disease. It causes clear consequences on the right heart, but because the severity of anatomical alteration is variable, the clinical course of the disease is also variable, ranging from intrauterine heart failure to mild manifestations beginning in adulthood. Case Report: Female, 66 years old, history of congenital heart disease, with interatrial communication closure in 2002, atrial block – total ventricular with pacemaker placement in 2012, evolved in early 2022 with dyspnea and edema of the lower limbs of progression of 2 months. He underwent outpatient follow-up with cardiology, diagnosed with Ebstein’s anomaly on the november 2021 echocardiogram, he showed important tricuspid regurgitation and valve alterations suggestive of Ebstein’s anomaly, with indication of surgical correction. Admitted electively on 02/06/2022 at Hospital Santa Casa, she underwent surgery with placement of bioprosthesis in a tricuspid valve on 02/08/2022. In the immediate postoperative period, she developed cardiogenic shock and was extubated after clinical improvement on 02/15/2022. Soon after, he developed fever, leukocytosis, pulmonary congestion. Collected cultures in the catheter tip culture was isolated Staphylococcus capitis, started antibiotic vancomycin. New echocardiogram 25/02/22 that suspected endocarditis or thrombosis in the bioprosthesis. After improvement of the congestive and laboratory condition, transesophageal echocardiogram was performed, seen in the lateral leaflet of the tricuspid thrombus bioprosthesis, discarded endocarditis. Started anticoagulation with xarelto and was discharged from the hospital on 03/10/2022 for outpatient control with cardiology and cardiac surgery. Conclusion: Despite being considered the longest natural evolution of all, often exceeding until the third or fourth decade of life, acquired phenomena such as volume overload and ventricular dysfunction right affect this trajectory to the point of increasing the operative risk and morbidity in the postoperative period with the advent of arrhythmias of difficult control, in addition to the greater deterioration of ventricular function. 109370 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES LUCAS CAUNETO SILVEIRA1, Rômulo Teixeira Vidal1, Flávio Visentin Pecci Maddalena2, Mariana Santos Gomes de Souza3, Marselha Marques Barral1 (1) Hospital e Maternidade Therezinha de Jesus – HMTJ; (2) Universidade Federal do Rio Janeiro – UFRJ/campus Macaé; (3) Faculdade de Ciências Médicas e da Saúde de Juiz de Fora – SUPREMA Introduction: Nonbacterial thrombotic or marantic endocarditis (NBTE) is a rare condition of non-infectious lesions of the heart valves (mainly aortic and mitral) due to platelet deposition and hypercoagulable state, associated with neoplasms. Epidemiological data show higher prevalence in people with cancer compared to the general population (1.25% versus 0.2%, respectively), most cases are diagnosed in autopsies, with rates ranging from 0.9% to 1.6% and when compared to malignancy, it occurs more in patients with adenocarcinoma. In addition, it affects all age groups, especially in the fourth to eighth decade of life, with no predilection for sex. Overall, treatment is based on systemic anticoagulation and the surgical indications are the same as for infective endocarditis. Case Description: 49 years old female patient was hospitalized due to ischemic stroke. Transthoracic echocardiogram (TTE) showed a mobile filamentary structure adhered to the ventricular face of the aortic valve, along with a moderate aortic regurgitation. The intracranial arterial computed tomography (CT) angiography demonstrated left frontoinsular hypodensity related to ischemic vascular event without further alterations; CT scan of the abdomen and pelvis detected voluminous ascites, multiple soft tissue peritoneal formations, enhanced by contrast, sparse throughout the abdominal cavity and suggesting peritoneal implant. Furthermore, liver hypocaptant images of irregular contours was showed, mainly in segment VII, measuring 2.2 × 1.5 cm; enlarged uterus with heterogeneous density and oval formations suggesting fibroids, large solid-cystic expansive formation in the pelvis, of well-defined edges, with the solid portions enhanced by contrast measuring about 11.0 × 11.8 × 9.2 cm diagnosed ovarian neoplasm. Carotid doppler USG and thoracic aorta CT angiography was normal. Peritoneal biopsy has shown clear cell carcinoma with omentum, peritoneum and pelvis implant; patient status worsened with septic shock and respiratory failure, requiring orotracheal intubation, vasoactive drugs and intensive care unit support. After ten days, brain death was diagnosed. Conclusion: NBTE is a rare disease with high mortality, finding in advanced stages of cancer. Treatment consists in anticoagulation. 109545 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES FERNANDA MARIA FRANCO CASTRO1, Ludmila Stephanie Julio Machado1, Mariana Fonseca Bittencourt1, Thiago Pinheiro Junqueira1, Eduardo Belisario Falchetto1 (1) HOSPITAL FELÍCIO ROCHO – HFR Constrictive pericarditis is a condition associated with the development of constrictive heart failure. It results from a thickening of the pericardium, with restriction of diastolic filling of the heart and consequent decrease in cardiac output. The cause is often unknown, although it may be subsequent to any illness that causes acute pericarditis. Symptoms are related to heart failure, and may also present changes such as severe cachexia, peripheral edema, ascites, pulsatile hepatomegaly associated with congestive liver disease and pleural effusion. Physical examination findings such as pulsus paradoxus, Kussmaul’s sign and pericardial knock may also be presente, but are not pathognomonic of this condition. We present the case of a 56-year-old male patient, melanoderm, hospitalized with a previous diagnosis of liver cirrhosis of undefined etiology, undergoing propaedeutic for liver transplantation. He was under irregular follow-up with a cardiologist, had a previous diagnosis of atrial fibrillation and heart failure, with a report of dyspnea that started 6 months ago, getting worse in the last 4 months associated with increase in ascites. He reported a history of tuberculosis associated with acute pericarditis for about 20 years ago. He performed a transthoracic echocardiogram whose image revealed a pericardium with increased echogenicity, mainly visceral, compatible with significant thickening, suggesting calcified constrictive pericarditis. Patient underwent surgical pericardiectomy with progressive improvement of heart and liver condition. Currently, there is no more indication for liver transplantation. Constrictive pericarditis is associated with elevated hepatic vein pressures, making patients with constrictive pericarditis prone to developing hepatic necrosis and cirrhosis. The clinical features are relatively nonspecific, making it easy to be confused with primary liver cirrhosis. Thus, a high index of suspicion for the diagnosis must be maintained. Some findings on physical examination may generate important clues (elevated jugular venous pressure, Kussmaul’s sign, pericardial knock, and pericardial calcification on chest X-ray). A correct diagnosis is essential, since pericardiectomy is curative if performed early. 109417 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ANDREY ALVES DE FARIA SILVA1, Henrique Nunes Pereira Oliva2, Mariana Brandão Soares Sousa1, Isabela Oliveira Oliva3, Rita de Cássia Oliveira Araújo3 (1) Universidade Federal dos Vales do Jequitinhonha e Mucurí; (2) Yale University School of Medicine and Unimontes PPGCS; (3) UNIFIPMoc Introduction: ALCAPA Syndrome, also called Bland-Altman-Garland syndrome, is a rare congenital disease characterized by the anomalous origin of the left coronary artery from the Pulmonary Artery (AP), whose probable cause of occurrence is an embryonic alteration that affects cells of the cardiac neural crest during the early stage of embryogenesis. The present article aims to describe the rare case of anomalous origin of the left coronary artery from the pulmonary artery. Case Report: Patient D. P. A, 35 years old, male, electrical engineer, comes to a regularly scheduled review. Electrocardiogram, showed Left bundle branch block. Echocardiogram were performed, and showed akinesia and hypokinesia in lower wall. It was decided to perform a computed angiotomography of the coronary arteries, which confirmed the diagnosis of ALCAPA Syndrome. (Figure 2. computerized tomography angiogram). The patient was submitted myocardial revascularization surgery, using a branch of the mammary artery to connect to the anterior descending coronary artery. In the postoperative period, the patient presented chest pain and dry cough, after 2–3 days he developed bilateral massive pulmonary thromboembolism and was hospitalized for 7 days. Pradaxa was prescribed twice a day. Conclusion: It is concluded that the ALCAPA syndrome in male adults is rare and is not well described in the literature. With the surgical correction employed, the patient evolved to improve his clinical condition. Therefore, knowledge of this condition is extremely important, since the early diagnosis and conduct provide a reduction in the patient’s morbidity and mortality. REFERENCES 1. Angeline P. Revisando a Síndrome de ALCAPA dos Tipos Infantil e Adulto: as Diferenças Estão nos Detalhes. Rev Bras Cardiol Invas. 2007; 15(4). 109416 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUAN GABRIEL PAESE1, Antônio Dejair Acosta Pazzini1, Sergio Antônio López1, Rômulo de Lima Moreno1, Rodney de Oliveira1 (1) Sociedade Hospitalar Angelina Caron HAC Introduction: The incidence of acute myocardial infarction (AMI) in pregnant women is low. Spontaneous coronary artery dissection is responsible for approximately 40% of AMI. Maternal mortality can reach 7%. It usually occurs in the third trimester or the immediate postpartum period. There is usually no benefit of planned cesarean delivery. The treatment is controversial and studies are still insufficient to draw definitive conclusions. Case report: Female patient, 34 years old, gestational age (GA) of 20 weeks, housewife, without comorbidities. Family history with smoker father with ischemic stroke at age 50. She was admitted to the emergency room due to chest pain, sweating and dyspnea. The electrocardiogram demonstrates sinus rhythm, subepicardial ischemia in the anteroseptal wall. Ultrasensitive troponin positive, in ascending curve. The transthoracic echocardiogram presented hypocontractility of the anterior wall, left ventricular (LV) ejection fraction of 46% and reduced global longitudinal strain (–13%). Cardiac catheterization revealed a 95% ostial lesion of the anterior descending coronary artery (AD), with thrombi throughout its extension, LV with anteroapical hypokinesia. She underwent primary angioplasty of AD with Orsiro drug-eluting stent 3.5 × 22 mm and was discharged with therapy for atherosclerotic disease (CAD). The classic presentation of AMI due to CAD is more severe in pregnant women. Unlike the high-risk population, the prevalence of atherosclerotic factors in this type of population is low. The ESC 2018 update guides the use of dual antiplatelet therapy with AAS and Clopidogrel, beta-blockers and nitrates, contraindicating the use of statins, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. It is recommended that delivery occur at least 2 weeks after the AMI, preferably vaginally. In case of a new pregnancy, it is recommended to wait a period of 12 months. Confirmed AMI in the pregnant woman, there is an indication for coronary angiography, using isosmolar contrast and protective measures. The definition of the use of balloon or stent-eluting is important. The risk of angiography is relatively small compared to its benefit for birth planning. Conclusion: The decision about the treatment to be performed is multifactorial taking into account the GA and the assessment of risks and benefits against the aforementioned drugs. The case had an good maternal evolution with hospital discharge without complications. 109422 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FRANCIELE LEIMANN1, Luiz Cláudio Danzmann2, Marciane Maria Rover1, Simone Louise Savaris1 (1) Instituto de Cardiologia de Porto Alegre; (2) Universidade Luterana do Brasil Background: Heart failure (HF) is a serious health problem with high morbidity and mortality. Among the several etiologies of HF, hypocalcemia is rare, however, it is potentially fatal. Case Reports: We described three cases of female patients aged 51, 58 and 38 years, respectively, with HF secondary to hypocalcemia. Calcium deficiency occurred due to idiopathic hypoparathyroidism in one of them and hypoparathyroidism secondary to total thyroidectomy in the other two. The patients presented with signs and symptoms of HF, besides suggestive clinic of hypocalcemia, with hypoesthesia and paresthesia of extremities. In etiological research findings, serum calcium levels below 5 mg/dL were found. In two cases, there was a prolongation of the QT interval to the electrocardiogram. Furthermore, it was identified a reduction in the ejection fraction of the left ventricle (LVEF) on the echocardiogram in the three cases – at the first case with 30%, second with 19% and third with 28%. After treatment for HF associated with calcium replacement, all the patients presented an improvement of the LVEF (50%, 46%, and 62%, respectively), as well as clinical and functional status. Conclusion: The cases here presented to illustrate this situation, highlighting the risk of hypocalcemia after total thyroidectomy procedure, and also demonstrate improved function systolic and clinical symptoms with correction of hypocalcemia associated with standard therapy for heart failure. 110136 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT PLÍNIO JOSÉ WHITAKER WOLF1, João Manoel Rossi Neto1, Marco Aurelio Finger1, Carolina Casadei dos Santos1, Victor Bemfica de Mello Mattos1 (1) Instituto Dante Pazzanese de Cardiologia Background: Long COVID is a condition characterized by long-term consequences persisting or appearing after the typical convalescence period of COVID-19. It may last several months but the duration is still matter of observation. The symptoms and the clinical manifestations are clinically heterogeneous including heart failure (HF) episodes. We describe a case series of 4 patients who had long COVID and evolved to heart transplantation (Tx) indication from January/2020 to February/2022. Description: Mean age was 38 ± 9.5 years, 75% were male and 50% were Caucasian. The whole sample was admitted in NYHA IV functional class. None of the patients had more than one previous comorbidity. All had positive troponin, NT-ProBNP (20234 ± 13373) and c-reactive protein (14,7 ± 13,1). Mean time from COVID-19 infection to admission date was 150 ± 113 days and mean time from admission date to outcome was 112 ± 62,1 days. Mean Ejection Fraction (EF) was 17 ± 3,7% and 75% used Intra-aortic balloon (IAB). Two patients underwent Tx (all still alive), one died prior to Tx and one is still in the waiting list. 75% of patients had thrombotic complications awaiting Tx and all transplant patients had treated cellular rejection and Cytomegalovirus infection (table). Conclusion: HF secondary to COVID-19 due to Long COVID can progress to refractoriness to clinical treatment and Tx is the therapy of choice, showing success, despite high mortality during waiting list. 109433 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES NATÁLIA GOMES CANDIAGO1, Luiza Maria Costi Menta1, Rafael Massuti1, Tais Regina Bisol1, Bibiana Guimarães Maggi1 (1) Hospital Geral de Caxias do Sul (HGCS) Introduction: Pericarditis is an usually mild disease with multiple possible etiological factors, including inflammatory diseases, immune responses after heart damages or viral infections, or several other chronic health conditions. Most of these cases, especially viral cases, remain undefined, as idiopathic. Case description: A 62-year-old male presented intermittent symptoms of asthenia, weight loss, thoracic pain, dyspnea, lower limb edema, vespertine fever, night sweats, and syncope on efforts. All symptoms started markedly after an infection of COVID-19, one month earlier. Patient had a familial history of leukemia, multiple sclerosis, and heart failure. The assistant physician team began a thorough outpatient investigation involving exams in search for rheumatic, collagen, neoplastic and infectious diseases. Chest tomography scans demonstrated parenchymal condensation, atelectatic striae, pleural effusion and pericardial effusion. An echocardiography demonstrated valve thickening and elicited suspicion of constrictive pericarditis. The patient evolved within months with recurrent symptoms and worst dyspnea, and so was admitted to the local hospital. A chest angiography demonstrated pulmonary thromboembolism and persistent pericardial effusion, and the patient was transferred to our hospital to assess the need for a pericardiocentesis. At our service, the diagnostic hypotheses of pericardial tuberculosis, other neoplasms and amyloidosis were raised, since the patient persisted with vespertine fever, tachycardia and night sweats during hospitalization. A new echocardiogram did not found constrictive pericarditis, with discrete improvement of findings. Patient evolved with empyema, and a cardiac magnetic resonance imaging found thickened pericardium, pericardial effusion exudate, and protein accumulation. A diagnostic videothoracoscopy was performed, with biopsy of the pericardial tissue and chest drainage. Pericardial biopsy was positive for tuberculosis, and suggestive of both chronic and acute pleuritis. Treatment with RHZE was instituted, and symptoms improved. After removal of chest drains, the patient was discharged, asymptomatic. Conclusion: Not all pericarditis are idiopathic, not even after viral infections. Tuberculosis reactivation after coronavirus infection have been frequent, and physicians must always elicit such diagnosis in light of compatible clinical histories, especially in countries of endemic tuberculosis such as Brazil. 109449 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARINA SOUTO DA CUNHA BRENDEL BRAGA1, Anabel Lima Vieira1, Amanda Valério Galindo1, Ândrea Virgínia Ferreira Chaves1, Dayse de Sena Moreira Alves1 (1) Hospital Agamenon Magalhães Introduction: Turner syndrome (TS) is one of the most common chromosomal abnormalities, caused by the partial or total loss of the X chromosome. Among the alterations, cardiovascular disease is more prevalent, and may be congenital or acquired. Mortality rate in young women increase due to associated malformations. In the literature, coronary ectasia associated with thrombosis is not well established. Case report: F.S.G., 44 years old, female, diagnosed with Turner Syndrome and hypothyroidism, without other comorbidities, sought a cardiologic emergency due to typical chest pain, without other symptoms, 12 hours from symptom onset. She denied previous episodes. Electrocardiogram (EKG) on admission showed no acute ischemic changes. Chest radiography without abnormalities. Serum troponin with positive curve (479 –> 1746; RV: 14). Cardiac catheterization was performed with evidence of diffuse coronary ectasia and thrombus in the middle to distal third of the Circumflex Artery. Transthoracic echocardiography (TTE) showed an ejection fraction of 47% with concentric left ventricular remodeling and segmental contractile deficit (basal and medial infero-lateral hypokinesia). It was decided to start full anticoagulation with a schedule of repeat the cardiac catheterization in 07 days, which kept the same pattern. After the result, acetylsalicylic acid and rivaroxaban were started for further reassessment in 01 year. Additional evaluation of atherosclerotic disease showed no changes – lipidogram within the normal range, carotid Doppler ultrasound without plaques, bone densitometry with android/gynoid ratio indicating low cardiovascular risk. Still, chest tomography showed normal aorta. Conclusion: The association of TS with cardiovascular changes, from bicuspid aortic valve and aortic aneurysm to stroke and acute myocardial infarction, is well established in the literature. Coronary ectasia associated with thrombosis was not described as a cause of acute coronary syndrome, as occurred in the case described. This complication corroborates the need for improved cardiological knowlegde of the syndrome, aiming at proper condution and treatment. 109488 Modality: E-Poster Young Researcher – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MATHEUS LIMA LULA GUIMARÃES1, Ananda Ribeiro Fretes1, Paula Maria Pinheiro Miranda1, Letícia Aparecida Braga da Silva1, José Sudário Cardoso Neto1 (1) Universidade Federal do Mato Grosso Introduction: Tuberculous pericarditis is an important complication of tuberculosis (TB) in developing countries. It affects up to 4% of cases of pulmonary TB and may evolve with fibrosis and calcifications causing chronic constrictive pericarditis. Case report: 58-year-old patient, male, alcoholic, smoker, under follow-up with hepatology due to alcoholic liver cirrhosis. Pathological history: untreated Atrial Fibrillation and treated pulmonary TB. He frequently complained of dyspnea on exertion, orthopnea, lower-extremity edema, ascites, occasional stabbing pain in the left hemithorax, fever, mental confusion, abdominal pain and nausea. Management of decompensated liver cirrhosis was performed, but with progressive worsening of ascites and orthopnea. Therapeutic paracentesis was performed on an outpatient basis, followed by lipothymia after the procedure, therefore, he was hospitalized for clinical compensation. On admission, he was hypotensive, tachycardic, irregular heart rhythm with hypophonetic heart sounds with splitting of the second heart sound, distended jugular veins, right-sided pleural effusion, ascites and lower-extremity edema. Echocardiogram showed preserved systolic function, enlarged hypocontractile right ventricle, significant biatrial enlargement, moderate mitral and tricuspid regurgitation, significant inferior vena cava ectasia, without respiratory variation, and hyperrefringent posterior pericardium. Chest tomography with cardiomegaly with diffuse calcifications in the pericardium, appearing to compress the ventricles and nonspecific mediastinal lymph nodes. Therefore, a diagnosis of constrictive pericarditis was made with echocardiographic criteria and clinical manifestations and of probable tuberculous etiology due to diagnosed pulmonary TB. Pericardiectomy was performed due to refractoriness to the proposed treatments (antituberculous and diuretic therapy). The patient evolved with significant improvement, with dyspnea only on great exertion, resolution of ascites and lower limb edema in subsequent consultations. Conclusion: There is a certain diagnostic difficulty in patients with liver disease due to the clinical similarity, but due to the high prevalence of tuberculous pericarditis and mortality if left untreated, we should consider such a diagnosis in patients with suggestive epidemiology. However, in a world scenario, tuberculous pericarditis is still among the often under-recognized and neglected cardiovascular diseases. 109486 Modality: E-Poster Young Researcher – Case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION JOÃO ANTÔNIO DA SILVA NETO1, João Antônio da Silva Neto1, Patrícia Alves de Oliveira1, Luciana Sacilotto1, Walace Magalhães Barbosa2 (1) Instituto do Coração INCOR; (2) Instituto Dante Pazzanese de Cardiologia IDPC Introduction: Sinus bradycardia (SB) at rest is considered normal in athletes and is determined by intrinsic heart rate (HR) reduction and modulations of the autonomic system. However, the possibility of progressive degeneration in association with genetic mutations that determine rhythm alterations should make the marked and persistent BS to be better investigated. Case report: Marathon runner, 28 years, SB with HR <30 bpm associated with first degree atrioventricular block (AVB) and left ventricular hypertrophy (LVH). Diagnosed with atrial flutter (AF) during a period of high-intensity training, which was treated with ablation. After 16 years of follow-up, he had an ischemic stroke (IS), treated with trombolisys, without neurologic sequelae. After developed episodes of sudden loss of consciousness, which was attributed to seizures resulting from the IS. However, the SB with pauses persisted, despite the improvement in the (LVH) pattern. HOLTER showed HR 36-48-80 bpm, BS rhythm, alternating with atrial ectopic rhythm, episodes of type I 2nd degree AVB and pauses of up to 2.7s. TILT-TEST with marked SB, 1st degree AVB and junctional escape, with accentuation of bradycardia and prolonged pauses to orthostasis, without HR response to sensitization with vasodilator, as well as with atropine. Suspecting genetic mutations that cause hereditary primary electrical diseases, a genetic test was collected. However, the patient had sudden death during sleep, before the result, which revealed a mutation of the SCN5A gene. Discussion: SB, prevalent in athletes, may be accompanied by AVB of varying degrees. In this case, in addition to marked bradycardia, the patient had AF. Despite the causal relationship between mutations in the SCN5A gene and the Brugada Syndrome phenotype, there are atypical clinical manifestations that include, in addition to the set of signs and symptoms of Brugada syndrome, AF, sinus node dysfunction, long QT syndrome, dilated cardiomyopathy, and others. Carriers of SCN5A mutations linked to Brugada syndrome or progressive cardiac conduction system degeneration are at increased risk of sudden death. Conclusion: Marked bradycardia in athletes should be investigated and the association with channelopathies leads to phenotypes with aberrant rhythms and progressive degeneration of the cardiac conduction system, which can determine fatal arrhythmias. 109497 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO MIRANDA MINSKI1, RODRIGO BODANESE1, RENAN LEOTTE DE SOUZA1, NADIA MAYER2 (1) Hospital São Lucas – PUCRS; (2) Prefeitura Municipal de Porto Alegre Introduction: Myocarditis is inflammation of the heart muscle resulting from exposure to external antigens or internal triggers, such as autoimmune activation. Due to the coronavirus pandemic and vaccination across the world, cases of vaccine reactions have been reported. Of these, myopericarditis corresponds to approximately 0.1% of complications. As a rule, these cases are related to messenger RNA (mRNA) vaccines. Case Description: A 17-year-old man comes to the hospital with chest pain that started 2 hours ago, with oppression, without irradiation or associated symptoms. Triggered at rest, with inspiration worsening. Previously healthy, without continuous use of medication. Two days before the onset of symptoms, he received the second dose of the COVID-19 mRNA vaccine (Pfizer). The diagnosis of COVID-19 was excluded by RT-PCR. On admission he presented with BP 109/50; HR: 101; SatO2: 98%; RR: 16; BT: 36°C. Cardiac and pulmonary auscultation without significant changes. Absence of signs of congestion. Electrocardiogram (ECG) with sinus rhythm (SR), HR 74, anterior, upper lateral and inferior ST elevation and T wave inversion in the lateral wall. High Sensitivity Cardiac (HSc) Troponin I: 16,752 (RV: 34.2). Given the hypothesis of myopericarditis, he was submitted to Cardiac Magnetic Resonance Imaging (MRI), which showed: extensive late enhancement of a non-ischemic aspect and diffuse mesocardial/subepicardial edema, mainly affecting the apical, medial lateral and basal walls. Pericardial edema, more evident in the middle lateral walls of the LV. EF 44%. Findings consistent with myopericarditis. During hospitalization, he remained asymptomatic, with good clinical evolution, receiving Angiotensin Converting Enzyme Inhibitor, Beta-Blockers and Colchicine. Discharged 5 days after admission, without complaints, with a sharp drop in HSc Troponin (202). Ten days after discharge, new ECG showing RS; HR 62; anterior, superior and inferior lateral T wave inversion. After 42 days ECG of normal pattern. After 90 days of the initial event, a new MRI, which showed: reduction in the extension and thickness of the late enhancement with a non-ischemic aspect in the mesocardium/subepicardium. Virtually complete resolution of myocardial edema and pericardial changes. EF: 50%. Conclusions: The description of adverse effects of the new vaccines is essential to quicly diagnose and manage them in the most appropriate way possible, preventing unfavorable outcomes. 109503 Modality: E-Poster Young Researcher – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RAMON OTT VARGAS1, José Carone Filho1, Schariff Moysés1, Roberto Ramos Barbosa1, Assad Miguel Sassine1 (1) Instituto de Cardiologia do Espírito Santo Introduction: Axenfeld-Rieger syndrome is an autosomal dominant genetic disease. Generally, described phenotypes consist in craniofacial and dental abnormalities, ocular anterior segment dysgenesis as well as congenital heart defects, mostly outflow tract diseases. Atrial and ventricular septal defects, valve stenosis and persistent truncus arteriosus are the most frequent cardiac presentations. The occurrence of premature coronary artery disease on these patients is not frequently documented. Case Description: Thirty year old male, diagnosed with Axenfeld-Rieger syndrome for the past twelve years, refers chest pain and oppression caused by mild efforts and daily activity, progressively worsening to limitations of any physical activities and, lately, angina at rest. Patient is eutrophic, practices physical exercise regularly, non-smoker, no diabetes or dyslipidemia and no family history for premature coronary heart disease. After being examined in clinic, laboratory exams were requested, as well as transthoracic echocardiography (TTE) and treadmill stress test. Lab results show normal cholesterol levels, glucose and triglycerides. Liver and renal functions were also normal. TTE showed no abnormalities, but the stress test was interrupted for excess tiredness and probably anginous chest pain on peak effort and recovery. Patient then presented a strong episode of angina at rest, and headed to the Emergency Room. On that occasion, a coronary angiography was requested, and showed a 90% obstruction on proximal left anterior descending artery (LAD). Percutaneous coronary intervention was performed, and a zotarolimus-eluting stent was uneventfully implanted. Patient performed well after the procedure, was discharged to home and returned to normal physical activities, maintaining follow-up in clinic. Conclusion: The occurrence of obstructive coronary lesions on young, low cardiovascular risk patients is always reason for questioning. In the described case, patient bears a rare syndrome, on which ophthalmologic and cardiac outflow tract malformations are frequently described. We hypothesize that the coronary lesion relates to this same syndrome. Further studies are necessary to evaluate this correlation. 109525 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FLÁVIO ROSA VIEIRA1, Jordana Pires Mendonça1, Tannas Jatene1, Rogério Lobo de Andrade Las Casas1, Vinicius Daher Vaz1 (1) Hospital do Coração Anis Rassi Bioprosthetic disfunction is a common late complication in patients submitted to surgical aortic valve replacement. Transcatheter Aortic Valve Implantation (TAVI) valve-in-valve is an option to treat this problem according to most recent guidelines. Although rare, coronary occlusion is a potentially fatal complication after TAVI. We present a case in which we used a technique denominated BASILICA (bioprosthetic or native aortic scallop intentional laceration to prevent iatrogenic coronary artery obstruction) to prevent this adverse event. A male patient, 75 years old, hipertensive, former smoker, with ongoing dyspnea NYHA III. Physical exam revealed sistolic and diastolic murmur (3/6+) in the aortic area. Past medical history of coronary artery bypass graft in 2007, surgical bioprosthetic aortic valve replacement in 2009, percutaneous coronary intervention with stent at first diagonal branch in 2012 and 2021 (in-stent restenosis). ECG shows sinus rhythm, first degree atrioventricular block, right bundle branch block and left anterior fascicular block. Transthoracic echocardiogram identified a severe regurgitation and stenosis on the bioprosthetic aortic valve (mean gradient 41 mmHg and peak velocity 4,5 m/s). Although STS Score was 6,14% and euroSCORE II 3,45%, the patient refused his third surgery. Angio CT showed high risk for coronary occlusion (Valsalva sinus mean diameter 24 mm, left coronary height 5,5 mm and VTC 3,5 mm). Heart Team opted for TAVI with the described BASILICA technique. The intraoperatory transesofagical echocardiogram (TEE) showed mean gradient 37 mmHg and severe aortic regurgitation due to rupture in the right coronary sinus leaflet. We used a 0.014 guide wire connected to an electrocautery to perforate and tear the left leaflet. Later after pre dilatation, we finally implanted a Sapien 3 (Edwards Lifescience) # 20 bioprothesis followed by post dilation at higher pressure with a #22 non compliant balloon. The TEE showed mean gradient of 12 mmHg and no paravalvular or central leak at the final procedure. Despite the risk, the left coronary occlusion did not occur. Patient remained for 2 days at the ICU due to an increase in the PR interval, which was sustained, and was discharged 3 days after the procedure. We concluded that BASILICA technique is a feasible alternative to the patients candidates to TAVI in Valve-in-Valve with high risk for coronary occlusion. 109538 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY PEDRO GUIMARÃES SILVA1, GUILHERME RAPOSO DE MEDEIROS1, MARILIA TAILY SOLIANI1, RONEY ORISMAR SAMPAIO1 (1) INSTITUTO DO CORAÇÃO DO HCFMUSP Introduction: Pectus exacavatum is one of the most iconic features of Marfan syndrome. In patients who have not undergone thoracic correction during childhood, surgical treatment of valvular heart disease faces an even greater challenge. The following case report presents a patient with significant mitral regurgitation due to previous infectious endocarditis who is submitted to valve replacement along with the correction of his severe acute pectus excavatum. Case report: An 18 year-old male diagnosed with Marfan syndrome at 10 years old presented to the emergency room (ER) in February 2022 with worsening dyspnea and fatigue. He had a severe acute pectus excavatum (Haller index: 8.8), arachnodactyly, and and a positive family history of Marfan syndrome (both his father and brother). Concerning the past medical history, in 2018 he suffered from an infectious endocarditis (IE), treated only with antibiotics at the time, which caused a rupture of the tendinous cords of the mitral valve and a prolapse of both its cusps, along with the formation of a significant vegetation on the atrial aspect of the mitral anterior leaflet. He continued his outpatient treatment for 4 more years, which consisted mainly on the management of the symptoms caused by the the worsening mitral regurgitation After the admission to the ER, a transesophageal echocardiography was performed, showing a significant mitral vegetation, similar to the one described in 2018, of 21 × 6 mm along with severe mitral regurgitation; a left atrium of 230 ml/m2; and an ejection fraction was 54%. There was no involvement of the aortic valve or aortic sinus. After thorough assessment, the hypothesis of a recurrent IE was abandoned (low C- reactive protein levels, no fever or other stigma of IE, and negative blood cultures) and the present condition was attributed solely to the worsening of mitral regurgitation. Chest computerized tomography presented a severe pectus excavatum with a pronounced mediastinal deviation to the left, and a total atelectasis of the left lower lung lobe Mitral valve replacement was then performed using a biological prosthesis. After closure of the left atrium, the pectus excavatum was then corrected using the Ravitch technique and Marlex mesh, without the Nuss bar (due to prolonged surgical time and detected intraoperative ventricular dysfunction). The patient evolved well during the postoperative period, with full recovery and relief of symptoms. 109570 Modality: E-Poster Young Researcher – Case Report Category: NURSING EVELYN GOMES OSÓRIO1, Kalliza Kary Rodrigues da Costa2 (1) Hospital Copa Star; (2) Hospital Copa Star Introduction: Heart transplantation (HT) is considered a therapeutic option for end-stage heart failure (HF) patients¹. The occurrence of rejection is one of the post-transplant complications. Although new immunosuppressive regimens have decreased the incidence of rejection, it still represents one of the key clinical concerns. It is estimated that 25% to 80% of recipients will experience at least one episode of rejection, requiring treatment during the first year after implantation. Rejection is the cause of approximately 10% of deaths in the first year after surgery². The nurse’s role in the post-transplant follow-up and the control of adverse events is of paramount importance to detect new complications early on. The early identification of rejection will impact the prognosis and the maintenance therapy decision for the transplanted patient3. Description of the Case: Male, 39 years old, diagnosed with dilated cardiomyopathy without defined etiology about 10 years. Last echocardiogram with ejection fraction (17%), listed for HT, he searched emergency room due to progression of symptoms. He was admitted with acute cardiac insufficiency, requiring inotropic and transfer to HT reference unit where was tested for SARSCOV-2 with a positive diagnosis resulting in removal from the transplant line for 30 days. He returned in line priority subsequently, was transplanted on the 34th day. On the 10th postoperative day, the patient showed rejection grade 3R (severe rejection). The patient evolved with significant clinical improvement but presented infection by cytomegalovirus (CMV), one of the main causes of morbidity and mortality in HT in cases of recurrent or difficult to control infection4. After hospital discharge, the patient continued to be followed up by the multi-professional team, made possible through telemonitoring, the nurse’s follow-up to detect early signs of aggravation, monitoring of the serum level in laboratory results to evaluate treatment accession. The treatment was based on oral corticosteroid therapy associated with immunosuppressive therapy. After 3 months of follow-up, the results of the biopsy showed an evolutionary improvement with results of 3R, 2R, and 1R. Conclusions: The outcome of the case was favorable. The advances in telemonitoring and the nurse’s role have shown to be important management and follow-up tools for transplant patients at home, seeking early detection of signs of aggravation and monitoring of vascular graft disease. 109597 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY SÂMIA BADWAN MUSTAFÁ1, Sâmia Badwan Mustafá1, Bácila Lunks Badwan Musa3, Yasmine Badwan Mustafá2 (1) Hospital Universitário de Santa Maria; (2) Grupo Hospitalar Conceição; (3) Clínica Médica Bácila Badwan Introduction: Myocardial bridge is a congenital anomaly, usually in the anterior descending artery (AD). It is one of the main differential diagnoses of coronary artery disease, especially when there is a low probability of atherosclerotic disease. It may manifest as angina and, rarely, acute myocardial infarction or sudden death. Case description: B.E, 39, male, military, typical anginal pain for 1 year. No comorbidities, performs physical activities. Physical examination normal. ECG: sinus rhythm, 70 bpm, left atrial and left ventricular overload. Chest X-ray normal. Echocardiogram normal. Prescribed Rosuvastatin 10 mg/day and Ramipril 5 mg. Ergometric test: ST depression. Coronary angiography: large intramyocardial bridge in AD. Clinical therapy was optimized: Nebivolol 10 mg/day, Amlodipine 10 mg, ASA 100 mg/day, suspension of Ramipril. After a year it returns with pain. Myocardial scintigraphy: no ischemic areas. Surgical evaluation requested. Conclusions: A diagnosis of a congenital condition was made based on a typical angiographic finding – systolic reduction in the diameter of the epicardial coronary artery. Although congenital, symptoms are observed later, since, due to the growth of the heart, there is an increase in the systolic tension of the myocardial wall and a reduction in coronary flow due to atherosclerotic processes. Optimized clinical treatment with beta-blockers was started, in order to reduce heart rate, increase diastolic time and reduce systolic compression of the vessel and, thus, improve anginal symptoms; antiplatelet drug to reduce the risk of cardiac events. Nitrates were avoided, as they worsen coronary artery systolic narrowing, which can worsen symptoms. Despite optimized clinical treatment, symptoms persisted, and the possibility of invasive treatment with stent implantation was discussed. However, studies have shown stent stenosis in almost half of patients undergoing angioplasty and need for reintervention in more than one third. Therefore, clinical treatment is the first line, and invasive treatment should only be considered if it is refractory to optimized clinical treatment. 109614 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY AMARILDO BORGES DA SILVA OLIVEIRA1, Allan Oliveira Macedo1, Ana Caroline Prado Pereira2, Jéssica Danicki Prado Fernandes1 (1) Hospital Regional de Ceilândia – Secretaria Estadual de Saúde do Distrito Federal (HRC – SES/DF)); (2) Instituto Master de Ensino Presidente Antônio Carlos – IMEPAC Itumbiara/GO Introduction: Ebstein’s Anomaly (EA) is a rare congenital heart disease, characterized by abnormalities of the tricuspid valve (TV) and the right ventricle (RV), with clinical presentation varying from neonatal period to adulthood. Some echocardiographic and anatomical features are essential for diagnosis: apical displacement of the insertion of the TV septal leaflet (≥8 mm/m2), redundant elongated anterior leaflet of the TV, and RV thinning, enlargement, and dysfunction. Other comorbidities associated with TV regurgitation and right heart chambers enlargement may be misdiagnosed as EA, such as tricuspid valve dysplasia (TVD), presented in this paper, which aims to correctly identify EA and report a TVD case. Case description: MSL, female, 53 years old, dyslipidemic and diabetic, was admitted to the Emergency Unit with headache, for 4 days, associated with hypotension, sweating, general malaise and diarrhea. Later, she was transferred to a secondary hospital with signs and symptoms suggestive of B-profile decompensated heart failure. During hospitalization, an echocardiography (ECHO) was perfomed, which showed TV with thickened leaflets, apical displacement of the septal leaflet, compatible with EA. Subsequently, a new ECHO was performed with a team specialized in congenital heart diseases, which revealed a TV with redundant and dysplastic leaflets, septal leaflet with normal implantation, however, with a septum attachment with an extension of 7.5 mm/m2, not meeting the criteria for EA but for TVD. Surgical correction with valve replacement was indicated. Conclusions: EA is a complex congenital cardiac malformation with a great difference in anatomical and clinical presentations, and may be suspected in cyanotic patients, with tricuspid regurgitation, heart failure and arrhythmias, which are possible manifestations in TVD. Thus, TVD may mimic EA and its management needs to be individualized. Knowing the anatomical and hemodynamic variations, as well as the associated comorbidities, is essential for the correct recognition of cardiac involvement and management of the case, thus optimizing the patient’s longevity. 109625 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARCIO RAFAEL MONTICIELO1, Stefano Ramos Farias Leite1, Paula Thais Birk1, Andrieli Cristina de Oliveira1, Leonardo Gheller Zanatta1 (1) Hospital de Caridade de Ijuí – HCI Acute Myocardial Infarction (AMI) is among the leading causes of death in the world. It occurs due to obstruction of coronary vessels, leading to perfusion dysfunction of cardiac areas and consequent myocardial injury, with probable failure if not treated in time. Case: Male, white, 35 year, reporting sudden-onset chest pain, approximately 30 minutes before admission, at rest, radiating to the left upper limb, malaise and sweating. He had previous systemic arterial hypertension and reported COVID-19 disease six months ago. He had been vaccinated for COVID-19 eight days before admission (OXFORD). No alcoholism or smoking. On physical examination, he had stable vital signs, cardiac and respiratory auscultation without alterations and well-perfused extremities. The electrocardiogram (ECG) showed ST elevation in leads D2, D3 and AVF, showing inferior wall AMI. Coronary angiography showed a distal thrombotic lesion in the right coronary artery (TIMI 3). At catheterization time, he was without chest pain and with preserved coronary flow (TiMI 3). We defined for pharmacological treatment with Tirofiban, Acetylsalicylic Acid, Clopidogrel and low molecular weight heparin in therapeutic dose. Some hours after, he returned with chest pain and ECG shows inferior wall AMI. A new catheterization was done and observed a significant reduction of previous thrombotic lesion but now distal occlusion of the posterior ventricular branch was verified. Once again it was decided to maintain conservative treatment due to the risk/benefit relationship. He was discharged from the hospital after complete relief of pain and decrease in the enzyme curve. He was oriented to maintaining double platelet aggregation treatment for a period of 1 year and we requested coronary CT angiography for the return. Conclusion: Studies have shown that patients with covid-19 as well as with vaccine for covid 19 are predisposed to thromboembolic events, such as peripheral and pulmonary thromboembolism, stroke, AMI and acute lower limb ischemia. The fact that these patients had no risk factors for CAD and did not have coronary atherosclerotic plaques raises the possibility that the thrombotic event is associated with the hypercoagulable state of the covid-19/vaccine infection. More aggressive pharmacological therapy, such as fibrinolytics, glycoprotein IIb/IIIa inhibitor and prolonged use of anticoagulants, in these cases, in view of the high thrombotic load, replacing angioplasty, should be considered. 109659 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY BRENO DE ALENCAR ANTÃO1, Raysa Ramos Santos Negromonte1, Anabel Lima Vieira1, Maria de Fátima Nunes de Oliveira1, Maria Dolores da Trindade Henriques Assunção1 (1) Hospital Agamenon Magalhães Introduction: A left ventricular pseudoaneurysm (LVP) it is the result of cardiac rupture contained by adherent pericardium or scar tissue. Unlike true aneurysm, it does not contain endocardium or myocardium. Most of the LVP occurs after transmural myocardial infarction but also occurs after cardiac surgery, trauma, or infection. We describe a case of LVP after blunt chest trauma incidentally occurring with valvular heart failure. Case report: J.S.S., a 60-year-old male was admitted with dyspnea on mild exertion, orthopnea, paroxysmal nocturnal dyspnea, and lower limb edema. He had a mitral bioprosthesis (MB) since 2013 due to a blunt chest trauma caused by motor vehicle accident that led to acute mitral regurgitation (MR) as result of ruptured chordae tendineae. Initial echocardiogram (ECHO) revealed preserved systolic function with moderate tricuspid regurgitation, moderate pulmonary arterial hypertension and dysfunctional mitral bioprosthesis. Pre-surgical catheterization revealed anterior descending artery, 2nd diagonal branch and circumflex artery with significant luminal reduction during systole, without signs of coronary atheromatosis. Coronary angiotomography was requested evidenced a pseudoaneurysm in the basal segment of the anterior wall of the left ventricle with slight extrinsic circumflex artery compression. Patient underwent MB replacement and the LVP, which was below the aortic valve plane, measuring 1.3 cm in diameter, was repaired with implantation of a bovine pericardium patch. There was a postoperative bleeding successfully managed. New ECHO revealed normal implantation and fixation of BM, normal LV systolic function with moderate right chamber insufficiency and without evidence of a pseudoaneurysm. Patient had clinical improvement after procedures and remains in outpatient follow-up. Discussion: According to the literature, up to 80% of blunt cardiac injuries are caused by motor vehicle collisions (MVC) widely ranging from LVP to the ventricular free wall rupture. Untreated pseudoaneurysms have rupture risk (30% to 45%) and high mortality rate (almost 50%) with clinical treatment. Patients with severe MR have a higher risk which requires mitral valve replacement, as in the case described. Correct diagnosis and appropriate surgical management lead to reduced mortality and better prognosis. 109722 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY KELSON KEMUEL CONFESSOR DE SOUSA1, Kelton Dantas Pereira1, Vinicius Pereira Dantas1, Ana Olivia Dantas2, Fabio Mastrocola1 (1) Onofre Lopes University Hospital; (2) Federal University of Rio Grande do Norte Introduction: Aortic dissection is marked by the delamination of the middle layer of this vessel due to the inflow of blood. According to the Stanford classification, the ascending aorta (type A) or the descending aorta (type B) may be affected. Some studies show that the mortality of patients with type A acute dissection treated surgically is 26%, while that of those not undergoing surgery, due to advanced age or comorbidities, was about 58%. There are no data on mortality in chronic dissections. Case report: A 78-year-old female patient, with obesity, chronic hypertension and frailty syndrome, started with a sudden loss of muscle strength and dysarthria, being diagnosed with ischemic stroke. During investigation of the present clinical scenario, the Transthoracic Echocardiogram showed a Stanford type A dissecting aortic aneurysm measuring 68 mm. An angiotomography of the thoracoabdominal aorta showed a fusiform aneurysm of the ascending aorta, measuring 7.3 cm with signs of chronic aortic dissection, extending from the aortic valve annulus to the infra-renal abdominal segment. The patient was then also submitted to coronary evaluation, which showed multivessel coronary artery disease. In view of the chronic dissection of the ascending aorta, which treatment currently established is to replace the affected aortic segment, and considering the multiple comorbidities peculiar to this patient at high surgical risk, the clinical and surgical team considered that the surgery would bring more risks than the potential benefit. Thus, the patient was discharged with adequate blood pressure and heart rate control. Conclusion: Acute type A aortic dissection is the cardiovascular lesion with the highest mortality if not treated immediately, reaching a rate of 80% in about 14 days, with surgical treatment being one of the only possible approaches for both acute and chronic cases. There are complex and challenging cases such as the one described above in which the risk versus benefit must be weighed, as the patient has already passed the high mortality phase. Furthermore, the current medical literature is not clear about the mortality estimate in the scenario of chronic aortic dissection, which led us to choose a conservative approach in a patient at high surgical risk. This patient has been under follow-up for more than 6 months, an outcome that could be different if the surgical procedure had been chosen at the initial moment. 109749 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING GABRIELA ASSIS RANGEL DE ABREU1, Lucas Feldman Paz de Lima1, Angelo Antunes Salgado1, Marcos Paulo Lacerda Bernardo1, Joaquim Henrique de Souza Aguiar Coutinho1 (1) Hospital Universitário Pedro Ernesto Introduction: Loeffler’s endomyocardial disease is a rare disease caused by eosinophil infiltration into the endomyocardial. Heart failure characterizes the clinical picture, secondary to the restrictive behavior of the ventricle, triggered by fibrosis and obliteration of the ventricular cavities. The treatment targets the etiology of eosinophilia, which often remains unknown. Case: Male, 62, started outpatient follow-up in hematology for eosinophilia and splenomegaly. The laboratory showed leukocytosis of 36,090/mm3 with eosinophils 21,645/mm3 (60%). An investigation was carried out but without etiological elucidation. A transthoracic echocardiogram (TTE) showed biatrial enlargement, preserved biventricular function, type 2 diastolic dysfunction (DD), and increased thickness of the apical segments of both ventricles, suggestive of infiltrative disease. After one month, he presented ascites and dyspnea requiring hospitalization. A new TTE revealed type 3 DD, right ventricular systolic dysfunction with severe tricuspid regurgitation due to coaptation failure, and worsening of ventricular infiltration, obliterating more than 50% of the cavities. During hospitalization, the patient evolved with low cardiac output refractory to pharmacological therapy without improving eosinophilia despite corticosteroid. He then underwent endocardiectomy and biological tricuspid valve replacement; nevertheless, he came to death one day after surgery. Conclusion: Loeffler’s endomyocardial disease is a subcategory of the hypereosinophilic syndrome, with 2D TTE being the primary imaging method for diagnosis and follow-up. In this case, the lack of etiology, corticosteroid refractoriness, and fast progression of biventricular apical obliteration made the case difficult to handle and a worse prognosis. 109814 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY PEDRO CASTILHOS DE FREITAS CRIVELARO1, Nadine Oliveira Clausell1, Felipe Homem Valle1, Livia Adams Goldraich1, Leonardo Hennig Bridi1 (1) Hospital de Clínicas de Porto Alegre HCPA Background: Transcatheter edge-to-edge mitral valve repair (TEER) has been utilized as a potential therapeutic strategy in selected patients with advanced heart failure. According to a multicenter retrospective registry, TEER may promote clinical improvement and pulmonary hypertension relief as bridge to transplantation or to candidancy. Report: A 57-year-old male with ischemic cardiomyopathy and recently worsened severe functional mitral regurgitation (MR) was admitted with advanced heart failure and listed for transplant. Clinical status worsening, with intravenous inotropic support increases and temporary mechanical circulatory support followed. Hemodynamic assessments showed heart failure progression with fixed severe pulmonary hypertension and remarkably poor cardiac performance. Transplant was then deemed to be of high risk, and the patient was inactivated on the waiting list. TEER was considered as a “bridge to candidancy” and it was carried with the implantation of 2 XTw MitraClip® (Abbott Vascular) devices. Remarkable reduction on both MR severity and left atrial V-wave pressure were achieved. Functional and hemodynamic status dramatically improved and the patient was reactivated on the heart transplant waiting list at 30-day follow-up. Conclusion: TEER may be a feasible option in selected heart transplant candidates to improve severe pulmonary hypertension. 109858 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY JOAO CARLOS MATOS PINTO JUNIOR1, Luiz Felipe M F Alves1, João Luiz Frighetto1, Lisley Riano da Silva Pestana1, Estevão Carvalho de Campos Martins1 (1) Hospital de Força Aérea do Galeão Introduction: Rheumatic valve disease still is a prevalent condition in our country. Even when young, affected patients evolve the need of mitral valve replacement. Subsequent valve dysfunctions, predominantly in mitral valves, generate structural and pathological consequences, primary or secundary. Thromboembolic phenomenons and the increased operative risk in the classical surgical approaches confers a higher risk of morbidity and mortality to those patients. Modern strategies and devices for percutaneous treatment emerge as an option for a better approach. Case description: 78 years old, female, with hypertension and permanent atrial fibrillation. Evolves with worsening of functional class, signs of congestion, requiring hospital admission and intravenous diuretic therapy. This patient have a history of biological mitral valve replacement due to rheumatic valve disease. The first one was in 1968 and the second one was in 2000. She had a subarachnoid hemorrhage event in 2020. Evolves with worsening functional class, with signs of system congestion, requiring the use of intravenous diuretic therapy. After clinical stabilization, a transesophageal echocardiogram was performed, revealing an LA enlargement and a severe degeneration of the biological mitral prothesis with calcified and poorly mobile bases including a eversion of one of these leaflets into the left atrium, causing a severe mitral regurgitation, with a transprosthetic gradient. maximum 21 mmHg and average 9 mmHg, Peak Velocity 2.3 m/s, PHT 179 ms, AEO 1 cm², and presence of thrombus in the left atrium Due high surgical risk, percutaneous treatment was chosen with a VIV – “Valve in Valve” strategy preceded by central nervous system protection with the Sentinel™ device on the brachiouscefalic trunk and the left internal carotid beside Left Atrial Appendage with the watchman™ flex. Conclusion: The treatment of valve degeneration in elderly patients with previous biological valve replacement is still challenging, with the advent of percutaneous treatment (Valve-in-valve), which has brought new perspectives to those patients. The use of devices such as sentinel™ and watchman™ help to prevent one of the main complications, which is embolism to the central nervous system. References: Little SH, Bapat V, Blanke P, Guerrero M, Rajagopal V, Siegel R. Imaging Guidance for Transcatheter Mitral Valve Intervention on Prosthetic Valves, Rings, and Annular Calcification. JACC Cardiovasc Imaging. 20. 109870 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY RAQUEL SILVA BRITO DA LUZ1, Adnaldo da Silveira Maia1, Arturo Adrian Jara1, Janayna Rabelato1, Antonio Flavio Sanchez1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: COVID-19 infection has a wide spectrum of clinical presentations, including cardiovascular, including myocarditis and pericarditis. The prevalence and events associated with this condition still remain under analysis, as well as the repercussion of such complications after the viral infection. Case Description: 74-year-old female patient, former smoker, bilateral carotid stenosis without hemodynamic repercussions, rheumatoid arthritis and diagnosis of viral pneumonia due to COVID-19 in January 2022. She was admitted, in March 2022, to a referral center in cardiovascular diseases, with a clinical picture of chest pain that worsened during inspiration with 2 months of evolution. Laboratory measurements of troponin and D-dimer within normal limits. Admission electrocardiogram suggestive of pericarditis. Transthoracic echocardiogram (TTECT) revealed preserved biventricular function and pericardial effusion with a greater depth of 11.0 mm. Despite the optimized clinical treatment for the pericarditis, the patient evolved with clinical worsening and rapid evolution of the pericardial effusion initially evident, with a 30.0 mm blade and signs of hemodynamic repercussion, requiring urgent drainage. On the 1st postoperative day in the intensive care unit, the patient evolved with severe acute biventricular dysfunction and need for vasoactive drugs, without response to the established clinical measures, progressing to death. Conclusion: The prevalence of underlying pericarditis and pericardial effusion in patients with COVID-19, as well as its clinical significance remains the subject of clinical research. Thus, knowing risk factors and the pathophysiology of cardiovascular complications related to COVID-19 are sine qua non conditions for understanding the clinical presentation, prognosis and therapeutic management. 109879 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY SEPIDEH DARBANDI1, Samuel Congello1, Ali Mardan1 (1) MercyOne North Iowa Medical Center Patient is a 62 year old male with past medical history of tobacco abuse who had presented with abrupt onset of pain and paralysis in his right arm. Of significance, he had suffered a motor vehicle accident two years prior but had refused medical treatment for the diagnosed right clavicle fracture at that time. On physical examination, patient had ischemic discoloration of his right arm and forearm and was noted to have absent pulses in that arm. CT angiogram of the right upper extremity was performed which showed an occlusion at the thoracic outlet with another obstruction down at the bifurcation of the distal brachial artery at about the level of the antecubital fossa. Embolectomy was initially performed. A EV3 Protege self-expanding stent was deployed within the distal subclavian artery and proximal axillary artery. The stent was postdilated with a EV3 EverCross balloon. Post stenting angiogram did not show any significant residual stenosis. There were no immediate post operative complications. To further investigate etiology of patient’s arterial thrombosis, transesophageal echocardiogram was performed that was negative for intracardiac thrombus, however atheromatous plaque lesion was present in aorta and CT angiogram of the chest was recommended for further characterization. CT angiogram of the chest showed new right subclavian artery stent placement with resolution of previous occlusion. Report of CT scan read as such: Stent is patent but buckled proximally with luminal narrowing in the area of buckling. He subsequently underwent examination of the right arm and shoulder under fluoroscopy and showed that the distal segment of the clavicle (due to untreated displaced fracture) impinged on the stent with motion. Interestingly enough, with patient’s arm hyperadducted, there was impingement on the subclavian stent, and patient would actually experience numbness in his right arm. As a result, patient underwent operation by orthopedics for right clavicle open reduction internal fixation, and his symptoms of right arm paresthesia was resolved as his stent remained patent without kinking. Conclusions: The incidence of subclavian artery thrombosis is quite uncommon, although this risk is increased with risk factors such as peripheral vascular disease, obesity, and diabetes mellitus. Stent impingement should be on the differential in a patient that is admitting to motional symptoms that is related to the regional blood supply. 110595 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM THIAGO BURIL FONTES1, Rudyney Azevedo1, Maria Gorett W. Matioli1, Regina Adler1, Andre Luis Mendes Martins1 (1) Hospital Servidor Publico Municipal de São Paulo Background: Acute coronary syndrome (ACS) is an event that happens because of rupture of an atherosclerotic plaque, leading to ischemia of the heart muscle. The Covid-19 pandemic raised questions related to the increase in the number of infarctions, perhaps due to the destabilization of plaques by the inflammatory substrate. Objectives: To make a correlation between the clinical variables of the presentation, complementary exams such as ECG, echocardiogram and catheterization and the concomitant presentation with Covid-19. Methods: We evaluated 62 patients consecutively admitted with the diagnosis of ACS to a public hospital in São Paulo in 2021, during the Covid-19 pandemic. Results: The mean age of the patients was 66.9 years and standard deviation 10,44, 51.6% were men, 66.12% were hypertensive, 33.87% had diabetes, 14.51% had a documented history of coronary artery disease (CAD), 35% with a high risk GRACE score, 6.45% with Covid-19, of which 75% with STEMI. In the ECG, we obtained 11.29% of anterior ischemia, 12.9% of posterior ischemia and 20.96% without alteration. On the echocardiogram, the mean LVEF was 0.57 quantified by the Simpson method, 12.9% of regional change in the anterior wall and 24.19% of the posterior wall. In catheterization, the predominance was involvement of the anterior descending artery with 16.12% and only 3.22% without obstructive CAD. Only one patient with Covid-19 had a normal ECG. Conclusions: In a population treated with the diagnosis of ACS during the Covid-19 pandemic, the prevalence of positive cases was low, however, the incidence of STEMI seems to be high in this population, which denotes greater attention to the care and clinical evolution of these patients. 109893 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY RAQUEL SILVA BRITO DA LUZ1, Adnaldo da Silveira Maia1, Jhonathan Gouveia da Mota1, Matheus Meirelles1, Mario Issa1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Takotsubo Syndrome is a recurrent diagnosis, however, it’s rarely seen post cardiac surgery. The disease usually occurs after physical or emotional stress, most prevalent in post-menopausal women. The clinical features may present similar to acute coronary syndrome, with chest pain and electrocardiogram (ECG) alterations, but with this diagnosis being excluded after the absence of obstructive lesions in coronary angiography and encountering typical echocardiogram findings, such as compensatory apical akinesia and basal compensatory hypercontractility. Case Description: A 56-year-old woman with a history of rheumatoid arthritis, active smoking and percutaneous mitral valvuloplasty in 2007, presents with functional class II dyspnea and transthoracic echocardiogram (TTECO) showing important mitral regurgitation. Patient was submitted to mitral valve replacement surgery with an mechanical prosthesis on March 2022, without complications. In the immediate postoperative care, the patient evolved with cardiogenic shock, requiring vasoactive drugs. The ECG presented with T-wave inversion of precordial leads and TTECO revealed hypercontractility of the basal segments and akinesia of medium-apical segments, with left ventricular ejection (LVEF) of 20%. During hospitalization the patient gradually improved from cardiogenic shock, with normalization of LVEF to 64%. Conclusion: Takotsubo Cardiomyopathy is a reversible condition, characterised by transient ventricular dysfunction, with rare cases described in the context of postoperative care due to valve surgery. The surgical intervention can be considered as a trigger for the development of this syndrome. 109916 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT PEDRO CASTILHOS DE FREITAS CRIVELARO1, Felipe Homem Valle1, Willian Roberto Menegazzo1, Anderson Donelli da Silveira1, Nadine Oliveira Clausell1 (1) Hospital de Clínicas de Porto Alegre HCPA Background: Functional mitral regurgitation (fMR) is associated with worse prognosis in heart failure (HF). Furthermore, fMR severity worsening during exercise has been associated with even worse prognosis in this scenario. Report: A 44-year-old female with post-chemotherapy HF and moderate fMR presented with progressive exercise intolerance. To further elucidate mechanisms of exercise intolerance, combined exercise invasive hemodynamic assessment and exercise echocardiography were carried. The procedure was performed by right brachial venous access with exercise at a cycle ergometer. Hemodynamics were evaluated at rest and during progressive incremental exercise, with combined transthoracic echocardiography at both stages. At exercise in a 20 Watts work-load, fMR worsening and increase in pulmonary artery pressures occurred. These findings suggest a potential role of fMR in exercise intolerance. Conclusion: Exercise pulmonary hypertension is associated with worse prognosis in fMR. Whether transcatetheter mitral valve interventions may alter prognosis in this scenario is yet to be determined. We herein present a case of exercise intolerance that is potentially related to fMR worsening during exercise. Our group is prospectively evaluating the role of combined invasive exercise hemodynamic assessment and exercise echocardiography to elucidate mechanisms of exercise intolerance in fMR. 109927 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES PEDRO GUIMARÃES SILVA1, MELINA DE OLIVEIRA VALDO1, GUILHERME RAPOSO DE MEDEIROS1, MARILIA TAILY SOLIANI1, JOSE CARLOS NICOLAU1 (1) INSTITUTO DO CORAÇÃO DO HCFMUSP Introduction: Certain habits and practices present in groups defined as low risk for cardiovascular disease may be associated with the occurrence of major clinical events, such as acute myocardial infarction (AMI) and stroke. The following report portrays the case of a low-risk patient who suffered from a STEMI followed by pericarditis and whose only risk factor would be recreational cannabis use. Case report: A 35-year-old male with no previous comorbidities or positive family history of cardiovascular disease reported the onset of angina at 03pm on 02/26/2022, only seeking medical attention due to unrelenting thoracic discomfort by 5am on 02/27/2022. He admitted having an average alcohol consumption of 2–3 doses a day on weekends, and a daily cannabis use (up to 10 cigarettes a day), with no use of other drugs in the last 10 years. During assessment, he was in Killip class II and the ECG showed an anterior wall ST elevation, with high sensitivity troponin levels >25000. With the diagnosis of anterior STEMI he was referred to the quaternary reference hospital for a primary angioplasty, where he arrived at midday. The coronary angiography showed significant thrombosis in the proximal segment of the anterior descending artery *(ADA) and a stenosis of approximately 40%. There was also an occlusion in the distal segment, with features suggestive of thromboembolism. Other vascular beds had no lesions whatsoever. Thromboaspiration and angioplasty of the distal segment were performed, without any success in achieving reperfusion. Therapy with intravenous GpIIb/IIIa inhibitor infusion (Tirofiban) for 24 hours was undertaken due to the high thrombotic burden, The next day he evolved with recurrence of severe pleuritic chest pain and underwent a new angiography, with no new findings. Despite that, it was decided to treat the remaining stenosis in the ADA’s proximal segment. Subsequent ECGs presented diffuse ST-segment elevation associated with PR segment depression, suggestive of pericarditis epistenocardica. Echocardiographic data showed an ejection fraction of 40% and mild pericardial and pleural effusions. An association of Aspirin + clopidogrel and colchicine 0.5 mg of 12/12h was prescribed for the management of both STEMI and pericarditis. The patient evolved with an inactive electrical zone in the anterior wall and regression of the ST elevation, in addition to a recovery of the ejection fraction (48% on 03/09/22) and lessening of the symptoms. 109910 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY HELENA ALVES DE ANDRADE RIBEIRO1, Bianca Sarria1, Vanessa Sanson Lani1, Karllayno Camatta Milleri1, Fernando Luiz Torres Gomes1 (1) Antônio Moraes University Hospital (HUCAM) – Federal University of Espírito Santo (UFES) Introduction: Carotid sinus syndrome (CSS) is a complication of head and neck tumors, resulting from baroreceptor hyperexcitation. It was first recognized by Weiss and Baker in 1933, who established the role of this complex. Since then, there have been reports of the association of neck masses related to syncope. The case below describes a patient who presented with symptomatic bradycardia secondary to compression exerted by a neck mass. Case-report: AJBS, 69 years old, female, was admitted to the emergency department with episodes of recurrent syncope, with intensification in the left lateral decubitus position, associated with profuse sweating, nausea, left peripheral facial palsy, and generalized weakness, starting one week before seeking for medical care. She had a large left neck mass and a history of evolution during 20 years, with progressive growth, especially 2 months before the symptomatic condition. The heart rate was 27 beats per minute, in sinus rhythm, with ventricular extrasystoles, with a good response to atropine and venous hydration. Neck computed tomography shows expansive formation with soft tissue density, necrosis, calcification and heterogeneous enhancement, affecting the left parotid, measuring 7.9 × 6.7 × 6.4 cm, with local bulging and parapharyngeal space involvement and multiple lymph node hypertrophy. As an immediate treatment, a transvenous pacemaker was implanted, with subsequent insertion of a definitive pacemaker. This intervention initially showed a positive impact, with heart rate stability, but there was a successive return of hypotension episodes, reaffirming the character of an independent local stimulus caused by the expansive formation. Fine needle aspiration puncture (FNA) of the lesion was performed, with material under analysis. The therapeutic under analysis is local treatment for mass reduction and interruption of the feedback caused by it. Conclusion: SSC caused by the presence of neck masses is due to compression and invasion of the carotid sinus and nerve branches. This syndrome has two subtypes: cardioinhibitory and vasodepressor, and the differentiation is not always clear, since the abnormal stimulation of baroreceptors encompasses different cardiovascular phenotypes. Early diagnosis associated with proper management alters the course and outcomes of this syndrome, mitigating unfavorable outcomes resulting from the condition and having a positive impact on morbimortality. 109912 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM VERÔNICA HOMEM DE CARVALHO E SILVA1, João Pedro Soares Costa1, Yasmeen Salah Mustapha El Qatta1, Victor Marçal Carvalho Mendes1, Alexandre Anderson de Sousa Munhoz Soares2 (1) Universidade de Brasília (UnB); (2) Cardiologia DASA Hospital Brasília Unidade Águas Claras COVID-19 infection triggers a cytokine storm that includes a high expression of interleukin-6 (IL-6). Therefore, anticytokine therapy in patients with acute heart failure associated with SARS-COV2 demonstrated to be a good option of treatment. The case report in question is a case in which the use of tocilizumab with myocardial revascularization was successful. A 48-years-old man with past medical history of obesity, systemic arterial hypertension, type 2 diabetes and non-dialysis chronic kidney disease. Presented with complaints of progressive dyspnea for 10 days, decubitus intolerance and low oxygen saturation. At admission, blood exams demonstrated NT-proBNP 6825 pg/ml, D-dimer 425 ng/mL and creatinine 3,04 mg/dl. Next day, troponin curve was on the rise (519–542–671), which led to the suspicion of myocarditis due to COVID-19. After discussing the risks with the family, he was started on off-label treatment of tocilizumab intravenous, and methylprednisolone 2 mg/kg. Over the next days, exams demonstrated negative RT-PCR, pulmonary congestion on CT (moderate right and small left pleural effusion with restrictive atelectasis and multifocal and bilateral ground-glass opacities), compatible to hypertensive pulmonary acute edema, possibly by myocarditis, therefore tocilizumab and corticoid was suspended and cardiac MRI requested. The MRI resulted in a pattern of inferior and lateral fibrosis suggestive of ischemia, thus coronary angiography was requested. The angiography concluded the presence of severe coronary atherosclerotic disease with a three-vessel pattern, indicating the need of myocardial revascularization surgery. Due to the immunosuppression that tocilizumab causes, the infectious disease team was consulted to discuss the risks/benefits of delaying the surgery, it was agreed that it would be after the half-life of the product, which is 11 to 16 days. The surgery took place without complications. After surgery, the patient needed to use norepinephrine, dobutamine and vasopressin, reacting well to the treatment. After the episode, he spent another 9 days in the ICU with hemodialysis as his chronic renal disease worsened but quickly recovered, without other noteworthy complications, being discharged after the period. We report the successful myocardial revascularization surgery after the use of tocilizumab without any infectious complication. The surgery schedule after the half –life period may be considered to the treatment of this complication. 109961 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS VICTOR ALEXANDRE DOS SANTOS VALSECCHI1, Paula Santiago Teixeira1, Thalita Ruolla Barros1, Marcos Damião Cândido Ferreira1, Adriano Caixeta1 (1) Universidade Federal de São Paulo Introduction: Spontaneous coronary artery dissection (SCAD) is more common in women, aged approximately 50 years. It accounts for approximately 15–20% of myocardial infarctions (MI) during pregnancy or the peripartum period. It is still a disease of unknown cause, but it is related to triggering factors such as emotional stress and pregnancy. Objective: To report a case that occurred in a university hospital and emphasize the diagnostic importance. Case Report: E.L.A., 36 years old, female, hypertension during a previous pregnancy. Three months ago during gestational period and infection by Influenzae presented coronary syndrome without ST-segment elevation (NSTEMI). Catheterization showed spontaneous coronary dissection of the anterior descending artery and circumflex both classified as Saw type 2 with conservative treatment. The patient was admitted in the third postoperative day of a cesarean section due to atypical chest pain. Electrocardiogram showed T-wave inversion in DI, AVL, V5 and V6. The curve of markers of myocardial necrosis was negative and COVID-19 was positive. Echocardiogram (ECO) without segmental dysfunction. During hospitalization for NSTEMI patient evolved with a new episode of chest pain and dynamic alteration of the ST segment. Catheterization showed new focal spontaneous dissection in the distal third of anterior descending artery and first diagonal dissection in all its extension, both with Saw type 2 classification. Improvement of circumflex and right coronary artery free of stenosis. In view of hemodynamic instability, coronary angioplasty of the LAD was performed with 1 drug-eluting stent, with angiographic success. The patient evolved with improvement of symptoms. New ECO showed anterior apical and septal apical akinesia. Conclusion: It is critical that in young women with chest pain, SCAD is suspected because it is an underdiagnosed disease. Accurate and rapid diagnosis is paramount as the management of MI caused by SCAD differs from that of an atherosclerotic cause. 109964 Modality: E-Poster Young Researcher – Case Report Category: CARDIO-ONCOLOGY AMANDA DE ANDRADE CAMPELLO GIROTTO1, Luisa Wagner do Rego Barros1, Juliana dos Santos Macaciel1, Jacqueline Sampaio Miranda1, Antônio Fatorelli1 (1) Instituto Nacional de Cardiologia Introduction: Heart transplantation is the definitive therapy for eligible patients with end-stage heart failure. This subgroup of patients often develops with concomitant kidney injury and, in this context, treatment options become limited. Combined heart and kidney transplantation is a growing practice in recent years in large centers, with increasingly promising results. Case Report: L.R.G, female, 22 years old, born in Rio de Janeiro, with dilated cardiomyopathy secondary to anthracycline cardiotoxicity, being followed up for heart failure with reduced ejection fraction with good functional class. Refers progression of the condition 3 months after pregnancy, when she started to present dyspnea on minor exertion. In use of Carvedilol 50 mg, Sacubitril/Valsartan 98/103 mg 2x, Spironolactone 25 mg, Furosemide 80 mg. Undergoing CPET: VO2 10.7|VEVCO2 36.3|R: 1.17, being referred for evaluation of heart transplantation. Loss of outpatient follow-up in the following months, when developed osteomyelitis in the left lower limb prosthesis, and the need to use broad-spectrum antibiotics. She presented Acute Kidney Injury with evolution to daily hemodialysis. Clinical intractability and need for transfemoral limb amputation were defined. She maintains anuria and anasarca despite dialysis therapy, with the need for inotropes for clinical compensation. Dobutamine was started for type 2 cardiorenal component test, with partial response. SHe evolves with clinical stability on INTERMACS III, with improvement of anasarca. After discussion in the Heart Team, was opted for a combined heart and kidney transplant. Submitted to the procedure on 06/22/21. She was discharged from the hospital with satisfactory diuresis and preserved systolic function. Conclusion: The clinical case reinforces the importance of clinical identification of patients with signs of advanced heart failure, to refer them as soon as possible to specialized centers, with early treatment being an important prognostic marker. Combined heart transplantation is a worldwide trend approach, and this strategy is increasingly used in relation to the sequential transplantation strategy (kidney after heart). The database is still limited and has some retrospective studies, highlighting the importance of new studies with high scientific rigor to guide complex cases. 109969 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO LINHARES AZEREDO CORRÊA1, Fábio Lucas Bassini e Silva1, Jaime Lobo FIgueiredo1, Renata Rodrigues Teixeira de Castro1, Fábio Akio Nishijuka1 (1) Hospital Naval Marcílio Dias Introduction: COVID-19 infection has become a multiorgan disease, which is already well documented in the literature. 2 years after the beginning of the pandemic, we are knowing the long-term effects. As if that wasn‘t enough, vaccines against COVID-19 still had important side effects on the cardiovascular system. Objective: The objective of this report is to discuss the chronic cardiological form of COVID-19, its pathophysiological mechanisms and propose ideas for an approach. Case report: A 30-year-old male, physically active and without comorbidities, with confirmed COVID-19 infection at the end of 2020 and in August 2021, both with a mild condition, was hospitalized with acute myocarditis in September 2021. 3 weeks after discharge, he received SARS vaccine -CoV-2 (Pfizer), when the chest pain intensified, becoming daily. Hospitalized in November 2021 for tachycardia associated with lipothymia. Investigated with 24-hour holter that did not demonstrate arrhythmias and exercise test without criteria for ischemia. Magnetic resonance imaging showed worsening of mesocardial enhancement from 2% (Sep21) to 4% (Jan22) and edema. At follow-up, he presented with progressive dyspnea on exertion with intolerance to physical exercise, in addition to atypical chest pain that led to several visits to the emergency, attributed to chronic pericarditis. In March 2022, he performed an exercise stress test reaching 10.2 METS, and an echocardiogram with strain, without changes in function or contractility. This is why this service referred the patient to cardiac rehabilitation in order to improve functional capacity. Conclusion: Understand the pathophysiological mechanisms and develop strategies to mitigate the chronic and/or late cardiological effects of COVID-19. The symptoms of long-COVID cause multiple emergency visit, in addition to several exams that financially burden the patient and the health system. Similar cases could generate an unprecedented impact on the functionality of the economically active population. 109979 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY VÍTOR BONIATTI NEVES1, Deborah Valente Ramos Rocha1, Vítor Boniatti Neves2, Juan Tiago Nunes Pagnussat1, Tiago Vendruscolo3 (1) Universidade Federal da Fronteira Sul – RS; (2) Faculdade IMED – RS; (3) Instituto do Coração de Passo Fundo – RS Introduction: Common atrium is characterized by complete absence of the interatrial septum, the presence of small strads of tissue presente at the superior atrial wall of the common chamber, absence of interventricular communication and accompanying atrioventricular cushion defect. Objective: Present a report of a rare case of interatrial communication with absence of interatrial septum and complications. Case Report: N.R.M.O, 54 years old, female. Hypertensive and chronic obstructive pulmonary disease. Arives at the emergency room with precordial pain for 1 year, worsening in the last 5 days, associated with dyspnea. Management for acute coronary syndrome was initiated and the patient was referred for invasive stratification. Physical examination: regular rhythm, 2 clicks, systolic murmur 3/6+ at pulmonary focus. Oxygen saturation: 88%. Digital clubbing, but labial cyanosis at rest was not observed. Laboratory data revealed an elevated ultrasensitive troponin – 1941. Electrocardiography: showed left atrial overload. Echocardiogram; complete absence of the interatrial septum. The right ventricle was markedly enlarged with mild pulmonary hypertension (estimated right ventricular pressure, 68 mmHg). Coronary cineangiography demonstrated a LMCA with moderate/severe proximal lesion. An angiography of the ostial left main coronary artery for probable extrinsic compression of the pulmonary artery was performed (IVUS) for anatomical evaluation of significant luminal reduction. The first measurement identified a minimum luminal area of 8.77 mm2, without evidencing atherosclerotic disease. The heart team still discussing the therapy plain. Conclusion: The difficult diagnosis, which begins with investigation of other pathologies, leads to clinical and myocardial worsening of the patient. In the case of this patient, the common atrium led to pulmonary hypertension, with significant dilation of the pulmonary artery, causing extrinsic compression of the left coronary trunk. There are few case reports of older patients with such condition and the therapeutic approach still a challenge. 110038 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ADOZINA MARQUES DE SOUZA NETA1, Victor Agueda Salles1, Luciana da Rocha Ferreira Lobbe Cotta1, Bruno Miranda Marques1, Jacqueline Sampaio dos Santos Miranda1 (1) HOSPITAL COPA STAR Hypereosinophilic syndrome (HES) is rare and defined as blood eosinophil count >1.5 × 109/L and eosinophilia-related organ damage. According to Mankad et al. in 2015, the heart is an affected organ with an incidence of up to 50% of cases. HES, it is classified into hereditary, undetermined significance, primary (clonal/neoplastic) and secondary (reactive) variants. Diagnosing and stratifying the disease is essential to start treatment at an early. Male patient, 65 years old, with dyspnea and with pulmonary congestion for three weeks. Previous history of lymphoproliferative disease, Waldenstrom’s Macroglobulinemia, treated with cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab R-CHOP in 2013. In 2018, due to disease progression, he used R-bendamustine + lenalidomide 6 cycles ending 04/15/2019. Currently in remission. Previous lab history of leukogram with eosinophilia ranging from 19.1% on to 47.3%. He sought the emergency department with a picture of decompensated heart failure, associated with elevated troponin and BNP, being referred for coronary angiography, which showed no significant coronary artery disease, however, ventriculography with left ventricular tip obliteration. In the 48 hours after admission, the patient hemodynamic instability, requiring inotropes and mechanical ventilation. Leukogram with eosinophil of 1,160/mm3 (10%). Cardiac NMR imaging showed mild myocardial fibrosis and left ventricular apex occupied by solid content. With the possibility of HES, he underwent pulse therapy with 1g methylprednisolone for 3 days and underwent surgery to remove the mass from the left ventricle. The histopathology of the material showed a fibrin thrombus with numerous eosinophils, thus confirming the SHE. Faced with the possibility of recurrence of the lymphoproliferative disease, a bone marrow biopsy was performed to rule out the presence of B lymphocytes and a negative karyotype for the FIP1L1-PDGFRA mutation. Hereditary causes and reactivation of the neoplasm were ruled out, it confirms the hypothesis of SHE secondary to chemotherapy. The patient evolved with recovery of ventricular function and he was discharged from the hospital with corticosteroids and anticoagulation. In view of this report, the importance of performing routine echocardiography in patients with persistent eosinophilia is evident, given the high prevalence of cardiac involvement in HES. Early diagnosis and treatment improve quality of life reduce mortality. 110055 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ARGILA GONÇALVES DE CARVALHO SANTANA1, Árgila Gonçalves de Carvalho Santana1, Patrícia Veiga Nascimento1, Simone Letícia Souza Querino3, Thayná Oliveira Militão1 (1) Universidade Federal do Recôncavo da Bahia; (2) Hospital Ana Nery; (3) Universidade Federal da Bahia Background: Covid-19 infection exposes patients to several complications, including a greater predisposition to Acute Myocardial Infarction (AMI). Thrombotic complications occur as a result of the inflammatory process, triggering platelet activation, endothelial dysfunction and stasis. Objective: To report a case of a young patient, with no associated comorbidities, with ST-segment elevation AMI who underwent Primary Angioplasty. Methods: Retrospective study research, based on chart analysis, case study type. Case report supported by the Ethics Committee with CAAE 44553421.0.0000.0056. Results: Patient, 45 years old, male, black, general services assistant, smoker and without other previous comorbidities, complaining of fever, malaise associated with 5 days, taking the 2nd dose of the vaccine against SARs CoV2 – Oxford even 2 days ago showing typical symptoms of Covid-19 infection. On the morning of 07/18 at 5:30 am, he presented oppressive chest pain associated with upper limb paresthesia, without respiratory distress. Admitted to the hemodynamics of the HAN on an emergency basis at 09:40h, coming together with the SAMU, TR for SARS CoV2 negative, ECG performed with supra inferior wall, V3R, V4R, triggered AMI protocol, performed 300 mg of ASS, 600 mg of clopidogrel and morphine 2 mg. Undergoing coronary cineangiography and primary angioplasty: Right Coronary Artery with 100% lesion in the middle third by a thrombus. At the end of the procedure, there was good angiographic appearance of the treated arteries, TIMI 3 flow. Echocardiogram: Simpson EF: 42%, with segmental alteration: Akinesia of the basal and middle segments of the inferior and inferoseptal walls. The patient evolved without complications and was discharged after 2 days of hospitalization. Conclusion: Despite a door-to-balloon time of 4h10min, due to the prolonged ischemia time and high thrombotic load, although asymptomatic, the patient evolved with left ventricular segmental dysfunction, which demonstrates that “time is myocardium”. 110067 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ANANDA RIBEIRO FRETES1, Matheus Lima Lula Guimarães1, Paula Maria Pinheiro Miranda1, Leticia Aparecida Braga da Silva1 (1) Universidade Federal do Mato Grosso Introduction: Coronary artery embolism is an uncommon cause of acute myocardial infarction and one of the most important causes of non-atherosclerotic ST-segment-elevation myocardial infarction. The most common causes are: Infectious Endocarditis (IE), Atrial Fibrillation and Prosthetic Heart Valve Thrombosis. Case Report: Patient, 24 years old, male, smoker, alcoholic, with no previous diagnosed pathologies, with moderate intensity retrosternal pain in the last 30 days, dyspnea on moderate exertion, daily fever, sweating, chills and weight loss of 7 kg in the period. Admitted to an emergency care unit with significant worsening of severe retrosternal pain, worsening with exertion and associated with dyspnea on minor exertion. Physical examination showed an abscess in the left upper molar, cardiac auscultation with a more audible systolic murmur in a 4+/6+ aortic focus radiating to the carotids. The electrocardiogram showed ST-segment elevation in V2, V3, V4, V5 and V6 and troponin: 210 (reference value: <0.03). After 5 hours of evolution, catheterization was performed demonstrating left anterior descending artery (LAD) occluded in the middle third, right coronary artery and circumflex coronary artery free of obstructive lesions, followed by primary angioplasty with implantation of 01 drug-eluting stent in the LAD. An echocardiogram was performed with evidence of vegetation in the aortic valve and an image of paravalvular abscess. Infective endocarditis was confirmed with initiation of antibiotic therapy. Subsequently, during the same hospitalization, aortic valve replacement surgery was performed with implantation of a metallic prosthesis, correction of paravalvular abscess with a pericardium flap and enlargement of the aortic root. The patient evolved in the postoperative period without intercurrences, being discharged after the end of antibiotic therapy, with resolution of the previously presented complaints. Conclusion: Embolic myocardial infarction is a rare but increasingly recognized complication of infective endocarditis and it has increased risk of heart failure and a high mortality rate. Septic embolization resulting in acute coronary syndrome has an incidence of 2.2%. It should be suspected in young patients with a diagnosis of endocarditis or suggestive clinical signs. 110223 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT DOUGLAS BORGES DA COSTA FILHO1, Euton Freitas de Castro Junior1, Ana Carolina Brito de Alcântara2, Danielli Oliveira da Costa Lino3, Ane Karoline Medina Néri4 (1) Universidade Federal do Ceará, Hospital Universitário Walter Cantídio; (2) Hospital Geral Dr. Waldemar de Alcântara; (3) Hospital de Messejana Dr. Carlos Alberto Studart Gomes; (4) Universidade de Fortaleza, Postgraduate Program in Collective Health Introduction: Although uncommon, carcinoid heart disease is one of the main causes of morbidity and mortality in the context of carcinoid syndrome and can occur as the initial presentation of this neuroendocrine tumour. Among the cardiac impairments are the thickening of valves and right chambers due to infiltration of the parenchyma by carcinoid plaques and the high circulating concentration of serotonin, which can lead to severe dysfunctions. The aim of this study is to share a case series of individuals with carcinoid heart disease and to discuss the main clinical features related to this condition. Case Report: Three cases of carcinoid heart disease were treated in reference hospitals in Cardiology in the city of Fortaleza, state of Ceará, Brazil. The patients were female, were in adulthood, had intestinal carcinoid tumor with liver metastasis, presented with tricuspid valve involvement and a clinical presentation of right-sided heart failure (HF) at diagnosis. The first patient, a 59-year-old female, had insufficiency of the tricuspid and pulmonary valves with the latter also presenting stenotic lesion demonstrated by transthoracic echocardiography (TTE). The second patient, a 48-year-old woman, sought medical assistance complaining of gastrointestinal discomfort. A neuroendocrine tumour was diagnosed in the terminal ileum, with secondary lung implants. TTE showed a tricuspid valve with thickened leaflets, reduced mobility, moderate regurgitation and mild stenosis. The third patient, a 55-year-old female, sought an evaluation complaining of abdominal discomfort and cervical and facial flush. She was diagnosed with a well-differentiated neuroendocrine tumour in the colon and a TTE showed a tricuspid valve with thickened leaflets and mobility restriction with significant coaptation failure and torrential tricuspid insufficiency. Conclusions: This study reported the cases of three patients with carcinoid syndrome with heart involvement, describing their main clinical presentations and aspects in common between them, such as female gender, adulthood, the presence of intestinal carcinoid tumor with liver metastasis, tricuspid valve involvement and a clinical presentation compatible with right-sided HF. Despite being infrequent, carcinoid heart disease deserves attention and should generally be screened for among patients with carcinoid tumors, especially in those individuals who present with a condition compatible with right-sided HF. 110330 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS DANIELA BARBOSA MATEUS1, António Carvalho1, Rita Gomes1, Rita Duarte1, Carlos Rabaçal1 (1) Hospital Vila Franca de Xira – Portugal Introduction: Chemotherapeutic agents, especially proteasome inhibitors, used in the treatment of Multiple Myeloma, have a high risk of cardiovascular disease. The authors report a case of ST-segment elevation secondary to vasospastic angina resulting from the use of carfilzomib. Case Report: 63-year-old man, melanodermic, with a personal history of IgG lambda multiple myeloma diagnosed in February 2012 (stage IIA), under chemotherapy with carfilzomib 120 mg weekly, since May 2021. Admitted in the emergency department due to retrosternal pain with left upper limb irradiation, which started at rest, ten hours prior to presentation. Hemodynamically stable, with an unremarkable physical exam. Initial investigation revealed: electrocardiogram with sinus tachycardia, ST elevation (>2 mm from point J) in V2-V4 with hyperacute T waves; echocardiogram with hypokinesia of the apex and apical segments of the septum, anterior wall and inferior wall; preserved systolic function; non-dilated cardiac chambers; valve structures without morphofunctional changes. The patient was transferred to a center of emergency cardiac catheterization. The coronary arteries presented no angiographically apparent lesions; ventriculography revealed good ventricular function with apical hypokinesia and functional study of the anterior descending artery demonstrated reactivity to acetylcholine, favoring the diagnostic hypothesis of epicardial coronary vasospastic angina. Subsequently, analytically, troponin I (high sensibility) was 5,4 pg/mL, reaching a maximum value of 58 pg/ml. Vasospastic angina secondary to carfilzomib was assumed. Vasodilator therapy was started with good clinical evolution, without recurrence of angor and and maintaining the previously described electrocardiographic changes. Discussion: The authors intend to alert to the harmful cardiovascular effects with the use of chemotherapeutic agents, in this case carfilzomib. 110267 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY BRUNO GONÇALVES GARCIA1, Thaíssa Santos Monteiro1, Fábio Akio Nishijuca1, Jamili Zanon Bonicenha1, Maria Carolina Terra Cola1 (1) National Institute of Cardiology (INC) – Rio de Janeiro, Brazil Introduction: Atrial septal defect (ASD) is a congenital heart disease (CHD) in which blood flows between the atria, primarily with a left-right shunt leading to pulmonary hyperflow and right ventricle (RV) volume overload and consequently pulmonary hypertension (PH) type I and RV enlargement and dysfunction. Eisenmenger syndrome occurs when, because of the PH and RV dysfunction, the shunt flow turns right-left and cyanosis appears. However, there are many other causes for PH, which is classified by 5 different types. Case Report: Female patient, 41 year-old, with low educational level, former smoker and history of asthma, was referred to an adult CHD center after being initially diagnosed with ASD with Eisenmenger syndrome. The patient had clubbing and central cyanosis, a palpable RV and systolic murmur in the tricuspid focus. Echocardiogram identified RV dysfunction, bidirectional shunt in the interatrial septum, tricuspide valve regurgitation, pulmonary artery systolic pressure of 128 mmHg and significant enlargement of the pulmonary artery (PA), with thrombus in situ. Although there was a shunt in the interatrial septum, the ASD observed was small. Angiotomography of the pulmonary arteries confirmed bilateral chronic thromboembolism. Rheumatological assessment was negative, as well the thrombophilic panel. Late syphilis was diagnosed by antitreponemic antibody, and treatment was instituted. PA catheterization was not performed due to risk of distal embolization of thrombus present in the PA. The heart team concluded it was a multifactorial PH, including type I and type IV. Syphilitic arteritis of the PA was also remembered, but could not be confirmed. Closure of the ASD was considered contraindicated because of the PH, and anticoagulation was initiated as well as sildenafil, and the patient followed with improvement of oxygen saturation and exercise capacity. Conclusion: Facing a patient with PH and a late diagnosis of ASD, could have mislead to the conclusion of this being a case of PH type I with Eisenmenger syndrome purely. ASD is one of the most common cause of adult CHD, as there are other common causes for PH. One should not be blinded by the combination of both, and a thorough assessment should be done to insure there is indeed a cause-and-effect relationship. 110369 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES JIAN CHU1, Nikitaa Nath1, Natasha Rana2 (1) Rush University Medical Center, Department of Internal Medicine; (2) Rush University Medical Center, Department of Internal Medicine, Section of Cardiology An 81-year-old man with ischemic heart failure and aortic valve-in-valve bioprosthesis presented with acute dyspnea. On exam, he was afebrile, but hypotensive, tachycardic, and tachypneic. Labs were notable for a BNP of 3056 pg/mL and venous lactate of 2.1 mmol/L. Due to suspicion for cardiogenic shock, he underwent emergent cardiac catheterization, which revealed elevated left cardiac filling pressures. He was started on inotropes and vasopressors with placement of an intra-aortic balloon pump. A transthoracic echocardiogram showed a LV ejection fraction of 10–15% and prosthetic aortic valve dysfunction with a peak velocity of 3.7 m/s and mean gradient of 32 mm Hg (markedly higher than baseline). A 9 × 17 mm echodensity was seen on the valve leaflet consistent with bioprosthetic valve thrombosis (BPVT). After multidisciplinary discussion with cardiothoracic surgery and interventional cardiology, he was administered systemic tissue plasminogen activator with a 10 mg load followed by 40 mg over 4 hours. Within 24 hours, he was able to wean off all pressors with improved echocardiographic parameters. The data behind fibrinolysis for aortic BPVT is largely derived from the PROMETEE study, which supports ultraslow fibrinolysis and the present case shows that faster infusions may be safe and effective in decompensated patients. However, more data is needed to guide choice of surgical vs. non-surgical management of patients with decompensation from BPVT. 110485 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES ADRIANO LOPES BATISTA1, Gracylma Guimarães Rocha1, Ingrid Loureiro de Queiroz Lima1, Marlúcia do Nascimento Nobre1, Augusto de Carvalho Bezerra3 (1) Hospital Universitário Getúlio Vargas – HUGV; (2) Universidade Federal do Amazonas – UFAM; (3) Hospital Universitário Francisca Mendes – HUFM Antiphospholipid syndrome is an autoimmune disease characterized by recurrent episodes of vascular thrombosis, predominantly in females. Infective endocarditis associated with this disease is uncommon, with more prevalent aseptic endocarditis. However, there is potential for this collagenosis to lead to pulmonary hypertension, which in turn causes cardiac dysfunction in the right chambers, predisposing the endocardium to infection and, consequently, the lung to septic embolism. The case report is of a 23 years-old male with a clinical history since adolescence of recurrent deep venous and arterial thrombosis, lower limb edema, and progressive dyspnea. Eight years after the onset of symptoms, he developed a condition of digestive hemorrhage associated with thrombocytopenia where, during blood product transfusion, he showed signs of bacteremia. Since then, daily, with mild symptoms of chills, low-grade fever, sweating, arthralgia, and progressive worsening of dyspnea. After 30 days, the patient evolved with pulmonary focus septic shock and was then transferred to our hospital. Chest tomography showed nodules with necrosis and central cavitation compatible with septic emboli(1). Echocardiography showed dilatation of the right chambers, significant pulmonary hypertension, insufficient tricuspid valve, perforated, and a mobile vegetative lesion measuring 1.7 × 1.4 cm adhered to the anterior leaflet compatible with endocarditis. He received antibiotic therapy and valve replacement surgery. All collected blood cultures since admission were negative. We are not aware of cultures performed at the hospital where he came from. He was discharged from the Intensive Care Unit (ICU) and was awaiting the results of investigative tests for the underlying thrombogenic disease. Suddenly, he presented dyspnea and chest pain, evolving into cardiac arrest due to pulseless electrical activity resulting in death. After death, we received the tests that were positive for Antiphospholipid Syndrome. However, for the definitive diagnosis of this collagenosis, the antibodies must remain positive after 12 weeks of the first collection. 110493 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FELIPE MONGE TEIXEIRA1, Paulo Vitor da Silva Gonçalves1, Diego Bianchi Braga Campos1, Marcelo Imbroinise Bittencourt1, Ricardo Mourilhe Rocha1 (1) Cardiology Service – Pedro Ernesto University Hospital – UERJ Introduction: In 2020, the WHO declared COVID-19 a pandemic. In the same year, the US Food and Drug Administration (FDA) granted emergency use authorization for the Pfizer/BioNTech and Moderna vaccines. Despite the benefits of vaccination, it can also pose potential risks such as myocarditis. Case Report: Female, 37 years old, without comorbidities, denies alcohol consumption, smoking, and recent infections. She presented mild COVID in January 2021, and after 7 months, she was vaccinated with 2 doses of Pfizer/BioNTech. After vaccination, she developed oppressive type A chest pain, VAS 8/10, radiating to the left upper limb and nausea, seeking emergency care. An ECG was performed, without significant changes, but with high ultrasensitive troponin values (12,500|RV < 5), motivating his hospitalization. She underwent transthoracic echocardiography (TE), which demonstrated septo-apical hypokinesia with preserved function. Coronary angiography was performed on the first day after admission, which showed no obstructions. She had a laboratory with still high troponin, but falling, in addition to a negative PCR for COVID. A new TE was performed, which showed no segmental lesions or changes. Cardiac resonance imaging was requested, demonstrating moderate myocardial fibrosis (11%) suggestive of myopericarditis, as shown in Figures 1A and 1B. In view of this, he started treatment with colchicine and ASA, which led to a drop in troponin after 7 days of hospitalization, and he was discharged asymptomatic. Conclusion: Vaccination for COVID-19 continues to be recommended for all people, clearly outweighing the risks in all populations. As observed in the case described, most individuals with myocarditis had a full recovery of ventricular function, with few reports of major complications. 110515 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING VINÍCIUS STÉFANO BARROS DA ROCHA1, Anita de Oliveira e Souza Fragoso1, Maria Luciana Zacharias Hannouche da Trindade2, Ana Clara Tude Rodrigues1 (1) Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP); (2) Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (InRad) Introduction: Leiomyosarcoma is one of the most common malignant tumors among retroperitoneal tumors, corresponding to approximately 15% of these. Involvement of the inferior vena cava (IVC) and right heart chambers is rare and related to a worse prognosis. Ultrasound-enhancing agent echocardiography (ECHO with enhancement) has been used to identify intracardiac tumors with high accuracy. However, this exam may eventually present limitations in relation to the definition of malignancy. Case Report: A 53-year-old female patient with diabetes and dyslipidemia sought medical care with left low back pain for about 5 months, associated with bulging in the right flank region and weight loss of 5 kg. Investigation with computed tomography (CT) of the abdomen from an external service showed a retroperitoneal mass in the IVC and right atrium (RA), and the biopsy with guided puncture was compatible with high-grade leiomyosarcoma. The enhanced echo showed partial perfusion of the mass in the RA, while the three-dimensional transesophageal echocardiogram showed a heterogeneous multilobulated mass extending from the most apical portion of the IVC to the RA, close to the tricuspid valve, with vacuolar areas in its interior, both tests suggesting a benign process. The patient was scheduled for vascular and cardiac surgery for definitive treatment. Conclusion: Despite studies showing good accuracy of echocardiography with enhancement for differentiating between malignant and benign tumors, according to the characteristics of the perfusion, malignant tumors with necrotic process inside them, such as leiomyosarcomas, may present with partial perfusion of the tumor, limiting the accuracy of the method. 110539 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES GABRIEL RIBEIRO ROSA1, Elisa Carolina de Almeida Negrello1, Alcirley de Almeida Luiz1, Pedro Henrique Bonifácio Shiino1 (1) Hospital Universitário do Oeste do Paraná (HUOP) Introduction: Systemic lupus erythematosus (SLE) is associated with severe cardiovascular manifestations, including myocardial infarction (MI), which is an important cause of premature mortality and morbidity. Accelerated atherosclerosis is the main mechanism involved. We report a case of a young male with an acute MI with ST segment elevation, secondary to coronary thrombosis, which led to the diagnosis of SLE-associated antiphospholipid syndrome (APS). Case Report: A 24-year-old male was admitted to the hospital with typical chest pain, lasting 6 hours. The electrocardiogram in the emergency room (ER) showed ST segment elevation in the anterior wall. The patient had a history of SLE, which was diagnosed when he was 15 years old, and was undergoing investigation for APS due to the positivity of IgG anti-β2 glycoprotein-1 antibody. He had traditional risk factors, such as hypertension and class I obesity. He received treatment in the ER and was referred to Interventional Cardiology. A coronary angiogram showed complete occlusion proximally in the left anterior descending artery. A percutaneous angioplasty with no stent was performed, due to angiographic signs of thrombosis without associated atherosclerotic disease in other vessels. The patient had a good recovery without other complications. Conclusions: SLE is an autoimmune disease commonly associated with APS. This association increases mortality, and the positivity of antiphospholipid antibodies in SLE patients’ serum is an independent risk factor for premature death. Deep venous thrombosis and stroke, differently than the reported case, are the most common cardiovascular events in these patients. 110546 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISABELA GALIZZI FAÉ1, Carolina Kuchenbecker Soares1, Luís Henrique Coelho Pinto1, Fernanda Araújo Sá1, Anderson Ferreira Leite1 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais Introduction: Left ventricular (LV) pseudoaneurysm is a rare complication after myocardial infarction (MI) characterized by rupture of the LV wall contained by pericardium, thrombus or scar tissue. If not treated properly, it can lead to cardiac tamponade and death. We aim to describe a case of LV pseudoaneurysm after MI treated with percutaneous closure. Case Report: A 57-year-old man presented with chest pain and extensive anterior ST-segment elevation with right bundle-branch block. Acute MI protocol and thrombolysis was performed, without reperfusion criteria. Rescue angioplasty was not available that day. Cineangiocoronariography in the next day revealed severe occlusion (90%) in descending artery (DA) ostium, so angioplasty with pharmacological stent in the left main artery (LMA) towards DA was performed. Echocardiogram showed ejection fraction of 38%, segmentary defect of contractility and an image suggesting pseudoaneurysm in apex of LV, which was confirmed in cardiac resonance and angiotomography. The opted approach was percutaneous transcatheter closure due to high risk of rupture. Surgery was dismissed, as the patient was clinically stable and had elevated risk surgery, owing to MI and recent angioplasty to LMA towards DA. An interatrial communication prosthesis was implanted, without any flow inside the pseudoaneurysm orifice. The patient remains asymptomatic after six months of follow-up. Conclusion: High suspicion is required for the diagnosis of post-infarction LV pseudoaneurysm. Non-invasive imaging is indispensable in stable patients. Surgical treatment is usually recommended, notwithstanding, percutaneous transcatheter closure is growing in importance as it has lower morbimortality compared to surgery, with multiple successful case reports being described. 110790 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING JULIANA JANGELAVICIN BARBOSA1, Otavio Augusto Oliveira de Carvalho1, Thiago Abizaid Kleinsorge1, Ruldney Ray dos Santos Oliveira1, Antonio Tito Paladino Filho1 (1) Hospital SEPACO (SEPACO) Introduction: Coronary artery fistula (CAF) is a rare abnormality characterized by the connection between one or more coronary arteries and a cardiac cavity or great vessel. Depending on several factors can present without clinical repercussions or cause significant hemodynamics complications. Case description: A 33-year-old male patient presented with chest pain and ST-segment elevation in leads DII, DIII and aVF at the ECG. Troponin was elevated (Pocket). Referred for urgent cardiac catheterization, we not identify obstructive coronary lesions, but observed the presence of a fistula with extravasation of contrast into a neo-cavity. Cardiac magnetic resonance imaging was performed with findings suggestive of acute myocardial infarction (AMI) in the inferior wall and a “mass” adjacent to the right atrium with an image of a thrombus in its interior. The investigation was complemented with coronary CT angiography, bringing the hypotheses of a pseudoaneurysm and evidencing the feeding origin from the right coronary fistula. This “mass” exerted a compression effect on the right coronary artery (RCA), resulting in inferior wall infarction. The patient was referred for surgical intervention, confirming the hypotheses of a right ventricular (RV) pseudoaneurysm with a thrombus inside it. Conclusion: The diagnosis of CAF is rare, and in the clinical case, it was responsible for the formation of a pseudoaneurysm that expanded, evolving with AMI due to compression of the RCA. The case brings together other findings – CAF and RV epicardial pseudoaneurysm – which, despite being rare, presented with a common condition in cardiac emergencies: AMI. Advances in cardiovascular imaging contribute in such types of clinical cases with early diagnosis and effective management, leading to favorable clinical outcomes. 110810 Modality: E-Poster Young Researcher – Case Report Category: CARDIO-ONCOLOGY LAÍSSE BARRETO FERREIRA REIS1, Maíra de Melo Ibrahim Nogueira2, Valmir de Freitas Costa1, Rafael Alves da Silva1, Douglas Celestino Fróes3 (1) Instituto do Coração/FMUSP; (2) Hospital Felício Rocho; (3) Centro Hospitalar do Município de Santo André Introduction: Pazopanib is a tyrosine kinase inhibitor whose main therapeutic targets are PDGF (platelet-derived growth factor) and, mainly, VEGF (endothelial growth factor). Its use in oncology is in the treatment of renal cell carcinoma, sarcomas and, less commonly, thyroid cancer. This medication has arterial hypertension as its main cardiovascular side effect, but it can also cause arrhythmias, QT interval prolongation and heart failure. Case description: A 64-year-old patient with a personal history of arterial hypertension, dyslipidemia and non-insulin-requiring diabetes, had no history of heart failure and had an echocardiogram with a left ventricular ejection fraction of 60%. Diagnosed with non-metastatic renal cell carcinoma, she started using pazopanib at a dose of 800 mg daily. After one month of use, he started to experience dyspnea on minor exertion, orthopnea and lower limb edema. She was hospitalized for evaluation and management of the condition, her NT-Pro-BNP was 4,500, the electrocardiogram had left bundle branch block and the echocardiogram showed an ejection fraction of 35% with an area of septal and inferior akinesia and hypokinesia of the other walls. In view of this finding, pazopanib was discontinued and the use of enalapril, carvedilol, spironolactone and furosemide was started. An investigation of the etiology of ventricular dysfunction was carried out with the following findings: negative serology for Chagas disease, cardiac catheterization showed no obstructive lesions, cardiac magnetic resonance imaging with no edema, late enhancement or perfusion defects at rest and stress. Therefore, pazopanib was considered the cause of her heart failure. After discontinuation of chemotherapy and initiation of treatment for heart failure, the patient showed clinical improvement and a new echocardiogram showed an ejection fraction of 45%. In a decision shared between the oncology and cardiology teams and with the patient, it was decided to keep the medications for ventricular dysfunction and pazopanib at a lower dose, 400 mg once a day. Conclusion: Pazopanib plays a very important role in the survival of patients with renal cell carcinoma, the incidence of ventricular dysfunction is low and it is a diagnosis of exclusion. Management is challenging and requires shared decision-making between oncology and cardiology, respect for patient values and preferences, and frequent clinical surveillance. 110861 Modality: E-Poster Young Researcher – Case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GABRIELA MELCHUNA MADRUGA1, Rodrigo Otávio Bougleux Alô1, Lucas Yuiti Mori1, Camila Mota Guida1, Marco Antônio Smiderle Gelain1 (1) INSTITUTO DANTE PAZZANESE DE CARDIOLOGIA Congenital third degree block (CHB) is a relative rare disorder. The manifestations of CHB can vary according to many aspects, including ventricular rate of the escape rhythm and ventricular function. The main therapeutic decision is the need for and the ideal timing of permanent pacemaker insertion. Patients with adequate ventricular heart rate increasing appropriately during exertion and no symptoms such as syncope or near syncope and a structurally normal heart and cardiac function can usually be followed without intervention, and therefore, they can selectively participate in competitive sports. Although, this is not a permanent recommendation, and these patients should be followed closely. Case Report: 13-year-old asymptomatic male patient, with a previous diagnosis of CHB, who practices competitive soccer, in yearly follow up at the sports cardiology outpatient clinic, who presented a normal echocardiography, without structural alterations, normal Left Ventricle (LV) dimensions, a Left Ventricle End Diastolic Diameter (LVEDD) of 48 mm, and preserved LV function. His exercise test showed an excellent functional capacity (15 METS) despite a chronotropic insufficiency. However, on the 2021 clinical evaluation, he presented a significant worsening of the tests results, although remained asymptomatic. The new echocardiography presented LV dilatation with a LVEDD of 63 mm, but with preserved ejection fraction. The exercise test showed a worse functional capacity of 11 METS and chronotropic deficit. Therefore, it was indicated the insertion of a pacemaker. New tests were performed two months after the procedure. The echocardiography showed normal LV dimensions (LVEDD: 46 mm). At the cardiopulmonary exercise test, the patient reached 103% of the predicted VO2, showing a normal functional capacity. Although all these improvements, the practice of soccer was contraindicated due to the high risk of thoracic trauma. Nevertheless, the patient still may participate of other competitive and high intensity activities with low risk of lead dislocation and thoracic trauma. In conclusion, the CHB is not a prohibitive condition of high intensity sports, as long as the patient remains without structural cardiac alterations, normal LV function, asymptomatic and with a normal functional capacity. However, these conditions can change in a relative short period, so the follow up must be done closely. 110891 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES FLÁVIA CORRÊA DE OLIVEIRA LIMA1, Marília Spolador Marrafão1, Camila Clara da Costa Geraldo1, Maria Beatriz Marcussi Moretti1, Rafael Cecílio de Lima1 (1) Hospital Regional de Presidente Prudente – HRPP Introduction: Libman-Sacks endocarditis (LSE) – thrombotic, non-bacterial, marantic or verrucous – is characterized by inflammation of the endocardium and sterile vegetations in the heart valves. It is present in 6 to 11% of patients with Systemic Lupus Erythematosus (SLE), with a significant correlation of duration and severity. The development possibly occurs by endothelial injury of circulating cytokines in a patient in a hypercoagulable state, which results in the deposition of platelet thrombi and inflammatory molecules in the valves. Case description: Female, 18 years old, was hospitalized with dyspnea, weakness and bruising for 30 days. Personal history of active SLE with grade III lupus nephritis, hypertension, severe depressive disorder and drug addiction. Patient evolved with persistent high fever, neutropenia, thrombocytopenia, blood pressure peaks and significant worsening of renal function; referred to the intensive care unit. Physical examination: diffuse edema; cardiac auscultation without murmurs and pulmonary auscultation with crackling rales in bilateral bases. Acute intubation with heart failure due to advanced hemodialysis pulse patient edema. Four blood cultures were performed within 30 days, all negative. At the entrance performed Transesophageal Echocardiography (TEECHO), inlet ventricular hypertrophy, mitral function, va a to bicuspid with thickening of the inferior important and echogenic image adhered, filamentous, with random movements, suggestive of endocardial selection. Due to severe thrombocytopenia, it was decided not to perform anticoagulation; clinical support and multidisciplinary follow-up. After 30 exams that showed increased ventricular hypertrophy, increased repetition of myopia, insufficiency and absence of vegetations. In medical records, 9 years ago, ET ECHO with important mitral indication found and leaflet in anterior leaflet with later normalization. At the clinic, a cardiac surgery equipment opted for the conservative approach. Conclusions: Cardiac complications are the ones that most attribute morbidity and mortality to SLE cases. The treatment of the underlying disease with an interdisciplinary approach that included prevention, health education (therapeutic adherence and health care), individualized treatments and diagnosis of complications, such as LSE, can lead to an early risk of negative cardiovascular problems and a better in patient survival. 110960 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS NOURHAN CHAABAN1, Shilpa Kshatriya2 (1) University of Kansas; (2) Heartland Cardiology Spontaneous coronary artery dissection (SCAD) has been reported to be a rare cause of acute coronary syndrome (ACS) and sudden cardiac death (SCD). We report a case here to emphasize on the benefit of early recognition of SCAD, and to highlight on its association with systemic lupus disease. A 37-year-old female patient known to have hypertension, systemic lupus erythematosus, a history of pre-eclampsia with 3 C-section deliveries, and an active smoking habit presented to the emergency department with chest pain. On arrival, the patient was hypertensive to 152/122 mmHg, with a normal heart rate and respiratory rate. An electrocardiogram (EKG) showed minimum ST elevation in lead V2. Initial troponin was 0.1 ng/mL (normal <0.4 ng/mL) with a peak of 54.5 ng/mL after 6 hours. Aspirin 325 mg was given, and the patient underwent an urgent cardiac catheterization. Intravascular ultrasound was performed that showed evidence of intimal flap (mid LAD) spontaneous dissection with subintimal hematoma. The angioplasty resulted in successful stent placement in the mid-left anterior descending artery. SCAD diagnosis is challenging and requires a high index of suspicion. We aim to elaborate on the association of SCAD with systemic lupus disease and add to the literature this unusual case. 110999 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES POLIANA FERREIRA STROLIGO DIAS1, Paula Bline Lazzari1, Bruno de Souza Paolino1 (1) Hospital Universitário Pedro Ernesto (HUPE) Introduction: Purulent pericarditis is a pericardium located infection characterized by pus. The most common pathophysiology is through continuity or hematogenous dissemination of another infection. It’s usually associated with pneumonia and other infection sites are rare. Although the disease is manifested by chest pain, differential diagnosis with acute coronary syndrome (ACS) is made to exclude one of the conditions, since the concomitant presence of pericarditis and ACS is even rarer. Case: 52-year-old smoker and alcoholic male with hypertension and hemorrhoids seeks Emergency Care (EC) with typical chest pain, is diagnosed with inferior STEMI and treated by thrombolysis. He reported anal pain with purulent discharge 15 days before and fever 2 days before but did not use antibiotics. Patient transferred to our institution, still febrile, for invasive stratification after 4 days of EC hospitalization. Coronary angiography showed thrombotic occlusion of posterior ventricular branch (PV), with no atherosclerosis in other vessels. PV angioplasty and thrombus aspiration performed without flow return. Evolution to hemodynamic instability, orotracheal intubation and cardiopulmonary arrest with pulseless electrical activity for 6 min, reversed after CPR. TTE showed pericardial effusion with cardiac tamponade signs, with purulent fluid’s output of 300 mL in pericardiocentesis. He was referred to the surgical center, where pericardial window evidenced smooth pericardium. There whas leukocytosis with deviation up to metamyelocyte and CRP elevation in admission exams. Pericardial fluid analysis showed 68600 leukocytes/μl, numerous polymorphonuclear and gram-negative bacilli. Ventilatory and hemodynamic supports were kept and empirical antibiotic therapy with Vancomycin and Meropenem was initiated. The condition worsened, with fever despite antibiotics and antipyretics, progressing to death after 3 days. Later, culture results showed multi-sensitive E. Coli growth in all blood and pericardial fluid samples. Discussion: Purulent pericarditis is a rare and severe disease with mortality up to 30% even with pericardial drainage and adequate antibiotic treatment. Possibly, the infectious condition triggered concomitant coronary thrombosis, another rare but plausible association. Rapid recognition and appropriate treatment are essential for better outcomes and to prevent constrictive pericarditis. 111188 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LARISSA SEBOLD1, Abiran Dalri Merizio1, Bárbara Dalri Andreghetoni1, Tiago Gonçalves Pereira1, James Alberton1 (1) Cardiological department – Santa Isabel Hospital Introduction: As a congenital heart disease, the univentricular heart may be related to other pathological conditions, such as: pulmonary artery stenosis and aortic obstruction. Within this complex group, the left ventricle hypoplasia (HLHS) is the most common disorder, occurring when there is an underdevelopment of the left ventricle, mitral valve, aortic valve and aortic arch. Typically, it presents in the first 24 to 48 hours of life, due to the constriction of the ductus arteriosus. Adult patients not submitted to repairs usually develop pulmonary stenosis or pulmonary vascular disease. Case Description: AS, 31 years old, male, with congenital heart disease: Single ventricle of left morphology, membranous interventricular communication (IVC), ventriculoarterial connection type “Transposition of the Great Arteries” (TGA) – with pulmonary artery connected to SV, and aorta connected to the hypoplastic chamber – and presence of patent foramen ovale, maintaining signs and symptoms of Heart Failure, with Transthoracic Echocardiogram demonstrating complex cyanotic congenital heart disease (single ventricle + IVC + TGA + Patent Foramen Ovale). Mitral Insufficiency and mild to moderate Tricuspid. Mild Aortic and Pulmonary Insufficiency. Late postoperative of Pulmonary Artery Banding, apparently effective. Moderate enlargement of both Atria. SV with significant enlargement and dysfunction (Ejection fraction:27%). Medication optimization was done and A.S. was listed for performing Heart Transplantation. Conclusion: According to the case above, the clinical manifestations of SV depend on associated cardiac anomalies: if obstructed pulmonary flow, Tetralogy of Fallot clinic, with important cyanosis and without HF; if unobstructed flow, clinical of transposition of great vessels with intraventricular communication, with minimal cyanosis and important HF. When non-operated, many patients succumb to HF from birth, however, the rapid evolution of successful palliation, with surgical approach in stages, has led to a population of adults with only one ventricle. 111026 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ANA LUÍZA SILVEIRA BORELA PELLIZZER1, Reno Leite Zamignan1, Kamilla Guimarães Dias1, Pedro Leonel de Paiva Neto1, Ana Paula Lindoso Lima Barreiro1 (1) Santa Casa de Misericórdia de Goiânia Background: Coronary thrombosis is a serious cause of chest pain and needs to be promptly diagnosed and treated in time, as it leads the patient to the risk of death. One of the causes of coronary thrombosis is the indiscriminate use of anabolic steroids, in most documented reports is the combination of various anabolic steroids. Stanozolol is an anabolic considered the safest among the others, with little probability of complications, for oral use, and which is even approved by the FDA and has grown widely in its use. There are no reports in the literature with the isolated use of Stanozolol associated with serious cardiovascular complications. Case Report: Patient, 25 years old, previously healthy, evolving with tight chest pain, irradiating to the left lower limb, during physical activity (bodybuilding), in which he sought medical attention, and an electrocardiographic diagnosis of acute myocardial infarction with ST-segment elevation on the anterior wall was made. He reports that he had been on regular use of Stanozolol 10 mg once a day for 1 month, denying the use of any other associated medication, such as testosterone, oxandrolone and/or GH. He underwent cardiac catheterization that showed an occluded anterior descending artery with proximal thrombus. Angioplasty and recanalization with intracoronary thrombus aspiration were performed. Due to the presence of residual thrombi and TIMI-II flow, Agrastrat was started. Control catheterization was performed after 5 days in which no intracoronary thrombi were observed, and was then discharged from hospital with outpatient return. Discussion and Conclusion: Stanozolol, the only medication used by the aforementioned patient, is widely used by nutrologists and endocrinologists due to its good efficacy in gaining lean mass and the low probability of complications. The complications documented in publications and articles are neurological changes and hepatotoxicity. This case is of paramount importance, since there are no reports of serious coronary complications in the presence of the use of Stanozolol yet, which should serve as a warning to the general population and health professionals for its use. 112359 Modality: E-Poster Young Researcher – Case Report Category: DYSLIPIDEMIA GIOVANNA BOLINI BRAZÃO1, Isabella Rocha Gonçalves1, Eduarda Gabriel Mafra1, Samuel Sabbá Fadul1, Ana Augusta Motta Oliveira Valente1 (1) Centro Universitário do Estado do Pará (CESUPA) Introduction: Familial hypercholesterolemia (FH) is a genetic disease resulting from the mutation of one of the critical genes for the formation or catabolism of Low-Density Lipoprotein (LDL). Transmission is autosomal dominant and can be heterozygous or homozygous. In about 80% of patients with a definitive diagnosis, mutation can be observed in one of 3 genes: LDL receptor gene (LDLR), gain-of-function mutation in the Protein Convertase Subtilisin/Cexin Type 9 gene (PCSK9) and mutation in the apolipoprotein B (Apo B) gene. Homozygotes will invariably need multiple drugs to control cholesterol, and receptor negative patients will not respond to drugs that act directly or indirectly on LDL receptors. Case details: A 15-year-old man with a diagnosis of homozygous familial hypercholesterolemia by C681X mutation in LDL receptor, as known as Lebanese mutation, one of the most prevalent in Brazil. He has a family history of hypercholesterolemia (father, mother and siblings) and grandmother and uncles who died after AMI under 50 years. On physical examination, corneal arches with cholesterol deposits and tuberous xanthomas on knees, elbows and heels were observed. The treatment was conducted with two distinct pharmacological combined therapy: Therapy 1: rosuvastatin 40 mg daily, ezetimibe 10 mg daily and mipomersen 200 mg every two weeks, for one year. Therapy 2: rosuvastatin 40 mg daily, ezetimibe 10 mg daily and evolocumab 140 mg every two weeks, for one year. The result suggests that the second therapy is better suited to reach the target of 50% reduction in LDL, even without plasmapheresis and in low doses. This corroborates with recent findings that defines C681X mutation as null-receptor mutation, where PCSK9 inhibitors have low efficacy. Conclusion: Studies should be carried out to accumulate data on the distribution of the C681X mutation throughout the Brazilian territory, to pressure public health agencies to create treatment protocols aimed at the Lebanese mutation. 111041 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MARIA CLARA SILVEIRA MARQUES PEREIRA1, Thaisa Paula Perini1, Mayara Aparecida da Silva Oliveira1, Juliana Tasso Cândido de Lima1, Lucas Simões de Mello1 (1) Hospital Universitário de Londrina – HU Londrina/UEL Introduction: The coronavirus disease-2019 (COVID-19) pandemic has quickly become a global health problem with high rates of mortality and morbidity. The improvement of the world scenario was possible after the development and wide application of vaccines. A possible complication after vaccination is myocarditis. We report a case of myocarditis after inactivated SARS CoV-2 virus antigen vaccine. Case Report: An 18-year-old male patient attended the emergency department complaining of self-limited episodes of retrosternal chest pain, unrelated to exertion or rest, without irradiation, without other associated symptoms, and lasting up to 1 hour. He denied fever, dyspnea, flu-like symptoms, syncope or other complaints. Personal history of obesity grade III. He received a vaccine for Covid-19 15 days before the onset of the condition. An electrocardiogram was performed on admission, showing sinus tachycardia, positive ultrasensitive troponin result (1.887 – reference value <50 ng/L), NT-pro-BNP 100 (reference value <450 pg/ml). Investigation with chest angiotomography without evidence of pulmonary embolism, chest tomography without alterations. During evolution, the patient showed improvement in chest pain episodes, but with a significant ultrasensitive troponin curve (reaching 3.459 ng/L), without dynamic changes on the electrocardiogram. Considering the hypothesis of post-vaccine myocarditis, cardiac resonance was performed, showing an ejection fraction of 62.9%; area of hypersignal in the inferoseptal wall of the apical segment of the left ventricle (edema); preserved biventricular function; normal-sized cardiac chambers; absence of signs of ischemia at rest or areas of fibrosis (Figure 1). Patient presented spontaneous improvement of pain, being discharged from hospital after investigation. Conclusion: Myocarditis after vaccination is a possible reaction, but with a very low incidence, being more reported among young males, with a good prognosis and a mild and self-limiting course. Therefore the benefits of vaccination outweigh the morbidity and mortality rates associated with COVID-19 infection. 111079 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KAREN KATCHVARTANIAN1, Luis Henrique Silveira Moreira1, Armindo Jreige Junior1, Renan Cintra de Alvarenga Oliveira1, Camila Vilela Vieira1 (1) Instituto do coração – INCOR-SP L.P., age 46, female, with a previous history of benign familial thrombocytopenia, entry into the InCor emergency room in 2017, receiving a primary diagnosis of acute myocardial infarction with ST segment elevation. Being submitted to an investigation of primary dyslipidemia, sitosterolemia was identified, a rare genetic condition in which lipid metabolism is altered. During hospitalization, the patient developed critical segmental dysfunction and left ventricular failure, with drug therapy being optimized for HF conditions and regular patient follow-up with a specialized team. During hospitalization, progressive worsening conditions evolved presenting multiple organic dysfunctions despite the inotropic drug administration. Chosen MCS (HeartMate 2) as destination therapy, since the patient presented heart transplantation contraindication, because of the elevated immunologic panel due to multiple transfusions by benign familial thrombocytopenia. Sitosterolemia is characterized by hyperabsorption and lowered biliary excretion of dietary sterols, including the phytosterol beta-sitosterol, by plant ingestion. Healthy individuals absorb only about 5% of dietary phytosterols, but sitosterolemia-affected individuals absorb around 15% to 60%. Thus, there is a higher atherosclerosis risk in these patients, and the diagnosis delay has limiting consequences, amongst them, acute myocardial infarction and the subsequent progression to advanced HF when early diagnosis is not achieved. Advanced HF is characterized by heart dysfunction associated with severe symptoms such as dyspnea and fatigue at rest or with minimal physical effort (NYHA III or IV), of life, even with optimized drug therapy, in which life expectancy might be less than two years. At this stage, advanced therapies are considered, including heart transplantation, continuous inotropic therapy, MCS, or palliative care. In 2020, around 3 thousand patients were treated with MCS, and, more than 3 thousand patients had heart transplantation done in the United States, with additional 3,5 thousand patients awaiting transplantation. The importance of support devices as a destination therapy for left ventricularfailure grows incessantly due to organ donor scarcity. However, despite its dissemination, MCS implants still represent a high-risk, burdensome procedure, related to serious adverse events such as right HF, stroke, infection, pump thrombosis, and hemolysis. 111083 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KAREN KATCHVARTANIAN1, Camila Vilela Vieira1, Luis Henrique Silveira Moreira1, Renan Cintra de Alvarenga Oliveira1, Armindo Jreige Junior1 (1) Instituto do coração – INCOR-SP Heart failure (HF) showed an exponential increase in the last years, affecting about 1–2% of the world’sadult population, and having a significant impact on morbidity and mortality of patients. At the advanced stages of the disease after optimized drug therapy, both a heart transplant and left ventricular assist device (LVAD) implant must be considered for treatment. L.P., age 46, female, with a previous history of benign familial thrombocytopenia, was treated in the Instituto do Coração de São Paulo (InCor) emergency room in 2017, with a diagnosis of acute myocardial infarction, with left ventricular dysfunction development during hospitalization. Was discharged from hospitalization with optimized drug therapy with the outpatient return. At the followup was submitted to an investigation of primary dyslipidemia and sitosterolemia was identified, a rare genetic condition in which lipid metabolism is altered. In 2021 returned to the emergency room due to HF decompensation. During hospitalization, progressive worsening conditions evolved presenting multiple organic dysfunctions despite the inotropic drug administration. Chosen LVAD (HeartMate 2) implantation as destination therapy, since the patient presented heart transplantation contraindication, because of the elevated immunologic panel due to multiple platelet transfusions received. Sitosterolemia is a genetic, autosomal recessive condition, with identified loss-of-function variants in ABCG5 and ABCG8 genes, leading to absorption increase and reduction in the biliary excretion of phytosterol and cholesterol, resulting in prominently elevated serum concentrations. Thus, these patients present tendinous or tuberous xanthomas and early atherosclerosis, similar to familial hypercholesterolemia. The association between the clinical condition of serum sitosterol increase to levels above 1 mg/dl, and pathogenic variant confirmation in the associated genes listed above confirms the diagnosis. The treatment consists of sterol ingestion reduction, so these patients must avoid ingestion of sterol-rich plants such as corn, sesame seeds and oil, peanut, soy, margarine, avocado, and chocolate, associated with standard drug therapy such as ezetimibe and bile acid sequestrants. Sitosterolemia is a rare disease, being of paramount importance its identification to avoid early atherosclerosis. Diagnosis delay has limiting consequences, amongst them, acute myocardial infarction and subsequent progression to advanced HF. 111095 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS SÉRGIO LAÉLIO PEREIRA DA SILVA JÚNIOR1, Bruno Gonçalves Garcia1, Mariana Mancilha Carvalho dos Santos1, Carlos Henrique Oliveira Frango da Silva1, Sérgio Laélio Pereira da Silva Júnior1 (1) National Institute of Cardiology - NIC Introduction: Spontaneous coronary artery dissection (SCAD) has a low incidence – it represents 0.1 to 4% of ACS cases. However, the cases are underdiagnosed, especially in the early stages of presentation. The evolution of this entity is unpredictable. Keywords: Abstract: A 42-year-old woman with low cardiovascular risk, hospitalized after typical chest pain, with an initial diagnosis of acute myocardial infarction with ST-segment elevation, not thrombolysed, Killip I. Transferred to tertiary hospital for invasive anatomical stratification two weeks after initial event. She presented contractile alteration in the anterior and anteroseptal walls, with global systolic dysfunction of the left ventricle. Invasive anatomical evaluation showed SCAD, involving the left main coronary artery (LMCA) up to the middle third of the anterior descending artery, with TIMI 2 flow. Due to the technical difficulty of percutaneous resolution, clinical and hemodynamic stability of the patient, conservative treatment was initially chosen. After the reappearance of angina at rest, during hospitalization, the Heart Team indicated coronary artery bypass graft surgery, considering the feasibility of the surgical technique of vascular graft anastomosis. The postoperative course was satisfactory, without complications. Conclusions: The diagnostic difficulty of SCAD is a big challenge for Heart Team. The clinical presentation of ACS and/or cardiac arrest in a young patient with low cardiovascular risk strengthens the hypothesis. The diagnosis is mostly angiographic. It includes differentiating iatrogenic and secondary entities to coronary atherosclerosis. The classic angiographic feature – the radiolucid intimal flap is present in less than 30% of non-atherosclerotic cases, which underdiagnoses the condition. The prognosis is a reflection of the degree of involvement of SCAD and according to the modality of proposed therapeutic intervention. Estimates are drawn from retrospective series evaluating outcomes of heart failure, 10-year death, infarction, or dissection recurrence. Cases treated conservatively or with revascularization surgery had better outcomes when compared to percutaneous treatment. 111126 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIANA FERNANDES GUIMARÃES1, Carolina Resende Mariz1, Joaquim Márcio Duarte e Silva1, Viviane Campos Barbosa de Sena1, Aline Reis Bereta1 (1) Instituto Nacional de Cardiologia (INC) Introduction: Kawasaki disease (KD) is an acute vasculitis of unknown etiology that affects medium-caliber arteries. Coronary artery aneurysm is the most feared complication. Case Description: F.N.C, female, 10 years old, diagnosed with KD at nine months. Received immunoglobulin on the 24th day after the onset of symptoms and did not use acetylsalicylic acid. Four years after diagnosis, she evolved with a giant coronary aneurysm. Intake echocardiogram with normal cavitary dimensions and contractility, dilated left coronary artery (LC), greater aneurysm diameter of 0.7 cm. Angiotomography of the coronary arteries with aneurysm (0.63 × 0.55 cm) of the anterior descending (AD). Ectasia in circumflex (Cx) ostium. Giant aneurysm in right coronary (RC) (2.3 × 2.0 cm) with thrombus and subocclusion. Initially, opted for clinical follow-up, since the patient was asymptomatic and scintigraphy did not show stress-induced myocardial ischemia. After five years, the patient developed atypical chest pain and a new scintigraphy suggestive of stress-induced ischemia was performed in the anterior segments of the left ventricle. Coronary angiography presented mild to moderate lesion in the trunk and aneurysm in the LC. Bifurcation of AD with Diagonal Artery presenting significant stenosis. Ectasia in Cx. Occluded RC, with recanalization of the posterior ventricular branch from the collateral branch of Cx. The team indicated myocardial revascularization surgery. Patient awaiting procedure. Conclusion: KD with coronary artery aneurysm is a cause of acute myocardial infarction in early adulthood. It is currently expected that more than 10,000 patients with cardiovascular sequelae have reached adulthood. The understanding of pediatricians and clinicians about this pathology is fundamental. 111155 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KAROLYNE MOURA RIQUE DE OLIVEIRA1, Fábio Augusto de Luca1, Ernaque Viana Malta1, Ítalo Bruno dos Santos Sousa1, Júlia Galvani Nobre Ferraz1 (1) Hospital São Luiz – Rede D’or Introduction: Cardiac tumors are benign or malignant neoplasms arising primarily in the inner lining, muscle layer, pericardium or the surrounding of the heart, and can be primary or metastatic. The incidence of cardiac tumors is observed to be 0.02%, based on 22 large autopsy data from a United Kingdom center. Almost 75% of all primary tumors of the heart are benign and include myxoma, papillary fibroelastoma, rhabdomyoma, fibroma, lipoma and hemangioma. The lipomas of the heart are encapsulated tumors that are composed primarily of mature fat cells and can originate either from subendocardium, subpericardium or myocardium. Clinical manifestations depend on tumor size and location. The main method for demonstrating lipomatous tumors consist in computed tomography (CT) and magnetic resonance imaging (MRI). Case Report: 42-year-old obese woman with dyspnea on exertion for 8 months, in addition to lower limb edema, orthopnea, and paroxysmal nocturnal dyspnea. A transthoracic echocardiogram detected a mediastinal mass with extrinsic compression of the right atrium and interatrial septum of approximately 72 × 54 mm. Subsequently confirmed by cardiac CT and MRI, suggesting cardiac lipomatosis. On examinations, cardiac chambers had preserved dimensions and biventricular systolic function, without areas of infarction or myocardial fibrosis. The patient underwent thoracotomy for resection of the mass, however, in the intraoperative period, it was visualized multiple tumors with a lipomatous appearance, infiltration in the wall of the right atrium and ventricle, right coronary artery, pulmonary artery trunk extending to the left atrium wall, unresectable. Histopathological examination confirmed cardiac lipomatosis. Thus, the patient had a benign but unresectable primary cardiac tumor with an indication for heart transplantation as the only possibility of a cure. Conclusion: Cardiac lipomas are observed rarely and mostly consist of benign tumors, frequently remaining asymptomatic during a patient’s lifetime, but our patient had heart failure-like symptoms. Surgical excision is the treat and has good long-term success and low morbidity. Unfortunately, our patient has an unresectable masses, requiring heart transplantation. It is imperative that cardiac tumors are treated early without making a benign-malignant distinction. In this case, although benign, the lipomatosis was not subject to resection due to the involvement of multiple vital structures. 111157 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING ANNA XIMENES ALVIM1, Maicon Kirchmaier2, Weliton Mendes Apolinario5, Marcos Emanuel Rodrigues2, Thais Ortega Teixeira Guerra6 (1) Hospital São Jose do Avaí; (2) Universidade Iguaçu Campus V – Itaperuna (UNIG); (3) Faculdade Metropolitana São Carlos (FAMESC); (4) Cursos de Urgências Emergências (CUREM); (5) Universidade Federal Fluminense (UFF); (6) Faminas Introduction: The estimated prevalence of coronary anomalies in the general population is 1.3%, however it is variable and has been described in the literature between 0.21% and 5.8%. In most cases, these are isolated findings, without clinical or hemodynamic repercussions. However, they may be associated with other heart defects, such as valvular heart disease and congenital heart diseases, which may be the cause of myocardial ischemia and sudden death, including sudden cardiac death in young athletes, so their diagnosis and early treatment are important. Case Report: In normal coronary anatomy, the right coronary artery arises from the right coronary sinus (RCS), and the left main coronary arises from the left coronary sinus. It crosses the pulmonary main posteriorly, bifurcating into anterior descending and circumflex. There are reports in the literature of several coronary anomalies, among them, anomalous origin of the circumflex or anterior descending arteries from the RCS. However, there is no description in the literature of cases in which both coronary arteries (right coronary, circumflex and anterior descending arteries) leave the RCS in separate ostia. M.A.O, 61 years old, female, referred by the cardiologist for coronary angiotomography due to atypical chest pain. Physical examination without major changes. The exam performed did not show obstructive plaques, but a coronary anomaly not yet described in literature was found. All coronary arteries emerging from the RCS and with separate ostia. The course of the coronary arteries did not show any characteristics of malignancy, but the RCS was dilated in comparison to the non-coronary and left coronary sinuses. The patient is being followed up by a cardiologist who was alerted to the anomaly found. Conclusion: Angiotomography of the coronary arteries has been essential in the propedeutic of patients with low to moderate cardiovascular risk to detection and exclusion of atherosclerotic plaque. In addition, it presents other highly relevant information, such as the evaluation of coronary anomalies, as described in the case above. 111169 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CAMILA DALCOMUNI DOS SANTOS1, Igor Henrique Silva Leite1, Rafael Vieira Fernandes1, Airton Salviano de Sousa Júnior1, Barbara Porto Valente1 (1) Instituto Dante Pazzanese de Cardiologia – IDPC Fungal endocarditis is an uncommon affliction with high mortality rate; it represents 2% of infectious endocarditis cases with a mortality rate of about 50% and many times diagnosed post-mortem.¹² It most frequently affects an adult’s aortic or mitral valves. Standardized treatment requires antifungal therapy associated with surgical treatment.3 If there is an uncontrolled fungal infection, an early valvular replacement is recommended (<7 days). Patients that have received only medical treatment have a 100% mortality rate.² 78-year-old male, diabetic, with a previous bioprosthetic aortic valve replacement due to severe aortic stenosis, attended the emergency department with fever for the past two months, initially once a week and recently twice daily, associated with nonspecific unwellness and chills. He brought transthoracic echocardiogram that suggested prosthetic valve endocarditis. The patient was hospitalized. Blood cultures were taken and a transesophageal echocardiogram done, which showed typical vegetation annexed to the prosthetic’s aortic ring (7 × 7 mm) with mobile component projecting out of the left ventricle and restricting the opening of the prosthetic valve in this region, causing an increase of its gradients. A multi-sensitive Candida parapsilosis was shown in the blood cultures and then Anidulafungin 200 mg/day was initiated for an estimated 42-day treatment. The case was discussed with Heart Team because of the high surgical risk and the early need of valve replacement. Initial course of action had us schedule surgery for the seventh day of the antifungal treatment. Patient maintained daily fever and blood cultures were collected every 72h showing increase in Candida parapsilosis in all samples and surgery was sped up. The patient was submitted to a bioprosthetic aortic valve replacement with amplification of the ring due to the presence of abscess evidenced in mid-surgery. The patient was admitted in critical condition to the ICU, hemodynamically unstable in use of ascending vasoactive drugs, in vasoplegic shock with refractory septic component. The patient remained refractory to instituted measures evolving to death 24h after surgery. Fungal endocarditis is a rare but extremely serious condition, and a multidisciplinary approach is extremely important. Discussing a case of reoperation with high surgical risk with all those responsible for the care is the role of Heart Team and was essential to decide the best time for the surgery. 111174 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CAMILA DALCOMUNI DOS SANTOS1, Rafael Vieira Fernandes1, Airton Salviano de Sousa Júnior1, Igor Henrique Silva Leite1, Barbara Porto Valente1 (1) Instituto Dante Pazzanese de Cardiologia – IDPC Introduction: A myocardial bridge (MB) is typically asymptomatic, but sometimes can lead to a myocardial infarction with several mechanisms such as: coronary spasm, thrombosis, coronary dissection, or the development of focal atherosclerosis immediately proximal to the MB. Sometimes more than one mechanism can be present. The use of intracoronary images modalities, as Optical Coherence Tomography (OCT) in patient with MB can accurately define the mechanism of the myocardial infarction and provide further guidance to management strategy. Case Report: 56-year-old male with past medical history of systemic arterial hypertension and active tobacco use, attended the emergency department with severe oppressive chest pain after a long car trip in the heat. The initial electrocardiogram showed ST-elevation in anterior leads. Coronary angiography revealed a myocardial bridge with a systolic constriction of more than 90% in the proximal third of the left anterior descending artery (LAD), OCT demonstrate a lesion <20% with no signs of rupture. The patient was diagnosed with MINOCA with multiple mechanisms such as supply and demand imbalance, cigarette-induced vasospasm and hypovolemia. The patient was treated with beta blockers and antiplatelet therapy and discharged 3 days later. There was no recurrence of chest pain at follow-up appointments. Conclusion: Myocardial bridge (MB) is a congenital anatomic anomaly whereby a length of the artery tunnels beneath a section of myocardium. It can potentially be associated with phasic arterial spasm and ischemia. About 67–98% of cases have their anatomical location in the LAD. The gold standard for diagnosis is autopsy, but angiography and OCT provides more information about MBs, such as assessment of vulnerable plaque and coronary morphology. Unlike classic atherosclerotic plaque that produces a fixed stenosis, MB produces a dynamic effect that varies with cardiac cycle, heart rate, and sympathetic tone. According to the classification by Schwarz 2009, this case is classified as type C, with altered intracoronary hemodynamics. The clinical management was maintained with pharmacological therapy, which is the mainstay treatment. Angioplasty would be suggested only for cases with refractory symptoms. The patient was conducted with medications for management of heart failure and general orientation, with no recurrence of chest pain. 111198 Modality: E-Poster Young Researcher – Case Report Category: ANTICOAGULATION NATANAEL MENDES DE ARAUJO1, Natanael Mendes de Araújo1, Ana Paula Otaviano1, André Schmidt1 (1) HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DE RIBEIRÃO PRETO DA UNIVERSIDADE DE SÃO PAULO Background: Prosthetic valve thrombosis (PVT) is an important and severe complication of valvar replacement. Until today, treatment guidelines are scarce. Methods: A consecutive series admitted to a tertiary university hospital is reported. All of them were presented in a Heart Team meeting and surgical intervention was denied by various reasons. Non-fractioned heparin was started and patients reevaluated five days later with Doppler echocardiography to identify signs of improvement. Patients with sustained leaflet impairment and/or significant transvalvar gradient, an ultra-slow thrombolytic infusion protocol (Alteplase 25 mg in 25hours) followed by non-fractioned heparin was started and after completion a new echocardiographic evaluation was performed. A descriptive analysis is presented. Results: Six patients (4 females), 53 ± 9 years with a diagnosis of PVT admitted in two consecutive years were evaluated. Three (50%) of them had a metallic prosthesis in the mitral position, two (33%) in the aortic and one (17%) in the tricuspid position. All of them were using warfarin but the INR values were infra therapeutic (1.12 to 1.88). Two of the patients with mitral prosthesis had a significant reduction of transvalvar gradient (51% mean reduction) and good leaflet mobility and did not received thrombolytic. In the other 4 patients, thrombolysis was started and after completion of the protocol a mean gradient reduction of 69% was observed. Only the patient with a prosthesis in the tricuspid position had thrombolytic protocol repeated due to incomplete recover of leaflet mobility. No hemorrhagic or embolic complications occurred. All patients were discharged alive. Conclusion: Thrombolytic treatment using an ultra-slow infusion protocol was safe and presented a high effectiveness, suggesting that large studies are needed to define its indications. 111200 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LETICIA MACACCHERO MOREIRAO1, Leonardo Giglio Gonçalves de Souza1, Gisele Messias Mattioli1, Alex dos Santos Felix1, Matheus Burigo Oliveira1 (1) Instituto Nacional de Cardiologia (INC) Introduction: Rupture of the interventricular septum (IVS) is a surgical medical emergency and it is part of mechanical complications of Acute Myocardial Infarction (AMI), which can occur between 2 and 14 days after the event. Like the other ones, it becomes increasingly rare in the “post reperfusion” era. An incidence of 0.2% is estimated in the US, mainly when associated with single-vessel transmural infarction or the first event. Even with surgical treatment, it is still followed by high rates of mortality, reaching 80% in the first 30 days if not corrected. Clinical Case: R.S.M., 62 years old, male, white. Carrier of Diabetes Mellitus and dyslipidemia, he reports a sudden episode in October/2020 of chest pain. Diagnosed with AMI at the time, not reperfused. In the following two weeks, the pacient developed dyspnea on exertion, orthopnea associated with worsening of functional class (NYHA II). On physical examination, he presented a holosystolic murmur in the lower left parasternal border. A transthoracic echocardiogram was performed, with diagnosis of ventricular septal defect measuring 1.8 cm associated with multiple communicating orifices, LV > RV shunt showing a gradient of 85 mmHg. Coronary angiography showed severe subtotal lesion in the right coronary artery. The patient was hospitalized after a year and a half of the acute event for evaluation of surgical treatment, maintaining the symptoms described. After discussion with Heart Team, we opted to correct communication with bovine patch. Conclusion: Rupture of the IVS is a rare mechanical complication of AMI, with high mortality rates if not corrected early. The illustrated case presents a patient with a late diagnosis of this complication, a large area of communication and a favorable outcome even without initial surgical correction, which is rare according to the literature. BIBLIOGRAPHY: ABDULA, A.D. et al. Mechanical Complications of Acute Myocardial Infarction: A Scientific Statement From the American Heart Association. American Heart Association. Jul/21. THIELE H., ABBOTT J.D. Acute myocardial infarction: Mechanical complications. UpToDate, 2022. 111205 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LEONARDO PAIVA OHASHI1, Luiz Augusto de Andrade Costa1, José Cícero Stocco Guilhen1, José Honório de Almeida Palma da Fonseca1, Célia Maria Camelo Silva1 (1) Escola Paulista de Medicina/Universidade Federal de São Paulo Aortic dissection is a serious disease with a high mortality, characterized by the delamination of the media and intima layers of the vascular wall, promoting a rupture in the endothelium of this vessel. Despite of being studied in the adult population, it is rare in the pediatric population and there are few findings in the literature, which makes the management of these cases quite challenging. In this case report, a patient 9 years and 9 months old, male, 28 kg, student, was admitted on 24 of april 2021 to the emergency department of a tertiary hospital with a complaint of abdominal pain within the last 3 hours. He was accompanied by his mother, who revealed that the patient woke up with severe abdominal pain in the upper abdomen region (9 on a scale of 10). The pain radiated to the back and substernal region, and was accompanied by nausea and three episodes of vomiting. He denied similar previous events and any change in bowel habits, as well as fever or urinary symptoms. At birth, he was diagnosed with polycystic kidney disease and evolved with the need for a kidney transplant performed at the of four. There was no family history of aortic disease or a family history of sudden death. On admission physical examination, the physical examination was innocent, except for a absent peripheal pulse on the left upper limb. The admission tests showed mild leukocytosis with no deviations, with other laboratory tests within the normal range. Acute abdome routine X-rays were performed that only showed the presence of mediastinal widening. Aortic CT was then performed (figure 01), with the presence of a dissection flap that extended from the aortic arch to the bifurcation of the common iliac arteries. After a hypertensive peak, the patient evolved for a cardiorespiratory arrest anda despite all efforts during the CPR the death was confirmed at 00:00. This pathology can occur at this age group in 22% of cases, but, classically is relates to congenital anomalies, mainly those of the connective tissue. This way, early diagnosis and treatment are essential for better clinical outcomes. 111213 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY LUÍS HENRIQUE COELHO PINTO1, Gabriela Zamunaro Lopes Ruiz1, Claudio Leo Gelape1, Luiz Guilherme Passaglia1, Maria Letícia Leão Lana1 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais – HC-UFMG Introduction: Konno-Rastan procedure is an anterior aortoventriculoplasty originally described for the correction of congenital aortic stenosis, whether subvalvular, valvular or supravalvular. We report a case of an adult patient evolving with a major mismatch of a mechanical aortic prosthesis after 5 invasive surgical procedures due to congenital stenosis of the aortic valve and left ventricular outflow tract (LVOT). Case Description: Female patient, 37 years old, with congenital aortic valve disease associated with LVOT stenosis, who underwent 5 other surgical procedures – aortic commissurotomy in 1996 and 1999, valve replacement for a biological prosthesis in 2009, valve replacement for a mechanical prosthesis in 2013 (No. 19 – Standard), exploration of the valve with 3 linear incisions in the fibrosis of the interventricular septum (subvalvular) – evolving with NYHA functional class III dyspnea, chest pain, palpitations and presyncope on exertion. Transthoracic echocardiogram (TTE) showed a 40 mm left atrium, 25 mm aorta, left ventricle with mild hypertrophy, normal cavity and preserved ejection fraction. Mechanical aortic prosthesis with thickened discs, preserved opening and mobility. Maximum gradient of 87 and mean of 52 mmHg, valve area of 0.9 cm2. The patient underwent cardiac surgery in January 2022, using the Konno-Rastan technique, in which a section of the valve annulus, enlargement of the annulus and LVOT with section of the interventricular septum and septal myectomy were performed, followed by reconstruction of the annulus and septum with bovine pericardium patch and St. Jude model Regent No. 21 mechanical aortic prosthesis implantation. There were 134 minutes of cardiopulmonary bypass and 116 minutes of aortic clamping. The patient evolved in the postoperative period with severe vasoplegia and complete atrioventricular block. Bichamber pacemaker implantation was performed 8 days after surgery. Postoperative TTE showed a reduction in the mean transprosthetic gradient to 10 mmHg. She was discharged from hospital 16 days after surgery. The patient remains under follow-up, asymptomatic, maintaining echocardiographic parameters within normal limits. Conclusion: The Konno-Rastan procedure is a complex tecnique that proved to be an adequate option for the correction of the LVOT stenosis and mismatch presented by this patient, since it involved valvular, subvalvular and supravalvular stenosis not corrected in previous approaches. 111221 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISABELLE MENDES RODRIGUES SALOMÃO1, Alessandra Arnez Pacheco1, Daniel Xavier de Brito Setta1, Julia Paulo Mourilhe Rocha1, Ricardo Mourilhe-Rocha1 (1) Hospital Pró-Cardíaco Introduction: Spontaneous coronary artery dissection (SCAD) is an uncommon cause of myocardial infarction (AMI), reaching approximately 1% of cases and may be associated and may be rarely associated with carotid dissection. In most cases, it is underdiagnosed and may evolve to worse prognosis and even sudden death. Case report: Female, 36 years old, white, with a history of arterial hypertension, gestational diabetes and overweight. She reported stabbing chest pain, 10+/10, for 40 minutes, at rest, without irradiation, associated with sudden dyspnea, nausea and sweating. Normal electrocardiogram (ECG) and positive troponin I (TnI). Reported improvement in her symptoms after aspirin and clopidogrel and was transferred to another hospital asymptomatic. A new ECG was performed, which showed T wave inversion in the anterior and inferior walls and TnI us = 3,950 ng/mL. Echocardiogram (ECO) with severe left ventricular (LV) systolic dysfunction, hypokinesia of the basal segments and akinesia of the middle and apical portions of the LV. Coronary angiography (CAT) was performed, which showed spontaneous anterior descending dissection (ADA), 1st. diagonal, septal and 1st. marginal with TIMI III flow. One day after the CAT, she evolved into a new chest pain with hemodynamic stability and ECG with a new ST elevation in the inferolateral wall, being maintained with ASA, clopidogrel, statin and associated with bisoprolol. Cardiac resonance was performed, which showed recent transmural AMI affecting the anteroseptal wall of the LV (7% infarcted mass) and subendocardial AMI in the inferolateral wall (3% infarcted mass). Coronary CT angiography with absence of atherosclerosis with images of dissections in the middle third of the ADA and moderate dissections in the distal third of the right coronary artery. Angiotomography of the neck and skull showed saccular aneurysmal dilatation in the ophthalmic segment of the right internal carotid artery (ICA) measuring 6 mm and dissection in the distal cervical segment of the left ICA. After clinical control, she was discharged from the hospital in NYHA functional class 1 with improvement in LV function by ECO. Conclusion: SCAD is more prevalent in young women with few risk factors for coronary artery disease and may be related to fibromuscular dysplasia, pregnancy and the puerperium. The association with vascular alterations in other sites must be resherched in those patients. Conservative treatment had been chosen in most cases. 111223 Modality: E-Poster Young Researcher – Case Report Category: NURSING EMILY JUSTINIANO1, Rejane Reich1, Juliana Kruger1, Dulce Daise Guimaraes1, Paola Severo Romero1 (1) Hospital de Clínicas de Porto Alegre Introduction: Aortic stenosis is the most prevalent acquired valvular heart disease in the Brazilian population and has high morbidity and mortality. Transcatheter aortic valve implantation (TAVI) is an established procedure for the treatment of symptomatic stenosis for patients considered inoperable, at high risk and intermediate surgical risk. Objective: To report the first case of balloon-expandable prosthesis implantation in a degenerated self-expanding prosthesis performed in a public university hospital in southern Brazil. Case description: An 86-year-old female patient with a history of TAVI CoreValve prosthesis in 2012 was hospitalized with an echocardiogram showing aortic prosthesis with severe stenosis and moderate insufficiency, and a new TAVI (valve in valve) was chosen. The catheterization laboratory team previously performed combinations with the operating room team due to the possibility of conversion to a surgical approach. In addition to alignments with regard to materials, a team of professionals developed strategies to deal with intercurrences. The procedure was performed in September 2021, under general anesthesia, with transesophageal echocardiography follow-up. A balloon-expanded prosthesis, Sapien 3 23 mm, was implanted inside a previous prosthesis, by puncture in the right femoral artery (7 and 14 French introducer) and left femoral artery (7 French introducer). Passed 6 French introducer in the right femoral vein for transvenous pacemaker and in the right radial artery for catheterization of the right coronary artery. Hemostasis was performed with a suture closure device (Perclose) in the right femoral artery and by compression in the other access sites. Patient transferred to intensive care unit extubated and without vasopressor. The procedure time was 215 minutes and the room time was 337 minutes. Four medical specialties participated in the procedure, a nurse, two nursing technicians, a radiology technician, a prosthetic representative, in addition to a perfusionist and an instrumentation specialist in cardiac surgery present at the unit. The patient evolved satisfactorily and was discharged from the hospital on the tenth day after the procedure. Conclusions: The nurses of the catheterization laboratory, together with a team of professionals, actively participated in the organization of material resources and the logistics outlined for the care of a possible surgical approach. The procedure, although complex, was uneventful. 111234 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES DANIEL FIORAVANTI GIMENEZ1, Elaine dos Reis Coutinho1, Eduardo Hadad Cherulli1, Marina Romano Capellini1, Aloísio Marchi Rocha1 (1) Hospital da PUC- Campinas Introduction: The Mucopolysaccharidosis is a group of rare autosomal recessive metabolic disorders which affects multiple systems, including cardiovascular. It’s characterized by deficiency of lysosomal enzymes (alpha-L-iduronidase) that leads to accumulation of glycosaminoglycans in tissues and in consequence, tissue dysfunction. Mucopolysaccharidosis type 1 (MPS1) has an estimated prevalence of 1:100.000 births. This case reports a patient with MPS1, Scheie phenotype, with severe aortic stenosis. Case Report: A 47-year-old woman with MPS1 diagnosis since birth and with preserved ejection fraction heart failure (echocardiogram 03/21: left ventricular ejection fraction (LVEF): 51%) was admitted at emergency room with low cardiac output accompanied by pulmonary congestion due to COVID and Bacterial Pneumonia. The echocardiogram revealed: LVEF:25%, globally reduced, absence of segmental disorders, left chambers enlargement with Left Ventricular hypertrophy, malformed aortic valve (quadricuspid aspect) with mild thickening and calcification, critical stenosis and subtle insufficiency. Also, there was an severe mitral insufficiency with ring dilatation, apical systolic closure and posterior leaflet prolapse. The cardiac surgery team was opposed to an aortic valve replacement procedure due to high surgery risk. TAVI was not possible due to an unfavorable anatomy for the procedure and iodine allergy. Regarding her decreased mobility and NYHA functional class I, she was discharged and keeps her follow up in the ambulatory with optimized clinical therapy. In less severe MPS phenotypes, there may be valvular disorders independent of existing risk factors as congenital or rheumatic atherosclerosis. Therefore, the cardiological follow up is important for those patients, as pointed out in the case, in which an adult patient presents severe aortic stenosis with ventricular function loss secondary to MPS 1. Conclusion: The cardiovascular disease emerges subtly in attenuated phenotypic presentations of MPS, contributing to the premature mortality of these patients. Besides being rare, MPS 1 may present late cardiovascular implications, even in broader cases, and severe consequences that compromise the patient’s lifestyle. The case is even more uncommon not only for its underlying disease, MPS 1, but for the aortic valve’s anatomic modification (quadricuspid), not often reported in literature in this syndrome. 111233 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES AMANDA AYAKO MINEMURA ORDINOLA1, Salomon Soriano Ordinola Rojas1, Caroline Spagnol1, Viviane Cordeiro Veiga1 (1) Beneficência Portuguesa de São Paulo Intracranial pressure (ICP) assessment is a method that can guide the hemodynamic management of patients undergoing procedures and conditions associated with neurological disturbances. In this paper, two cases were reported. The first case is a 40-year-old male with Marfan’s syndrome, admitted with aortic endocarditis which underwent valved conduit exchange with extracorporeal circulation (ECC) 182 minutes, the patient presented one episode of tonic-clonic seizure. Non-invasive ICP (NIICP) showed altered brain compliance (figure 1). The management included increase in sedation and prevention of secondary cerebral ischemia. New NIICP showed well response to the neuroprotection management (figure 2). The second is a 64-year-old female diabetic patient admitted for cytoreductive surgery due to colon adenocarcinoma. Edema and facial plethora were presented on admission, associated with high central venous pressure and high systolic volume variation. The hypothesis of superior vena cava syndrome was raised and confirmed by evidence of thrombosis related to catheter. The NIICP showed altered brain compliance (figure 3), with improvement after cerebral protection measures. In all cases reported, NIICP identified morphological changes in the ICP waveform and throughout patient management in the intensive care unit. Noninvasive method assisted in clinical decision-making regarding the optimization of protocols adapted for neuroprotection. 111236 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM YURI CAVALCANTI ALBUQUERQUE TENORIO1, Priscila Alves da Silva2, Stephanny Isabelly Pessôa Neri de Araujo2, Igor Vieira Lima Alexandre2, Francisco de Assis Costa2 (1) Hospital Veredas; (2) Centro Universitário Tiradentes Introduction: Several cardiac complications have been described after the beginning of pandemic Covid-19, such as myocardial ischemia, acute cardiac heart failure, arrhythmia and myocarditis. Case description: M.J.F.S., female, 43 years old, denied any chronic disease or use of medication, admitted to the ward of a high complexity hospital presenting cardiac heart failure (NYHA IV) and oedema in the lower limbs. The patient had Covid-19 infection two months before and presented a normal routine transthoracic echocardiography (TT-ECHO). After the admission, a new TT-ECO presented global dilatation of cardiac chambers, specially left atrium (LA) and signs of coronary sinus of valsalva rupture and Aortic-Right Atrium (Ao-RA) fistula. These findings were confirmed in a transesophageal echocardiography, that presented left ventricle ejection fraction of 69%, rupture of sinus of valsalva aneurysm at aortic root promoting strong shunt from aorta to right atrium and cardiac chambers dilatation. Thoracic angiotomography described bigger dimensions of the right atrium and signs of pulmonary thromboembolism. Therapeutic decision included surgical correction of Ao-RA fistula, which occurred with complication of complete heart block requiring transitory pacemaker and cardiac arrest successfully reverted to systemic circulation. Definitive cardiac pacemaker was implanted and the patient was discharged in a stable clinical situation after 59 days of hospital stay. Conclusion: The clinical report presents rupture of sinus of valsalva aneurysm leading to Ao-RA fistula and pulmonary thromboembolism two months after Covid-19 infection. As a consequence, the authors suggest linkage between Covid-19 and thrombogenic state and inflammatory process leading to endomyocardial inflammation. 111245 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES ARIANE ZONHO WOGEL1, Andrei Carvalho Sposito1, Marcelo Sousa1, Renata Muller Couto1, Silvério Fernandes1 (1) Universidade Estadual de Campinas (UNICAMP) Introduction: Prosthetic valve thrombosis is a rare and highly lethal complication, more frequently found in mechanical valves and in the mitral position. The treatment involves systemic anticoagulation and valve replacement surgery. Thrombolysis appears as a second option, due to its complication rates and low efficacy. Case Report: A 65-year-old man who underwent mitral bioprosthesis implantation for severe secondary mitral regurgitation returns after 3 months with dyspnea and, on transthoracic echocardiography (TTE), prosthesis dysfunction due to thrombosis (Image 1: A-C). There was also a global dilation and left ventricular systolic dysfunction (ejection fraction of 17%). Due to a prohibitive surgical risk (EUROSCORE 34%), alteplase 25 mg was administered for 6 hours followed by full anticoagulation with intravenous unfractionated heparin. TTE 24 hours post thrombolysis showed thrombus reduction, improvement in valve motion, and a drop in the transvalvular gradient (Image 1: D–E). The same dose of alteplase was repeated after 48 hours, resulting in improvement in dyspnea, and in valve opening and mobility on TTE (Image 1:F–G). There was no bleeding or adverse effect and he was discharged with Warfarin. Conclusion: Fibrinolytic therapy for prosthetic biological valve thrombosis remains controversial due to the lack of evidence of its benefit and safety. Hence, its use has been sporadic and restricted to critically ill patients who are at high surgical risk. The case presented here stands out for the efficacy and safety of fibrinolysis in severe thrombosis in a mitral bioprosthesis. This report deserves consideration as a therapeutic possibility either in clinical research or in similarly unfavorable clinical conditions. 111265 Modality: E-Poster Young Researcher – Case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION PALOMA DE BORBA SCHNEIDERS1, Heloíse Benvenutti1, Ane Glauce Freitas Margarites1, Mauren Porto Haeffner1, Fernanda Cecília dos Santos de Vasconcellos1 (1) Hospital de Clínicas de Porto Alegre Introduction: Heart Failure is the final pathway for heart diseases. Considering limiting situations such as lack of donors and contraindications to the Heart Transplant (HTx) new strategies are needed. Mechanical Ventricular Assistance Devices allow patients to be able to perform physical exercises in Cardiac Rehabilitation. Description of Cases: Case 1: FPB, male, 54 years old, ischemic etiology. Ejection Fraction 32%, HTx contraindicated due to immunological hypersensitivity. After HeartMate II, had reduced respiratory muscle strength – RMS (PiMax: 18/PeMax: 37) and cardiorespiratory fitness – CRF (distance in six meters walking test: 261 m). Preserved peripheral muscle strength – PMS (28 KgF) and no self-care limitations (SCL). Case 2: JSP, female, 50 years old, chagasic etiology. Ejection Fraction 16%, HTx contraindicated due to immunological hypersensitivity. After HeartMate II, had reduced RMS (PiMax: 34/PeMax: 46) and CRF (383 m). Preserved PMS (18 KgF). After hospital discharge showed improvement in CRF (429 m). Case 3: MHPD, female, 61 years old, Dilated Cardiomyopathy etiology, worsened after chemotherapy. Ejection Fraction 13%, HTx contraindicated due to pulmonary hypertension. After HeartMate III, had reductions in RMS (PiMax: 50/PeMax: 56), PMS (17 KgF), CRF (273 m) and SCL (Katz: 2). Case 4: LBF, female, 63 years old, Dilated Cardiomyopathy etiology. Ejection Fraction 16%, HTx contraindicated due to pulmonary hypertension. After HeartMate II, had SCL (Katz: 2) and present reductions in RMS (PiMax:75/PeMax:109), PMS (28 KgF) and CRF (390 m). Case 5: AAAV, male, 61 years old. Dilated and non-ischemic etiology. Ejection Fraction 20%. After HeartMate II, had some complications (driveline infection, massive pleural effusion, multifactorial anemia) and prolonged stay (154 days). At hospital discharge, he could walk more than 30 m, but failed to perform the walking test; SCL (Katz: 3); adequate PMS (25 kgF). Conclusion: Hospital Cardiac Rehabilitation was performed with aerobic, respiratory/peripheral strength and balance training. All patients had reductions in RMS and PMS. Two of them had functional SCL and two of them had low exercise tolerance (<300 m). These patients‘ CRF may be reduced, due to low adaptive capacity to increase cardiac output during physical exercise. Potentially reversible extracardiac factors such as muscle atrophy and deconditioning seem to improve patients‘ functionality. 111274 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISMAR JUNIOR PEINADO LIJERON1, Larissa Kaline Santana Diniz1, Marina Albanez Albuquerque de Medeiros1, Soraia Rachid Youssef de Campos1, Valeria Mozetic de Barros1 (1) Instituto Dante Pazzanese de Cardiologia IDPC Introduction: Aneurysmal dilatation of the coronary arteries is found in up to 5% of the patients undergoing coronary angiography, with higher incidence in men and proximal segments of the coronary artery. The right coronary artery (RCA) is more affected (40%), followed by the left Anterior Descending (AD) (32%). Atherosclerosis, vasculitis, genetic susceptibility and post infection are reported as possible etiologies. The presence of coronary aneurysm or ectasia has been associated with a worse long-term prognosis. Case Description: Female, 48 years old, with no previous diseases, presented with an unprecedented typical chest pain after performing moderate physical exertion. On the electrocardiogram it was observed sinus rhythm with electrically inactive area in the inferior wall and positive troponin. Performed coronary angiography that showed important ectasia of RCA, AD with mild ectasia in the proximal third and other arteries with discrete parietal irregularities. Echocardiogram shows akinesia of the inferolateral wall and of the middle and basal segments of the inferior wall and ejection fraction 58%. The angiotomography of coronary arteries confirmed ectasia of RCA, measuring in its largest diameters 10.8 × 10.4 mm and thrombosis in the distal third. Started anticoagulation with warfarin and clopidogrel together with antianginal drugs and after treatment she got angina improvement and had no new thromboembolic events. Conclusion: Aneurysmal dilatation of the coronary vessels is an unusual. Antiplatelets and anticoagulation are the most addressed therapy, but there are possibilities of performing percutaneous coronary intervention or surgical resection. The treatment is still controversial, requiring more randomized studies to define the best and safest treatment strategy. 111515 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO FELICIANO FATORELLI1, Vitor Agueda Salles1, Elisangela Cordeiro Reis1, Letícia Macacchero Moreirão1, Jacqueline Sampaio dos Santos Miranda1 (1) Instituto Nacional de Cardiologia Introduction: Cardiac amyloidosis (CA) is a systemic disease caused by tissue deposition of fibrillar and insoluble protein aggregates in different organs, including the heart, leading to organic dysfunction. Different subtypes of amyloidosis can give rise to overlapping clinical manifestations. The importance of carrying out the genetic test is due to the need to correctly characterize the precursor protein for the institution of specific treatment. Thus, the present report aims to describe the case of two patients diagnosed with transthyretin-linked (TTR) AC and discuss the importance of genetic testing, as well as early diagnosis for a better prognosis. Case Report: These are two elderly patients, 71 and 83 years old, with a clinical picture of advanced heart failure, who had indicators in the anamnesis, physical examination and complementary exams of CA and whose etiology was confirmed during the workup. Both had immunoglobulin heavy and light chain form (AL) ruled out with investigation for monoclonal light chains by means of serum and urinary immunofixation, in addition to measurement of the serum kappa/lambda ratio. On pyrophosphate scintigraphy, grade III uptake was identified, one of them with extensive involvement of the right ventricle, corroborating an advanced stage of the disease. Despite the high prevalence of wild type ATTR CA in the elderly population, both were tested for mutation of the ATTR V122l gene. This mutation is well established as a pathogenic variant, with heterozygosity being the most common. It presents after the age of 60 with predominantly cardiological involvement, with a higher frequency in Afro-descendants, which possibly makes it frequent in our country. Specific therapy was instituted with selective stabilizers of TTR tetramers with good tolerance and the patients are in follow up. Conclusion: Thus, CA could be considered an underdiagnosed condition, rather than a rare disease. The patient’s journey to diagnosis is long; it is estimated that there is a delay of more than 2 years from symptom onset to diagnosis, with the involvement of an average of five different professionals. Thus, it is essential to disseminate knowledge about CA; clinicians and cardiologists must give greater consideration to this entity, aiming at earlier diagnosis and adequate therapeutic guidance, thus improving patient prognosis and survival. 111931 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY LEONARDO PAIVA OHASHI 1, Diego Felipe Gaia dos Santos1, Paulo Matheus Sanches de Souza1, Aislan Henrique Bezerra Pinheiro1, Pedro Reges Pereira Meira1 (1) Escola Paulista de Medicina/Universidade Federal de São Paulo Pericardial effusion is a common finding in clinical practice with a spectrum manifestations ranging from asymptomatic mild effusions to cardiac tamponade. As a minimally invasive form of treatment, video-assisted pericardial window is an alternative intervention. A patient, 73 years old, male, referred to a tertiary hospital for investigation of Asthenia, dyspnea, chronic low back pain, loss of 10 kg in 3 months and solid expansive lesion in the pre-sacral region. The patient had a history of hypertension, Kyphoscoliosis, Pectus Carinatum, Alcoholism for 15 years and previous treatment of gastric ulcer. Laboratory and imaging findings at admission and during hospitalization culminated in a diagnosis of Multiple Myeloma. During the institution of chemotherapy, an echocardiographic follow-up was performed, which showed growth of the pericardial effusion lamina, which progressed from asymptomatic to the presence of signs of ventricular restriction and symptoms of cardiac tamponade. As a result, it was decided to perform a pericardial window before hospital discharge. However, the presence of previously described thoracolumbar deformities, added to the massive abdominal hernia caused by diastasis of the rectus abdominis, precluded the classic subxiphoid surgical approach or through lateral thoracotomy. It was then decided that the only therapeutic possibility would be the use of videothoracoscopy (figure 1). In perioperative care, multiparametric monitoring, invasive blood pressure acquisition through the right radial artery puncture, central venous access in the right Femoral Vein, and Orotracheal Intubation were performed. Intraoperatively, 400 ml of serohematic fluid was released, with a drain in the chest on the right. After the procedure, the patient improved with dyspnea, maintaining chest drainage for 5 days. This is a patient diagnosed with Multiple Myeloma who evolved during hospitalization with significant pericardial effusion and signs and symptoms of cardiac tamponade. Due to the chest deformity that made conventional drainage unfeasible, a video-assisted intervention was chosen. 111330 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES RAIANE FONSECA SILVA HERDY1, Douglas Fernandes de Souza1, Nathalia Targa Silva1, Alina de Souza dos Santos1, Marcia Barbosa de Freitas1 (1) Instituto Nacional de Cardiologia Introduction: Mechanical circulatory support has been increasingly used in refractory cardiogenic shock associated with conventional treatment in patients with severe heart failure (HF). The literature indicates use as a bridge to recovery for transplantation (Tx) for decision or target therapy. We report the case of using venous-arterial extracorporeal membrane oxygenation (ECMO) in a young patient with cardiac Tx indicated already in use intra-aortic balloon (IAB) and maintenance of low cardiac output that recovered his clinical condition and subjected to Tx. Case Description: A 38-year-old male patient with dilated cardiomyopathy for 2 years developed severe heart failure in NYHA III–IV despite optimized medical therapy. He was indicated cardiac Tx due to disease progression. The patient was hospitalized several times due to HF decompensation in a cold and wet hemodynamic profile, requiring high-dose inotropic support. In the current hospitalization, even with inotropic, his case deteriorated, with several parameters of poor tissue perfusion (lactate increase, renal function worsening, increased transaminases and bilirubins). Already with association of 2 inotropic (Dobutamine and Milinone), as well as vasopressor (norepinephrine and vasopressin), was indicated a device of short permanence circulatory support, IAB. The next day there was maintenance of critical state, which led to an indication of ECMO. There was a significant improvement of oliguria, significant decrease in lactate and plasma creatinine. Two days later, a compatible organ emerged, the patient underwent Tx, extubated six days after the procedure and recovers well from necrotizing pneumonia acquired during hospitalization. Conclusions: ECMO is indicated as a bridge to another device or to Tx in severe heart failure when the patient has refractory cardiogenic shock. Therefore, patients with non-recoverable cardiac function should not be selected. The success of the therapy involves a multidisciplinary team trained with experience in this technology. In the case presented, the use of ECMO associated with systematized care was fundamental to keep the patient viable until the emergence of a compatible organ. Thus, heart transplantation was possible and the patient recovered from the baseline clinical condition, illustrating a good example of the successful use of ECMO as a bridge to cardiac Tx. 111332 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES FELIPE KESSLER PEREIRA1, Luanna Damasceno Amaral de Sousa1, Julia Paulo Mourilhe Rocha1, Daniel Xavier de Brito Setta1, Ricardo Mourilhe-Rocha1 (1) Hospital Pró Cardíaco Introduction: Infective endocarditis of the right side (IELD) of the heart is an entity where there is microbial involvement of the tricuspid or pulmonary valves and is responsible for 10% of all cases of infective endocarditis (IE). Case Report: M.B.B, 59 years old, male, with arterial hypertension, dyslipidemia, type I diabetes mellitus, hypothyroidism, coronary artery disease, was admitted to the hospital with atypical chest pain associated with chills, fever, and vomiting. Acute coronary syndrome was ruled out and the hypothesis of myopericarditis was raised, and pharmacological treatment with colchicine was initiated. Despite the established treatment, the patient continued to experience chills and vomiting. Blood cultures were collected and antimicrobial therapy with vancomycin was administered empirically. There was growth of vancomycin-sensitive Staphylococcus caprae, but in the investigation of IE with transesophageal echocardiography, there was no evidence of vegetations. A scintigraphy with labeled leukocytes was then performed to screen for an infectious focus and the tricuspid valve was the only point of evidence of an inflammatory process, confirming the diagnosis of IE, subsequently being treated with antibiotic therapy for 6 weeks. Discussion: Staphylococcus caprae is part of the group of coagulase negative staphylococci (CoNS) and was isolated for the first time in goat milk. Later, it was described as a component of the microbiota of healthy human skin, nails, and nasal mucosa, living symbiotically with its host. S. caprae is a rare cause of infective endocarditis, even more so of native valve. In the literature review, we found a single case report, published in 1995 by Vandenesch et al, of infective endocarditis caused by S. caprae. The case was reported in situ mitral valve involvement, which was treated with intravenous antibiotics for 2 weeks and early surgical removal of the vegetation, preserving the valve, with a good clinical outcome. The main predisposing factors for S. caprae infection are recent use of antibiotics, malignancies, diabetes mellitus, chronic renal failure, open fractures, immunodeficiency, and immunosuppression associated with systemic or local corticosteroids, chemotherapy, radiotherapy. Seng et al. observed that 21 of the 25 patients with S. caprae infection were male, supporting the sex preference hypothesis previously made by Shuttleworth et al. 111348 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS THALES CARDOSO WHATELY1, João Gabriel Monteiro Junqueira1, Ana Salomé Eurico1, José Ary Boechat e Salles1, Esmeralci Ferreira1 (1) Hospital Universitário Pedro Ernesto; (2) Universidade do Estado do Rio de Janeiro Introduction: The main etiology of acute ST-segment elevation myocardial infarction (STEMI) is the rupture of atherosclerotic plaque with thrombus formation. However, other less common etiologies, such as vasospasm, COVID 19 and coronary embolism, should be considered and investigated when there are risk factors, being important to define the best treatment for each patient. Case Report: Female, 56 years old, former smoker, with systemic arterial hypertension, ischemic stroke (CVA) for one month with motor sequelae (right hemiparesis) and aphasia. Sought emergency for typical anginal chest pain. An electrocardiogram was performed, which showed atrial fibrillation rhythm and ST-segment elevation from V1 to V6, DI and AVL (extensive anterior wall STEMI). She was not submitted to fibrinolytic therapy due to absolute contraindication for stroke in less than 3 months. She was referred to the hemodynamics department, and occlusion of the anterior descending artery was evidenced with a negative image, suggestive of a thrombus, without severe obstructive lesion. As reperfusion therapy, mechanical thrombus aspiration was performed followed by implantation of a drug-eluting stent, reestablishing the coronary flow. Transesophageal echocardiogram showed severe left ventricular dysfunction with anterior hypokinesia and presence of thrombus in the auricle. Cranial resonance was performed with ischemic images suggestive of thromboembolism. In view of the results of the complementary exams, the most likely hypotheses for acute coronary syndrome are atherosclerosis associated with coronary embolism caused by thrombus in the auricle related to unkown atrial fibrillation. Dual antiplatelet therapy (clopidogrel and ASA) and full anticoagulation were initially started, with outpatient and echocardiographic follow-up to assess cardioversion and adjusts in antiplatelet therapy. Conclusion: The authors describe a patient with STEMI due to high thrombotic load, recent stroke and presence of atrial thrombus, whose treatment dilemmas begin with the impossibility of using fibrinolytic therapy, the decision to perform thrombus aspiration and the definition of the best post-reperfusion pharmacolocgical therapy. 111756 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES CATHARINA TUROLLA DE ANDRADE 1, Roberta Dutra Fortes1, Luiza Gondim Toledo4, David Nigri3, Erverton Gregório de Andrade2 (1) Hospital de Força Aérea do Galeão; (2) Clinica São Vicente da Gávea – Rede dOr São Luiz; (3) Hospital Copa Star – Rede dOr São Luiz; (4) Universidade Federal do Rio de Janeiro – UFRJ Introduction: Obstructive sleep apnea (OSA) is a disorder characterized by obstructive apneas, hypopneas or respiratory effort-related caused by repetitive collapse of the upper airway during sleep. The estimated prevalence is 30 percent in males. It is essential to recognize OSA as a predisposing factor for cardiovascular disorders. This case report aims to show the occurrence of coronary vasospasm (CV) in a patient with OSA. Case Report: A 67-year-old male, body mass index 26, ex-smoker, systemic arterial hypertension, chronic coronary artery disease (three previous stents), with a known high rate of admissions in the emergency room for acute pulmonary edema (APE), has been readmitted at the hospital with similar acute symptoms started during sleep. The vital signs showed desaturation, tachypnea and high blood pressure. Respiratory auscultation with bibasal crackles. The patient has been hospitalized for clinical surveillance and further investigation for APE. Troponin was elevated in one dosage, electrocardiogram showed no acute alterations, transthoracic echocardiogram was normal and a chest computerized tomography with a pattern of congestion. Despite the initial treatment, the patient continued to have episodes of hypoxemia and dyspnea, especially during sleep, and further complementary tests were requested. The patient underwent coronary angiography, which revealed vasospasm in the circumflex artery and marginal branch with a focal lesion of 70 percent, reversing during the exam with intracoronary mononitrate infusion [figure1]. Polysomnography showed severe OSA. The patient received specific treatment – continuous positive airway pressure and oral nitrate – with significant clinical improvement, remained asymptomatic after discharge. Conclusion: This report presented demonstrates CV as a cardiovascular repercussion possibly caused by severe OSA. Recognizing OSA as predisposing factor for cardiovascular dysfunction is essential to provide adequate therapy, prevent complications, reduce symptom exacerbation and morbidity for these patients. 111377 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CAROLINA KUCHENBECKER SOARES1, Claudio Leo Gelape1, Robson de Souza Almeida Junior1, Geraldo Brasileiro Filho1, Luiz Guilherme Passaglia1 (1) Hospital das Clinicas da UFMG Introduction: Whipple’s disease (WD) is a rare chronic multisystem illness caused by the bacterium Tropheryma whipplei. Most patients have polyarthritis, malabsorption and anemia. Up to 50% can present with cardiac manifestations, being infective endocarditis (IE) with negative blood culture considered the most frequent and aortic valve the most affected. Case description: A 63-year-old woman, previously healthy, is admitted in the Emergence Department with sudden left hemiplegia. Head tomography confirmed stroke showing acute hypodensity in right corona radiata. Carotid doppler was normal and transthoracic echocardiogram showed a filamentous structure in the ventricular face of the right coronary leaflet of the aortic valve, 9.3 mm long, with moderate regurgitation (confirmed by transesophageal exam). There were no clinical or laboratory signs of infection. Serial blood cultures, rheumatoid factor, abdominal ultrasound, and urinalysis were normal. In view of the possible differential diagnosis of IE, fibroelastoma and Lambl excrescences, as well as the concern for further embolization, cardiac surgery and excision of the material was chosen for anatomopathological diagnosis. She was then submitted to aortic valve replacement with a bioprosthetic valve, an uneventful procedure. The slide evaluation showed mononuclear cell infiltrate with foamy macrophages containing PAS-positive granulations, diagnosing WD. Conclusion: Although rare, WD can be prone to recurrences and its recognition is important to initiate target therapy with parenteral streptomycin and benzylpenicillin for 14 days, followed by oral trimethoprim-sulfamethoxazole for 1 year. Unlike common IE, WD occurs mainly in native valves, without underlying disease and the anatomopathological pattern of inflammation is fundamental, as Trophyerma whipplei cannot be cultured using standard techniques. The presence of prodromes such as gastrointestinal symptoms or arthritis should also be considered in the suspicion. 111911 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING ERICA SILVA1, Bernardo Santos Resende1, Ingred Hellen de Resende Andrade1, Henrique Patrus Mundim Pena1, Carlos Eduardo Ornelas1 (1) Rede Mater Dei de Saúde Introduction: Infective endocarditis (IE) is a high mortality rate (30–50%) disease with many clinical features. Thus, some diagnoses may be challenging. Increase in the use of prosthetic materials and intracardiac devices (ICD) follows the importance of complementary tests, as 20% of EI patients have valve prosthesis or ICD. Duke criteria, traditional in IE diagnosis, are based on the presence of typical microorganisms in hemoculture and suggestive valve alteration in echocardiogram. However, in over 20% of cases, these may be inconclusive, requiring extended propaedeutics with more accurate exams. Thus, in other suspicious cases, more exams are essential for a precise diagnosis, such as Positron emission tomography (PET-CT). Case Report: F.J.S.M., 59 years old, 15 years with an aortic biologic prosthesis, was admitted to ER reporting paresthesia of left upper limb, lips and tongue, of short duration and spontaneous recovery. Neurology assessed the case and nuclear MR of the skull showed cerebral ischemia area suggesting embolic etiology. Cardiological follow-up and complementary propaedeutics were started for etiologic investigation. Transesophageal echocardiogram image suggested aortic biologic valve periprosthetic vegetation. Patient had no symptoms of infection. Serial hemoculture results showed no bacterial growth. So, under Duke criteria, IE diagnosis was inconclusive (1 major, 2 minor criteria). PET-CT with radiopharmaceutical FDG-18F aimed at cardiac assessment confirmed hypothesis showing hypercaptation of the radiopharmaceutical, circumferential, in the aortic position biologic prosthesis, compatible with valve infective endocarditis. Directed treatment initiated with empiric antibiotic therapy and heart surgery for valve replacement. Conclusions: As the use of prosthetic materials and intracardiac devices (ICD) advances, it is necessary to perform exams other than the traditional doppler echocardiography. Such information follows the European IE Treatment Guidelines (ESC 2015), which cites use of PET-CT in patients with clinical suspicion, when the evidence of valve periprosthetic abnormal activity is a major criterion in IE diagnosis. In the case described, PET-CT was essential to confirm IE diagnosis in patient with valve prosthesis, when Duke criteria were inconclusive. It is a high mortality disease, so, when it is suspected, even with atypical signs, investigation must be exhaustive to minimize risk of improper treatment. 111434 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ANNE KATHRINE MØLLER NIELSEN1, Lars Allan Larsen2, Vibeke Hjortdal1 (1) Department of cardiothoracicsurgery, Rigshospitalet, Copenhagen, Denmark; (2) Institute of cellular and Molecular Medicinen, University of Copenhagen, Denmark Background and aims: Patients with atrial septal defect (ASD) have increased mortality and morbidity. This can only be partly explained by hemodynamic changes caused by the ASD, suggesting additional underlying causes. Patients with an ASD have an increased burden of pathogenic gene variants in ASD related genes, indicating genetics as an important etiologic factor. This study aimed to investigate genetic associations in familial ASD and comorbidities. Methods: In a cohort with familial ASD we identified a family with ASD present in 12 family members in three generations. We performed whole exome sequencing on five family members older than 18 years to detect the causative gene variant in this family and evaluated phenotype. Results: We identified a novel pathogenic variant within the T-box domain of TBX5 (F232L) in all affected family members over 18 years of age. They presented with diverse cardiac phenotypes including heart failure and arrhythmias. Two carriers needed a pacemaker. Skeletal malformations where subtle with small hands as the only visible malformation. These findings propose Holt-Oram syndrome. Conclusions: We report a novel variant in TBX5 in a family with Holt-Oram Syndrome, characterized by septal defects, severe cardiac arrhythmias, and mild skeletal malformations. Clinical awareness of family history, arrhythmias, and heart failure in patients with familial ASD is important and may lead to timely treatment and uncover patients with Holt-Oram Syndrome. 111478 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES HENRIQUE TROMBINI PINESI1, Vinicius Esteves1, Francisco Monteiro de Almeida Magalhães1, Rafael Alves Franco1, Carlos Vicente Serrano Junior2 (1) Rede D’OR São Luiz; (2) Instituto do Coracao, Faculdade de Medicina, Universidade de Sao Paulo Introduction: The percutaneous approach of valvular diseases has gained increasing importance in recent years, especially in frail, elderly and high surgical risk patients. However, procedures in the tricuspid valve are still rare. Case Description: Female, 87 years old, previously independent for instrumental activities of daily living, with a history of dextrocardia, dual-chamber pacemaker and difficult-to-manage atrial fibrillation requiring atrioventricular node ablation. She presented with progressively worsening dyspnea up to NYHA class III/IV, fatigue, and progressive swelling of the extremities in the last 4 months, with two hospitalizations due to acute heart failure in this period. Transthoracic echocardiogram identified preserved biventricular function, with significant biatrial and right ventricular dilatation. The Mitral valve showed significant regurgitation with prolapse and the tricuspid valve severe regurgitation with failed coaptation and torrential reflux secondary to annular dilation. A transesophageal echocardiogram identified dilatation of the valve annulus with a slight thickening of the cusps in addition to prolapse of segments A1/A2 of the anterior cusp and P1/P2 of the posterior cusp, with significant reflux. After optimization of medical treatment, she remained symptomatic with signs of congestion, associated with worsening of renal function. After discussion with the Heart Team, considering the patient’s elevated surgical risk (STS mortality of 17,29%), a less invasive treatment was chosen. A combined procedure was successfully performed, with a percutaneous mitral valve edge to edge repair (MitraClip™) and heterotopic tricuspid treatment with percutaneous bicaval valve implantation (TricValve™). The procedure was a success despite the difficulties caused by the simultaneous multivalvular approach in a patient with dextrocardia. The patient evolved with clinical improvement and normalization of ventricular function. She was discharged with a great enhancement in functional capacity. Conclusion: We present a rare and up to the moment, the first description case of an elderly patient with dextrocardia, high surgical risk and refractory heart failure who underwent successful percutaneous treatment of the mitral and tricuspid valves simultaneously. 111481 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES RAPHAELA TEREZA BRIGOLIN GAROFO1, GUILHERME COELHO FORTES1, ISABELLE DE OLIVEIRA PARAHYBA1, CAMILA VIEIRA VILELA1, RAYZA KARLLA SALLES ARAUJO1 (1) HOSPITAL DO CORAÇÃO – ASSOCIAÇÃO DO SANATÓRIO SÍRIO Introduction: The diagnosis of infective endocarditis is often difficult. We found diagnostic limitations especially in patients with prosthetic valves and implantable cardiac electronic devices. Case Report: A 37-years-old man, diagnosed at birth with tricuspid hypoplasia, significant right ventricle hypoplasia and wide atrial septal defect with bidirectional shunt. He has been submitted to Blalock-Thomas-Taussig surgery on the right at 2 years old and on the left at 20 years old. Between 2019 and 2020 he was diagnosed with pneumonia on 4 occasions. After six months he continued to present with weight loss, low fever at night and asthenia, with elevated serum inflammatory markers. In July 2020, he sought the Emergency complaining of ventilatory-dependent chest pain and worsening of asthenia. Chest tomography showed a consolidation area in the right lung and antibiotic therapy was initiated. Even after the end of treatment, the patient remained subfebrile and with elevated C-Reactive Protein. Blood cultures were positive for Lactobacillus casei, paracasei and Rhamnosus. Due to the clinical suspicion of an undetermined infectious focus and with a negative investigation for Infective Endocarditis, an 18FDG PET/CT was performed, which showed an increase in glycolytic metabolism in a focal area next to the vascular prosthesis interposed on the left in the pulmonary systemic anastomosis. The diagnosis of prosthetic valve infective endocarditis was confirmed. After antibiotic therapy guided by blood cultures, the patient showed clinical and laboratory improvement. He was discharged asymptomatic, with a negative blood culture and normalized inflammatory markers. Lactobacillus are commensal microorganisms of humans and are recognized as a rare cause of endocarditis in people with previous comorbidities. When questioned, the patient reported daily ingestion of Lactobacillus-based probiotics. Conclusion: The FDG-18F PET/CT should be used when there is a high clinical suspicion without diagnostic confirmation with traditional methods in a patient with a prosthetic valve or intracardiac device. 111485 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RAPHAELA TEREZA BRIGOLIN GAROFO1, GUILHERME COELHO FORTES1, THAÍS DOS SANTOS VIEIRA1, LAURO MARTINS NETO1, CAROLINA TOSI PIRES1 (1) HOSPITAL DO CORAÇÃO – ASSOCIAÇÃO DO SANATÓRIO SÍRIO Introduction: Chest pain in young patients without comorbidities is a challenge. This report is relevant since the patient in the case presented with acute myocarditis associated with acute coronary thrombosis. Case Report: A 20-year-old male patient without previous diseases and with a history of myocarditis 4 years ago. He went to the emergency room with type C chest pain for four hours. An electrocardiogram was performed with no changes. Laboratory tests with troponin curve (0.638 and 2.570 – Reference 0.034). Due to the hypothesis of myocarditis, a Cardiac Magnetic Resonance (CMR) was performed, which showed anterior and anterolateral hypokinesia associated with myocardial edema, and non-ischemic late mesoepicardial enhancement in these segments. It was decided to perform coronary CT angiography due to the segmental alteration on CMR, which showed proximal occlusion in the Anterior Descending Coronary Artery (ADCA) and its diagonal branches. Coronary Angiography was performed in association with Optical Coherence Tomography, which showed acute thrombosis in ADCA without signs of atherosclerosis. The thrombus was aspirated and a stent was implanted. After these findings, the patient reported a dose of mRNA vaccine for COVID-19 21 days before admission. The coronavirus causes a condition of hyperinflammation and hypercoagulability, leading to dysfunctions of various organs, including the heart. Among the cardiac dysfunctions we can mention myocarditis and thromboembolic events. Myocarditis should be considered in young people with chest pain after mRNA vaccine. He had an episode of acute myocarditis and ADCA occlusion with high thrombotic burden without evidence of atherosclerosis, other etiologies for myocarditis and rheumatologic disease. Due to its recent vaccination history for COVID-19, it is likely that the vaccine was the protagonist of the activation of the coagulation cascade and the inflammatory response. Conclusion: This case reinforces the importance of differential diagnoses in young patients with atypical chest pain in order to promote adequate treatment. 111500 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES RODRIGO BINDA DE MAGALHÃES LOIOLA 1, Emerson Oliveira Lise3, Laura Barbosa David4, Gisele Tezolin Menezes5, Alexandre Lobato Rodrigues Guimarães6 (1) Faculdade Metropolitana de Manaus; (3) Faculdade Metropolitana de Manaus; (4) Faculdade Metropolitana de Manaus; (5) Faculdade Metropolitana de Manaus; (6) Maternidade Estadual Balbina Mestrinho Amniotic fluid embolism (AFE) is a rare and exclusive complication of pregnancy, and is characterized by the sudden onset of maternal impairment. It usually involves the cardiorespiratory and hematological systems, and can progress to cardiac arrest and coagulopathy, leading to death. The estimated incidence of AFE is 1:15,200 and 1:53,800 births in North America and Europe, respectively. The mortality rate associated with AFE is 30%. The objective of this report is to emphasize the need for knowledge of this serious clinical entity. Patient 21 years, G2P2A0C2, GA 38 weeks and 1 day, admitted to the maternity hospital. Ultrasound with Doppler was requested, by which signs of fetal centralization were found, and cesarean section was indicated. On the 1st day post cesarean section (PCS), in the operating room, she suddenly developed acute respiratory distress and cardiorespiratory arrest (CRA), with return of spontaneous circulation after the first cycle. Sent to the ICU, where she presented 2 more CRAs, both with return in the first cycle. On the same day, the patient developed cardiogenic shock, requiring the use of vasoactive drugs for hemodynamic compensation. Wells criteria: with a high probability of PTE. Laboratory tests and echocardiogram (ECG) were requested, which revealed an increase in right chambers, pulmonary artery systolic pressure (PASP) 68 mmHg. On the 2nd day PCS, ECG showed dilatation of the right ventricle (RV) and right atrium (RA), left artery collapse, dilated inferior vena cava, dilated superior vena cava, presence of pulmonary hypertension with PASP: 60 mmHg. On the 3rd day PCS, she suffered 2 more CRAs, with return in the first cycle. On the 4th day PCS, weaning of vasoactive drugs and sedation was initiated, ECG was requested, dilated inferior vena cava not collapsing with inspiration, RV in “D” due to high pressure, PASP: 60 mmHg, RA increased in size. On the 5th day PCS, extubation was performed. On the 6th day, patient was hemodynamically stable without the use of vasoactive drugs, in good general condition. Presented edema of upper limbs+++/4+ and lower limbs ++/4+. Started on furosemide. On the 8th day, she started captopril due to BP of 149/85 mmHg. On the 9th day PCS, the patient was discharged from the ICU. Although rare, AFE should be maintained as a differential diagnosis for intensivists and obstetricians. Their knowledge is of paramount importance for the early recognition and proper management of the patient. 111528 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BÁRBARA MARIANA DOS SANTOS SILVA1, Carlos Eduardo Lucena Montenegro1, Fiamma Ferreira Nogueira1, Raquel Cristina Farias de Medeiros Queiroz1, Claudia Carolina Mendonça Campos1 (1) Pronto Socorro Cardiológico de Pernambuco (PROCAPE) Introduction: Cardiac complications such as post-vaccination myocarditis from BioNTech-Pfizer have been reported. There is prevalent in young men, evolving with fever, chest pain and dyspnea in the first days after the vaccine. Active infection is ruled out by negative RT-PCR and in all cases MRI showed delayed myocardial enhancement after contrast. Most of the reported cases showed complete resolution of symptoms and absence of sequelae after supportive therapy. Cases of myocarditis have a difficult etiological diagnosis, but they should be considered as part of the clinical diagnosis. Case Report: A 42-year-old woman, previously diagnosed with hypertension, dyslipidemia and overweight, with no other comorbidities, presented to the emergency department with progressive dyspnea started about 2 months ago, progressing to dyspnea on minimal exertion, orthopnea and paroxysmal nocturnal dyspnea. On physical examination, the patient presented with signs of pulmonary and systemic congestion and cardiac auscultation without alterations. She reported onset of symptoms after 1 week of taking the BioNTech-Pfizer COVID-19 vaccine, reporting only a few episodes of associated retrosternal chest pain. She had normal laboratory tests, ECG with sinus rhythm and left bundle branch block pattern, chest X-ray that showed signs of pulmonary congestion, transthoracic echocardiogram with left ventricle showing moderate enlargement, walls with diffuse hypokinesia and markedly reduced systolic function (SIMPSON ejection fraction 25%), cardiac catheterization without evidence of atherosclerotic disease and cardiac MRI with late mesocardial enhancement after gadolinium infusion, with signs of non-ischemic fibrosis in the septal-lower segments and inferior basal and middle, suggestive of sequel of previous inflammatory/infectious process. Optimized treatment was started for heart failure with reduced ejection fraction (sacubitril-valsartan, bisoprolol, spironolactone and furosemide), the patient evolved with significant symptomatic improvement. The patient has had no recurrent symptoms since hospital discharge and is currently under the care of the heart failure center. Conclusion: Post-vaccination myocarditis is a rare event, but it should be considered a differential diagnosis after vaccine for COVID-19, especially when it has a temporal relationship with the BioNTech-Pfizer vaccine. Its evolution is still uncertain and clinical follow-up in specialized centers is necessary. 111532 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ANDRÉ DIAS NASSAR NABACK1, Anderson Ferreira Leite1, Lucas Queiroz Fernandes Campos1, Larissa Barroso Mayrink2, Thales Moura De Battisti1 (1) Hospital das Clínicas da Universidade Federal de Minas Gerais, HC-UFMG; (2) Centro Universitário de Belo Horizonte, UniBH Introduction: Left ventricular (LV) thrombus is one of the complications after acute ST-segment elevation myocardial infarction (STEMI), mostly in cases of anterior wall STEMI with LV ejection fraction <50%. For its diagnosis, transthoracic echocardiography (TTE) is the initial method of choice. Clinical case: Male, 76 years old, diagnosed with STEMI of the anterior wall and submitted to primary angioplasty of the left anterior descending coronary artery (LAD) three and a half hours after the onset of symptoms. One day after the procedure, he presented with transient mental confusion and rotational vertigo. Cranial computed tomography showed a dubious image of an ischemic area in the posterior fossa region. ECOTT did not show intracavitary thrombus, despite an LV ejection fraction of 44%. Nine days after the STEMI, he again presented chest pain and V2–V6 ST-segment elevation on the electrocardiogram. Coronary angiography showed proximal thrombosis of drug-eluting stent in LAD requiring balloon angioplasty and tirofiban infusion. One day later, he recurred with chest pain and worsening of the ST-segment elevation, requiring a new balloon angioplasty due to a new stent thrombosis in the LAD. Changed P2Y12 receptor inhibitor. Two new TTE did not show intracavitary thrombus. It was decided to perform magnetic resonance imaging (MRI) of the heart, which confirmed an image compatible with a thrombus adhered to the apex of the LV, measuring 18 × 10 × 4 mm. Brain MRI confirmed cerebellar ischemic area. Then, anticoagulation with warfarin was started. Conclusion: Intracavitary thrombi are commonly underdiagnosed. Although TTE is the most accessible test, its sensitivity is 35%. The use of ultrasound contrast is an alternative to improve the performance of the exam (sensitivity of 64%). However, despite being less accessible and more expensive, MRI is the gold standard for the diagnosis (sensitivity of 82%–88% and specificity close to 100%), being indicated for patients with high probability of LV thrombus and TTE without thrombus. 111938 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY ELISA ITO MENDES DE ANDRADE1, José Carone Filho1, Márcio Luiz Roldi1, José Carone Júnior1, Carlos Alberto Sancio Júnior1 (1) Hospital Evangélico de Vila Velha Introduction: Acute type A aortic dissection secondary to trauma is a potentially fatal cardiovascular emergency that needs to be diagnosed early and treated effectively. Redirection of blood flow to the true lumen with prosthetic grafts is the mainstay of treatment. Method: A 58-year-old man was admitted to the hospital in his city after a power pole fell. A CT scan was requested due to a suspected fracture of the facial bones. In addition to confirmed bone lesion, he showed acute type A (Stanford) aortic dissection secondary to trauma. The patient was transferred to the referral service in cardiovascular surgery, where computed tomography angiography (CT Angio) of the thoracic aorta showed “Flap in the aortic arch and emergence of the brachiocephalic trunk, with flow maintained in them. The other segments of the thoracic aorta had normal caliber, walls and flows”. He underwent transsternal thoracotomy, and aortic derivation from the proximal aorta to the carotids with a bifurcated graft (Debranching) and implantation of an endoprotheses with antegrade approach without cardiopulmonary bypass (CPB). He is discharged from the Intensive Care Unit (ICU) on the fourth day after surgery, being transferred for surgical treatment of mandibular fracture. Returns to the emergency room 49 days after discharge, complaining of atypical chest pain, which started one week ago, with significant worsening that day. A new CT angiography of the aorta showed the presence of an endoprosthesis that extended from the emergence of the brachycephalic trunk to the descending portion of the thoracic aorta, with contrasted content outside the proximal portion of the endoprosthesis and in apparent continuity with the intimal flap, suggesting an II/IA ? endoleak. He underwent transfemoral implantation of a 36/36/150 endoprosthesis in the ascending aorta and arch, requiring the use of an accommodation balloon with excellent results. The patient progressed satisfactorily in the postoperative period, being discharged on the third day after the approach. Discussion: It is estimated that about 80% of patients with acute type A aortic dissection secondary to trauma die even before hospital care. Endovascular treatment is not the first option for type A aortic dissection, but in this case the patient was at high risk of serious complications if submitted to heparinization and cardiopulmonary bypass. A study with the objective of long-term follow-up of patients undergoing endovascular treatme. 111565 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES BÁRBARA MARIANA DOS SANTOS SILVA1, Diana Patricia Lamprea Sepulveda1, Raquel Cristina Farias de Medeiros Queiroz1, Suéllen Lídia da Silva Barbosa1, Déborah Rodovalho de Menezes1 (1) Pronto Socorro Cardiológico de Pernambuco (PROCAPE) Introduction: Infective endocarditis has different forms of presentation. Signs and symptoms result from either local destructive effects of the cardiac endothelial surface, metastatic embolization of infected fragments, hematogenous seeding of other sites or antibody formation. Typical clinical features are fever, weight loss, malaise, pulmonary or peripheral congestion and new or changing heart murmur. In rare cases, it may be associated with cutaneous vasculitis or hepatitis. Case Report: A 36-year-old man with a rheumatic heart disease, has undergone mitral and aortic valve replacement in 2008 and aortic valve replacement with tricuspid valve repair in 2018, referred progressive dyspnea for about 3 months associated with febrile episodes and jaundice. His initial laboratory panel showed a marked increase in leukocytes with neutrophilia, thrombocytopenia, elevated CRP, liver dysfunction (AST 528/ALP 357) and hyperbilirubinemia (TB 15,1/DB 12,5). Abdominal imaging exams without changes. On examination, the patient was emaciated, with moderate intensity jaundice, diffuse purpuric lesions, signs of peripheral embolization and jugular distension. On cardiac auscultation, important systolic and diastolic murmurs in the mitral focus and systolic murmurs in the aortic and tricuspid areas. Pulmonary auscultation with crackling rales in lung bases. Transesophageal echocardiography revealed moving filamentous images suggestive of vegetation on the atrial face of the mitral bioprosthesis, in addition to signs of mitral, aortic and tricuspid valve dysfunction. Antibiotic treatment with oxacillin, ceftriaxone, gentamicin and rifampicin was started, with gradual improvement in a few days of the purpuric lesions and the icteric syndrome. Initial blood cultures with negative results. The patient underwent a new surgery to replace the aortic and mitral valves (mechanical) and tricuspid repair. During hospitalization, positive control blood culture samples for Candida albicans and Candida haemulonii were evidenced. Antibiogram-guided treatment with micafungin was performed. Patient was discharged clinically stable and with improvement of all initial clinical and laboratory findings. Conclusion: Infective endocarditis can appear in an atypical manner and can be a diagnostic challenge in such cases that initial signs are these rare complications, such as hepatic failure or vasculitis. Late diagnosis worsens prognosis and leads to increased mortality. 111579 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY NICOLAS BIONI STEFANO 1, Elisa Souza Lini2, Alexandre Quadros1, Renato Kalil1, João Carlos Vieira da Costa Guaragna1 (1) Hospital Divina Providência; (2) Pontifícia Universidade Católica do Rio Grande do Sul Introduction: Coronary artery single ostium cardiac anomaly represents a rare congenital alteration of unknown etiology. This condition presents clinically from asymptomatic forms to cardiac ischemia and manifestations of sudden cardiac death, especially in young adults. The incidence of this pathology is variable, affecting 0.03% to 0.4% of patients undergoing cardiac catheterization. Case Report: Patient LVAG, 69 years old, male, hypertensive, dyslipidemic, heart failure, hypothyroidism and COPD. Hospitalized for salmonella colitis. During hospitalization, he presented anginal chest pain. He already had similar episodes at home with small efforts, with gradually worsening in the last few weeks. ECG showed sinus rhythm and septal inactive zone, there was elevation of troponin levels, being taken to coronary angiography. During evaluation, an anomalous coronary path was visualized, with the anterior descending artery and circumflex artery originating from the right coronary sinus, together with the right coronary artery. There was also an ulcerated lesion in the middle segment of the circumflex artery, but with a complicated anatomy and path to perform angioplasty. Coronary computed tomography angiography showed a malignant path of the circumflex artery, with passage between the aorta and the pulmonary artery. Echocardiogram showed mild systolic dysfunction with an ejection fraction of 42%. Heart Team defined coronary artery bypass graft surgery for definitive treatment. Conclusion: Congenital coronary artery anomalies are subdivided into seven categories, highlighting the single-ostium coronary artery anomaly, in which four paths can be followed: retroaortic, septal, anterior and interarterial. Its clinical presentation includes angina, ventricular arrhythmias, dyspnea, syncope, heart failure and sudden death. The occurrence of ischemia associated with this anomalous condition, in this case, consists of the path of the vessel between the pulmonary artery and the aorta, which would eventually suffer extrinsic compression. In the present case, the patient had an anomalous interarterial course (between the pulmonary artery and the aorta), considered a malignant type. In these cases, the patient may initially undergo coronary angioplasty and, later, surgical treatment, through reimplantation or ligation of the coronary arteries, followed by grafting of the saphenous vein or internal thoracic artery. 111591 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MATHEUS RAMOS DAL PIAZ1, Caio Menezes Machado de Mendonça1, Bruno Mahler Mioto1, Luis Henrique Wolff Gowdak1 (1) Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) Introduction: Severe stenosis of the left main coronary artery (LMCA) in patients with stable symptoms is uncommon and usually associated with a dominant right coronary artery (RCA) and rich collateral circulation. The incidence of total occlusion has been reported to be between 0.04% to 0.43%. The impact on the left ventricle (LV) function is often significant with systolic dysfunction, especially with significant involvement of the RCA. Surgical myocardial revascularization remains the best treatment for chronic total occlusion (CTO) of the LMCA. Case Report: A 69-year-old woman with hypertension, dyslipidemia, and obesity, without previous acute myocardial infarction, presented with CCS III angina, associated with dyspnea on exertion for a year. A myocardial perfusion scan revealed extensive stress-induced ischemia in the anterior, anterolateral, lateral, and inferolateral walls. A transthoracic echocardiogram showed a globally preserved ventricular function (LVEF = 65%). We found a CTO of the LMCA and a 70% stenosis of the middle RCA during an elective coronary angiography, with grade III collateral circulation from the RCA to the LMCA. The patient was admitted for surgical myocardial revascularization. A successful triple bypass was performed (left internal thoracic artery-left anterior descending artery, radial artery-left obtuse marginal branch, and saphenous vein graft-posterior descending artery). Conclusion: CTO of LMCA is a rare manifestation in patients presenting with stable symptoms. Two conditions are related to a higher probability of patient survival and maintenance of LV function: a dominant RCA without significant stenosis and an extensive collateral circulation to the left system, conditions only partially found in our patient. Despite having significant stenosis of the RCA, LV function was preserved, and the patient had stable symptoms for a year. 111598 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LARISSA XAVIER ALVES DE OLIVEIRA1, Debora Rodrigues1, Mauricio Lopes Prudente1, Adriano Gonçalves de Araújo1, Giulliano Gardenghi1 (1) Hospital Encore Introduction: Pulmonary arteriovenous malformations (PAVMs) consist in an abnormal communication between an artery and a vein, causing clinical manifestations as chronic hypoxia or embolic events. Most cases of PAVMs are represented by hemorrhagic telangiectasia, a rare congenital disease. In the past, pulmonary fistulas were treated only by open surgery, with an expressive rate of complications. In the 70s, the first percutaneous catheter embolization was performed with coils. Currently percutaneous embolization has become the treatment of choice. We report three different cases of percutaneous correction of PAVMs using coils and vascular plugs. Cases Description: First patient, 47 years old, had an 8 mm diameter fistular sub pleural sac. Complete embolization was performed without residual shunt. The second patient, 48 years old presented a pulmonary fistula associated with chronic hypoxemia. The fistula came from several branches of the right pulmonary artery. After embolization was shown immediate improvement of central oxygen saturation. Third patient, 30 years old, the PAVM had a pseudoaneurysm raising next to the first branches of the left pulmonary artery to the upper lobe of the left lung, and two important fistulas in the right inferior lobar arteries forming a shunt right to left. Embolization of the PAVM and pseudoaneurysm was performed successfully. Conclusion: The percutaneous treatment of PAVMs is effective and safe in these cases. 111629 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CAIO MENEZES MACHADO DE MENDONÇA1, Matheus Ramos Dal Piaz1, Antônio Carlos Fonseca de Queiroz Filho1, Matheus Barbosa Gastaldo1, Luis Henrique Wolff Gowdak1 (1) Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) Introduction: Post–myocardial infarction pericarditis occurs in approximately 5% to 6% of patients who receive thrombolytic agents. It develops in the first 2 to 4 days following infarction, mainly in the first 24 hours, and usually produces only mild symptoms. Nonetheless, rare complications include hemopericardium, cardiac tamponade, and constrictive pericarditis. Treatment is primarily supportive, with appropriate use of nonsteroidal anti-inflammatory agents. However, pericardiocentesis is indicated to treat a large pericardial effusion, particularly if accompanied by tamponade. Case Report: A 63-year-old male patient with a history of hypertension and smoking was transferred to our hospital 24 hours after an extensive anterior ST-elevation myocardial infarction (STEMI). The patient was initially treated in another facility with ASA + clopidogrel and underwent thrombolysis with tenecteplase, with signs of reperfusion. The patient was brought to the cath lab, significant 3-vessel disease was found, and surgical myocardial revascularization was recommended. Clopidogrel was withdrawn, and full anticoagulation with enoxaparin was initiated. The patient remained asymptomatic, but a transthoracic echocardiogram on the second-day post-MI revealed a pericardial effusion of 25 to 30 mm with signs of ventricular filling restriction with no evidence of left ventricular (LV) dysfunction or LV free wall rupture. The patient developed severe hypotension on the same day, requiring vasoactive drugs. Urgent pericardiocentesis was performed, and 900 mL of hematic fluid was recovered. Right after, the patient evolved with significant hemodynamic improvement. Histopathological examination of a pericardial fragment revealed the diagnosis of acute fibrinohemorrhagic pericarditis. Conclusion: Acute hemorrhagic pericardial tamponade after infarction rarely occurs, at a reported incidence of only 1% of STEMI patients treated by thrombolysis, often within the first 24h. Despite being a rare complication, the present case calls attention to the fact that hemopericardium and cardiac tamponade can occur in patients who have suffered an acute MI and should be included in the differential diagnosis in those presenting with hemodynamic instability. Prompt echocardiography assessment may lead to early diagnosis and better outcomes in these patients. 111883 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING MAYARA DE SOUZA VASCONCELOS1, PEDRO RAFAEL VIEIRA DE OLIVEIRA SALERNO1, LUCIA MARIA VIEIRA DE OLIVEIRA SALERNO1, EVELINE BARROS CALADO1 (1) HOSPITAL DAS CLÍNICA DA UNIVERSIDADE FEDERAL DE PERNAMBUCO Introduction: Intracardiac masses are rare. Causes include neoplasic and non-neoplasic conditions. Diagnosis is challenging due to nonspecific clinical presentation and commonly occurs incidentally. Echocardiography (ECHO) is usually the initial method of assessing the size, location, fixation, mobility, and hemodynamic repercussion of the mass. Computed tomography (CT) and magnetic resonance imaging (MRI) further complement the mass evaluation. Case Description: A 54-year-old female presented with facial edema and conjunctival hyperemia that progressed to diarrhea, lower limbs edema, ascites, abdominal pain, jaundice, and dyspnea at moderate efforts. After a syncopal event with no post-ictal period, she was admitted for investigation. Past medical history included a stroke in 2019 and schistosomiasis that was treated in 2000. After admission, discrete leukocytosis, with significant eosinophilia, elevated C-reactive protein, high aminotransferases, and high lactate dehydrogenase were identified. Blood cultures were negative. Urine culture was positive for Escherichia coli and ceftriaxone + metronidazole were initiated. Transthoracic and transesophageal ECHOs displayed a large right ventricle (RV) mass that occupied most of the chamber and was attached to the tricuspid valve (TV), enlargement of the right atrium, inferior vena cava, and hepatic veins were also seen. Cardiac MRI confirmed the mass that measured 21 × 28,5 × 33,4 mm, attached to the TV and projecting towards the RV outlet. Metastatic disease screening with imaging studies provided non-significant findings. Lower limbs USG with Doppler identified bilateral deep venous thrombosis. Tumor markers and investigation for thrombophilia and collagen disease were negative. Cardiac surgery for the removal of the mass was done, intraoperative findings included a mass involving the papillary muscles and the leaflets of the TV with a caseous appearance. After excision, a biologic prosthesis was inserted in TV position. Histopathological analysis showed a material containing fibrin and leukocytes, and endomyocardial necrosis without significant eosinophil infiltration—no signs of neoplasia, bacteria, or fungus. After discharge, previous blood tests were identified and showed chronic fluctuating eosinophilia (largest count 4.190/mm3, 27,3%). Conclusion: Unique features of this case are the large thrombus-like RV mass causing obstructive symptoms and the presence of persistent eosinophilia perhaps suggesting Loeffler’s endocarditis. 111622 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING THIAGO BURIL FONTES1, Pedro Henrique Pereira Inglez1, Rudyney Azevedo1, Cássia Cardoso Costa1, Carolina Frezzatti de Andrade Neves1 (1) Hospital do Servidor Público Municipal de São Paulo Introduction: Infective endocarditis (IE) is a cardiovascular disease. The etiology is predominantly gram-positive cocci, but it has other infectious agents: HACEK group, fungi and other atypical germs. IE caused by Serratia marcescens is rare, documented in less than 1% of cases. Pathophysiology is multifactorial. Case Report: E.G.V., 58 years old, history of aortic valve disease due to rheumatic fever and permanent pacemaker (PM). PM, on 03/05/22, evolving after ten days of the exchange with phlogistic signs, fever and fluctuation in the generator store region. Opted for cleaning the PM pocket with secretion collection for culture. Febrile peaks remained,37.7°C, after PM cleaning. White blood cell 13 (4.8–10.8) with absolute neutrophils of 11 (1.4–6.5). Cultures with the presence of Serratia marcescens in secretion of PM store and in blood culture. Treatment for bloodstream infection with Meropenem and Teicoplanin. The electrodes were then removed from the PM, with laser, and sent for culture. In the culture, 04/01/22, there was growth of Serratia marcescens on PM electrodes. Because of this, a 2D TEE was performed, showing a mass adhered to the tricuspid valve, which may correspond to vegetation. Changed Daptomycin and maintained Meropenem. The definitive PM generator implanted on 04/19/22, maintaining afebrile condition on the 6th PO and obtaining negative results from the 6 control blood cultures. 3D TEE was performed on 05/11/22 showing persistence of the image of the mass in the right ventricle, this time being interpreted as a residual image due to the removal of the PM. Conclusion: It is a rare case of Serratia marcescens infection and the challenge of interpreting an image of an intracardiac mass, the 3D TEE can help in the follow-up of the case. 111626 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY SABAH ABAZAR YOUSIF MOHAMED ALI1, Paulo Rafael Gonçalves da Silva Von Zuben1, Gabriel Queiroz de Abreu1, Ricardo Ribeiro Dias2, Georgina del Cisne Jadán Luzuriaga2 (1) Centro universitário São Camilo (CUSC); (2) Instituto do coração do Hospital das Clínicas (InCor-HC) Aortic dissection is a disease, which presents with chest discomfort, acute hemodynamic compromise and high risk of mortality, requiring surgical management, with endovascular repair of the aorta. However, the prosthesis used in the procedure can be considered a possible etiology of aortoesophageal fistula. LF, female, 64 years old, with a history of systemic arterial hypertension (SAH) for 6 years, hysterectomy for 30 years, with diagnostic hypotheses of aortic aneurysm and chronic dissection of the ascending aorta (55 mm) and descending (50 mm). Replacement of the aortic branch and reimplantation of supra-aortic island branches was performed. Three months after the first surgery, the patient reported daily low-grade fever, malaise, dyspnea on minor exertion, and hyporexia for two months, in addition to hemoptysis, cough, and diffuse chest pain. According to tomography angiography, a large thrombus was identified in the aorta associated with a dissection blade in the abdominal aorta, from the celiac trunk to the bifurcation, extending along the entire length of the superior mesenteric artery. Upper gastrointestinal endoscopy (UGE) showed ulceration in the esophagus with a fistulous orifice in the upper thoracic esophagus. An aortic endoprosthesis was implanted. Two weeks after the second surgery, the aortoesophageal fistula was repaired. The surgeries were a success, however, the patient developed urinary tract infection, acute respiratory failure, pneumothorax, bacteremia and acute kidney injury. The condition evolved with septic shock due to mediastinitis caused by Pseudomonas aeruginosa due to the aortoesophageal fistula, resulting in death. In the literature, aortoesophageal fistula in the postoperative period of chronic aortic dissection is rarer than aortic dissection. Based on the findings of this study, it was possible to observe the severity of such a complication, such as mediastinitis secondary to bacterial infection, which rapidly progresses to death. The situation reported, similarly to previous studies, describes presentations with the same outcome of death, which had some factors similar to that of this patient, such as chronic dissection, uneventful postoperative period and need for reintervention. Patient with an uncommon complication of aortoesophageal fistula after correction of chronic aortic dissection, however, with extreme severity of infectious complications, such as mediastinitis and death. 112133 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY ANAÍS CONCEPCION MARINHO ANDRADE DE MOURA1, Cítara Trindade de Queiroz1, Dr. Marcos Antonio Ferreira Lima2, Dr. Marcel Delafiori Hikiji2, Dra. Aline Hofmann Baiao2 (1) Universidade Potiguar; (2) Hospital do Coração de Natal Introduction: The coronary arteries‘ anomalous origin is rare, with a frequency of reported cases of 0.64% in neonates and 0.17% in asymptomatic children and adolescents who were discovered through an echocardiogram. Its late discovery may occur in symptomatic patients with hemodynamic repercussions, therefore, early intervention is necessary and performed with surgical correction. Case Report: A 69-year-old female, diabetic, hypertensive, and with chronic coronary artery disease was diagnosed with an anomalous origin of the right coronary artery (RCA) with a malignant path. Symptoms of typical angina and recurrent syncope started in 2012. In 2013, after an anginal condition, she underwent an elective angioplasty, with a complication that led to coronary dissection and brief cardiorespiratory arrest, requiring implantation of 2 stents in the proximal third and mean RCA, however, the artery anomaly was not described in the procedure report. Even after the intervention, the patient maintained the same clinical picture with optimized therapy for 9 years. After a coronary angiotomography in 2021, the anomalous origin of the RCA emerging in the upper portion of the left coronary sinus and its interatrial course, associated with ostial cleft compression, was described, and then coronary artery bypass graft surgery was indicated. The surgical approach was performed in 2022, through an aortic bypass graft to the middle third of the RCA and closure of its proximal third, where the previous stent was located. The patient developed with a total improvement of angina and syncope. Conclusion: Patient with a rare ACD anomaly with a malignant path, which was only discovered when she was elderly, because of symptoms of ischemia that occurred through compression of the artery, which passed between the ascending aorta artery and the pulmonary trunk artery, during ventricular systole. It is worth emphasizing the importance of early diagnosis and therapeutic intervention for better patient survival because of the high risk of sudden death. 111646 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MURYELLE ROSA COELHO1, Rogerio Las Casas1, Vinicius Daher Vaz1, Fabricio Las Casas1, Tannas Jatene1 (1) Hospital do Coração Anis Rassi (HCAR) Introduction: Acute Myocardial Infarction (MI) occurs due to atherosclerotic plaque rupture, and consequent coronary thrombosis. A large amount of intracoronary thrombus is a challenge for the interventional cardiologist as it difficult to restore TIMI III flow and predisposes embolization to distal microcirculation (no-reflow). Coronary thromboaspiration reduces the thrombotic load, improves coronary perfusion and reduces fibrosis area. Case Description: Male, 72 years old, smoking history, hypertension, dyslipidemia, diabetes mellitus and chronic kidney disease. Admitted at emergency room with ST-segment elevation myocardial infarction on inferior wall and 7-hour pain. A loading dose of acetylsalicylic acid and ticagrelor was administered followed by cardiac catheterization that revealed acute total occlusion in the middle third of the right coronary artery. After balloon recanalization, high thrombotic load was observed. A glycoprotein IIbIIIa inhibitor was administered, followed by unsuccessful conventional mechanical thrombus aspiration and TIMI I distal flow. In the face of failure, a Penumbra CAT RX mechanical thrombus aspiration system was used. It contains a catheter with a larger internal lumen and coupled to a pressure aspirator, unlike the conventional modality that uses a manual suction syringe system. TIMI III flow was reestablished and drug-eluting stent was successfully implanted, followed by transfer to the coronary care unit. The evolution was uneventful, echocardiogram showed preserved ejection fraction and inferior wall hypokinesia. Hospital discharge after four days of hospitalization, using dual antiplatelet therapy, statin and beta-blocker. Conclusions: Although TAPAS trial suggests a reduction in major cardiovascular events with routine mechanical thrombus aspiration in MI, larger randomized trials such as TASTE and TOTAL turned this therapy a class III recommendation, because it did not demonstrate clinical benefits and increased the risk of stroke. As the favorable results of mechanical thrombus aspiration on TOTAL trial subgroup with high thrombotic load, the Penumbra CAT RX system was developed. This system contains features as continuous vacuum, making aspiration more effective and with a lower incidence of stroke. The ongoing CHEETAH trial exclusively evaluates patients with high thrombotic burden using the Penumbra CAT RX, as in the case described, to answer the real benefit on mechanical thrombus aspiration. 111654 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY RAMON OTT VARGAS1, Elisa Ito Mendes de Andrade1, José Carone Filho1, Assad Miguel Sassine1, José Carone Júnior1 (1) Hospital Evangélico de Vila Velha Introduction: Acute aortic dissection is a rare, but lethal entity during pregnancy. Difficulties in early diagnosis and small number of studies available in this group of patients are complicating factors. Case Description: Female, 30 years old, hipertensive, at 35 weeks 6 days of pregnancy, coming from high-risk pregnancy referral service, where she was hospitalized due to restricted intrauterine growth. One week after admission, she developed severe chest pain and syncope. Electrocardiogram and lab work showed no myocardial ischemia. Transthoracic Echocardiogram was performed, suggesting ascending aortic dissection affecting the aortic valve, with mild regurgitation. A computed tomography scan confirmed the diagnosis of Stanford type A aortic dissection. Patient was transferred for cardiovascular surgery, accompanied by all the necessary equipment and medical personnel to perform emergency delivery. Admitted to this service with chest pain, using sodium nitroprusside. In the operating room, patient was submitted to orotracheal intubation with care to avoid cardiorespiratory depression of the fetus. Emergency C-section was performed, neonate with good vitality, Apgar 8/9. Immediately after the C-section, aortic surgery began. Dissection of the common carotid artery was performed for arterial cannulation. Deep hypothermia was induced in the patient. After aortotomy, a dissection flap was detected in the non-coronary sinus. The diseased aortic segment was excised, and both ends were reinforced with teflon matresses, and ultimately anastomosed to one another, obtaining excellent results. Patient resumes spontaneous rhythm after opening of the aortic clamp. Cardiopulmonary bypass time was 100 minutes and aortic cross clamp was 80 minutes. Patient developed sinus tachycardia and hypertension, requiring use of beta-blockers and intravenous vasodilators. Extubated after 48 hours. Patient progresses well, discharge from the Intensive Care Unit on the 6th day after surgery and hospital discharge seven days later. Conclusion: Despite the dismal diagnosis of acute type A aortic dissection in pregnancy and the challenges of keeping both mother and fetus alive in the context of a complex surgery such as the one described, both the patient and the neonate showed good outcome after the approach. Accurate diagnosis and timely multidisciplinary action were decisive for the result obtained in this case. 111663 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY JOSÉ CARONE FILHO1, Roberto Ramos Barbosa1, Elisa Ito Mendes de Andrade1, Ramon Ott Vargas1, Roger Alain Pantoja Ribera1 (1) Hospital da Santa Casa de Misericórdia de Vitória – ES Introduction: Acute cardiac failure is a high mortality syndrome with varied ethiology. Mitral regurgitation following acute coronary syndrome is one of its causes and necessary management requires exceptional attention to haemodynamic patient balance. This paper describes the in-hospital events of a patient on which a intra-aortic ballon pump was implemented, after acute mitral insufficiency and cardiogenic shock, until surgery for valve replacement. Case Description: Woman, 84 years old, hypertensive, presenting with oppressive chest pain, radiating to the jaw, even at rest. Physical exam: diaphoretic, regular general state. Irregular cardiac rhythm, normal S1 and S2, holosystolic murmur heard on cardiac apex (2+/6+). Pulmonary auscultation with bilateral rales, respiratory rate of 32 and pulse oximetry saturation of 76%. Acute miocardial infarction (without elevated ST segment) was diagnosed (Killip 3, Timi Risk 4). Transthoracic echocardiography (TTE) was performed and showed chordae rupture resulting in severe regurgitation. Left ventricular ejection fraction was 62%. Patient evolved with cardiogenic shock, respiratory failure with endotracheal intubation, requiring vasopressor. Case was discussed between a multidisciplinary team and intra-aortic balloon pump (IABP) implantation was indicated for salvage and support for percutaneous coronary intervention. Patient was at hight operative risk at that moment (EuroSCOREII: 28%). On the eighth day after admission, percutaneous coronary intervention was performed, with implantation of 3 stents (anterior descending, circumflex and right coronary arteries). After 20 days in-hospital, mitral valve replacement surgery was performed, and a bioprosthesis (size 29) was implanted. IABP was removed after termination of cardiopulmonary by-pass. Patient was discharged 40 days after admission. Control TTE showed functioning bioprosthesis and no sign of intracavitary thrombus. Patient is clinically well, receiving optimized medical therapy and no signs of decompensation. Conclusion: Presence of increasingly high risk patients with multiple comorbidities and complex conditions is rising by the day in our Cardiology practices. This case shows how staged hybrid approach can be performed in high operative risk patients obtaining satisfactory results. 111666 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RAQUEL CRISTINA FARIAS DE MEDEIROS QUEIROZ1, Bárbara Mariana dos Santos Silva1, Isly Maria Lucena de Barros1, Rafael Dayves Medeiros de Queiroz1, Suéllen Lidia da Silva Barbosa1 (1) Pronto Socorro Cardiológico de Pernambuco – PROCAPE/UPE Introduction: Some patients presenting with ST elevation myocardial infarction had a syndrome named myocardial infarction with non-obstructive coronary arteries (MINOCA). This diagnosis requires an AMI and absence of obstructive CAD on angiography and may present with or without ST segment elevation on the ECG. Thrombosis may be a mechanism and the prevalence is low. It may arise from thrombotic disorders, atherosclerotic plaque disruption or coronary dissection with associated thrombosis or coronary emboli. To aid in diagnosis, we can use angiography with intravascular ultrasound or OCT (40% of patients with MINOCA have some evidence of plaque disruption). In this scenario, the inpatient treatment usually involves anticoagulation and the outpatient treatment may vary depending on the underlying etiology, but we know that DAPT (dual antiplatelet therapy) may be useful in those cases where plaque disruption is possible. Case Report: Male patient, 39-year-old, with no previous comorbidities and denying the use of illicit substances or medications. He sought emergency care complaining of intense retrosternal pain, which had started forty minutes before, associated with sweating and vomiting. Electrocardiogram showed ST-segment elevation in anterior leads. The patient was referred for invasive stratification and received DAPT. The coronary angiography showed an image suggestive of thrombus presence in the proximal segment of the anterior descending artery, with Thrombolysis in Myocardial Infarction – TIMI 2 – distal flow, and all the vessels were free of significant stenosis. It was decided not to perform a percutaneous intervention at that time; abciximab was administered by 24 hours and anticoagulation was maintained with unfractionated heparin, as well as the concomitant use of DAPT. The new catheterization ten days later did not reveal the same image compatible with thrombus presence and showed a small plaque in the same proximal segment of the anterior descending artery. He showed clinical improvement and was discharged with a prescription of DAPT. Conclusion: This case illustrates the importance of identifying the causes of MINOCA to facilitate more definitive and specific treatment strategies, such as the use of DAPT. Multimodality imaging with OCT and CMR can help in our understanding of the underlying pathophysiological mechanisms within and beyond the context of coronary atherosclerotic disease, however, are not available in our practice. 111687 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING GABRIELA RIBEIRO PRATA LEITE BARROS1, Juliana Pato Serra Souza1, Kevin Rafael De Paula Morales1, Jürgen Beuther1, Diana Rodrigues de Araujo1 (1) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo. Introduction: Kawasaki disease (KD) is an acute vasculitis that preferentially affects medium-sized arteries, particularly the coronary arteries. Patients with coronary artery aneurysms (CAA) are at risk for cardiac events including coronary artery thrombosis or stenosis, myocardial infarction and death. Coronary computed tomography angiography (CCTA) has been shown to be superior to echocardiography for the detection of CAA in distal portions of the vessel. It also a reliable modality for follow-up of CCA, with close correlation with catheter angiography. Case report: A 9-year-old patient with previous diagnosis of kawasaki disease treated with immunoglobulin and history of giant coronary aneurism seven years ago, presented to the pediatric cardiology consult complaining of chest pain on exertion. Previous CCTA showed coronary dilatation in of the right coronary artery (RCA) measuring and left anterior descending artery (LAD), both without luminal reduction. A new CCTA was performed. The LAD showed a large aneurysmal dilatation in the proximal segment, measuring 10 × 10 mm with proximal luminal evaluation limited by intense calcification (Figure A). The RCA showed a fusiform aneurysmal dilatation measuring 8 × 7 mm, with mural thrombi and diffuse parietal calcifications, highlighting an area of critical luminal reduction right at the origin of the aneurysm (Figure B). Cardiac catheterization was performed, proving significant luminal reduction in RCA, and balloon angioplasty was successfully performed. Conclusion: Aneurysms in Kawasaki Disease can evolve with difficult-to-treat stenosis. Coronary computed tomography angiography is an attractive alternative to catheter angiography for the assessment of coronary manifestations of the disease. 111690 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LARISSA MARIA VOSGERAU1, Roberto de Ávila Martins1, Pedro Felipe Gomes Nicz1, Paulo Roberto Cruz Marquetti1, Eduardo Leal Adam1 (1) Hospital de Clínicas da Universidade Federal do Paraná (HC-UFPR) Introduction: Chest pain in patients with pulmonary arterial hypertension (PAH) is usually attributed to the imbalance oxygen supply demand to the right ventricle, which can lead to subendocardial hypoperfusion. It is known that the pulmonary artery (PA) can induce extrinsic compression of the left main coronary artery (LMCA) in these patients, and it is described that a PA trunk greater than 40 mm in diameter with typical angina is correlated with compression of LMCA in most patients. This luminal reduction can lead to acute myocardial infarction, left ventricular dysfunction, arrhythmias and even sudden death. The evaluation of coronary arteries is often not performed routinely in these patients, so the incidence of LMCA compression by the PA is not well established in literature. There is no consensus about the best therapeutic strategy, but percutaneous coronary intervention is a viable option to relief symptoms and improve outcomes with a high success rate. Case Report: A 30-year-old female patient diagnosed with idiopathic PAH for 9 years and with optimized treatment seeks medical service for progressive chest pain with minimal efforts and quality-of-life impairment. During investigation, a coronary computed tomography was performed and showed a 56 mm PA trunk with a left main coronary artery ostial angulation (17.9°) and significant luminal reduction (>70%) due to extrinsic compression (figure 1). Coronary angiography shows a critical stenosis in the LMCA. A percutaneous angioplasty with implantation of two drug-eluting stents (stent-in-stent) was successfully performed and the patient evolved with complete improvement of chest pain after revascularization. Treatment of PAH was associated with dual antiplatelet therapy with subsequent outpatient reassessment. Conclusion: Extrinsic compression of the LMCA should be considered as a differential diagnosis in patients with PAH and angina, especially in patients with a PA trunk greater than 40 mm. Percutaneous coronary intervention is a safe and effective option to improving symptoms and reduce cardiovascular outcomes with a low rate of complications. 111725 Modality: E-Poster Young Researcher – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MATHEUS BURIGO OLIVEIRA1, Gisele Messias Mattioli1, Fernanda Nascimento Dourado1, Leticia Macacchero Moreirão1 (1) Instituto Nacional de Cardiologia Introduction: Coronary embolism is a rare cause of myocardial infarction (MI). Unlike type I MI (plaque instability), its pathophysiology and etiology are not yet well defined. Some studies show it is the underlying cause of 3% of MI, but often neglected. Case Report: A 31-year-old male diagnosed with dilated cardiomyopathy and hypertension was admitted with typical chest pain and Q waves in leads V1–V6 on the electrocardiogram and submitted to coronary angiography, which showed thrombi in the left anterior descending artery and second diagonal branch without other obstructive lesions. Investigation ruled out the presence of risk factors for atherosclerosis (coronary calcium score 0 and normal lipid profile), left ventricular thrombus, atrial fibrillation and paradoxical embolism. Research for thrombophilia showed antinuclear antibodies (ANA) 1:180 of pathological pattern (nucleolar homogeneous) which did not meet the criteria for diagnosis of lupus or antiphospholipid antibody syndrome (APS). We opted for conservative therapy with enoxaparin 1 mg/kg twice a day. A new coronary angiography 1 week after therapy showed a considerable reduction in thrombi. Conclusion: MI caused by coronary embolism should be considered in young patients with few risk factors for atherosclerotic disease. This case illustrates the need to search for triggers of this event, notably arrhythmias, severe left ventricular dysfunction, thrombophilia and rheumatological diseases. The lack of randomized trials on this subject makes it difficult to decide on the type of anticoagulation and the duration of therapy. Finally, the underlying diagnosis is impaired by acute MI either by changes in the coagulation cascade or by the need for rapid use of anticoagulation, which may alter the results of laboratory tests. 111737 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES ARMINDO JREIGE JÚNIOR1, Karen Katchvartanian1, Luis Henrique Silveira Moreira1, Renan Cintra de Alvarenga Oliveira1, Bruno Biselli1 (1) Instituto do Coração do Hospital das Clínicas da FMUSP (InCor/HCFMUSP) Introduction: Rheumatic fever is a possible late complication secondary to group A streptococcus infection. Repeated episodes of acute outbreaks can progress to rheumatic heart disease, which is associated with significant morbimortality in children and young adults. Case Description: A 46-year-old male patient with history of rheumatic fever underwent valve replacement with a caged-ball metallic prosthesis in 1992 due to combined aortic stenosis and regurgitation. He remained asymptomatic until 2020, evolving after that with symptoms of heart failure (HF). The initial hypothesis was dysfunction of the metallic prosthesis. Transesophageal echocardiogram did not reveal prosthetic alterations, showing the presence of ventricular dysfunction, reduction of ejection fraction to 25% (previously normal), dilatation of left cavities and areas of segmental alteration. Coronary angiography showed no obstructive lesions and serology for Chagas disease was negative. Magnetic resonance imaging (MRI) of the heart showed areas of late transmural enhancement in the apical and subendocardial segments in the lateral, anterior and middle wall segments. In view of the MRI findings and the previous diagnosis of rheumatic fever, the main hypothesis was active rheumatic myocarditis. Gallium-67 scintigraphy identified the presence of increased uptake in a projection of the cardiac area with a diffuse pattern and mild degree. This result corroborated the existence of an active cardiac inflammatory process. Patient currently under outpatient follow-up, undergoing clinical evaluation for corticosteroid therapy or inclusion in the heart transplant waiting list. Conclusions: The treatment of rheumatic carditis lacks consensus, with little evidence. Regarding the diagnosis and treatment of rheumatic myocarditis, the scenario is even worse, with only a few observational studies. The therapy currently proposed for severe and refractory cases, as described, is based on the use of corticosteroids. There is great variation between the data in the literature, related to the dosage of the drug to be used, treatment duration and associated pulse therapy. Heart transplantation is also a possibility for cases of terminal and refractory HF. Due to the high prevalence of rheumatic fever in our country and the clinical severity associated with rheumatic myocarditis, further studies must be done for adequate therapeutic management and reduction of morbimortality secondary to this clinical entity. 111742 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY CAROLINA RESENDE MARIZ1, Catharina de Almeida Serra Faria1, Julia Colonese Serra1, Viviane Campos Barbosa de Sena1, Joaquim Marcio Duarte e Silva1 (1) INSTITUTO NACIONAL DE CARDIOLOGIA Introduction: Since the beginning of SARS-CoV-2 pandemic in 2020, several consequences resulting from this infection have been reported, including myocarditis. The mechanism’s of myocardial injury are not fully understood yet, but seems to be related to direct damage to cardiomyocytes, systemic inflammation, interstitial fibrosis, mediated immune response, and exaggerated cytokine response. Case Description: G.D.M, male, 4 months old, previously healthy, admitted to Intensive Cardiac Unit in acute congestive heart failure, requiring invasive mechanical ventilation, inotropic and diuretic support for 15 days. Echocardiogram revealed severe mitral insufficiency, left ventricular(LV) dilation and severe dysfunction, with ejection fraction (EF) of 18%. Angiotomography ruled out coronary artery anomalies and cardiac resonance magnetic revealed evidence of myocarditis. Laboratory tests showed elevated BNP, D-dimmer as well as troponin. SARS-CoV-2 PCR and IgM were negative, but SARS-CoV-2 IgG was positive. Also, respiratory syncytial virus and Influenza PCRs were also negative. After the diagnosis of Myocarditis related to SARS-CoV-2 infection was made, therapy with immunoglobulin, enoxaparin and acetylsalicylic acid was initiated. After this therapy, the patient improved clinically as well as laboratory and was discharged with medications. Follow up made with echocardiogram at 2 months showed significant LV dilation, with only mild improvement of EF (Teicholtz and Simpson, respectively, 27% and 24%). Conclusion: Pediatric post viral myocarditis usually has a benign and self-limited course, but in some cases, it results in severe cardiac dysfunction. Given the ongoing pandemic, patients with cardiovascular dysfunction should raise suspicion for SARS-CoV-2 infection and its complications. 111744 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING GABRIELA RIBEIRO PRATA LEITE BARROS1, Juliana Pato Serra Souza1, Kevin Rafael De Paula Morales1, Max Walter Reyes Barrenechea1, Eduardo Kaiser Ururahy Nunes Fonseca1 (1) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo. Introduction: Left ventricular pseudoaneurysm (LVP) results from a cardiac-free wall rupture contained by adherent pericardium or scar tissue without any involvement of myocardium or endocardium. It frequently occurs in the setting of myocardial infarction, cardiac surgery and endocarditis. Patients with LVP can report symptoms of heart failure, dyspnea, or chest pain. Left ventriculography is the gold standard for diagnosis, but coronary computed tomography angiography has been increasingly used as noninvasive diagnostic technique. Case Report: A 68-year-old female patient, with a previous history of mitral valve replacement, developed dyspnea and atypical chest pain. During investigation, a transthoracic echocardiogram was performed and showed an oval image with flow inside and aparent communication with the right atrium. Heart and coronary tomography showed a multiloculated pseudoaneurysm (*) with origin in the basal inferoseptal wall of the left ventricle, close to the mitral valve prosthesis. This formation maintained ample contact with the middle and distal segments of the right coronary artery, measuring about 64 × 32 × 27 mm. The image corresponded to a left ventricular pseudoaneurysm of apparent late postoperative etiology, determining compression of the of the venous sinus and in intimate communication with the right coronary artery. Conclusion: Heart and coronary computed tomography is a non-invasive method for diagnosis and evaluation of LVP. 111768 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES CAROLINA JANUARIO DA SILVA1, Diana Patricia Lamprea Sepulveda1, Flávio Hilton Feijó Cavalcanti Silva1, Raquel Cristina Farias de Medeiros Queiroz1 (1) PRONTO SOCORRO CARDIOLÓGICO DE PERNAMBUCO – PROCAPE Introduction: Lutembacher Syndrome (LS) is a rare condition and is defined as a combination of ostium secundum atrial septal defect (ASD) and mitral valve stenosis and both can be congenital or acquired. The hemodynamic repercussions of this syndrome result from the severity of the valve stenosis and ASD sizes. This can lead to numerous symptoms and complications, such as intolerance to exertion, palpitation, pulmonary hypertension, left atrium and right chamber dilatation, atrial arrhythmias and tricuspid valve failure. The treatment can happen through open surgical correction (with ASD closure and mitral commissurotomy or valve replacement) or percutaneous therapy (with balloon mitral valvuloplasty and septal occlusion with a prosthetic device) in eligible patients. We report a case in a mild age male, with late diagnosis and complicated by atrial fibrillation and pulmonary hypertension. Case Report: Male patient, 46-year-old, with previous diagnosis of systemic arterial hypertension. In 2011, he sought emergency care complaining of dyspnea on exertion and orthopnea. The echocardiogram showed an important mitral stenosis; ostium secundum atrial septal defect with a shunt from the left atrium to the right atrium; preserved left ventricle function and moderate left atrium dilatation. Based on the clinical and imaging findings, LS was diagnosed and was opted for the isolated mitral valve repair, with subsequent correction of interatrial communication – which occurred in 2013. In april 2022, the patient was hospitalized for the same symptoms he had 11 years before. The new echocardiogram showed mitral valve with reduced opening and thickened cusps, average gradient of 5 mmHg and valvular area calculated by the planimetry of 1 cm², representing an important mitral stenosis associated with a moderate mitral regurgitation; important left and right atrium dilatation; preserved left ventricle function and a moderate pulmonary arterial hypertension (with pulmonary artery systolic pressure estimated at 47 mmHg). The electrocardiogram showed a previously unknown atrial fibrillation. Clinical treatment was optimized and the patient was referred for mitral valve replacement. Conclusion: Early diagnosis with correction of mitral stenosis and closure of ASD indicate a good prognosis. However, in the cases that the diagnosis occurs late, the presence of heart failure, atrial fibrillation and pulmonary hypertension is more common and the prognosis is reserved. 111776 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CAMILA DALCOMUNI DOS SANTOS1, Rafael Vieira Fernandes1, Airton Salviano de Sousa Júnior1, Igor Henrique Silva Leite1, Barbara Porto Valente1 (1) Instituto Dante Pazzanese de Cardiologia – IDPC Aneurysmal dilation of the coronaries arteries has a prevalence rate between 1–3%(1). It can be classified in aneurism or ectasia. The first, defined as a focal dilation of about 1.5× the normal diameter and the second as a diffuse dilation The right coronary is the most targeted with 40% of cases, followed by 32% of the left anterior descending (2). Fifty percent are associated with atherosclerosis, the rest is caused by congenital anomalies and inflammatory diseases, such as Kawasaki’s (1). Most are clinically silent and are only detected incidentally during coronary angiography or computed tomography. Due to the lack of evidence, management is challenging. Usually, these patients are more susceptible to presenting complications like local thrombosis, distal embolization and myocardial infarction (5). The use of anticoagulants in the context of the acute coronary syndrome (ACS) is implicated in the reduction of major adverse cardiovascular events (MACE) in observational studies. Similar evidence sustains the use of dual antiplatelets (2,4). We present here a serie of 9 cases with aneurysm/coronary ectasia with median follow up of 3,5 years. All 9 patients had Myocardial infarction with non-obstructive coronary artery, 7 with ST-Elevation, due to aneurysm/coronary ectasia, of which 6 were men and 3 women, median age 54,5 years. Two patients had a family history of early coronary artery disease and previous infarction. Three patients had diabetes, history of smoking and obesity. Four patients were dyslipidemic and six had history of hypertension. One patient had atrial fibrillation/Flutter. The ejection fraction ranged from 40 to 60%, with an average of 52.7%. In the treatment, all patients were using statins and beta-blockers. Regarding antiplatelet and anticoagulation of these patients, 2 were using warfarin associated with P2Y12 inhibitors (iP2Y12), 1 was using warfarin alone, 1 was using direct-acting oral anticoagulant alone, 1 was using aspirin alone, 1 was using warfarin associated with aspirin and iP2Y12, and 2 used aspirin associated with iP2Y12. One of the patients evolved with dyspneia and 3 with angina. Three patients had another ACS. No deaths were recorded. Aneurysmal dilation of coronary vessels is a challenging and misdiagnosed entity, with lack of evidence, supporting an individualized management. 111812 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FELIPE ROSSI LORO1, CARLOS ALBERTO CAMPELLO JORGE2, GUILHERME DE MORAES FAVERO1, GABRIELLE MOLINA PINTO1, FERNANDA FURTADO REGATIERI1 (1) UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL – UFMS; (2) Hospital Militar de Área de Campo Grande – HMILACG Vasospastic angina, also known as Prinzmetal angina, corresponds to a form of angina pectoris related to coronary artery spasm, which can lead to transient ST-segment elevation on the electrocardiogram. We documented a case of a 62-year-old male, a former smoker, with hypertension, with a history of atypical chest pain during exercise for 11 years, and a previous investigation with catheterization showing no findings. In the meantime, he was also diagnosed with Crohn’s Disease due to gastrointestinal symptoms. He presented to our emergency department due to typical chest pain, which had been lasting 45 minutes and started after a positive treadmill exercise stress test for ischemia. The electrocardiogram and echocardiogram performed in the emergency room showed no abnormalities. He performed catheterization that showed severe stenoses in the right coronary and the left anterior descending arteries, but while preparing for angioplasty, the stenoses resolved after intracoronary nitroglycerin injection, thereby confirming the vasospasm. The case represents an atypical presentation of variant angina. Our hypothesis is that vascular hyperreactivity caused by Crohn’s disease acted as a precipitating factor for the patient coronary spasms. 111822 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JOÃO DE AZEVEDO1, Marcelo Kirschbaum1, Silvio Marques Póvoa Júnior1, Breno Oliveira Almeida1, Claudio Cirenza1 (1) Hospital Israelita Albert Einstein Introduction: Complications of the post-acute phase of COVID-19 can manifest in several organs and systems, mainly in the lung, but also in the cardiovascular and other organs. This is an uncommon clinical case of a patient with previous history of coronary artery disease and recent angioplasty, who was infected by COVID-19. This patient evolved with rapid and significant progression of underlying atherosclerotic disease resulting in an acute coronary syndrome and sub occlusion of the main left coronary artery. Case Report: A 58-year-old male patient was admitted to the emergency room with intermittent left arm pain, worsening on exertion and improving at rest, starting 24 hours before presentation. He had a history of coronary artery disease with elective angioplasty of the left anterior descending artery 2 months before in the context of stable angina, with no other coronary lesions. Later he had mild COVID 19 infection. He presented a dynamic change in the EKG, with ST-segment depression greater than 2.0 mm in DI, DII, AVF, V2–V6, in addition to ST-segment elevation in AVR, ultrasensitive troponin of 19 (VR ≤ 5). He was referred to the hemodynamics department for early invasive stratification and underwent angioplasty of the left main coronary artery due a sub occlusive lesion in that artery. Conclusion: Cardiovascular risk may increase in post-acute phase of COVID-19. This is an important issue that should draw health system attention to improve assistance either for mild or aggressive post-infection patients. 111828 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY EVELYN AMBROSANO ANTONIO1, JOSÉ EDUARDO DE LIMA BORRELLI FILHO1, LUCIANE FRANCISCHINI GOTTSCHALL ODONE1, FERNANDA MENDES1, VINICIUS MEARIM ODONE1 (1) HOSPITAL DOS FORNECEDORES DE CANA Despite the technological and therapeutic advances, infective endocarditis (IE) remains a pathology with high morbidity and mortality. On the other hand, there is a change in its epidemiological profile, with an increase in cases related to the presence of invasive devices and valve degeneration, with staphylococcus being the main infectious agent. We report a successful case in the treatment of a severe aortic valve IE that resulted, in addition to multiple sites of embolization and cardiogenic shock resulting from acute and severe aortic insufficiency (AOI), in coronary leaflet perforation, extensive valve abscess and consequent ventricular septal defect (VSD) subaortic COP, 73, male, with indolent lymphocytic lymphoma and non-dialytic chronic renal failure. Admitted with poor general condition, fever, abdominal pain and dizziness. Submitted to infectious screening and initially diagnosed with urinary tract infection, without improvement despite empirical antibiotic therapy. After neurological worsening, associated with septic shock of abdominal focus, multiple sites of embolization and hematogenous dissemination were visualized on tomography, including pyelonephritis, splenic abscess, cerebellar ischemia and skin lesions characterized as Janeway lesions. Identified in paired blood cultures S. Aureus infection. He underwent transesophageal echocardiography, which confirmed filamentous vegetation in a 10 mm native valve associated with valve dysfunction – moderate aortic insufficiency. On the 45th day of treatment, he presented rapid clinical deterioration to cardiogenic shock and cardiorenal syndrome, secondary to acute and severe AOI, and urgent surgical treatment was chosen. He underwent aortic valve replacement with a biological prosthesis, with intraoperative visualization of right coronary sinus perforation, extensive valve abscess and contiguity to the septum, resulting in a VSD approximately 1 cm below the aortic valve, which was corrected at the same surgical time. Maintained in intensive support and prolonged antibiotic therapy, with resolution of the infection, cardiac compensation and recovery of renal function We report the case of successful clinical and surgical treatment in a severe IE of native aortic valve, with positivity in all diagnostic criteria, multiple embolizations and rapid evolution to valve insufficiency, associated with abscess and consequent subaortic VSD, an uncommon but serious and potentially fatal local complication. 111870 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FELIPE HADDAD LOVATO1, Arthur Antunes Silva Castro1, Maria Julia Palitot de Melo2, Isabella Secol Corteze1, Isabella Souza Oliveira1 (1) Hospital Sírio-Libanês (HSL); (2) Pontifícia Universidade Católica de Campinas (PUCCAMP) Background: Takotsubo cardiomyopathy (TCM) is a reversible left ventricular dysfunction triggered by emotional or physical stress, which closely mimics acute coronary syndrome (ACS) and can presents with chest pain and dyspnea, dynamic ST/T changes, elevated myocardial necrosis biomarkers and and regional left ventricular (LV) wall motion abnormalities, without significant coronary artery disease. It is particularly frequent in 65–70 year-old female patients and should be considered as a differential diagnosis in episodes of chest pain. We describe the case of a 69 year-old woman who presented with chest pain while under stress and was then diagnosed with a focal variant of TCM, which represents 1.5% of patients in the International Takotsubo Registry study. SUMMARY M.E., 69 year-old female, with previous diagnosis of hypertension, presented to the emergency department with a 3 hour long chest pain during emotionally stressful event. On admission, ECG showed ST-segment depression in the inferior wall and V4–V6, with asymmetric negative T-wave in V2–V3, and laboratory tests showed high troponin. With the suspection of ACS, cardiac catheterization was indicated and showed no obstructive lesions. Echocardiogram showed hypokinesia in the anterior basal region. During investigation, the myocardial MRI showed a focal akinesia of the anterior middle segment of the LV. After these findings, we diagnosed TCM with focal involvement. Conclusion: TCM is an underdiagnosed cause of chest pain. It should be considered a differential diagnosis especially in postmenopausal women presenting with symptoms similar to ACS. This cardiomyopathy is often delayed, so it is important that physicians maintain a high index of suspicion for TCM, mainly in patients with few cardiovascular risk factors. 111877 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY MARIANA SILVEIRA DE ALCANTARA CHAUD1, Laís Olivo Rossi1, Felipe Lopes Malafaia1, Pedro Gabriel de Melo Barros e Silva1, Mucio Tavares de Oliveira Junior2 (1) Hospital Samaritano Paulista; (2) Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo Introduction: Left ventricular (LV) pseudoaneurysm is a rare disorder. It results from the rupture of the LV free wall and leads to hemorrhagic process contained by pericardium or scar tissue. LV pseudoaneurysms are most commonly caused by myocardial infarction. It can also be identified after cardiac interventions, infections or trauma. We describe a rare case of LV pseudoaneurysm secondary to myopericarditis with recurrences after first approach. Case Report: A 36-year-old man, with past medical history of hypertension, presented to our hospital with a 3-year complaint of chest pain, worsening in the last thirty days. Discomfort was described as sharp and radiated to left shoulder. It was associated to dyspnea on moderate exercion. Admission tests revealed negative troponin. Coronary computed tomography angiography (CTA) had no obstructive lesions. Echocardiogram confirmed LV pseudoaneurysm. At magnetic ressonance imaging, LV pericardial and mesoepicardial thickening was observed, with delayed enhancement suggesting myopericarditis, in addition to apical thrombus image. Coronary angiography confirmed the diagnosis, and aneurysmectomy was indicated. Surgery was uneventful and he was discharged in good condition twelve days after surgery. Five months after surgery, patient presented with chest pain and fever. Chest CTA showed a saccular formation at the apex of the LV. The patient was admitted for re-approach. Aneurysmal sac was sewn with anchored stitches and it was closed over the orifice without complications. He was discharged on the seventh postoperative day with good clinical evolution. Six months after the second intervention, patient had chest pain again. Echocardiogram showed LV rupture and communication to a neocavity with thrombus formation, which led to a new surgical approach with a vascular plug. On the fifth postoperative day, he was discharged with a well-positioned apical plug, obliterating the orifice. Six months later, a control echocardiogram was performed with an apical occluder device without shunt, apical hypokinesia and preserved LV ejection fraction. At one-year follow-up the patient remains asymptomatic and practicing physical activity. Conclusion: The case demonstrates that the presence of LV pseudoaneurysm resulting from myopericarditis, although rare, must be identified soon with multimodality tests. Surgical correction must be performed before long in order to prevent its rupture and provide quality of life for the patient. 111879 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY ANA LUIZA DE OLIVEIRA QUEIROZ TEOFILO1, WILSON ANDRÉS MENA SILVA1, Amanda de Miranda Constantino1, Luiz MInuzzo1, Daniel Dantas1 (1) INSTITUTO DANTE PAZZANESE DE CARDIOLOGIA Introduction: Tricuspid insufficiency (TI) is thought to be a rare complication of blunt, non-penetrating chest trauma. The right ventricle (RV) is immediately behind the sternum, predisposing it to an anteroposterior compression type of injury, especially during the end diastolic phase. Case Description: A previously healthy 39-year-old male without cardiac history was admitted to the emergency room with symptoms of heart failure and atrial flutter. Transthoracic echocardiogram showed significant tricuspid regurgitation, with an image suggestive of chordae rupture related to the anterior leaflet, with significant enlargement of the right chambers (right atrium indexed volume 115 ml/m²), moderate RV systolic dysfunction. After clinical stabilization, a history of high-energy automobile trauma occurred 20 years ago was identified, which resulted in traumatic brain injury and blunt abdominal trauma, requering abdominal surgeries; did not have any cardiovascular symptoms. Since then, he has not performed any cardiovascular follow-up. However, about three years ago, after starting to practice intense sports, he noticed of fatigue and dyspnea. He noticed progression of symptoms, seeking care in the emergency room with dyspnea on minimal exertion, palpitations and chest pain. In the heart team it was indicated surgical correction for severe TI. In the intraoperative, rupture of the chordae was observed, with calcification of the related papillary muscle and significant dilation of the tricuspid ring (60 mm);he was underwent valve replacement for a biological prosthesis in the tricuspid position. Postoperative echocardiogram showed the prosthesis in tricuspid position without reflux and maximum gradient of 8 mmHg. Conclusion: We report a case of traumatic TI, manifesting symptoms 20 years after the trauma. The interval between the traumatic event and the appearance of symptoms is variable, possibly due to the variability of possible associated valve lesions. While papillary muscle rupture is associated with more severe and acute symptoms (weeks to months), chordae rupture more commonly causes a slower course –10 to 25 years. The timing and appropriate treatment of traumatic TI depends on the natural course of the disease. Most cases do not require surgical intervention in the immediate period after the event. However, in the medium and long term, surgical correction of the tricuspid valve -by replacement or valve repair- is indicated to improve symptoms and avoid permanent impairment of the RV. 111887 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARIANA DE PAULA PERES1, Clarissa San Thiago1, Eduardo Rosa da Silva1, Camila Bussolo Schmitt1, Evandro de Campos Albino1 (1) Instituto de Cardiologia de Santa Catarina Interventricular communication (IVC) after acute myocardial infarction (AMI) is more common after anterior wall infarction, in the apical septal region, but, less commonly, it can occur in inferior wall AMI, at the base of the septum. It is a serious and potentially fatal complication that can suddenly lead to cardiogenic shock. Female patient, 56 years old, cleaning lady, is admitted to the emergency department with burning chest pain, of strong intensity, beginning 2 hours ago, irradiated to the left upper limb. She had arterial hypertension (SAH), diabetes mellitus, dyslipidemia and obesity, in addition to being an active smoker, with early family history of acute coronary syndrome. On admission, she was hemodynamically stable, respiratory and cardiac auscultation without alterations, as well as the remainder of the physical examination. The electrocardiogram (ECG) presented a tracing with sinus rhythm and ST-segment elevations in leads referring to lower walls. Coronary angiography visualized an obstruction of the distal third of the right coronary artery (RCA). Primary angioplasty of the lesion in the RCA was successfully performed. Transthoracic echocardiogram (TTE) showed aneurysm in the inferior wall of left ventricle (LV), LV concentric remodeling, grade I diastolic dysfunction and ejection fraction preserved despite segmental myocardial involvement and mild mitral regurgitation. Five days after hospitalization, she evolved with the presence of a 4+/6+ systolic murmur in the lower left sternal border, and a new TTE was requested, which showed IVC in mid-inferobasal segment from the base of the aneurysm, neck diameter estimated in 1.5 cm and diameter of the outlet in the right ventricule estimated at 0.6 cm, with mild hemodynamic repercussion. After one day, the patient started with symptoms of pulmonary congestion and showed clinical improvement with furosemide. Correction of post-AMI IVC with bovine pericardium was successfully performed twice weeks after identification via TTE. A new TTE was performed, and showed an intact interventricular septum (IVS). Currently, the surgery is indicated even in hemodynamically stable pacientes and with preserved left ventricular function, due to the possibility of abrupt expansion of the local rupture, resulting in sudden hemodynamic collapse. The shutdown surgery remains the procedure of choice, but the timing is controversial, and it should be performed as early as possible in cases of cardiogenic shock. 111893 Modality: E-Poster Young Researcher – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES FLÁVIA DE MORAES PEDRO MOISÉS1, Bernardo Cleto Teles e Silva1, Andrea Haddad1, Alessandra Godomiczer1, João Felipe Tamiozzo Reis1 (1) Hospital Unimed Rio Histoplasmosis is an infection caused by inoculation of the fungus Histoplasma capsulatum into soil or by dust contaminated with bird or bat feces. Prolonged and intense exposure, age and immunosuppression are risk factors. It may be restricted to the lung or spread hematogenously to other organs. Most cases are asymptomatic or mild, progressing without diagnosis. Cardiac manifestations are endocarditis, mediastinal granuloma, fibrosing mediastinitis, pericarditis and cardiac tamponade. Patient 27 a, male, without comorbidities, denies drinking and smoking, delivery man at a food distributor. He started having an afternoon fever for 1 month, dry cough, hyporexia, sweating, weight loss and pain in the right hypochondrium. Tachycardic and feverish, pulmonary auscultation reduced in bases. Chest CT with consolidations and nodules with subpleural soft tissue density in the right lower lobe, right pleural effusion with a heterogeneous mass in the right paratracheal chain and voluminous pericardial effusion. CT abdomen with lymph node enlargement at the height of the hepatic hilum. Laboratory without leukocytosis, lymphopenia, hepatogram without alterations, normal ESR and C-reactive protein. Echocardiogram with significant pericardial effusion, signs of tamponade with diastolic restriction, inferior vena cava without variation. Drainage 750 ml of pericardial fluid and make a pleuropericardial window with biopsy. Pericardial fluid with hemorrhagic appearance, lactate dehydrogenase 689, glucose 74, proteins 5.4 g/dl, adenosine deaminase 69 g/dl, BAAR negative, cytology without cell atypia, PCR for M. tuberculosis and negative fungal culture. Serology for dengue, human immunodeficiency virus, hepatitis B and C and COVID negative. A lymph node biopsy, right paratracheal mass and parietal pleura showed a granulomatous chronic inflammatory process involving mediastinal and pleural fibroadipose connective tissue with areas of central necrosis, presence of rounded and minute structures in the necrotic central portion of the granulomas suggestive of fungal yeasts from Histoplasma sp with absence of AFB. Although the patient did not present risk factors, did not present pulmonary lesions and presented a non-specific condition, the diagnosis was suspected due to paratracheal lymph node enlargement with a granulomatous aspect, increased incidence in the state and after exclusion of common causes of tamponade. Stable discharge for outpatient follow-up. 111958 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY BARBARA VIDIGAL DOS SANTOS1, Laís Villela Costa Vazquez1, Rayane Fontoura Koch1, Mariane Higa Shinzato1, Maria Júlia Silveira Souto1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Patent foramen ovale (PFO) is the persistence of the opening between the atrial septum primum and atrial septum secundum at the location of the fossa ovalis. It is present in about 25% of adults and is a casual finding without hemodynamic repercussions. However, studies prove an association between PFO and potentially severe clinical conditions, such as ischemic stroke, pulmonary embolism (PE), and, more rarely, acute myocardial infarction (MI). Despite well-documented consequences of paradoxical embolism through the PFO, the passage of venous clot from a patent foramen has been rarely described in the literature. We report a case of a patient with PFO and manifestation of three concomitant embolic events: venous thromboembolism, transient ischemic attack, and MI. Case Description: A 49-year-old woman with a history of idiopathic pulmonary arterial hypertension, asthma, PFO, and two previous episodes of PE sought emergency care with dyspnea on mild exertion for 2 weeks and oxygen desaturation on room air. She was admitted for compensation and clinical investigation. Chest CT angiography revealed dilatation of the pulmonary trunk and filling defects in the segmental and subsegmental arteries bilaterally. Full anticoagulation was started with enoxaparin. On the fourth day of hospitalization, the patient presented sudden burning chest pain, frontal headache, decreased consciousness, and tachypnea. ECG showed ST-segment elevation inferior MI and complete heart block, which spontaneously reverted to sinus rhythm. Patient also developed sudden left-sided hemiparesis and ipsilateral hemineglect. Non-contrast brain CT ruled out hemorrhage and aortic angiotomography excluded dissection. Due to the reported PFO, an embolic etiology was hypothesized and it was decided not to perform a coronary cineangiography and also to suspend antiplatelet drugs. The patient had complete reversal of the deficits within hours and no changes were noted in a subsequent brain CT. Cardiac magnetic resonance imaging confirmed inferior acute MI, and echocardiogram revealed PFO with small right-to-left shunt. After 18 days, the patient was discharged under oral anticoagulation and outpatient follow-up revealed no further complications. Conclusion: We report a case of paradoxical embolism manifested in distinct sites in a young patient with PFO, with good outcomes with anticoagulant therapy. 111964 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JOÃO VICTOR LIMA DANTAS 1, Hellen Dutra Passos1, Josefa Camila Menezes Reis Carvalho1, Antonio Carlos Sobral Sousa1, Joselina Luzia Meneses Oliveira2 (1) Hospital São Lucas – HSL; (2) Universidade Federal de Sergipe – UFS Takotsubo’s cardiomyopathy is defined as a transient abnormality of left ventricular wall movement, triggered by severe emotional or physical stress, in the absence of coronary obstruction. It is estimated that the syndrome is present in 2% of all patients who come to the emergency room for suspected ACS. Multiple variants have been described, classified according to the site of involvement of the left ventricular wall. The inverted form (Inverted Takotsubo), which corresponds to basal hypokinesia/akinesia, associated with apical hyperkinesia has a prevalence of 2.2%, occupying the 3rd place among the 4 described forms, therefore, a rare condition. We report the case of a previously healthy male patient, 56 years old, hospitalized due to left tibial fracture after falling from his own height, which evolved with hemodynamic instability followed by CRP during anesthetic induction to correct the trauma. The patient was submitted to the investigation and received the diagnosis of inverted Takotsubo. Although the inverted variant is more prevalent in women and young people, the greater knowledge of the pathology has shown increased incidence in men, with physical stress being the main triggering factor. The pathophysiological process of inverted Takotsubo is similar to its typical variant and not completely understood, but there is considerable evidence that sympathetic stimulation is the central mechanism of its pathogenesis. 111970 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY AMANDA DE MIRANDA CONSTANTINO1, Vivian De Biase1, Beatriz Moura Sucupira Tajra1, Alana Osterno Moreira Linhares1, Mardelson Nery de Souza1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: Scimitar syndrome (CS) is a rare congenital cardiac malformation that occurs in 2/100,000 live births with a 2:1 predominance in females, it belongs to the group of partial anomalous pulmonary venous connections, which has numerous described variations, being a subtype that constitutes only 3–5% of congenital cardiac anomalies. It is classified as a heart disease with increased pulmonary and cyanogenic flow, its diagnosis is usually early, but we will highlight the importance of attention to this pathology in adult patients without a previous diagnosis. Case Report: Female patient, 39 years old, with dyspnea for 3 years, with progressive worsening of functional class. On physical examination, pulmonary auscultation was normal with 98% saturation. On cardiac auscultation, regular rhythm, with fixed split S2 and ejective systolic murmur 2+/4+ in the middle right sternal border. A chest X-ray was performed during the investigation, which showed a slightly arched tubular structure towards the right atrium with dextrocardia. In transthoracic echocardiogram evidenced anomalous pulmonary venous connections: the right pulmonary veins drain into the right atrium at the mouth of the inferior vena cava, with free flow, and the left into the left atrium. Ostium secundum interatrial communication of 13.5 mm with bidirectional flow, significant dilation of the right cavities, with right ventricular myocardium with mild hypertrophy, with preserved function. PSVD 51 mmHg and hypoplastic right pulmonary artery. Discussion: SC is a rare disease characterized by anomalous venous drainage from the lung directly into the inferior vena cava. The disease may be associated with right pulmonary hypoplasia, dextrocardia, cardiac abnormalities, and systemic pulmonary collaterals. The present case report draws attention to the late discovery of a congenital heart disease, with the patient already symptomatic and with possible important hemodynamic repercussions, highlighting the importance of always evaluating the clinical picture of dyspnea and heart failure in adults during the routine investigation. the presence of a congenital heart disease. 112001 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING YURI RHIOS ARAÚJO SANTOS1, Luiz Henrique de Oliveira Bernardino1, Yuli Mendes de Souza1, Victor dos Reis Cunha1, Thaiz Ruberti Schmal1 (1) Universidade Federal de Juiz de Fora, UFJF Introduction: Sarcoidosis is an idiopathic systemic inflammatory disease marked by the formation of non-caseating granulomas, most prevalent in middle aged women and afro descendants. Diagnosis is made based on clinical, imaging, and histological findings after excluding other causes of granulomatous diseases. Although rare, Cardiac Sarcoidosis (CS) is the second cause of death in affected patients. Poor prognosis is related to ventricular dysfunction and severe arrhythmias. Although endomyocardial biopsy allows final diagnosis, guidelines using clinical and imaging criteria have been proposed to avoid invasive procedures. Case Report: A 41-year-old black woman complaining of headache develops amaurosis, hypoacusis, cutaneous lesions, dyspnea, hypotension, and tachycardia. Meningeal biopsy revealed non necrotizing granulomatous inflammation, leading to the diagnosis of neurosarcoidosis. In face of cardiac symptoms, cardiac involvement was also suspected. The electrocardiogram showed sinus tachycardia. Echocardiogram revealed moderate left ventricle (LV) systolic dysfunction, reduced Global Longitudinal Strain and hypokinesia of multiple wall segments. Magnetic Resonance Imaging exhibited mild LV global dysfunction and segmental wall dysfunction of multiple segments. Specific T2 weighted sequences showed small regions of myocardial edema and the specific sequences for late enhancement demonstrated subepicardial fibrosis of the septal apical and anterolateral basal segments and transmural fibrosis of the inferolateral medial wall as seen in the annexed image. The diagnosis of sarcoidosis with meningeal and cardiac involvement was made and imunossupressor systemic therapy was started with standard heart failure medication, leading to clinical stabilization. The patient was discharged and continued its follow-up as an outpatient. Conclusion: The current report shows that it is possible to diagnose CS in patients with a pre-established diagnosis of sarcoidosis through clinical and imaging findings. As a result, adequate treatment may be started as soon as the diagnosis is made, avoiding disease progression. 112012 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES RAYANNE KALINNE NEVES DANTAS1, Dieison Pedro Tomaz da Silva1, Ramon Felix de Avila1, Eduardo Caetano Abujamra1, Luciano Vaccari Grassi1 (1) Hospital de Base São José do Rio Preto; (2) Faculdade de Medicina de São José do Rio Preto Introduction: Systemic lupus erythematosus (SLE) is often associated with cardiovascular lesions, and may evolve mainly with pericarditis and myocarditis. Aortitis is a rare complication in SLE and can present itself in a variety of ways, from minimal aortic valve regurgitation to aneurysm with risk of dissection. This is an extremely rare complication, being more commonly found in patients with arterial hypertension and prolonged use of corticosteroids, and, in many cases, they are not diagnosed early and are documented during the perioperative period or by autopsy. Case Report: Female patient, 49 years old, with a previous diagnosis of systemic lupus erythematosus, systemic arterial hypertension and membranous nephropathy, using prednisone 5 mg/day, hospitalized after an outpatient consultation due to pancytopenia, arthritis and worsening of renal dysfunction, hemodynamically stable. One day after hospital admission, he developed tight chest pain, started at rest, of strong intensity and with spontaneous improvement. An electrocardiogram showed diffuse changes in ventricular repolarization and, on laboratory examination, he showed an ascending troponin curve. The possibility of lupus myocarditis was questioned and the decision was made to start pulse therapy with methylprednisolone. The patient evolved with orthopnea and jugular distension, without improvement with intense diuretic therapy, muffled heart sounds and desaturation, requiring supplemental oxygen, and was transferred to the intensive care unit. An emergency transthoracic echocardiogram was performed and confirmed the diagnosis of cardiac tamponade, probably due to lupus pericarditis. During the surgical approach for pericardiocentesis, an important acute aortic dissection was visualized. Surgical approaches for both conditions were uneventful, and today, the patient is clinically and hemodynamically stable from a cardiovascular point of view. Conclusion: Thoracic aneurysms and vascular dissections, mainly aortic, in SLE have a greater correlation with disease activity, in addition to a high risk of fatal outcome. Given this scenario, it is important to remember that aortitis is a cardiovascular manifestation in these patients, aiming for timely treatment to reduce death and disabling sequelae. 112028 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES ANDRESSA DE MEDEIROS PULCHERIO TOSETTO1, Joana Carolina Junqueira Brum1, Vanessa Grings1, Luiz Claudio Danzmann1 (1) Hospital São Lucas PUCRS – HSL PUCRS; (2) Santa Casa de Misericórdia de Porto Alegre Introduction: The withdrawal of inotropic drugs in patients with heart failure with reduced ejection fraction (HFrEF) is frequently challenging. In this group of patients, peripheral vasodilatation contributes for clinic and hemodynamic improvement (1). For this reason, the vasodilatory properties of neprilysin inhibitors can be of great value in this scenario. However, there’s little data available in regard to the use of neprilysin inhibitors in patients receiving inotropic drugs. Case Report: A 71-year-old male patient with previous diagnosis of HFrEF with ischemic etiology (Simpson EF 31%) was admitted in a hospital in south of Brazil after cardiac arrest in a public street. The cardiac arrest rhythm was identified by first responders as ventricular fibrillation. Patient was immediately referred to coronary angiography which showed total occlusion of the circumflex coronary artery, subocclusion of the right coronary artery and severe stenosis of the anterior descending coronary artery. Performed angioplasty of the anterior descending coronary artery and intra aortic balloon and Swan-Ganz catheter insertion. Simultaneously, he needs hemodynamic support with noradrenaline, vasopressin and dobutamine. After four days of intensive care he presents hemodynamic and laboratory improvement, withdrawal of noradrenaline and vasopressin and maintenance of dobutamine at moderate doses. At this moment, sacubitril-valsartan is added to the therapeutic regimen. Initially, there were some episodes of hypotension (systolic arterial pressure 80–85 mmHg), but after 48 hours of medication was observed improvement in lactatemia and central venous oxygen saturation without hiperkalemia or worsening of renal function. In this context, it was possible to withdrawal dobutamine and discharge patient from ICU. Later, patient was discharged from the hospital with an optimized dose of sacubitril-valsartan and after 2 months, he maintained clinical stability and functional class NYHA II. Conclusion: The PIONEER-HF study (2) showed the safety of starting neprilysin inhibitors in a hospital setting, excluding patients in use of vasoactive drugs. In this case report, it was decided to start the medication in a patient while in use of an inotropic medication. This approach was taken considering data of a small retrospective analysis of 25 patients with HFrEF in use of vasodilators and/or inotropes (3). In this study, the use of neprilysin inhibitors was well tolerated and impro. 112057 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MILENA JOLY KULICZ1, Alana Ohashi1, Isadora Daleffi Zocoler1, Daniel Hideki Tashima1, Talita Beithum Ribeiro Mialski1 (1) Complexo Hospital de Clínicas UFPR (CHC-UFPR) Introduction: The ischemic heart disease is the main cause of death in Brazil and worldwide. The following case illustrates a diagnoses of ST-elevation myocardial infarction (STEMI) with two culprit arteries. Case Report: A 53-year-old man, obese and active smoker (40 pack-years), admitted at a tertiary hospital with typical chest pain for 10 hours. The admission electrocardiogram showed right bundle branch block and ST-elevation on anterior wall, DIII and aVF leads. The patient received loading doses of aspirin and clopidogrel and was referred to invasive coronary angiography, which demonstrated proximal occlusion of the right coronary artery (RCA), 99% stenosis with thrombus inside of proximal anterior descending coronary artery (LAD), and 80% stenosis of distal left coronary artery (LCA). Percutaneous coronary intervention (PCI) was performed with stent placement in the RCA and LAD. The patient was referred to the cardiac ICU requiring doses of vasoactive drugs for 24 hours, presenting significant clinical and hemodynamic improvement. After Heart Team discussion, it was performed PCI with stent placement in the LCA. The patient was discharged asymptomatic from hospital, with left ventricular ejection fraction of 39%, in addition to septal, apical, mid anterior and inferior akinesia, receiving STEMI and heart failure treatment. Discussion: Multiple Culprit Arteries are rare in STEMI and associated with worse outcomes, death and high incidence of complications, including cardiogenic shock and ventricular arrhythmias. The presented case highlights the STEMI with two culprit vessels (RCA and LAD), severe stenosis of distal LCA and good evolution after PCI. 112060 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING HEBER HENRIQUE ALVES LESSI1, Santiago Andres Castro Vintimilla1 (1) Instituto do Coração Left ventricular pseudoaneurysm is a very rare complication after acute myocardial infarction, which results from ventricular wall rupture. Although rare, it can be catastrophic due to imminent risk of rupture and death. The differential diagnosis with true aneurysm is very important because it implies the patient’s prognoses. We report an interesting case of presentation of left ventricular giant pseudoaneurysm after myocardial infarction in a 72-year-old man, with a history of acute coronary syndrome eighty days before he sought our service, admitted to the emergency unit with new chest pain that had started 24 hours ago. About 48 hours after admission, the patient underwent cardiac catheterization, showing multivessel disease and inferior aneurysm on cardiac ventriculography. A transthoracic echocardiogram was performed that showed an pseudoaneurysm in the basal segment of the inferior wall. The inferobasal pseudoaneurysm has a neck of 2.8 cm, a length of 4.7 cm and a distal width of 3.8 cm. Cardiac Magnetic Resonance confirmed the diagnostic in the left ventricle with increased dimensions at the expense of a giant pseudoaneurysm in the basal segment of the inferior wall and the presence of intracavitary thrombus measuring 16 × 5 mm, in the lower basal wall. The patient underwent surgical correction of the pseudoaneurysm with geometric reconstruction of the left ventricular due to free wall rupture and coronary arterial bypass grafting. A favorable hemodynamic evolution and discharge from the intensive care unit on the third postoperative day. About one week after discharge from the ICU he developed infectious complications and required readmission to the intensive care unit and died 30 days after cardiac surgery. 112066 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING LEONCIO BEM SIDRIM1, Simone Cristina Soares Brandão2, Diana Patrícia Lamprea1, Thais Araújo Nóbrega1 (1) Pronto Socorro Cardiológico de Pernambuco – PROCAPE/UPE; (2) Hospital das Clínicas – UFPE Introduction: Positron emission tomography with 18F-fluorodeoxyglucose (FDG PET/CT) is increasingly used in cases of Prosthetic Valve Endocarditis (PVE). However, the specificity of the method still represents a limitation. It is recognized that an uptake in the perivalvular area can occur in the absence of infection. The persistence of uptake in the periprosthetic area can be explained by the persistence of a healing process around the seam ring, with endothelization. Such a process can even trigger marked fibroblast proliferation, leading to obstructive pannus in the most severe cases. Case Description: Male, 51 years old, with a history of rheumatic heart disease, with two biological aortic and mitral valve prostheses implanted 2 years ago. Admitted with paresthesia and labial commissure deviation for 3 weeks. He denied fever. Cardiac auscultation without murmurs. Cranial tomography showed hypodensity in the caudate nucleus related to the sequel of an ischemic insult. A transesophageal echocardiogram was performed, which showed a dysfunctional mitral bioprosthesis with a filamentous image measuring 3.0 cm, adhered to one of the leaflets. Blood cultures did not show bacterial or fungal growth. FDG PET/CT showed homogeneous hyperuptake in mitral and aortic perivalvular topography (maximum SUV of 5.49 and 5.28, respectively). The patient underwent surgery for valve replacement due to prosthetic dysfunction. The intraoperative finding was of vegetating lesions adhered to the prosthesis. The culture of the prosthesis was negative. Conclusion: Caution is needed when interpreting FDG PET/CT in suspected PVE, and special attention should be paid to the CVP uptake pattern and implantation time. Finally, the need to integrate FDG PET/CT results with the clinical context is highlighted. 112081 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BRUNO LINHARES AZEREDO CORRÊA1, Jaime Lobo Figueiredo1, Sabrina Pedrosa Lima1, Fabio Akio Nishijuka1, Renata Rodrigues Teixeira de Castro1 (1) Hospital Naval Marcílio Dias Introduction: Lupus is an autoimmune disease with multiple presentations, several signs and symptoms and involvement of various organs and systems. Like Lupus, Coronavirus-19 is no longer an acute lung disease to become a multiorgan disease. The involvement of multiple systems is already well described in the literature. 2 years after the beginning of the pandemic, we are knowing its long-term effects. The cardiological spectrum has been gaining prominence after the chronification of the disease because it has a great impact on the quality of life of patients. Case Report: Female, 33 years old, no previous comorbidities, with a history of COVID-19 in April 2020 without hospitalization. Started home treatment with symptomatic and oral corticosteroids with partial improvement of symptoms. Evolving with exercise intolerance, palpitation, chest pain and symptoms of postural orthostatic tachycardia syndrome (POTs). In March 2021, after remission, she takes 2 doses of Coronavac, and returned with cardiovascular symptoms, associated of low output. She performed cardiac magnetic resonance imaging (cMRI) in April 2021 with a pattern of inflammatory injury in the pericardium. The 24-hour holter resulted sinus rhythm, sinus arrhythmias, and mean heart-rate of 104. Treated with ibuprofen for 30 days and colchicine for 6 months. In October 2021, the control cMRI showed significant improvement in pericarditis. In January 2022, a new oligosymptomatic SARS-Cov-2 infection. In March 2022 she returned with symptoms. In a new study with cMRI, the pattern of pericarditis returned. Ibuprofen was restarted for 30 days and colchicine for 6 months, maintaining medical follow-up until the present moment. Conclusion: Time has consolidated COVID-19 as another multiorgan disease. In this context, it’s important to understand the pathophysiological mechanisms in the heart, generated by the long term COVID-19, as well as to develop strategies to mitigate the impacts on the functionality and quality of life of the economically active population. 112087 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY AMANDA DE MIRANDA CONSTANTINO1, Vivian De Biase1, Beatriz Moura Sucupira Tajra1, Thauan Paulúcio de Oliveira1, Mardelson Nery de Souza1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: The Atrioventricular Septal Defect (AVSD) corresponds to 3% of congenital heart diseases, being associated with genetic syndromes and other congenital malformations. One of the anatomical alterations present in AVSD is the posterior displacement of the atrioventricular node, located between the coronary sinus and the annulus of the atrioventricular valve, increasing the risk of injury to the same during mitral cleft repair, generating atrioventricular block (AVB), Such a complication may evolve with the need for definitive implantation of a pacemaker. Abstract: A 27-year-old female patient with complex heart disease (left atrial isomerism, partial AVSD, muscular interventricular communication, single atrium, pulmonary hypertension), underwent atrial septation surgery, mitral cleft repair and ventriculoseptoplasty in the first year of life, evolving in the postoperative period with total AVB, requiring implantation of a permanent pacemaker 1 month after the surgical procedure. In follow-up, she demonstrated heart rate reversibility, being submitted to extraction of the pacemaker generator and plasty of the abdominal pocket at age 22, remaining with the endocardial electrode. The patient evolved with symptoms of right heart failure (dyspnea, with worsening of the functional class). During the current diagnostic investigation, a significant pulmonary gradient was evidenced by the transthoracic echocardiogram, but without valvular lesion. An angiotomography of the heart and basal vessels was performed, which showed extrinsic compression of the right pulmonary artery by the pacemaker wire, which generated the pulmonary stenosis evidenced by the echocardiogram. Conclusion: Extrinsic compression by endocardial pacemaker lead is a rare phenomenon that can be difficult to recognize. In the case reported, due to the left atrial isomerism and the need to implant a permanent pacemaker during childhood in the abdominal cavity, the electrode was implanted through the azygos venous system. With the development and growth of the patient, the fixed electrode implanted in the right ventricle pulled the azygos vein under the right pulmonary artery, generating extrinsic compression and pulmonary stenosis evidenced both by imaging methods and by the patient’s clinical condition. It is clear the importance of late follow-up of patients who still have implanted electrodes, even without the use of a pacemaker, in complex anatomical situations. 112084 Modality: E-Poster Young Researcher – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MONIQUE ALMEIDA VAZ1, Gustavo Lara Moscardi1 (1) Hospital Universitário de Brasília (HUB-UnB) Introduction: The permanent pacemakers (PPM) are most commonly implanted via an epicardial approach in pediatric patients, due to transvenous route limitations. The advent of generators and thinner leads has increased the implantation of PPM with transvenous leads over the last three decades. Growth of children causes stress on leads, which do not tolerate ongoing linear strain. Aiming to reduce conductor fractures, some redundancy should be left as slack to avoid tension, or the use of an atrial loop. However, the extra loop of lead can result in mechanical and hemodynamic complications, as lead prolapse through the pulmonary valve (PV) and its insufficiency. Case Report: A 16-year-old male with a history of congenital complete heart block, underwent dual chamber epichardical pacemaker implant on the first day of life. At age 10, transvenous atrial and ventricular leads were implanted along with pulse generator in thoracic site, left an atrial loop. During routine follow-up, he was asymptomatic and with normal average growth. Over the next year, transthoracic echocardiogram (TTE) showed prominent lead slack prolapsing across the PV, into the pulmonary artery (PA), mild tricuspid regurgitation, mild pulmonary regurgitation and normal left ventricular systolic function (56%). At age 13, TTE elucidated prolapsed lead into pulmonary valve, causing moderate regurgitation, and signs of interventricular dyssynchrony. During device interrogation, at age 16, evidence of lead fracture was found. A new TTE showed right ventricular (RV) hypertrophy and dysfunction, and redundant lead with a long loop, located inside the PA, causing double pulmonary valve lesion. Removal and replacement of the ventricular lead was indicated. Conclusion: Prolapse of transvenous lead through the PV can occur when excess slack is left for growth. The tip of ventricular lead fixated in the apex may facilitate its prolapse into PA when it is closer to the outflow tract, because of alterations in contractility and vortex flow patterns. Therefore, it is recommended to place RV leads in septal locations to avoid that complication. The treatment consists in repositioning leads, through extraction and replacement, particularly if adherent to valve apparatus. However, the pulmonary valve disfunction may be irreversible. Periodic radiographic and echocardiographic monitoring is essential in pediatric patients, allowing a preclinical detection of valvular dysfunction secondary to lead prolapse. 112120 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR IMAGING JÉSSICA MAYARA DE FIGUEIRÊDO OSÉAS1, Nestor Rodrigues de Oliveira Neto1, Jessione de Carvalho Lima Neto1, Matheus Fontes Leite1, Camila Albuquerque Coelho Lopes2 (1) Universidade Federal do Rio Grande do Norte, UFRN; (2) Universidade Potiguar, UnP This study aims through a case report of an unusual pathology to recall the importance of considering the importance of differential diagnosis in clinical reasoning in the care of patients with acute myocardial infarction, especially considering the possible etiologies of a type 2 infarction. A 42-year-old male patient, with no history of comorbidities, was admitted to UPA 30/02/2022 due to severe precordial pain, in sudden onset grip when performing physical exercise. Requested propaedeutics of infarction. ECG with lower wall ST segment unevenness and elevated troponin, compatible with AMI. He was referred to the university hospital for cardiac catheterization on the same day, which did not show any athertortotic lesions in coronary arteries. However, due to the abnormal vascular path observed on the examination, hemodinamycrist suggested the presence of tumor lesion in the left atrium. On 4/6, a transesophageal echocardiogram showed a mass in both atria of 6.4 × 2.6 cm, suggestive of myxoma. Surgery was performed on 4/29, and complete resection of the myxoma was performed in need of resection of the entire interatrial septum. The patient evolved clinically stable and discharged from the hospital on 05/05/2022 for outpatient follow-up with clinical treatment after AMI. This report of a type 2 infarction by an atheroembolic event due to atrial myxoma in a young patient without comorbidities exemplifies why we should not limit the diagnosis of infarctions to type 1, even if it is the most common. 112111 Modality: E-Poster Young Researcher – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY AMANDA DE MIRANDA CONSTANTINO1, Vivian De Biase1, Beatriz Moura Sucupira Tajra1, Thauan Paulucio de Oliveira1, Mardelson Nery de Souza1 (1) Instituto Dante Pazzanese de Cardiologia Introduction: ALCAPA Syndrome (Anomalous Left Coronary from the Pulmonary Artery) is the anomalous origin of the left coronary artery from the pulmonary artery. Also known as Bland-White-Garland Syndrome, it is a rare congenital heart defect, present in 0.25–0.5% of patients with congenital heart defects. It can occur in isolation or be associated with other birth defects. Usually the diagnosis is early, made in the prenatal period and the need for its correction is immediate. Case Report: A 63-year-old female patient sought emergency care with chest pain, dyspnea on moderate exertion, and palpitations. Myocardial scintigraphy was positive for ischemia, corresponding to the territory of the anterior descending artery. Cardiac catheterization was requested, in which an anomalous left coronary artery was observed in the pulmonary artery trunk. The transthoracic echocardiogram showed the following findings: akinesia of the apical and anteroapical septum, with hypokinesia of the anterolateral wall and of the mid-basal segments of the anterior wall, moderate left ventricular systolic dysfunction (ejection fraction of 37%), pulmonary artery systolic pressure of 62 mmHg. It has magnetic resonance imaging of the heart, which shows the areas of infarction mentioned in the echocardiogram, without myocardial viability. Discussion: ALCAPA syndrome is difficult to diagnose, and should be suspected in children with dilated cardiomyopathy. About 85% of cases manifest up to 2 months of age, but symptoms can be misinterpreted and the disease may be underdiagnosed. The disease requires surgical treatment to restore the flow of oxygenated blood to the myocardium. The procedure of choice consists of reimplanting the coronary artery in the aorta and it is estimated that the surgical mortality is less than 5–10%, with a good prognosis after reimplantation. The peculiarity of the case reported is due to the fact that the diagnosis was extremely late and with important irreversible hemodynamic repercussions: the patient has an ischemic cardiomyopathy without myocardial viability, a condition usually related to chronic coronary artery disease or acute coronary syndromes, rarely related to ALCAPA, since due to the severity of the syndrome, its diagnosis is early. Currently, due to the lack of myocardial viability, surgical correction would not bring benefits or recovery of heart muscle, opting for clinical treatment for cardiac remodeling. 112145 Modality: E-Poster Young Researcher – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY ANDRES FELIPE VALENCIA RENDON1, Marcello Augustus de Sena2, Murilo Castro3, Bernardo Kremer1 (1) Federal University of Rio de Janeiro; (2) ITPAC Palmas; (3) Palmas Medical Center 43 years old female patient, with controlled Hypertension, without any other known comorbidities. Two years ago, she was submitted to an open biological Mitral valve replacement due to severe rheumatic mitral valve stenosis. During the procedure, a highly redundant atrial septum was noted, hence an atrioseptoplasty was performed using bovine pericardium. In the recent year, she evolved with worsening dyspnea, platipnea, with a Trans Thoracic Echocardiogram showing a severe Mitral stenosis with a mean gradient of 20 mmHg. The patient declined another open-heart surgery, therefore a transcatheter approach was selected. Under general anesthesia, under guidance of Trans Esophageal Echocardiography (TEE), a Brokenbrough catheter with needle was advanced though the right femoral vein reaching the right atrial septum. Not having a clear reference of the Fossa Ovalis due to the previous atrioseptoplasty, the anatomical navigation for the septal puncture was performed under the TEE landmarks. The penetration of the bovine pericardial patch presented a high resistance with the classical approach; hence the needle was connected to an electro surgical scalpel console using 30 mV, having success at the first attempt, with no anatomical alterations or hemodynamic instability. A sapiens 3 valve was advanced to the mitral valve position with no issues after deployment, presenting a minimal atrio ventricular pressure gradient. To our knowledge, this is one of the first reports of a mitral valve in valve intervention through a previous a bovine pericardial septal patch. 112151 Modality: E-Poster Young Researcher – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GIOVANNA BOLINI BRAZÃO1, Fábio Vasconcellos Brazão3, Maria Angélica Bolini Brazão2, Patrícia Brazão Cohen4 (1) Centro Universitário do Estado do Pará (CESUPA); (2) Universidade Federal do Pará (UFPA); (3) Laboratório Ruth Brazão, LTDA; (4) Universidade do Estado do Pará (UEPA) Introduction: In the last two years, several post-infectious cardiac repercussions have been observed in SARS-CoV-2 infections. In severe cases, the infection can cause pneumonia, severe acute respiratory syndrome, cardiomyopathies, kidney failure and even death, with an impact on public health. Case Description: Female, 51 years old, without comorbidities, remained in prolonged hospitalization due to COVID-19 in a private hospital in São Paulo (SP). Upon admission, developed a Severe Acute Respiratory Infection, requiring admission to the ICU-Adult and later orotracheal intubation and mechanical ventilation (MV). Remained on MV for a long time and presented complications such as septic shock from ventilator-associated pneumonia, bronchitis obliterans with organizing pneumonia, which required high-dose corticosteroids, in addition to a short-term catheter-related bloodstream infection [antimicrobials used: Ceftriaxone; Cefepime; Meropenem + Vancomycin; Ceftazidime + Vancomycin; Polymyxin + Amikacin; single dose ivermectin]. At that time, presented a chest computed tomography with a report showing more than 90% of pulmonary involvement. Due to the prolonged time on MV, she needed a Tracheostomy to wean off sedatives and MV. After adequate awakening, started a process of respiratory, cognitive, muscular and sensory rehabilitation due to the critically ill patient’s polyneuromyopathy. About a month after awakening, during the rehabilitation process, had an episode of chest discomfort investigated for suspected acute coronary syndrome. A transthoracic echocardiogram was performed, which showed mild hypokinesia of the anterior wall and segmental myocardial involvement of the left ventricle – non-existent changes in an exam performed one month before – in addition to the dosage of troponin (18 pg/mL) and CPK (21 U/L). The investigation and risk stratification showed no high cardiovascular risk, since they related changes to psychological stress due to the death of the patient’s mother in this interval. Therefore, was released for activities and for hospital discharge. She was discharged to continue outpatient follow-up and home rehabilitation, which lasted for about 6 months. Conclusion: Studies should be carried out to accumulate more data on the cardiac repercussions of the new coronavirus and its variants in Brazil and in the world, in order to pressure public health agencies to create protocols aimed at the prevention and treatment of such cardiomyopathies. 112177 Modality: E-Poster Young Researcher – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BRUNO LINHARES AZEREDO CORRÊA1, Sabrina Pedrosa Lima1, Fabio Lucas Bassini e Silva1, Fabio Akio Nishijuka1, Renata Rodrigues Teixeira de Castro1 (1) Hospital Naval Marcílio Dias Introduction: Heart failure is not a pathology that usually affects young adults. However, once manifested, ischemic etiologies or those associated with infectious myocarditis should be ruled out. In parallel, hereditary etiologies must be considered, in which genetic tests are of enormous importance, and which are gaining more and more space in current medicine. Case Report: A 22-year-old man presented with progressive dyspnea until minimal exertion, associated with paroxysmal nocturnal dyspnea and orthopnea. Admitted with heart failure with reduced ejection fraction (EF) (admission echocardiogram EF 09% by Teichozl) hemodynamic profile B. MAGGIC Score 29 points. After the condition was compensated, triple therapy with beta-blockers, spironolactone and enalapril (ACEI) was started. Subsequently, the ACEI is replaced by the angiotensin and neprilysin receptor inhibitor (INRA) for hospital discharge. 3 weeks after discharge, she was readmitted due to decompensation associated with poor adherence to treatment. There was no water restriction. A new drug adjustment was performed, with sequential nephron block and inclusion of dapagliflozin, with a good response. During the diagnostic investigation, there was a positive family history of dilated cardiomyopathy on the part of the mother at age 25 years. Cardiac magnetic resonance imaging (cMRI) showed late enhancement with a pattern of myocardial injury of non-ischemic origin with EF 10%. Six months after admission, a new cMRI showed the same pattern, but with EF 21%, and ergospirometry with VO2max of 35.36 ml/kg/min. The genetic test showed the presence, in heterozygosity, of the variant described as NM_001458.5(FLNC):c.6976C > T;p.(Arg2326Ter), classified as pathogenic, in the FLNC gene (Filamin C), associated with Familial Restrictive Cardiomyopathy of the type 5 (OMIM:617047), of autosomal dominant inheritance. Conclusion: In young patients with heart failure, in addition to investigating the ischemic etiology or related to infectious myocarditis, one should also consider hereditary etiologies. At this point, genetic research provides important information about the definitive diagnosis and therapeutic choice. However, the high cost of genetic sequencing makes this test little available. Making it accessible will allow testing the source patient’s family members and offering patient-directed therapy, ensuring improvement in symptoms and, consequently, quality of life. 112226 Modality: E-Poster Young Researcher – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MILENE FERNANDES FARIAS1, Adriel Alves de Paiva; Emídio Almeida Júnior Jr; João Marcos Bemfica Barbosa Ferreira; Katia do Nascimento Couceiro.2 (1) Fundação Hospital do Coração Francisca Mendes – Amazonas, Brasil.; (2) Universidade do Estado do Amazonas Introduction: Myocardial infarction without obstructive coronary atherosclerosis (MINOCA) is a syndrome with many causes, representing 10 to 15% of all diagnoses of acute myocardial infarction (AMI). Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by the plasma detection of antiphospholipid antibodies, such as anticardiolipin and lupus anticoagulant, which clinically manifests itself mainly as recurrent arterial and/or venous thrombosis. It is the main acquired cause of hypercoagulability, occurring in 2% of the general population and has high morbidity and mortality. Myocardial involvement, however, is rarely described in this pathology. Case Report: The present report describes the case of an elderly, non-sedentary, hypertensive patient, using Nebivolol 5 mg, who, after a flight to Switzerland, presented on the seventh day in that country with typical chest pain, without dyspnea, syncope or associated palpitations, electrocardiogram with millimeter elevation of V3, with flat T waves, even negative in aVL, elevated troponins, being diagnosed with acute coronary syndrome without ST-segment elevation. Echocardiogram showing normal ventricular ejection fraction (estimated at 60%), mild inferior hypokinesia. After coronary angiography, thrombosis of the posterior ventricular branches was evidenced with thrombus aspiration by microcatheter and thrombectomy with a stent retriever. Lower limb venous thrombosis was ruled out by angiographic evaluation, and treatment with low-molecular-weight heparin was initiated. Upon returning to Brazil, three months after the event, the patient was treated at a referral hospital, and a diagnostic investigation was carried out, including APS, deep vein thrombosis of the lower limbs and patent foramen ovale (PFO). Conclusion: We report the favorable outcome of primary APS with cardiological complications, which after its diagnosis determined the institution of full anticoagulation with oral anticoagulant. Clinical management of hypertension was maintained with beta-blocker therapy, with no recurrence of chest pain. Therefore, he continues to be followed up with a hematologist and cardiologist, with the aim of reducing the morbidities that may eventually exist. 112232 Modality: E-Poster Young Researcher – Case Report Category: CARDIOVASCULAR SURGERY ALBERT SALVIANO DOS SANTOS 1, Ana Luiza Scholl Giaretta1, Ana Maria Rocha e Pinto1, Valquíria Pelisser Campagnucci1, Camila de Queiros Mattoso Archela dos Santos1 (1) Irmandade da Santa Casa de Misericórdia de São Paulo Introduction: Primary aortic thrombosis is a rare condition, with incidence of 0.45% in aortas with no prior lesions. It’s usually presented as embolization and has a high mortality rate. Its correlation with COVID-19 was described in six cases in the literature and in our service, in three patients. Cases: The first case a 57-year-old man, a former smoker, with DM2 and thyroid cancer. Diagnosed a thrombus in the ascending aorta, received parenteral anticoagulation and after 13 days the thrombus has completely resolved. The second patient, 47 years old, smoker, hypertensive, dyslipidemic, with a history of stroke a year prior, sought medical attention for a subocclusion of the axillary artery and thrombus in the aortic arch. The patient was anticoagulated and evolved with the resolution of the thrombus. The last case, a 56-year-old female, smoker, arrived at the ER with acute myocardial infarction and acute arterial occlusion of the left upper extremity. A thrombus was found in the ascending aorta and aortic arch. The patient underwent emergency surgery. The patient progressed with biventricular dysfunction, need for an intra-aortic balloon, high doses of vasoactive drugs, and died from mixed shock and electrical instability 24 hours after surgery. Conclusion: The formation of thrombus in COVID-19 is related to endothelial inflammation due to hypoxia, increased concentration of clotting factors, and its high specificity in binding to ACE-2 receptors present in the endothelium. Primary aortic diseases are the most frequent risk factors for thrombus formation. In COVID-19-related cases, smoking was a common risk factor. Our patients had additional risk factors that, associated with the thrombogenicity of SARS-CoV-19, increased the risk of aortic thrombosis even in the absence of lesions, such as plaques or ulcers. In the surgery of the 3rd case, we observed thrombi pedunculated in smooth endothelium, without lesions. There is no consensus on treatment. Most authors advocate anticoagulation, with surgery being reserved for cases of persistence, recurrence of the thrombus or embolization. Others argue that aortotomy should be the immediate treatment to prevent complications. There is a trend toward early surgical treatment in young patients, thrombi larger than 3 cm, location in the ascending aorta or arch, recurrence of embolic events, and high thrombus mobility. 107750 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY HENRIQUE ULISSES DUARTE DE CASTRO1, Caio Caldas Couto1, Bruno Almeida Rezende1 (1) Faculdade Ciências Médicas de Minas Gerais (FCMMG); (2) Universidade Federal de Minas Gerais (UFMG) Introduction: Heart failure is an extremely important clinical condition whose symptoms are the result of a structural or functional disorder of the heart. Heart transplantation is a complex and high-risk procedure, but when successfully performed demonstrates a considerable improvement in the clinical condition and in the patient’s quality of life. However, the vascular changes of such a procedure are not fully known. Objective: Collect and analyze data related to arterial stiffness in patients undergoing heart transplants from March 2020 to January 2021 at the Hospital das Clínicas de Belo Horizonte. Methods: The Mobil-O-Graph 24H device was used, which makes it possible to measure hemodynamic parameters of arterial rigidity that is estimated by evaluating the variables pulse wave velocity (PWV) in the brachial artery, augmentation index (AIx) adjusted for a heart rate of 75 beats per minute (Aix@75), central and peripheral arterial pressure. The measurements were made immediately before surgery and 60 days after. Results and Discussion: There was an increase in blood pressure levels after transplantation surgery for both systolic (SBP), diastolic (DBP), and mean (MAP) blood pressure (p < 0.031). The values of central arterial pressure also increased significantly (p < 0.031). Cardiac output and cardiac index increased (p < 0.031 and p < 0.036). PWV was also increased after surgery (p < 0.031). The increase in PWV can be related to the medications used to control the rejection of the transplanted organ and hemodynamic changes. The data obtained in the study showed that there is a trend in the increase of Aix@75 after heart transplantation (which happened with all the patients studied so far). However, the inconstancy of this variable made it not possible to demonstrate that this increase was statistically significant due to the low sampling. Possibly, with the increase in the sample, it will be possible to show a significant increase in Aix@75 in the short term after heart transplantation. Conclusion: The non-invasive assessment of hemodynamic parameters can contribute to better control of the evolution of patients, in order to prevent cardiovascular events related to vascular remodeling. As it is an easily applicable method, it could be used for the continuous monitoring of the patient in different phases of the post-transplant recovery process. A longer follow-up period and a larger sample are needed to reinforce the applicability of the proposed method. 107773 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES LUCAS SOUSA SALGADO1, Renato Lott Bezerra2, Yago Machado da Silva3, Gustavo Guimarães Rocha Figueiredo3, Raimundo Matos Bezerra Filho3, Eduardo Luís Guimarães Machado3, Isabel Cristina Gomes3, Ângelo Geraldo José Cunha1 (1) União Educacional do Vale do Aço, UNIVAÇO, Ipatinga, MG.; (2) Hospital João XXIII, Belo Horizonte, MG; (3) Complexo de Saúde São João de Deus, 3 Divinópolis, MG – Brazil Introduction: Infective endocarditis (IE) is a high morbimortality disease with increasing incidence. With medical improvement in diagnosis and treatment, several epidemiological changes have been reported over time, however, most of the studies were conducted in developed countries, and it is questionable whether developing countries are susceptible to this epidemiological transition. Objectives: It was sought to describe the epidemiological profile, mortality predictors, and analysis of possible microbiological transition from patients admitted to three tertiary centers in Brazil. Methods: In this cross-sectional and retrospective study, data from 211 patients with definite or probable IE were analyzed according to the modified Duke criteria, between 2003 and 2017. The association between categorical variables was assessed using Chi-square or Fisher’s exact test and binary logistic models were built to investigate the death outcome. We considered p < 0.05 as statistically significant. Results: Median age of the sample was 48.00 (33–59) years old, 70.6% were men and the most prevalent pathogen was Staphylococcus spp. (19%). Mortality was 22.3% and it was observed that increasing age is the leading risk factor for death (p = 0.028). With regard to the location of the disease, native valves were the most affected, with aortic valve being more affected in men when compared to women (p = 0.017). The average number of cases of Staphylococcus spp. (τ = 0.293, p = 0.148) and Streptococcus spp. (τ = –0.078, p = 0.727) has remained stable over the years. Conclusion: No trend towards a reduction or increase in mortality between 2003 and 2017 was evident. Although Staphylococcus spp. was the most prevalent pathogen, it was not possible to observe the expected epidemiological transition. 107794 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY Maria Luísa Souza Santana1, Rodrigo Carvalho de Menezes2, Isabella Bonifácio Brige Ferreira3, Kaique Vinicius da Cruz Santos Aguiar4, Raissa Barreto Lima3, Ana Luísa de Aguiar Almeida Silva5, Elias Soares Roseira5, Luiz Carlos Santana Passos6, Rodrigo Morel Vieira de Melo6, Nivaldo Menezes Filgueiras Filho1 (1) Medicine, Salvador University, C ampus Teacher Barros, Salvador, Brazil; (2) Human pathology, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (3) Medicina e saúde, Bahia School of Medicine and Public Health – C abula, Salvador, Brazil; (4) Gemini, Grupo de Estudos em Medicina Intensiva, Salvador, Brazil; (5) Medicine, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (6) Medical board, Hospital Ana Nery, Salvador, Brazil Introduction: Prolonged use of mechanical ventilation has been associated with poor clinical outcomes, and about 10% of patients who undergo heart surgery are part of this group. Identifying those who are at a higher risk for late weaning may assist in the management of these patients, decreasing the risk of death and complications. Objectives: To determine factors associated with late extubation (>6 hours) after cardiac surgery. Methods: Retrospective cohort study, with secondary data analysis from Ana Nery Hospital’s, Bahia, Brazil, patients who underwent cardiac surgery between 2018 and 2021. The D‘agostino test was used to assess normality. Continuous variables were assessed using the Mann-Whitney test, categorical variables using Fisher’s exact test. Variables that showed statistical relevance in the univariate analysis were included in a binary logistic regression model. Results: A total of 1594 patients were admitted to the study, with a mean age of 56 years (45–66) and a slight predominance of male patients. The most performed surgery was coronary artery bypass grafting (635 [40.1%]). Median Society of Thoracic Surgeons (STS) score of 1.17 (0.70–2.08) and a EUROscore of 1.30 (0.86–2.15). Among admitted patients, 860 had late extubation (>6 hours) MV, with a median of 12 (9–18) hours on the machine while patients who had early extubation had a median of 4 (2.5–5) hours on MV. 74.2% had type 2 diabetes. They also had more noradrenaline use – 55.3% – less vasopressin use (6.86%), with a median peak troponin of 3.51 (1.67–8.24). Conclusions: The presence of congenital valvulopathy, history of endocarditis, troponin and lactate values, presence of previous type 2 diabetes, and use of vasopressin and debutamine were independently associated with late extubation. 107813 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ANA CAROLINA RIOS CARVALHO1, Maria Luisa Souza Santana1, Rodrigo Carvalho de Menezes2, Isabella Bonifácio Brige Ferreira3, Kaique Vinicius da Cruz Santos Aguiar4, Raissa Barreto Lima3, Ana Luísa de Aguiar Almeida Silva5, Elias Soares Roseira5, Luiz Carlos Santana Passos6, Rodrigo Morel Vieira de Melo6, Nivaldo Menezes Filgueiras Filho1 (1) Medicine, Salvador University, C ampus Teacher Barros, Salvador, Brazil; (2) Human pathology, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (3) Medicina e saúde, Bahia School of Medicine and Public Health – C abula, Salvador, Brazil; (4) Gemini, Grupo de Estudos em Medicina Intensiva, Salvador, Brazil; (5) Medicine, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (6) Medical board, Hospital Ana Nery, Salvador, Brazil Introduction: Cardiac surgery aims to restore the functional capacity of the heart and reduce symptoms. Complications have a negative and variable effect on outcome after cardiac surgery. Therefore, the measurement of its effect is of fundamental importance. Objectives: To determine patient characteristics associated with complications post-cardiac surgery. Methods: Retrospective cohort study, with data analysis from Hospital Ana Nery’s, Bahia, Brazil patients who underwent cardiac surgery between 2018 and 2021. The D‘agostino test was used to assess normality. Continuous variables were evaluated using the Mann-Whitney test, and categorical variables evaluated using Fisher’s exact test. Variables that showed statistical relevance in the univariate analysis were included in a binary logistic regression model. Results: A total of 1594 patients were admitted to the study, with a mean age of 56 years (45–66) and a lower predominance of male patients. The most performed surgery was coronary artery bypass grafting (635 [40.1%]). The study population has a median Society of Thoracic Surgeons (STS) score of 1.17 (0.70–2.08) and a EUROscore of 1.30 (0.86–2.15). 279 patients had intra-ICU complications. These were older (58 [48–67] vs. 56 [45–65]), median 85 (64–110) min on cardiopulmonary bypass, 62.2% used dobutamine, 64.4% used noradrenaline. Among complications, 183 (65.6%) had infection, 103 (37.3%) required reoperation, 56 (20.1%) had acute kidney injury, 11 (9.73%) had stroke, and 23 (8.30%) required dialysis. Conclusions: Cardiopulmonary by-pass time, debutamine use, presence of type 2 diabetes, chronic kidney disease, and use of intra-aortic balloon pumps were identified as independent risk factors for the development of postsurgical complications. 107814 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ANA CAROLINA RIOS CARVALHO1, Maria Luísa Souza Santana1, Rodrigo Carvalho de Menezes2, Isabella Bonifácio Brige Ferreira3, Kaique Vinicius da Cruz Santos Aguiar4, Raissa Barreto Lima3, Ana Luísa de Aguiar Almeida Silva5, Elias Soares Roseira5, Luiz Carlos Santana Passos6, Rodrigo Morel Vieira de Melo6, Nivaldo Menezes Filgueiras Filho1 (1) Medicine, Salvador University, C ampus Teacher Barros, Salvador, Brazil; (2) Human pathology, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (3) Medicina e saúde, Bahia School of Medicine and Public Health – C abula, Salvador, Brazil; (4) Gemini, Grupo de Estudos em Medicina Intensiva, Salvador, Brazil; (5) Medicine, Federal University of Bahia Ondina C ampus, Salvador, Brazil; (6) Medical board, Hospital Ana Nery, Salvador, Brazil Introduction: Risk prediction models have been developed to provide information about risk for both physicians and patients, as well as to guide decision making. Likewise, knowing a patient’s risk may allow the implementation of individualized strategies, aimed at preventing mortality. Objectives: To determine patient characteristics associated with post-cardiac surgery Mortality. Methods: Retrospective cohort study, with secondary data analysis from Ana Nery Hospital’s, Bahia, Brazil, patients who underwent cardiac surgery between 2018 and 2021. The D‘agostino test was used to assess normality. Continuous variables were evaluated using the Mann-Whitney test, and categorical variables evaluated using Fisher’s exact test. Variables that showed statistical relevance in the univariate analysis were included in a binary logistic regression model. Results: A total of 1594 patients were admitted to the study, with a median age of 56 years (45–66) and a lower predominance of male patients [RCdM1] [MS2] The most performed surgery was coronary artery bypass grafting (635 [40.1%]). The study population has a mean Society of Thoracic Surgeons (STS) score of 1.17 (0.70–2.08) and a EUROscore of 1.30 (0.86–2.15). Among the patients who did not survive, it was observed that a median age of 66 (53.3–71), BMI with a median index of 24.4 (21.5–27.6), higher rate of CKD and 41.6% had previous AMI. They had a higher EUROscore – 2.58 (1.45–3.76) – and STS – 2.05 (1.31–3.75), needed more supportive medications, and had more postoperative complications, measuring up to 51.1%. Conclusions: BMI, peak 24-hour lactate, CKD, presence of complications in the intensive care unit, use of norepinephrine and vasopressors were identified as risk factors associated with postoperative patient mortality. 107822 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY IVANA BECKER1, Bruna Strube Lima1, João Pedro Dutra2, Samantha Cristiane Lopes1, Susane Fanton3, Franciani Rodrigues da Rocha1, Marcelo Vier Gambetta1, Luiz Eduardo Bacca1, Caroline de Oliveira Fischer Bacca1 (1) UNIDAVI – Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí; (2) CEPON – Centro de Pesquisas Oncológicas; (3) FURB – Universidade Regional de Blumenau Background: The introduction of new chemotherapy drugs has improved overall survival for patients fighting breast cancer. They might however be the cause of secondary structural or functional alterations to the heart, defined as cardiotoxicity. Objectives: To identify the incidence of secondary cardiotoxicity in breast cancer treatment while emphasizing its demographical, clinical, and echocardiographic characteristics. Methods: Observational, analytical, and cross-sectional study in the oncology sector of a reference hospital in Alto Vale do Itajaí, Santa Catarina, Brazil. We analyzed 238 patient’s records of women under medical echocardiographic supervision from August/2018 until June/2021. It was defined as cardiotoxicity the presence of left ventricular dysfunction, represented by the reduction of at least 10% in the ejection fraction (LVEF) to values of lower limits of normality (LVEF <50%). It was employed the SPSS software, Kolmogorov-Smirnov normality, and Fisher exact tests. The chosen level of significance was p < 0.05. Results: The sample counted on 118 women who met eligibility criteria. Average age was 52.73 ± 12.35 years. The incidence of cardiotoxicity was 2.5% (4 patients), meaning significance to the association between treatment with trastuzumab and radiotherapy (p < 0.05). There wasn’t any statistical relevance in the analysis of sociodemographic parameters, as well as in the clinical data related to CT outcomes. Conclusion: The incidence of cardiotoxicity in women under medical echocardiographic supervision during breast cancer treatment was 2.5%, with statistical relevance to the association between treatment with trastuzumab and radiotherapy. 107900 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY JULIA DE CONTI PELANDA1, André Vinícius de Oliveira1, Annelise de Jesus Oliveira1, Mateus Rodrigues Alessi2, Alexandre Alessi1 (1) Universidade Federal do Paraná (UFPR); (2) Universidade Positivo Introduction: Patients who underwent mastectomy as cancer treatment are often advised not to measure their blood pressure (BP) in the ipsilateral arm and to avoid many daily-life activities, in order to prevent lymphedema. These recommendations are based on a small number of studies with widely controversial results. Hence, a thorough review of the literature is imperative to better evaluate the adequacy of the current guidelines. Methods: A cross-sectional online survey with cardiologists and mastologists, and a semi-systematic literature review were performed. Data were cross-linked to assess the degree of concordance between clinical practice and guidelines. Survey results were evaluated by Chi-Square test, with a significance level of 95%. Results: From the 334 physicians surveyed, 206 (62%) were mastologists and 128 (28%) were cardiologist. Whereas cardiologists contraindicated measuring the BP after mastectomy (Chi² 20.1, p < 0.001), sentinel lymph node biopsy (Chi² 25.6, p < 0.001) and radiotherapy (Chi² 18.5, p < 0.001), mastologists did not. According to the literature, there is no data supporting the maleficence of such procedure in mastectomy patients. Conclusion: These results highlight the discrepancy of recommendations among physicians of different specialties, which can increase patients’ illness anxiety. In this study, mastologists showed greater awareness of risks and benefits of measuring the BP in the ipsilateral arm of the procedure. 107945 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION SOFIA HELENA VITTE1, Joaquim Barreto1, Andrei C. Sposito1 (1) Universidade Estadual de Campinas Background: Cardiovascular risk estimates assist healthcare providers in targeted selection of type 2 diabetes (T2D) individuals who may benefit the most from stricter control of risk factors. Subclinical cardiovascular disease (SCD) either as carotid plaque or coronary calcifications is a surrogate marker of increased incidence of cardiovascular events and is thus employed as a classifying feature. Whether screening SCD significantly reclassifies the estimated risk of T2D individuals assessed by risk engine equations at the primary prevention level remains unexplored. Objectives: We sought to determine whether SCD significantly reclassified the UKPDS-based risk estimates in T2D individuals at primary prevention. Methods: This is a cross-sectional study with data collected by the national prospective cohort Brazilian Diabetes Study. Briefly, in this cohort, patients with DM2, aged ≥30 years, underwent carotid Doppler ultrasound for common carotid intima-media thickness (CIMT) measurement and computed tomography angiography to calculate coronary calcium score (CAC). SCD was considered in those who had at least one of the following: (i) carotid atherosclerotic plaque; (ii) CIMT > percentile 75; (iii) CAC > 0 Agatston. The UKPDS risk engine was used to estimate the 10-year risk of CV events classified as low (<15%), intermediate (15–30%) and high-risk (30%). Receiver operator curve (ROC) was used to evaluate the predictive value of UKPDS value. The proportion of individuals at each risk class were compared between the UKPDS and the UKPDS + SCD groups by chi-square test. Results: Among 371 patients included in this analysis (Mean age: 58 + 7.5 years; T2DM duration: 9 + 6.7 years; 59.8% male), 302 (81.4%) had SCD defined by either CIMT (72.4%) or CAC score (71.6%). The UKPDS had an area under the curve of 0.66 (95%CI: 0.60, 0.74; p < 0.001). Adding SCD data to the UKDPS risk engine significantly change the proportion of individuals classified as low- (80.32% vs 17.25%), intermediate (15.36% vs 1.08%) and high-risk (4.31% vs 81.67%) (p < 0.001 for all). Only 5% of SCD individuals were classified as high-risk by UKPDS. 108555 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREA NATHALLIA GOMES DE SOUZA1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá UNIFAP Introduction: Malignant Neoplasm of the Heart, Mediastinum and Pleura (MNHMP) is considered a rare condition, which could only be better studied after improving diagnostic methods. Heart tumors originate mainly in internal tissues, such as the muscular layer and the pericardium, presenting with symptoms common to other cardiovascular diseases, requiring a differential diagnosis. Objective: To analyze the epidemiological aspects and number of people affected by MNHMP, in the years 2019, 2020 and 2021, observing the variation of numbers over the years and main epidemiological findings. Methods: We used the data available in the PAINEL-ONCOLOGIA, from the Department of Informatics of the Unified Health System (DATA-SUS), which comes from the Outpatient Information System (SIA). Results: It was observed, a reduction in the number of cases of people diagnosed with MNHMP over the years. In 2019, 1420 cases were recorded, in 2020, there were 1273 and in 2021, the decrease was more pronounced, totaling 621 cases. Concerning gender, it was found that the frequency in females is slightly bigger, in 2019, 50.53% were females, in 2020, they corresponded to 53.18% of cases and in 2021, they represented 52.17%. Furthermore, another important epidemiological aspect is the age group of these patients, where it was possible to observe that in 2019, in the age group from 0–19 there were 103 cases, and a larger group, which comprises 45–79 years old, represents 58.87% of the cases that year, where those aged 65–69 reached 178 cases. In 2020, the age group from 50–74 years old stood out, with greater attention to 55–59 years old, with 180 cases. In 2021, with the decrease in the total number, no age group exceeded 100 cases, with the highest number, 77 cases, patients between 60 and 64 years old. It was also possible to analyze the distribution of these cases throughout Brazilian territory, some states maintained higher numbers of MNHMP diagnoses during the analyzed period: Bahia, Minas Gerais, Rio de Janeiro, São Paulo and Rio Grande do Sul. Conclusions: It is possible to sketch epidemiologically, from the data, that MNHMP affects more females than male, from the fifth decade of life, who live mainly in the states of Bahia, Minas Gerais, Rio de Janeiro, São Paulo and Rio Grande do Sul. It is necessary to pay attention to the sudden decrease that occurred from 2020 to 2021 and not rule out the possibility of underreporting, mainly because of the current pandemic. 108595 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LARISSA DE OLIVEIRA BELTRÃO1, Ricardo Henrique Freitas Tavares2, Maria Keyllane Vasconcelos de Miranda2, Isabella Carla Barbosa Lima Angelo2, Ieda Fernanda da Silva Santos2, Henrique Pessoa Tseng2, João Henrique Neves Ferreira2, Carlos Henrique Pereira da Silva2, Matheus Guilherme de Assunção França2, Maria Luisa Lopes Rodrigues2, Erinaldo Siqueira de Medeiros2, Pedro Rafael Salerno2 (1) Faculdade Pernambucana de Saúde (FPS); (2) Universidade Católica de Pernambuco (UNICAP) Introduction: Systemic Arterial Hypertension (SAH) is a chronic and non-communicable disease of multifactorial origin, characterized by sustained elevation of systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, when it has no clearly identifiable cause, it is called essential hypertension. It was estimated that almost a quarter of the Brazilian population has hypertension. Objectives: This study aims to compare the mortality rate from essential hypertension and relate it to years of study between the Northeast Region and Brazil. Results: It was observed that there were 241,797 deaths resulting from essential hypertension in Brazil from 2010 to 2019, with 78,134 cases in the Northeast region, characterizing it as the second region with the most deaths due to this pathology, representing 32.31%. In addition, regarding education, in individuals who had no years of study, there were 33,641 deaths in the Northeast, representing a rate of 51.31%. Furthermore, among patients with 1 to 3 years of study in the Northeast, there were 16,062 deaths, with 26.42% of the total in Brazil. As for the population with 4 to 7 years of study, 8,049 deaths were obtained in the Northeast, representing 20.44%. In relation to 8 to 11 years of study, 3,704 deaths were recorded in the Northeast, constituting 19.61%. In addition, in individuals with 12 or more years of study, the Northeast represented a total of 906 deaths, with 16.17% of the total. Finally, in some notifications, the degree of education was not reported, which represented 15,772 deaths in the Northeast, representing 30.57%. Conclusion: The schooling factor is a variable to be considered when assessing death from SAH, highlighting the Northeast region of Brazil. It was observed that mortality rate was inversely proportional to the level of education, with half of the national cases occurring in individuals who did not have training, demonstrating the relevance of this variable. Thus, health prevention and promotion measures are needed for patients with low schooling to control essential hypertension in order to reduce complications resulting from this disease. 107996 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT HITESH BABANI 1, Hitesh Babani1, Giovana De Oliveira Sarubi1, Marjorie Bindá Leite1, Guilherme Henrique Souza1, Rania Gabriele Said1, Ornella Aquino Da Silva1, Lorrana de Oliveira Teixeira1, Adria Melissa Silva Campos1, Jéssica Alessandra Cruz Dos Santos1, Rodrigo de Souza Leitão2 (1) Centro Universitário Fametro; (2) Fundação Hospitalar de Hematologia e Hemoterapia do Amazonas (HEMOAM) Background: Heart failure (HF) is a progressive life-limiting condition affecting 1–2% of the general adult population in high-income countries. By 2030, it is estimated to affect 8 million people, consuming more than $69 billion in medical costs. Patients with advanced HF have a risk of premature death and report physical symptoms, psychosocial burdens, and spiritual needs, making necessary the interventions of PC (palliative care). PC offers total care for a person with an incurable disease which may still respond to disease-modifying treatments, but is nonetheless progressive and life-shortening and is not limited to a specific diagnosis, nor to a particular prognosis. Aims: This document aims to identify outcomes of PC intervention in patients with HF and ascertain the efficacy of management. Methods: A systematic literature review was developed in three steps: Development of the research question, search for scientific articles in the Pubmed database and critical analysis of included articles. The search was conducted in March 2021, and articles between 2019 and 2022 were selected, for a total of 74 articles, of which 19 were used. Results: PC interventions improved quality of life, symptom burden, physical symptoms, mental symptoms, and patients were able to end their life with dignity. The PC needs to be based on multidisciplinary team management for end-stage HF with the purpose of improving quality of life and reducing healthcare costs. In addition, the presence of a multidisciplinary team was associated with multiple positive results. The outcomes of improving access to palliative care for people with advanced HF might reduce their suffering and of their loved ones, as well as decrease hospital readmissions. Conclusions: PC aims at integral care and approaches the patient as a whole (spiritually, socially, physically, and emotionally) these interventions should be provided alongside optimal cardiologic management. This study indicates that there are associations between PC and positive outcomes for patients with end-stage heart failure, resulting in a substantial improvement in quality of life as well as comfort and dignity whilst dying. 108575 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ISADORA BUELONI GHIORZI1, Maurício Rouvel Nunes1, Mateus dos Santos Taiarol1, Marina da Rocha Besson1, Eliaquim Beck Fernandes1, Adriano Louro Moreira1, Juliana Bergmann1, Giulia Righetti Tuppini Vargas1, Konopka1, Valberto Sanha1, Thais Vanessa Salvador1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre Introduction: Patients with trisomy 21, or Down syndrome (DS), have an increased risk of presenting congenital heart defects (CHDs). They have been described in around 40–50% of the patients. Objective: To detect the frequency and types of CHDs in a sample of patients with DS. Methods: The sample was composed by patients seen at a Genetics Clinic Department, in a period between 1994 and 2008. Data was collected retrospectively, emphasizing the cardiological assessment. All patients underwent cytogenetic evaluation through GTG-Banding karyotype. Results: Among the 299 patients from the sample, 166 (55.5%) were male. The mean age at first evaluation was 23.4 days. The predominant karyotypic abnormality was trisomy 21 (96%). Structural abnormalities were found in 3% of patients of the sample, and mosaicism was found in 1% of them. Two hundred and thirty two patients (77.6%) underwent cardiological assessment, and 160 of them (69%) presented abnormal physical examination findings. Among them, 151 underwent echocardiography, and malformations were detected in all of the patients. Among the 72 patients with normal findings (31%), 41 underwent echocardiography, and malformations were detected in 3. Therefore, CHDs were detected in 154 patients among the 232 who underwent cardiological assessment (66%). The most common CHDs observed were: atrioventricular septal defect (n = 61; 39.6%), atrial septal defect (n = 60; 39%), ventricular septal defect (n = 38; 24.7%), and patent ductus arteriosus (n = 38; 24,7%). Other CHDs consisted of Fallot tetralogy (n = 12; 7.8%), pulmonary stenosis (n = 7; 4.5%), pulmonary atresia (n = 1; 0.65%), and Ebstein abnormality (n = 1; 0.65%). Conclusions: Data of this study confirm the increased prevalence of CHD in patients with DS. Therefore, as opposed to what was observed in this sample, in which 22.4% were not assessed, this study reinforces the importance of cardiological assessment in every child with DS, since it may have a significant impact on their prognosis. 108730 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY LUISA PEREIRA DE OLIVEIRA ZANETTI GOMES1, Ana Carla Schmidt1, Camila Martins Marinelli1, Ricardo Zanetti Gomes1 (1) Universidade estadual de ponta grossa; (2) Universidade da região de Joinville Chronic venous insufficiency (CVI) is a common disease that causes calf muscle pump dysfunction and has repercussions for the hemodynamics of the structures involved. To analyze the effects on venous hemodynamics of exercises to strengthen the calf muscles in patients with CVI. This non-randomized clinical trial analyzed 25 lower limbs with CVI, classified from C1 to C5 according to CEAP, Were included if they had achieved at least 60% attendance in the calf strengthening group session. The variables analyzed were collected at at baseline, after 1 month and 2 months and at the end of the exercise protocol. The isometric dynamometry, goniometry, leg circumference and adipometry of the calf The intervention duration 24 session. Both open and closed kinetic chain exercises were performed, involving plantar and dorsal flexion of the ankle joint. Initially, a descriptive analysis of patient variables was conducted, estimating simple frequencies. The variables were analyzed with estimation of mean, median, standard deviation, and 25th and 75th percentiles for each data collection period. Differences between different data collection times were tested with the Friedman test followed Bonferroni correction. In this study were included 25 limbs, comprised of women, who had been diagnosed with CVI for a mean of 6.4. The results of the comparison between the first and the last section showed that there was an increase in the muscular strength of 0.5 kgf (p = 0,2) In the dorsiflexion and plantar flexion measurements increase by 5º (p < 0,0001). The leg circumference increases 0,6 cm (p = 0,215). The Adipometry reduce in 5 mm (p < 0.001). At the analysis of the dynamometry results, there were no statistically significant. The dorsiflexion and plantar flexion there was an increase of 5º at the C3 in the dorsiflexion, at the C2 group in the plantar flexion (p < 0,002). The leg circumference increase 1,2 cm in the group C1 (p = 0.045). The adipometry results showed a reduction at the C3 classification of 1 mm (p = 0,004), Administration of exercise protocols should be considered as a treatment option for CVI, since it has a positive impact on risk factors and on the functions that are impaired by this pathology. A positive impact was observed after the 3 month at the muscular strength, adipometry, range of movement in the patients. 108010 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY ANTONIO DOSUALDO NETO1, Aline Mara dos Santos2, Ana Paula Samogim2, Isabela Aparecida Moretto2, Gabriel Macherini Quaglia2, André Alexandre de Thomaz2 (1) Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS); (2) Universidade Estadual de Campinas (UNICAMP) Introduction: Cardiovascular complications of anticancer therapies are emerging as a major public health problem, considering that the cancer survival rate has increased considerably in recent years. Cardiotoxicity resulting from the action of chemotherapeutic agents, such as Doxorubicin (doxo), is a serious condition that can progress to chronic cardiomyopathy, congestive heart failure and patient death. Studies focusing on signaling activated by antineoplastic therapies have demonstrated the importance of focal adhesion kinase (FAK) for cell survival and resistance to this treatment, however, this signaling remains poorly understood. The present work aimed to characterize the effects of treatment with the chemotherapy drug doxo on the subcellular redistribution of FAK in H9C2 cardiac myocytes. Furthermore, we investigated the interaction between FAK and DNA damage response (DDR) related proteins after doxo treatment, such as DDX5, XRCC5 and DNA-PK which were previously identified in FAK co-immunoprecipitation experiments. Objective: Investigate whether FAK is modulated by Doxo in cardiac myocytes and whether this protein co-localizes with DNA damage response (DDR) proteins by means of super-resolution microscopy. Methodology: Culture of ventricular cardiomyoblasts (n = 80 cells), treatment with doxorubicin, immunostaining of proteins with subsequent analysis by Super Resolution Microscopy were performed. Finally, the T-Student distribution was used for the statistical data and the comparative effect between the control and chemotherapy groups was detailed by the ImageJ program. Results: Among the main findings, it was found that there is an accumulation of activated FAK in the nucleus and the approximation between this kinase and the DDR proteins during genotoxic stress by doxo, indicative of mutual action to maintain cell viability. Furthermore, doxo stress did not cause a significant gain in fluorescence intensity of DDX5, but a subnuclear redistribution accompanied by FAK. XRCC5 and DNA-PK increased their intensities in cells treated with chemotherapy, with concomitant approximation of FAK. Conclusion: The findings of this project contributed to the understanding of the mechanisms by which FAK promotes cell survival against treatment with doxo and, in addition, may contribute to the establishment of new therapeutic modalities for tumor treatment with reduction of the deleterious effects on cardiac function. of the patient. 108048 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANA RAQUEL WHITAKER FILIPE1, Ana Raquel Whitaker Filipe1, Sofia Helena Vitte1, Daniel Campos de Jesus1, Joaquim Barreto1, Andrei C. Sposito1 (1) Universidade Estadual de Campinas (UNICAMP) Introduction: Orthostatic hypotension (OH) is a surrogate marker of poor prognosis in type 2 diabetes (T2D) and results at least partially from arteriosclerosis, autonomic dysfunction, and glucotoxicity; all features involved also in the progression of atherosclerotic cardiovascular disease. Objective: To determine whether OH is related to subclinical coronary artery disease (SCD) in T2D individuals. Methods: This was a cross-sectional, predefined analysis of the Brazilian Diabetes Study, a single-center, prospective cohort of T2D. After 3-min resting sited with their arms at their heart level, participants had their blood pressure (BP) measured three times with a 1-min interval between each measurement and the mean value of the last 2 was considered. Orthostatic BP was then measured as the BP obtained after 1-min standing. OH was defined as an orthostatic systolic or diastolic BP drop greater than 20 mmHg and 10 mmH, respectively, when compared to sitting BP. SCD was a coronary calcium score above zero in individuals without prior acute coronary syndrome, coronary revascularization, or stable angina. Logistic regression was used to evaluate the influence of the independent variable, OH, on the dependent variable, SCD, and further adjustment by age, gender, and LDL-C was performed. To assess if OH was related to SCD severity, ordinal logistic regression was performed using OH as a predictor of higher CAC groups (0, 1–99 and > 100 Agatston). Results: Among 366 individuals (mean age, 57 + 8 years; 58% male; T2D duration, 9 years), included in this analysis, 41 (11.2%) had OH and 260 (71%) had SCD. Compared with controls, HO group were older (57 + 7.4 vs 60 + 7.8 years; p = 0.034), had higher levels of LDL-C (106 + 38 vs 121 + 40.1, p = 0.033) and displayed a higher prevalence of SCD (69.2% vs 85.4%, p = 0.032). HO was related to SCD with an OR of 2.59 (95%CI: 1.06, 6.36; p = 0.037), which remained significant after adjustment (OR: 2.87; 95%CI: 1.03, 8.03, p = 0.044). OH was significantly related to the chance of being classified at a higher CAC group in logistic ordinal regression, with an OR of 2.34 (95%CI: 1.09, 5.01, p < 0.001). Conclusion: T2D individuals with HO are at an increased risk of SCD. This should be borne in mind of healthcare providers when assessing CV risk in this population. 108065 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PRISCILA PEREIRA ALBUQUERQUE1, Kassielly Melissa Ribeiro Rodrigues1, Nathalia Bianco Fabris1, Rodrigo Lage Carneiro1, João Gabriel Pacetti Capobianco1 (1) Pontifícia Universidade Católica de Minas Gerais campus Poços de Caldas Introduction: There is a growing number of transgender women who use hormone replacement therapy (HRT), based mainly on the use of estrogen, so it’s necessary to understand their effects on the individual’s health. The main goal of this treatment is to adjust secondary sexual characteristics to be congruent with your gender. The impact of hormone therapy on cardiovascular health is not widely known. Objective: The study aims to perform a systematic review on what are the cardiovascular risks caused by hormone therapy in transgender women. Methodology: The search was performed using the following electronic databases: Pubmed, Scopus, BVS, Scielo, Web Of Science, Science Direct, Sage Journals, DOAJ, Oxford Journal, Lippincott Williams & Wilkins Journals from 2015 to 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology using the terms “Hormone treatment AND Transgender AND Cardiovascular diseases”. Only primary studies were included, which investigated the cardiovascular effects of HRT in transgender women. Reviews, meta-analyses, non-primary studies, animal studies, and adolescent studies were not included. Results: Of the 228 articles found, after applying the inclusion criteria, 7 articles were selected for analysis. The results were heterogeneous and showed that cross-sex hormone therapy impacts the health of transgender women in several aspects, such as: changes in blood pressure (BP), lipid profile, body mass index (BMI) and vascular compromise. Regarding BP, 2 studies showed an increase in blood pressure levels and 1 observed reduction. As for changes in the lipid profile, 2 studies didn’t observe changes, however, 2 other studies reported a decrease in these parameters. Only 1 study showed an increase in BMI as a consequence of the use of HRT. Concerning, vascular compromise, 1 study reported the occurrence of deep vein thrombosis, on the other hand, another study with 676 transgender women showed only one case. Conclusion: The heterogeneity of the results found in relation to the effects of HRT indicates the need for studies with larger samples that longitudinally follow up individuals involved in this process. The recent beginning of the use of this therapy and the neglect that this population group suffers from society are the factors identified by the authors as responsible for the lack of robust data regarding the safety of HRT for the health of transgender women. 108066 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION NATÁLIA CHICANI ZORZETO 1, Sofia Helena Vitte1, Daniel Campos de Jesus1, Ana Raquel Whitacker Filipe1, Joaquim Barreto1, Andrei Carvalho Sposito1 (1) Universidade Estadual de Campinas – UNICAMP Introduction: Sensory-motor diabetic polyneuropathy (DP) is driven by axonal lesion, which is chiefly mediated by the activation of receptors for advanced glycation end-products (RAGE). RAGE are also expressed in the endothelial layer, where it intensifies the progression of atherosclerosis. Henceforth, plausibly DP may be a surrogate marker of atherosclerotic cardiovascular disease among T2D individuals. Objective: We sought to investigate whether DP is related to the risk of atherosclerotic cardiovascular disease in T2D individuals. Methods: This was a prespecified analysis of the Brazilian Diabetes Study dataset. T2D subjects were screened for DP using the Michigan Neuropathy Screening Instrument (MNSI), which was deemed altered in all participants scoring 4 or more points in a 15-questions questionnaire, or in those scoring 2 points in a directed foot examination. Carotid doppler ultrasound was performed for intima-media thickness (C-IMT) measurement, and carotid disease (SCD) was considered for all with C-IMT higher than the 75th percentile validated for our population by the ELSA-Brasil Study. The relationship between SCD and DP was estimated by binary logistic regression, adjusted by the covariates: age, gender, A1c, LDL-C, hypertension, and prior cardiovascular disease. Results: Among 324 individuals (age: 58 + 7.1 years; 85% hypertensive; T2D duration: 9 + 6 years; 58.6% male; HbA1c: 7.8 + 1.6%), 134 (41.4%) had DP and 220 (67.9%) had SCD. Compared to non-DP, DP group had a higher prevalence of SCD (61.1% vs 77.5%; p = 0.002) and higher values of C-IMT (0.71 + 0.16 mm vs 0.78 + 0.19 mm; p < 0.001). DP was related to a SCD risk, with an OR of 2.21 (95%CI: 1.34, 3.64; p = 0.002), which remained significant after adjustment by age and gender (OR: 2.31; 95%CI: 1.28, 3.53; p = 0.003), and when further adjustment by A1c, LDL-C, hypertension and prior CVD was performed (OR: 3.38; 95%CI: 1.46, 7.81; p = 0.004). Conclusion: In T2D individuals, DP is independently related to SCD. Healthcare providers should thus consider DP screening as a potential tool when assessing individuals at higher risk of cardiovascular events. 108069 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY LUCAS YUJI SONODA1, Maria da Graça Lepre Hawerroth1, Jean José Silva2, Walter Alvarenga de Oliveira2 (1) Minas Gerais State University; (2) Santa Casa de Misericórdia de Passos Introduction: Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia and shares several common risk factors with end-stage renal disease (ESRD), as advanced age, hypertension, diabetes and vascular disease. Objective: To assess the clinical-epidemiological profile, risk of stroke and bleeding in patients with AF on renal replacement therapy undergoing hemodialysis in a regional hospital in Minas Gerais. Methods: A cross-sectional study, carried out in the nephrology sector of a hospital in the southwest of Minas Gerais, which included patients on renal replacement therapy on hemodialysis from January to March 2020. Patients with glomerular filtration rate (GFR) > 15 mL/min/1.73 m2, hemodialysis treatment for < 3 months, age < 18 years, and patients on peritoneal dialysis were excluded. All patients underwent a 12-lead electrocardiogram (ECG) during the hemodialysis session. The risk of stroke and major bleeding were calculated using the CHA2DS2VASC and HAS-BLED scores, respectively. Results: 247 patients from the hemodialysis sector were screened, of which 17 (6.8%) had AF on the ECG. Among patients with AF, 76% were male, the mean age was 72.5 ± 7.6 years. The mean GFR was 6.8 ± 3.1 mL/min/1.73 m2. The most common comorbidities were hypertension 14 (82.4%), smoking history 13 (76.4%), heart failure 11 (64.7%), diabetes 5 (29.4%), coronary artery disease 5 (29.4%) and stroke 3 (17.6%). The CHA2DS2VASC was ≥ 2 in 94.1% (mean 3.8 ± 1.5) of these patients. HAS-BLED scored ≥ 3 in 76.4% (mean 3.2 ± 1.1) of the cases. 3 (17.6%) of the patients were taking oral anticoagulants. Discussion: The prevalence of AF in patients with ESRD is approximately 11.6%, with a range from 4.5 to 27%. These patients have several conditions which increase the risk of stroke and also the risk of bleeding. However, to date, there is a lack of evidence on the safety and efficacy of oral anticoagulants in hemodialysis patients, remaining a dilemma in the management of these patients. Conclusion: In the present study, there was a prevalence of 6.8% of patients with AF on hemodialysis, with multiple comorbidities, high risk for both stroke and major bleeding and most were not receiving oral anticoagulation. 108079 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANA RAQUEL WHITAKER FILIPE1, Ana Raquel Whitaker Filipe1, Sofia Helena Vitte1, Daniel Campos de Jesus1, Joaquim Barreto1, Andrei C. Sposito1 (1) Universidade Estadual de Campinas (UNICAMP) Introduction: Orthostatic hypotension (OH) is a surrogate marker of cardiovascular morbimortality in type 2 diabetes (T2D). The prevalence of OH is influenced by characteristics that significantly vary across populations. Objective: To determine the prevalence of OH and its risk factors in T2D Brazilian individuals. Methods: This was a cross-sectional, predefined analysis of the Brazilian Diabetes Study, a prospective ongoing cohort of T2D. Blood pressure (BP) was measured after 3-min resting using appropriately sized cuffs and the mean value of the last two out of three measurements were considered. Enrolees stood for 1-min for orthostatic BP. OH was defined as systolic or diastolic BP drops greater than 20 mmHg and 10 mmHg, respectively, or any OH-related symptoms. Baseline demographic and anthropometric data were recorded, and blood samples collected for biochemical analysis. The median family income was considered to classify study sample as low- or high-income, whereas LDL-C > 100 mg/dL was deemed as high. Results: Among 1030 patients (mean age: 57 years; T2D duration: 9 years; 59% male), 104 (10,09%) had HO. Compared with non-HO, HO group was older (58 + 8.1 vs 60.4 + 7.5 years; p = < 0.001) and had a higher prevalence of female individuals (38.7% vs 58.7%; p < 0.001), high LDL-C levels (53.0% vs 65.8%; p = 0.038), low-income (15.2% vs 35.6%; p < 0.001), and of individuals with less than primary school education (15.1% vs 27.2%; p < 0.001). In binary logistic regression, the characteristics related to HO were age (OR: 1.046, 95%CI: 1.02, 1.07; p < 0.001), female gender (OR: 2.25, 95%CI: 1.49, 3.39; p < 0.001), high LDL-C (OR: 1.70, 95%CI: 1.02, 2.84; p = 0.040), whilst high-income (OR: 0.33; 95%CI: 0.17, 0.62; p < 0.001) and secondary education (OR: 0.31; 95%CI: 0.17, 0.57; p < 0.001, compared with less than primary education) were protective. After adjustment by covariates, female (OR: 2.16; 95%CI: 1.08, 4.34; p = 0.030) and secondary education (OR: 0.29; 95%CI: 0.09, 0.85; p = 0.025) were independently related to HO risk. Conclusions: OH affects 10% of Brazilian T2D subjects and its prevalence is higher among female and less educated individuals. This should be borne in mind when assessing OH status in this population. 108703 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ESTEPHANY DE JESUS SILVA1, Aline Goneli de Lacerda3, Claudio Tinoco Mesquita1 (1) Universidade Federal Fluminense – UFF; (2) Fundação Euclides Cunha – FEC; (3) Pós Graduação em Comunicação – UFF Introduction: During the COVID-19 pandemic, the spread of fake news on social media took on such proportions that it was described by the World Health Organization as an “infodemic‘‘. Sudden deaths of artists and athletes or by stroke have become associated with Covid-19 vaccines in social networks and message groups. As YouTube is the largest online video platform, our goal is to evaluate the spread of Fake News linking vaccines and sudden death on YouTube in Portuguese language videos. Methodology: We conducted a search for videos on Youtube Data Tools by the “Video Network”, from the junction of the keywords “vaccine” AND “sudden death”, from the crawl depth 0 of the tool to identify the most relevant actors (videos) on the topic on this platform. Then we used the software Gephi 0.9.2, for network analysis and visualization. Finally, we analyzed the content of the 99 most relevant videos to group them into 2 categories: “fake news” and “non-fake news”. Results: Of the 99 videos, 17 were considered as fake news videos (18%), with a total of 2,289,355 views. The most watched fake news (673,175 views) related the death by stroke of a young man with the use of the COVID vaccine, although the hypothesis was ruled out by the health team in less than 6 months. In contrast we found 82 non-fake news videos, with the most viewed one accounting for 1,410,154 views. Videos with fake news often did not feature health professionals, and some were from mainstream media vehicles such as television newspapers. Conclusion: One in five of the most watched videos on YouTube linking vaccines and sudden death contain fake news. Although most videos are scientifically based, the dissemination of misinformation can contribute negatively to the health of the population by increasing hesitancy to vaccinate. Strategies to increase scientifically based cardiology content should be encouraged. 108356 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION VITOR QUEIROZ DE CASTRO SOUZA1, Victória Valadares Andrade1, Thais Velasques Dias1, Carlos Roberto Brites Alves2, Eduardo Martins Netto2 (1) Faculdade de Medicina da Bahia (FMB/UFBA); (2) Laboratório de Pesquisa em Infectologia (LAPI), Hospital Universitário Professor Edgard Santos (HUPES), Salvador, Brazil Introduction: People living with HIV (PLHIV) are twice as likely to develop cardiovascular disease. The identification of high cardiovascular risk (CVR) is essential to manage prevention strategies and can be performed through prediction equations. However, there is no validated CVR score for Brazilians living with HIV. Objective: To assess the agreement between three CVR scores in PLHIV. Methods: Cross-sectional study carried out with 265 Brazilians living with HIV, aged 40 to 74 years, using antiretroviral therapy. Ten-year CVR was calculated using two scores for the general population, the Framingham Risk Score (FRS) and the American College of Cardiology/American Heart Association (ASCVD), and one specific for PLHIV, Data Collection on Adverse Effects of Anti-HIV Drugs Cohort reduced (D:A:Dr). Multivariate logistic regression was performed to investigate the association of high CVR with statistically significant variables. Agreement between scores was assessed using the weighted Kappa coefficient and the Bland-Altman plot. The preventive recommendation with statins was analyzed, according to the Brazilian Clinical Protocol and Therapeutic Guidelines, through the use of scores. Results: The median age was 52 years (47–58), 58.9% were men, 46% brown, 11.3% were smokers, 34% were hypertensive, 9.1% were diabetic and 50.2% were overweight/obese. The median time of antiretroviral use was 15 years (7–21), 8.3% had a detectable viral load. Multivariate analysis showed that age, current smoking and diabetes mellitus were independent predictors for high CVR in the three scores. The agreement between the D:A:Dr and the FRS was perfect (k = 0.82; 95% CI 0.77–0.87; p < 0.001), and substantial between the FRS and the ASCVD (k = 0, 74; 95% CI 0.69–0.79; p < 0.001) and between D:A:Dr and ASCVD (k = 0.70; 95% CI 0.64–0.76; p < 0.001). The Bland Altman plot revealed greater discordance between scores as the RCV increased. The use of statins would be recommended for 25 patients when using the FRS, 8 with the ASCVD and 23 with the D:A:Dr. Conclusion: A high CVR was found in this study. The agreement between the FRS and the D:A:Dr suggests that both scores may be suitable for classifying the CVR in this population. There is still a need to develop calibrated equations for Brazilians living with HIV. Our results generate insights for the incorporation of D:A:Dr as an alternative to the FRS in the Brazilian protocol, as well as other international guidelines have been advocating. 108373 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY BEATRIZ HACHUL DE CAMPOS1, Denise Tessariol Hachul2, Cristiano Pisani3, Tan Chen Wu3, Alberto Ferraz3, Mauricio Ibrahim Scanavacca2 (1) FICSAE – São Paulo – São Paulo – Brasil; (2) Hospital Israelita Albert Einstein – São Paulo – São Paulo – Brasil; (3) INSTITUTO DO CORAÇÃO DO HCFMUSP – SP – BRASIL Introduction: Catheter ablation of atrial fibrillation (AF) presents esophageal injury as a possible post-procedure complication. It is important to know its prevalence, to analyze the efficacy of protection techniques and establish early diagnosis and preventive treatment. Objective: To identify the prevalence of esophageal lesions in patients who underwent AF catheter ablation at Hospital Israelita Albert Einstein in a determined period. Methods: This is a retrospective study, with clinical follow-up based on the survey of medical records related to the perioperative period, of patients who underwent AF catheter ablation from 2016 to 2019. The esophageal mucosa evaluation was made by upper digestive endoscopy, performed between 24 and 72 hours after the procedure. Patients who showed severe injuries were selected to undergo a second endoscopy in the following week. The lesions observed were standardized by the Kansas City classification (KCC). Results: Fifty-seven patients were included in the study. Most of them were male (75.4%) with a mean age of 60.1 years old. Of the patients included, 75.4% had paroxysmal AF. In most cases (86%), a linear thermometer was used to avoid critical increase in the temperature and the occurrence of injury. In the other cases, esophagus displacement with a transesophageal Echo probe was carried out. Considering the esophageal injuries, 36 (63.2%) patients had no lesions in the endoscopy. Among the lesions observed, 1 (1.8%) had erythema (KCC1), 7 (12.3%) had erosion (KCC2A), 3 (5.3%) had hematoma-ecchymosis and 8 (14%) had traumatic injury. The overall prevalence of esophageal lesions was 34.5%. In the control endoscopy, performed in the following week, most of the injuries healed spontaneously, and no patient had complications during the medium and long terms follow-up. Conclusion: Despite protective measures, a considerable number of esophageal lesions was observed. Thus, this study suggests the need to improve the techniques of left atrium ablation and of esophageal protection. 108375 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION GABRIELA CORREIA MATOS DE OLIVEIRA1, Luis Matos de Oliveira2, Luis Jesuino de Oliveira Andrade3 (1) Faculdade de Medicina – UniFTC – Salvador – Bahia – Brazil.; (2) Faculdade de Medicina – Salvador – Bahia – Brazil.; (3) Colegiado de Medicina – Universidade Estadual de Santa Cruz – Ilhéus – Bahia – Brazil. Background: The low available of Glut-4 transporters in sarcolemma of the cardiac cells is what characterizes the myocardial insulin resistance (MIR), which is triggered separately of generalized insulin resistance. Insulin receptors are quite evident in the heart muscle and vessels, and mitochondrial activity performs a significant function in MIR preserving cellular homeostasis by cell reproduction, cells livelihoods, and energy generation. Objective: It is to evaluate the MIR mechanism, and through the signaling pathway design. Methods: PubMed database was employed to search for reviews publications with MIR. The referenced data of the signaling pathway was chosen aggregating references of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A signaling pathway was designed based on MIR research manuscripts, where we show several mechanisms included in the MIR. The KEGG server was employed to exploit the interrelationship protein-protein, and elaborate signaling pathway diagram. The signaling pathway mapping was carried out with PathVisio software. Results: We selected 42 articles from a total of 450 articles in the PubMed database that presented a significant association between the terms “insulin resistance myocardial” AND “signaling pathway”. Founded on database-validated research papers, we choose well-founded pathways and we succeeded representative description of these pathways. The reproduction contigs taken from the KEGG database designed the signaling pathway of the bio-molecules that lead to MIR. Thus, the acting among multiple mechanisms releases factors that participate of the development of MIR. Conclusion: The interaction among various mechanisms and molecular interactions are important factors in development of MIR. 108661 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GABRIELA DE OLIVEIRA SALAZAR1, José Icaro Nunes Cruz1, Cláudia Bispo Martins-Santos1, Mayara Evelyn Gomes Lopes1, Ullany Maria Lima Amorim Coelho de Albuquerque1, Marília Marques Aquino1, Juliana Maria Chianca Lira1, Nathalia Luiza Silva Sobral1, Lucas Villar Shan de Carvalho Cardoso1, Antônio Carlos Sobral Sousa2, Joselina Luzia Menezes Oliveira2, Eduardo José Pereira Ferreira2 (1) Federal University of Sergipe; (2) Rede D’Or São Lucas Hospital; (3) Primavera Hospital Introduction: The symptomatic manifestation of aortic stenosis (AoS) represents a considerable increase in the risk of death and indicates valve replacement, either by transcatheter route (TAVR) or by conventional transthoracic surgery (SAVR). Recent studies seek to compare the results and complications between these procedures, with attractive results for TAVR, especially after the development of more modern prostheses. Objective: To compare hemodynamic outcomes, mortality and in-hospital complications in patients with severe AoS treated in SAVR and TAVR in Sergipe (Brazil). Methods: Retrospective, observational and analytical cohort study. It included data collected between 2013 to 2021 based on medical records. Fifty-five patients were included, divided into groups according to treatment for severe aortic stenosis, SAVR (17; 30.9%) or TAVR (38; 69.1%). Statistical analysis was performed using the Student’s T test, indicated by the Shapiro-Wilk test, in addition to the chi-square test or Fisher’s exact test. A significance level of 5% was adopted. Results: The mean age of the sample was 76.6 ± 10.93 years. Of the total, 59.3% were male. Of the TAVRs (38), all were via transfemoral access and the most used prostheses were EVOLUT R (64.7%), followed by COREVALVE (17.6%) and SAPIEN 3 (8.8%). In SAVR (17), bovine pericardium bioprosthesis was the most used (80%). Patients in the TAVR group had a higher surgical risk profile according to the EuroSCORE I, II and STS scores (p < 0.05). The mean length of hospital stay was 11 days for both groups and hospital mortality was 8.5% for TAVR and 5.9% for SAVR (p > 0.05). There were no differences regarding the percentage of improvement in the echocardiographic parameters after the procedure, regarding the development of conduction disturbance or cardiac rhythm alteration (p > 0.05). However, there was more mild and moderate paravalvular regurgitation in the TAVR group (p = 0.03) and more major or minor bleeding complications (p = 0.046) and acute renal failure (p = 0.017) in the SAVR group. Conclusion: Patients with severe AoS treated by SAVR and TAVR had similar results in terms of valvular hemodynamic performance, mortality rate and mean hospital stay. Patients in the TAVR group had a higher rate of paravalvular regurgitation and those in the SAVR group had more bleeding complications and acute kidney injury. 109089 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BRUNA GUIMARÃES AGUIAR1, Ana Christina Beltrão de Souza Guerra Curado2, José Felipe Valois Ribeiro Silva1, Paulo Borges Santana3 (1) Centro Universitário Maurício de Nassau (UNINASSAU); (2) Faculdade Pernambucana de Saúde (FPS); (3) Hospital Agamenon Magalhães (HAM) Introduction: The use of traditional cigarettes (TC) is still a major public health problem, having negative effects on the cardiovascular system (CVS), despite incentives to reduce its use. The electronic cigarette (EC) appears aiming to replace the TC, releasing nicotine without containing the smoke of the latter. However, ECs are not pharmacologically controlled products, leading them to have an even higher nicotine content than CT. Objectives: Analyze the cardiovascular effects of EC use, as well as the harm caused by the chemical products present in cigarettes. Methodology: A systematic review was carried out using a search for scientific articles on Scielo and PubMed, in March 2022, without language and year restrictions, using the descriptors: “Cardiovascular System”, “Cardiovascular Disease”, “cigarette electronic” associated to the Boolean operator AND. Results and Discussion: 98 articles were found, of which 83 were discarded by reading the titles, 10 by reading the abstracts, since they were not related to the research purpose, being selected 5 articles for complete reading, and all were used for the research. ECs work by heating and releasing vapors that produce toxic substances, including nicotine. Its use is related to high cytotoxicity and changes in cell morphology, which negatively affects endothelial function and the supply of nitric oxide, resulting in an increased risk on CVS. These users, when compared to non-smokers, have twice the risk of having a myocardial infarction, and may have worsened CVS ongoing pathologies, since nicotine, which is also related to dependence, has repercussions on hemodynamics, coronary blood flow, angiogenesis, hypertension and inflammation. Paradoxically, the concentration of nicotine in EC is higher than in CT, and can vary from 16 to 21 mg/mL. In addition, the use of EC increases the risk of atherosclerosis, due to changes in low-density lipoprotein (LDL). Conclusion: Smoking is a relevant health problem, being an important risk factor for heart disease. EC may contain an even higher nicotine content than CT, due to the lack of pharmacological control. The risk of myocardial infarction is greater in smokers than in non-smokers. In addition, nicotine affects hemodynamics, angiogenesis and worsens atherosclerosis. Furthermore, further studies on this topic are needed to broaden the understanding of the EC’s repercussions on public health. 108460 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM EDUARDO AUGUSTO QUIDUTE ARRAIS ROCHA1, Luis Gustavo Bastos Pinho3, Juvencio Santos Nobre3, Pedro Sales Pereira Gondim1, Arthur Holanda Dantas1, Pedro Barbosa Duarte Vidal3, Aston Alves de Freitas1, Vitor Olímpio Coimbra1, Davi Sales Pereira Gondim2, Bruna Sobreira Kubrusly3, Fernanda Pimentel Arraes Maia3, Francisca Tatiana Moreira Pereira4 (1) Centro Universitário Christus (Unichristus); (2) Universidade de Fortaleza (Unifor); (3) Universidade Federal do Ceará (UFC); (4) Centro de Arritmia do Ceará (CACE) Introduction: The COVID-19 pandemic determined great difficulty or even the impossibility of ambulatory follow-up for several patients, including those with heart disease and those with implanted electronic cardiac devices (IECD). These patients need special monitoring for testing and programming their devices. The groups with remote monitoring (RM) could be followed during the pandemic at a distance. It is important to analyze the results of this population, including the possibility of expanding this form of monitoring in Brazil. The aim of this study was to analyze the findings of IECD followed by RM during the COVID-19 pandemic. Methods: Cohort, observational, prospective study involving 119 patients. Events detected in the RM were: presence of atrial and ventricular arrhythmias, defibrillator therapies, changes in the battery, electrode or heart failure parameters. Comparisons were performed using the chi-square test and paired student t test, with p < 5% considered significant. The numbers of events during the pandemic (Group 1-G1) and before the pandemic (Group 2-G2) were compared, with the patient initially being used as his own control. Then, the event rates of G-1 were compared with the group followed just before the pandemic period (Group 3-G3). Results: The patients were 30.2% female, with a mean age of 72 ± 14.2 years, ejection fraction of 55% (34.5/57%), and these types of devices: 30.2% pacemaker; 42, 8% with defibrillator, 3.3% biventricular pacemaker and 22.7% with defibrillator/biventricular pacemaker. 58.8% of the patients in the study had events in the pandemic. Group 1 hadn’t a higher number of events when compared to its own control (G2), RR 1.03 (CI 95% 0.83–1.28), or in relation to group 3, RR 1.02 (CI 95% 0.81–1.30). The groups followed during the pandemic and outside the pandemic showed no differences in characteristics such as age (p = 0.86), ejection fraction (p = 0.08) or functional class > II (p = 0.25). Conclusions: During the COVID-19 pandemic, patients with IECD followed by RM had a high number of monitored events. The COVID-19 pandemic hasn’t determined increases in arrhythmic events in the population of this study. The RM should be considered as an additional form of follow-up for patients with IECD in Brazil. 108718 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION JOSÉ ICARO NUNES CRUZ1, Gabriela de Oliveira Salazar1, Cláudia Bispo Martins-Santos1, Juliana Maria Chianca Lira1, Edvaldo Victor Gois Oliveira1, Lara Teles Alencar Duarte1, Ullany Maria Lima Amorim Coelho de Albuquerque1, Myllena Maria Santos Santana1, Lucas Villar Shan de Carvalho Cardoso1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital Introduction: Cardiorespiratory fitness (CF), quantified in Metabolic Equivalent of Task (MET), has an inverse relationship with cardiovascular morbidity and mortality. It is estimated that an increase of 1 MET to the CF reduces mortality from all causes from 11.6% to 15.0% and from 16.1% to 19.0% mortality from cardiovascular causes. Objectives: To determine the predictors of low CF in patients with myocardial ischemia. Methods: Cross-sectional, analytical study, whose inclusion criterion was the diagnosis of myocardial ischemia on exercise stress echocardiography. The criterion for low CF was MET < 7.9. A multivariate analysis using binary logistic regression was performed to determine risk and protective factors for low CF in patients with myocardial ischemia. The Nagelkerke pseudo R-squared for the model was 39.3%. The analyses were performed using SPSS Statistics software, version 22.0. Results: 1443 patients were included in the study, of which 56.1% were male (809) and 37.8% had low CF (545). Predictors of low CF were smoking or ex-smoking, chronotropic incompetence, female sex, sedentary lifestyle, typical chest pain, systemic arterial hypertension (SAH), diabetes mellitus (DM) and age. Individuals with asymptomatic myocardial ischemia were 57.3% less likely to have low CF. Left ventricular ejection fraction (LVEF) was also a protective factor – an increase of 1% in LVEF provided 4.2% greater protection against low CF (Table 1). Conclusions: Smoking, female sex and sedentary lifestyle are strong predictors of low cardiorespiratory fitness. It has also been demonstrated that chronotropic incompetence, typical chest pain, SAH and DM are risk factors for low CF. In addition, the absence of symptoms and preserved LVEF confer a lower risk of low CF in ischemic patients. 108927 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION TOMÁS DE SOUZA MELLO1, Karine da Silva Guimarães1, Gabriela Gama Zagni Jardim1, Ana Rachel Bucar Cervasio1, Clara Avelar Mendes de Vasconcellos1, Juliana Camara Garcia1, Rodrigo Eugenio Vinuto Borges1, Natalia Rossilho Moyses Ushijima1, Luiza Brandão Catharina1, Luísa Leite Vaz da Silva1, Ana Cristina Tenório da Costa Fernandes1, Elizabeth Silaid Muxfeldt1 (1) IDOMED – Universidade Estácio de Sá, Medicine School, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil. Objective: To establish the relationship between cardiovascular (CV) risk profile and detected risk of Obstructive Sleep Apnea (OSA) in two questionnaires – STOP-BANG (SB) and Epworth Sleepiness Scale (ESS), in a young population of adults registered in a Primary Health Care unit in Rio de Janeiro, Brazil. Design and Methods: This cross-sectional population study enrolled adults between 20–50 years old, registered in a primary healthcare unit in Rio de Janeiro. A database is being developed including sociodemographic and anthropometric data, and CV risk factors. Office blood pressure and Home Blood Pressure Monitoring (7-day protocol) (Omron-705CP). Moreover, OSA was investigated by SB and ESS. Patients with a high risk for OSA in either of these two questionnaires were subsequently assigned for polysomnography. Results: A total of 562 subjects were evaluated [40% males, 38.9 ± 8.8 years], where 151 (26.9%) were identified as high risk for OSA by the SB questionnaire and 210 (37.4%) by ESS. The most common CV risk factor was physical inactivity (43%), followed by dyslipidemia (38%) and obesity (28%). By Office blood pressure, the prevalence of hypertension was 13.4% while by Home Blood Pressure Monitoring was 18.6%, with a low concordance between them (kappa = 0.472). Subjects with a high risk at SB are older, with a higher prevalence of obesity, hypertension and higher Office blood pressure and Home Blood Pressure Monitoring. On the other hand, individuals with high-risk by ESS were more obese, with increased waist circumference, higher prevalence of dyslipidemia and metabolic syndrome. Nevertheless, there was no difference in Office blood pressure levels. Among the subjects submitted to polysomnography, 46% had a diagnosis of OSA (AHI ≥ 5/hour) and 23% of moderate/severe OSA (AHI > 15/hour). The best predictor of AOS was SB, positive in 100% of subjects with moderate/severe OSA, while ESS was positive in only 20% of them. Conclusion: This young and apparently healthy population presented a high prevalence and risk for OSA. The SB had a higher association with higher Office blood pressure levels, while ESS was associated with a worse metabolic profile. SB questionnaire seems to be the best predictor for moderate/severe OSA in this young adult population. 108480 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ALINE PETRACCO PETZOLD1, Fernanda Lourega Chieza3, Luiz Cláudio Danzmann2, Luiz Carlos Bodanese1 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS); (2) Universidade Luterana do Brasil (ULBRA); (3) Hospital São Lucas da PUCRS Introduction: Gender differences in the epidemiology of heart failure (HF) have been suggested. Findings reinforce that women respond differently to left ventricular ejection fraction (LVEF) values and HF treatment. Objectives: To compare survival between sexes in HF patients, stratified by LVEF values, in a new proposal (< 40%, between 40–59% and > 60%), in a 5-year cohort follow-up. Methods: This is a retrospective cohort study in which the 5-year mortality rates of 385 patients, was analyzed. The sample was divided into three groups: LVEF ≤ 40% (n = 133), LVEF 40–59% (n = 145) and LVEF ≥ 60% (n = 107), differentiating the sexes in each group. Inclusion criteria were age > 18 years, previous diagnosis of HF and transthoracic echocardiogram. The logrank test and Kaplan-Meier curve were used. Results: Patients with lower LVEF were younger, for both sexes. High mortality among all groups was observed. The main HF cause was, for women, hypertension, and, for men, ischemic heart disease. In a 5-year analysis, the mortality rates for each LVEF group and sex were compared (Table 1). The mortality rates found were similar between sexes, thus our results go against women responding differently to LVEF values and to HF treatment. Conclusions: No statistically significant difference was found in survival between the sexes, comparing the different LVEF groups. There is a need for better assessment of the HF population, since all mortalities found were high. 108512 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES DANIEL MARQUES DA SILVA1, SEVERINO PEIXOTO NUNES NETTO2, LETÍCIA STEPHANIE DE SOUZA ARAÚJO3 (1) UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE (UFRN); (2) UNIVERSIDADE FEDERAL DE UBERLÂNDIA (UFU); (3) UNIÃO SOCIAL CAMILIANA (USC) Introduction: During the COVID-19 Pandemic’s first three months in Brazil, a decrease in Elective Cardiology Diagnostic Tests and Procedures (ECDTP) was reported, which can be associated with deficient planning of cardiovascular disease (CVD) management and may have contributed to the increase of in-hospital fatality due to CVDs. Objective: To analyze the impact of the Pandemic on the amount of ECDTP performed at Brazilian Unified Public Health System (SUS) in 2020 and 2021. Methods: We evaluated the number of ECDTP performed at SUS between March and December of each year from 2015 to 2021 from the Brazilian Outpatient Information System (SIA/SUS). The data until 2019 was used to project the number of ECDTP expected in 2020 and 2021 by a linear regression using a statistical significance level of 0.05 (P-value = 0.0026 for both angular and linear coefficients). The projection was compared to the real data from 2020 and 2021. Results: The linear regression until 2019 presented a R-squared of 0.967, which showed a rising trend of ECDTP performed per year before the Pandemic. The data recorded in 2020 was 38.65% lower than the predicted. It showed that the cardiovascular care provision was notably affected by the pandemic due to SUS rearrangement to attend COVID-19 requirements. In 2021, after the mass immunization campaign in Brazil, the ECDTP returned to a rising trend, with 21.39% increase compared to 2020. Despite the improvement, in 2021 the real data was 29.14% lower than the amount predicted. Conclusions: Rearrangements to address COVID-19 requirements affected the amount of ECDTP performed. This effect was mainly noted in 2020, but was still remarkable in 2021 even with the immunization campaign. This situation may have resulted in the neglection of cardiovascular diseases. 108536 Modality: E-Poster Scientific Initiation – Non-case Report Category: ANTICOAGULATION ESTEVÃO OLIVEIRA CARVALHO1, João Ricardo Nogueira Perez1, Gabriela Billafan Ferreira1, Lucas Lagares Bragança Magami1, Luan de Castro França1, Leandro Guedes Santos1, Diana Aristotelis Rocha de Sá1 (1) ESCOLA SUPERIOR DE CIÊNCIAS DA SAÚDE (ESCS) Introduction: The coronavirus disease 2019 (COVID 19), caused by Sars-CoV-2 virus, according to stipulations, claimed about 18,2 million lives until December 31, 2021 [1]. The COVID-19 generates a hypercoagulability state. Therefore, the main guidelines [2–3] recommend thromboprophylaxis in hospitalized COVID-19 patients. However, doubts remain about the use of low-molecular-weight heparin (LMWH). Aim: To analyze the incidence of venous thromboembolism in hospitalized patients with COVID-19 in the regime of thromboprophylaxis with LMWH. Method: This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) rules. EMBASE and PubMed were the selected databases. The following health descriptors were used: “venous thromboembolism” “COVID-19”, “coronavirus disease 2019”, “anticoagulant” and “anticoagulants agent”. Two authors independently performed a systematic literature search in databases. A search encompassed that was published between 01/01/2019 until 31/12/2021. The studies eligible for inclusion had a population over 18 years old, with confirmed diagnosis of COVID-19, hospitalized, and all population did use exclusively LMWH Articles were excluded if they did not provide data about prophylactic anticoagulation regimen (PAR) and if they are case reports. Two authors (E.O.C and J.R.N.P) realized the data collection process, in an independent form. Results: Our search found 202 articles, of which 80 were duplicates. A total of 122 titles were excluded after reading the title and abstract. Therefore, 17 articles were selected for reading the full-text. In this step, 5 titles were excluded, because they don’t give information about the PAR and 3 because they don‘t use LMWH exclusively. So, we selected 9 titles [4–12]. A total of 1614 participants, from 8 countries, submitted to prophylaxis with LMWH. Being that 160 participants (9,91%) had a venous thromboembolic event. 6 articles [6–11] gave information about bleeding that occurred with 43 participants (3%). Conclusion: The proportion of thrombotics events in hospitalized patients diagnosed with COVID-19 is 18% to 37%, without prophylactic anticoagulation [13]. Our research demonstrated that less than half of the population submitted to prophylaxis anticoagulation evolved to venous thromboembolic events. 109325 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LEONARDO SANTANA ANDRADE1, Emerson Santana Santos1, Braúlio Cruz Melo1, Antônio Guilherme Cunha de Almeida1, João Victor Andrade Pimentel1, Larissa Rebeca da Silva Tavares2, Vinícius Barbosa dos Santos Sales1, Júlia Souza Diniz1, João Paulo Dias Costa1, Irlaneide da Silva Tavares1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe; (2) Universidade Tiradentes Introduction: Transthyretin amyloidosis (ATTR) is one of the hypertrophic cardiomyopathy (HCM) phenocopies. It is caused by pathogenic variants in the transthyretin (TTR) gene and occurs in five per cent of patients with unexplained HCM. Aims: Estabilish the prevalence of ATTR in a population previously diagnosed with HCM. Methods: A prospective study was conducted and a 168 genes panel related to cardiomyopathy was performed. HCM was diagnosed according to the guidelines of the American Heart Association (AHA). Results: The TTR gene was sequenced in 74 index patients referred to our cardiogenetics outpatient clinic between January 2021 and March 2022. Among the included patients, 4 had positive diagnosis for ATTR and other major clinical findings are summarized on Table 1. Conclusion: The prevalence of ATTR was similar to literature. Our study highlights the importance of early diagnosis for ATTR since treatment has higher rates of effectiveness when administered in the early stages, it also reinforces that phenocopies, such as the ATTR, should always be included in differential diagnosis investigations for patients with unexplained HCM. Keywords: Transthyretin amyloidosis, Hypertrophic Cardiomyopathy, Genetic Panel. 108685 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ARTHUR MAROT DE PAIVA2, Gabriel Baêta Branquinho Reis2, Luiz Fernando Sposito Ribeiro Baltazar2, Pedro Guimarães Moreira da Silva2, Walace Chaves dos Santos1, Maurício Lopes Prudente1, Abissay Francisco Dias1, Marcos Vinícius Pires Rodrigues1, Thainá Lopes de Souza1, João Alberto Pansani1, Artur Henrique de Souza1, Giulliano Gardenghi1 (1) Hospital ENCORE – Aparecida de Goiânia/GO; (2) Universidade Federal de Goiás- Goiânia/GO Introduction: Delirium is an acute mental status change, with a fluctuating course and a high incidence in cardiac surgery post-operative (PO) period. Delirium can lead to both short and long-term consequences. Objective: Describe the prevalence of delirium in patients summitted to cardiovascular surgery. Methods: This is a descriptive cross-sectional study with patients summitted to cardiac surgery between January 2020 and February 2022. The assessment of delirium was performed on the 1st PO in the Intensive Care Unit using the CAM-ICU and RASS scales. The evaluation of epidemiological data, previous comorbidities, use of pre and post-operative medications, anthropometric measurements and information related to the surgery were collected from TASY® electronic medical record. Data collected and tabulated using specific spreadsheets with Excel 2010® software. Results: 74 patients (39 men) were included. The prevalence of delirium was 16.2%. In patients with delirium, 50% were males and in those without delirium, 53.3% were males. The mean age in those with delirium was 72(±7.9) years old and the mean of BMI was 25.6(±2.89), while those without delirium was 57.2(±12.9) and 27.8(±4.1), respectively. In patients with delirium, 58.3% was summitted to general anesthesia associated with spinal anesthesia and in 41.6% general anesthesia was used alone. In patients without delirium, 61.3% underwent spinal anesthesia plus general anesthesia and in 38.7% only general anesthesia. In patients with delirium, the mean cardiopulmonary bypass (CPB) time was 100.7(±31.34) minutes, and the mean aortic clamping time was 70(±27.3) minutes. In those without delirium, the mean time of CPB was 97.5(±26.6) minutes and the aortic clamping time was 69.4(±22.6) minutes. Regarding comorbid diseases, the mean was 5.08(±2.27) in patients with delirium and 3.2(±2.2) in those without delirium. With respect of the number of medications used, in patients with delirium, the mean home medications used was 5(±2.1) and the mean of post-operative medications was 7.8(±1.9). While, in patients without delirium the mean number of medications used was 4.1(±2.1) and post-operative used was 7.5(±1.9). Conclusion: The prevalence of delirium found in the sample was consistent with current literature data. Considering patients with delirium, multiple risks factors were observed, such as advanced age, greater number of comorbidities and pre and post-operative polypharmacy. 108757 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION TAINÁ SILVEIRA ALANO1, Larissa Xavier Neves da Silva3, Guilherme Lucca Casali3, Jayne Santos Leite3, Andresa Conrado Ignacio3, Linda Ariene dos Santos Cardoso1, Daniel Umpierre2 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (2) Hospital de Clínicas de Porto Alegre (HCPA); (3) Universidade Federal do Rio Grande do Sul (UFRGS); (4) Prefeitura Municipal de Porto Alegre (PMPA) Background: The elderly are the most medicalized age group in society. Due to their metabolism changes, many commonly used drugs are no longer appropriate, with their risks outweighing the benefits. When the patient already has a chronic disease, the prescription should be even more responsible. The Beers Criteria for Potentially Inappropriate Medications (PIMs) is a systematic cataloging of the existing evidence in this field, aiming to improve the healthcare of the elderly. Objectives: Assess the prevalence and characterize the use of PIMs in older adults with hypertension enrolled in research studies in the city of Porto Alegre. Methods: Cross-sectional study of secondary data. The population was composed of individuals aged 60 years or older and diagnosed with systemic arterial hypertension (SAH). The data were retrieved from 2 studies carried out in the city of Porto Alegre between 2018 and 2020. Based on the answers to a General Health Questionnaire, the prevalence of PIMs was analyzed using the 2015 Beers Criteria. Drugs with minimum dosage requirements were excluded from the analysis. The data analysis was performed using Python 3.9. The pre-analysis plan, statistical codes, and dataset used for this study are available at osf.io/ksfg2. Results: There were 318 hypertensive patients (71% female) included, with a mean age of 69 years old (standard deviation 6.4). A total of 32 different PIMs were found, with the most prevalent being omeprazole, used by 30 (9%) patients. A total of 88 (28%) patients were using at least one PIM. Considering sex, 23% of males and 30% of females were using PIMs. Patients aged from 60–69, 70–79, and 80–89 years old had a prevalence rate of using at least one PIM of 28%, 27%, and 30%, respectively. Conclusions: There is still a notable usage of PIMs in the elderly with SAH, with the highest rates being in females and octogenarians. The existing literature on this subject is inconclusive, with prevalences ranging from 24% to 82%. The results reflect the need for better pharmacological and geriatrics training for healthcare professionals and the empowerment of the patient in the prescription process. This study presents some limitations, such as the absence of the full illness history and a possible selection bias, since the participants were already involved in health-related scenarios. Further studies could explore interventions of education on responsible prescribing and self-medication risks in hypertensive elderlies. 108639 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LETICIA VIEIRA SENGER1, Rafael Fabiano Machado Rosa1, Helena Guedes da Rocha1, Helena Marcon Bischoff1, Grasiele do Amaral Martins1, Georgia Marques Jardim1, Alexandre Perin Decol1, Matheus Ribeiro Fretes1, Bianca Brinques da Silva2, Estefany Karenine Rodriguez Casanova1, Carolina Feijó Bombana1, Carolina Andreatta Gottschall2 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (2) Universidade Luterana do Brasil (ULBRA) Introduction: In children with Down Syndrome, there is a high prevalence of congenital heart diseases, which characterize anomalies associated with hospitalization and surgical correction. Objectives: To investigate incidence, clinical presentation and cytogenetic characteristics of patients with Down syndrome (DS) and congenital heart disease (CHD) who are admitted to a cardiac Intensive Care Unit (ICU) of a pediatric hospital. Methodology: A prospective and consecutive cohort of patients hospitalized for the first time by CHD in the Cardiology ICU of the Santo Antônio Children’s Hospital was evaluated for 1 year. For each patient, a standard clinical protocol was applied, with abdominal ultrasound and karyotype examination. Results: 207 patients composed the sample and karyotype could be performed in 204. Chromosomal alterations were observed in 29 individuals, 24 of them (12%) with DS (23 by trisomy free of chromosome 21 and 1 by isochromosome 21q). The main CHDs observed in these patients were septal defects (n = 18), especially the atrioventricular septal defect (AVSD), present in half of the cases. DS was the cause of AVSD in 55% of the cases of the sample, and the association of this defect with DS was statistically significant. There was no difference in the frequency of alterations detected on abdominal ultrasound, length of hospital stay and deaths among patients with DS and normal karyotype. Conclusions: The frequency of DS found in our study, as well as the types of chromosomal abnormalities identified in these patients, were in accordant with the literature. In our series, patients with DS presented a good evolution, similar to patients with normal karyotype, as described in other studies. 108640 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LEO CHRISTYAN ALVES DE LIMA1, Leo Christyan Alves de Lima1, Elaine Largura Biazati1, Willian Kubo1, Hildeman Dias da Costa2, Alexandre Venturelli da Silva3, Fabiana Cristina Schabatoski Passos3, Luiz Henrique Gasparelo3 (1) Liga Rondoniense de Cardiologia Clínica e Cirúrgica (LICAR); (2) Universidade Federal de Rondônia; (3) Angiocenter Introduction: Interatrial communication (IAC) is one of the most common congenital heart diseases, affecting one in 800 births, characterized by a defect in the closure of the interatrial septum. Due to its slow evolution, it is usually diagnosed late in adulthood, when symptoms result from right heart failure, pulmonary hypertension with cor pulmonale or atrial arrhythmias. Objectives: This is a descriptive study of the registration of cases of IAC correction performed in a high-complexity service in the Western Amazon. Methods: Data obtained through research via medical records and complementary exams were used. The database selection was through Pubmed and Scientific Electronic Library On-line (SCIELO). Results: Five patients were followed, one male and four female, all of them with symptoms of dyspnea on exertion and fatigue. The transthoracic echocardiogram showed an ostium secundum (OS) IAC, with signs of right chamber overload and pulmonary hypertension (mild to moderate). The prostheses were implanted through the right femoral vein and with the aid of intracardiac echocardiography. Although vascular complications are known, in this small serious there were no complications during the procedures. The prostheses used were Coccum® in one case and Amplatz® in four cases. The patients had a hospital stay of less than two days and in the outpatient follow-up they showed a favorable evolution. Discussion: The indication for percutaneous IAC closure are patients who have ostium secundum (OS) +96 with favorable anatomy associated with symptoms or with any degree of pulmonary hypertension, right chamber overload, as described in this work. In addition, percutaneous closure is a less invasive alternative and is associated with fewer thromboembolic complications compared to the traditional surgical method. Conclusion: Despite a smaller number of cases than most large centers, positive results can be seen in this type of treatment. The percutaneous treatment of OS type IAC is a safe procedure and performed in the state without the need to transfer to other locations, improving the patient’s quality of life with a relatively low risk and increasing their life expectancy. 108643 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY JOSÉ ICARO NUNES CRUZ1, Gabriela de Oliveria Salazar1, Cláudia Bispo Martins-Santos1, Mayara Evelyn Gomes Lopes1, Ullany Maria Lima Amorim Coelho de Albuquerque1, Marília Marques Aquino1, Lucas Villar Shan de Carvalho Cardoso1, Edvaldo Victor Gois Oliveira1, Joselina Luzia Menezes Oliveira2, Enaldo Vieira de Melo1, Eduardo José Pereira Ferreira2, Antônio Carlos Sobral Sousa2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital Introduction: Aortic stenosis (AoS) is the restriction of blood outflow through the aortic valve leaflets during ventricular systole. When symptomatic, AoS can be treated with valve replacement, which can be performed percutaneously (TAVR) or by conventional transthoracic surgery (SAVR). Objectives: To evaluate the influence of clinical variables and surgical risk on therapeutic indication for AoS in Sergipe – the smallest Brazilian state. Methods: This is an observational, cross-sectional, analytical study, conducted in two private hospitals in Sergipe (Brazil). The sample included patients who underwent TAVR or SAVR after diagnosis of severe AoS defined by echocardiographic criteria and symptomatological assessment, between 2013 and 2021. The TAVR and SAVR groups were compared in terms of comorbidities and surgical risk scores. Categorical variables were analyzed using Fisher’s exact test, whereas quantitative variables were analyzed using Student’s t-test or Mann-Whitney U test. The statistical analysis was performed using SPSS Statistics software, version 22.0. Results: The sample included 55 patients, of which 38 underwent TAVR and 17 underwent SAVR. The TAVR and SAVR treatment groups did not demonstrate differences in sex, height and weight distributions, however age was higher among those who underwent TAVR (88 vs. 62 years; p < 0.001). The TAVR group exhibited more patients in functional class III or IV (New York Heart Association) compared to the SAVR group (88.6% vs. 40.0%; p < 0.01), as well as higher prevalence for diabetes mellitus (46.2% vs. 5.9%), dyslipidemia (76.0% vs. 31.3%) and chronic kidney disease (78.6% vs. 33.3%) (p < 0.05). The four surgical risk scores assessed were also higher in the TAVR treatment group: logistic EuroSCORE I (15.6% vs. 6.7%; p < 0.05); EuroSCORE II (7.0% vs. 2.9%; p < 0.01); STS mortality score (5.3% vs. 1.7%; p < 0.05); and STS morbidity or mortality score (29.5% vs. 11.5%; p < 0.05). Conclusions: Patients treated with TAVR presented older age, greater severity of heart failure, greater frequency of comorbidities, in addition to exhibiting a greater surgical risk profile according to the 30-day mortality prediction scores. Although the guidelines recommend TAVR for patients at higher surgical risk, this treatment was also expanded to intermediate-risk patients in Sergipe. 108657 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING JOSÉ ICARO NUNES CRUZ1, Cláudia Bispo Martins-Santos1, Gabriela de Oliveira Salazar1, Juliana Maria Chianca Lira1, Myllena Maria Santos Santana1, Ana Luísa Lisboa Prado1, Giulia Vieira Santos1, Letícia Luiza Gomes Marques1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2, Luiz Flávio Galvão Gonçalves2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital Introduction: Coronary angiography is the gold standard test for the diagnosis of coronary artery disease (CAD). In recent years, with the advent of coronary computed tomography angiography (CCTA), the possibility of non-invasively diagnosing CAD has emerged. However, in order to validate CCTA locally, it is necessary to assess its diagnostic accuracy in a northeastern brazilian state, where the use of CCTA has recently been expanded. Objectives: To determine the diagnostic accuracy of CCTA in a sample from Sergipe, considering coronary angiography as the gold standard test. Methods: This is an observational, cross-sectional, analytical study. The sample included patients with cardiovascular complaints who underwent CCTA and coronary angiography at an interval of less than six months, in Sergipe (Brazil). The identification of plaques associated with luminal narrowing of any degree was considered positive for CAD. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy and Receiver Operating Characteristic (ROC) curve were evaluated to determine the diagnostic potential of CCTA for CAD, considering coronary angiography as the gold standard method. Results: 130 patients were included in the study. The median age was 62 years (IQR: 57.0–67.25). The prevalence of CAD determined by the gold standard test was 76.9% (100). The CCTA showed a sensitivity of 95.0%, specificity of 80.0%, PPV of 94.1%, NPV of 82.8% and accuracy of 91.5%. The area under the ROC curve demonstrated significant discriminatory capacity (0.875; CI95% 0.79–0.96; p < 0.001) (Figure 1). Conclusions: The study results are consistent with those observed in meta-analytic studies and clinical trials. The high prevalence of CAD in the sample determined a higher PPV than NPV. It is concluded that, in Sergipe, CCTA has significant accuracy for the diagnosis of CAD, being recommended for the assessment of chest pain in patients at intermediate risk for CAD. 108672 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM SARA CRISTINE MARQUES DOS SANTOS1, Luan Tardem Veloso Teixeira1, Thaís Lemos de Souza Macedo1, João Pedro de Resende Côrtes1, Ivan Lucas Picone Borges dos Anjos1, Paula Pitta de Resende Côrtes1, João Carlos de Souza Côrtes Júnior1, Eduardo Tavares de Lima Trajano1, Carlos Eduardo Cardoso1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: In COVID infection, vascular stiffening may be induced due to indirect damage caused by the virus and its systemic inflammatory state and binding to angiotensin-converting enzyme 2, which cause cellular injury1. These mechanisms may contribute to the progression of atherosclerosis and increased cardiovascular risk, arterial stiffness, and augmentation index in previously infected individuals. The objective of the present study was to seek correlation between pulse wave analysis data in individuals up to 30 years of age who tested positive for COVID at least 15 days before the exam and individuals who were not infected. Methodology: Observational and cross-sectional study, carried out from May to July 2021 in students aged over 20 and under 30 years, in accordance with the current hypertension guideline of the Brazilian Society of Cardiology. Of 59 participants, 4 were excluded for absence of answers about COVID infection and 10 for age >30 years. After selection, participants were divided into 2 groups: prior COVID (CoP) and non-infected (NI). We used an anonymous questionnaire and the values provided by the Arteris device by means of the oscillometric method: PWV, AIX@75, heart rate (HR), central systolic pressure (CSBP) and central diastolic pressure (CDBP). The mean, maximum and minimum values were calculated using Excel software. Evaluation of sample normality (Shapiro-Wilk) and unpaired Student’s T test (with Welch correction) were performed for parametric samples and Mann Whitney for non-parametric samples, with confidence level of 95% through GraphPad Prism Software version 9.2. Results: In the CoP group, the means were: PWV 4.65 m/s (5.4 ± 4.2); AIX@75 23.22% (40.7 ± 9.3); HR 89.5 bpm (119 ± 71); CSBP 97.72 mmHg (118 ± 80); CDBP 76.68 mmHg (98 ± 61). While in the NI group, the means were: PWV 4.58 mmHg (5.2 ± 3.1); AIX@75 21.85% (41.7 ± 5.3); HR 86.3 bpm (128 ± 60); CSBP 97.6 mmHg (113 ± 80) and CDBP 73.8 mmHg (54 ± 91). There was no statistical difference between PWV (p = 0.95) and AIX@75 (p = 0.63) values between CoP and NI group. Conclusion: Although higher values were observed for the CoP group in several hemodynamic and arterial stiffness parameters, no statistical difference was obtained between this group and NI group. However, it is valid to emphasize the importance of further studies in the area to be able to affirm or rule out the influence of the SARS-COV-2 virus on vascular integrity. 108678 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ARTHUR MAROT DE PAIVA2, Gabriel Baêta Branquinho Reis2, Luiz Fernando Sposito Ribeiro Baltazar2, Pedro Guimarães Moreira da Silva2, Thais Vieira de Araújo Rodrigues1, Lays de Souza Albuquerque Oliveira1, Abissay Francisco Dias1, Marcos Vinícius Pires Rodrigues1, João Alberto Pansani1, Maurício Lopes Prudente1, Artur Henrique de Souza1, Giulliano Gardenghi1 (1) Hospital ENCORE – Aparecida de Goiânia/GO; (2) Universidade Federal de Goiás- Goiânia/GO Introduction: Minimally invasive cardiac surgery has becoming popular in the last two decades, associated with a better aesthetic effect and less surgical trauma, which may be related to a lower prevalence of pain or a different neuropathic component in pain. Objective: The aim is to compare the prevalence of pain in the first postoperative day (PO1) of patients undergoing valve replacement between conventional and minimally invasive techniques. Methods: Analytical cross-sectional study with patients undergoing heart valve replacement surgery between January 2020 and February 2022. Pain assessment was performed on the PO1 using the Visual Analog Pain Scale (VAS) and the Leeds Assessment of Neuropathic Symptoms and Signs (LANNS) Scale. For the analysis of independent samples, the t-student test was performed with 5% significance. Results: 41 patients were included in the study. 33 patients underwent conventional heart valve replacement surgery (CONV group) and 8 patients underwent minimally invasive valve replacement surgery (MINI group). The mean age of the CONV group was 55.5 ± 12.5 years versus the MINI group: 62.9 ± 16.1 years (p = 0.19). Regarding pain intensity by VAS, the mean of the CONV group was 4.3 ± 3.4 points versus 4.1 ± 3.4 of the MINI group (p = 0.89). Regarding the number of painful areas, the CONV group had a mean of 1.3 ± 1.0 points versus 1.1 ± 0.8 of the MINI group (p = 0.59). In the CONV group, the main painful area was the sternal region and in the MINI group it was in the right thoracic region. According to the LANNS Scale in the CONV group, 72.7% of the patients had a score lower than 12, 15.2% did not report pain and could not be classified with the scale, and 12.1% had a score higher than 12. In the MINI group, 75% of patients scored less than 12 and 25% reported no pain, which means that no one in this group had a neuropathic pain. In the statistical analysis, there was no significant difference between the groups (p = 0.81). Conclusion: There was no difference between conventional and minimally invasive surgeries in terms of pain assessed in the present sample. 108683 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ARTHUR MAROT DE PAIVA2, Gabriel Baêta Branquinho Reis2, Luiz Fernando Sposito Ribeiro Baltazar2, Pedro Guimarães Moreira da Silva2, Thais Vieira de Araújo Rodrigues1, Abissay Francisco Dias1, Marcos Vinícius Pires Rodrigues1, Thainá Lopes de Souza1, Walace Chaves dos Santos1, João Alberto Pansani1, Artur Henrique de Souza1, Giulliano Gardenghi1 (1) Hospital ENCORE – Aparecida de Goiânia/GO; (2) Universidade Federal de Goiás- Goiânia/GO Introduction: Minimally invasive cardiac surgery has been gaining popularity in the last two decades, associated with better aesthetic effect and decreased surgical trauma. Objective: The present study aims to compare patients undergoing valve replacement between conventional method and minimally invasive cardiac surgeries. Methods: Analytical cross-sectional study with patients undergoing heart valve replacement surgery between January 2020 and February 2022. Patients undergoing microsurgery were included from September 2021 onwards. The assessment of delirium was performed at first postoperative (PO1) day in an Intensive Care Unit (ICU) using the CAM-ICU and RASS scales. The evaluation of epidemiological data, previous comorbidities, use of pre and postoperative medications, anthropometric measurements and information related to the surgery were collected from TASY® electronic medical record system. The collected data were tabulated and calculated directly using specific spreadsheets with Excel 2010®. For the analysis of independent samples, the t-student test was performed. Results: 41 patients were included in the study. 33 patients underwent heart valve replacement surgery by the conventional manner (CONV group) and 8 patients underwent minimally invasive cardiac surgery (MINI group). When comparing patients undergoing different surgical procedures, the mean age of patients in CONV group was 55.5 (±12.5) and in MINI group was 62.9 (±16.1) (p = 0.19). The mean BMI of CONV was 26.6 (±3.8) and of MINI 27.7 (±6) (p = 0.51). The mean of cardiopulmonary bypass (CPB) time in CONV was 99.2 (±19.8) minutes and the mean clamping time was 71 (±15.7) minutes and the mean CPB time of MINI was 115.4 (±32.8) minutes (p = 0.08) and the mean clamping time was 76.9 (±30.2) (p = 0.45). The mean number of comorbidities in CONV was 3.1 (±2) and in MINI was 3.9 (±2.5) (p = 0.33). In CONV, the mean of home medications was 4.1 (±2.3) and in the postoperative period was 7.7(±2.3), while in MINI the mean of home medications was 5.1(±2, 9) (p = 0.29) and of postoperative medications was 7.1 (±1) (p = 0.47). The overall incidence of delirium in the CONV was 15.2% and 12.5% in MINI (p = 0.85). Conclusions: The observed delirium prevalence findings were consistent with the current literature in both groups. Risk factors and triggers classically related to delirium, such as age and polypharmacy, was not statistically significant between the two groups. 108694 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JARBAS RYGOLL DE OLIVEIRA FILHO1, ALESSANDRO MENEGHETTI ANVERSA2, ISABELLA KLAFKE BRIXNER3, ANDRESSA DUARTE SEEHABER3, NATÁLIA DA SILVEIRA COLISSI3, ALESSANDRA REBELATTO BOESING3, MATHEUS WERLANG DONADEL3, BRUNA SANTI DOS SANTOS3, ALEXANDRA SEIDE CARDOSO3, ALEXANDRE SHAAN DE QUADROS3, MATEUS DINIZ MARQUES3, ANIBAL PEREIRA ABELIN3 (1) Universidade Federal da Fronteira Sul (UFFS), Campus Passo Fundo, BRASIL; (2) Instituto de Cardiologia do RS/FUC, Porto Alegre, RS, BRASIL; (3) UFSM, Santa Maria, RS, BRASIL Background: ST-elevation myocardial infarction (STEMI) is a myocardial injury characterized by coronary artery occlusion. Treatment is rapid myocardial reperfusion performed through primary percutaneous coronary intervention (pPCI) or fibrinolytic therapy. Off-hours hospital presentation may impact the choice of reperfusion strategy, therefore increase cardiovascular outcomes. Objeticve: The aim of this study was to compare the effect of different times of presentation (on- and off-hours) on reperfusion strategy and in-hospital outcomes. Methods: Prospective cohort study, including patients diagnosed with STEMI between September/2016 and February/2020 in a public university hospital in southern Brazil. Patients were divided into on- and off-hours admission. Off-hours presentation was defined as hospital admission between 18:00 p.m. and 8:00 a.m. from Monday to Friday and weekends. In-hospital outcomes were evaluated. Results: A total of 150 patients (56%) were admitted during off-hours, and 118 (44%) during on-hours. Baseline characteristics were balanced between the two groups, except that hypertension was more prevalent in the off-hour group. Patients admitted off-hours had a higher rate of non-reperfusion strategy (31.3% vs 22.9%), lower rate of fibrinolytic therapy (9.3% vs 22%) and similar pPCI (p = 0,011). Median door-to-balloon time was similar between groups (on-hours group: 119 min (IQR 56–175) vs. 109 min (IQR 59–161)) (p = 0,640). No difference was found in the rates of in-hospital mortality (p = 0,621) and MACCE (p = 0,16). Conclusions: In our hospital we observed a difference in the reperfusion estrategy between patients admitted on and off-hours, with a higher rate of non-reperfusion. However, there was no difference in-hospital outcomes. 109405 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JARBAS RYGOLL DE OLIVEIRA FILHO1, EVA BRENDA SANTOS SILVA1, THIAGO EMANUEL RODRIGUES NOVAES1, TASSO KFURI ARAÚJO MAFRA1, JOSSIMARA POLETTINI1, GUSTAVO OLSZANSKI ACRANI1, SHANA GINAR DA SILVA1, RENATA DOS SANTOS RABELLO1, IVANA LORAINE LINDEMANN1 (1) Universidade Federal da Fronteira Sul (UFFS), Campus Passo Fundo, BRASIL Introduction: The new viral infection, caused by Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV2), called Coronavirus Disease 2019 (COVID-19), presents different clinical conditions and levels of severity, like patients who develop Acute Respiratory Syndrome. Severe (SRAG). Such cases are mostly elderly, considered at risk because they are more vulnerable due to physiological changes inherent to the aging process. Objectives: To identify the prevalence of hospitalization in the Intensive Care Unit (ICU), use of ventilatory support and case evolution in elderly patients diagnosed with COVID-19, carriers and non-carriers of chronic cardiovascular diseases (CVD). Methods: Observational study based on data analysis from the Influenza Epidemiological Surveillance Information System (SIVEP-Gripe) in Passo Fundo, RS. The study population consisted of SARS cases due to COVID-19, confirmed and notified from January 1 to December 31, 2020. Sociodemographic and health data were collected from individuals aged 60 years or older. The use of ventilatory support, admission to the ICU and the evolution of the case (death/cure) were used as outcomes of interest, and the exposure analyzed was the diagnosis of CVD. The prevalence was verified with a 95% confidence interval (95%CI) of the outcomes and their distribution according to the predictor variable (chi-square test, admitting an α error of 5%). Results: The sample consisted of 585 participants, most of them women (52.3%), aged up to 75 years (63.4%) and with CVD (59.0%). Of the total, 31.4% were admitted to the ICU (CI95 28–35), 74.2% used ventilatory support (CI95 71–78) and 41.2% (CI95 37–46) died. A higher prevalence of ICU admission was observed among patients with CVD (34.3%, p = 0.031), but no statistically significant difference was identified in the relationship between CVD and death (38.0%, p = 0.242) and use of ventilatory support (75.5%, p = 0.320). Conclusions: Elderly people diagnosed with SARS by COVID-19, carriers of CVD, were more exposed to ICU admission, which reinforces that having CVD increases the complexity of the case. Although the mechanisms are not yet fully understood, it is possible that the fact is related to the action of the angiotensin-converting enzyme 2, the main gateway of SARS-COV2 in the body and also the target of treatments in patients with CVD. With regard to lethality and use of ventilatory support, it was not possible to establish a relationship. 108707 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JARBAS RYGOLL DE OLIVEIRA FILHO1, SILVIO LUIZ SIMOES ANCINES FILHO1, SABRINE AGUIAR DE SOUZA1, ALAN ROBSON FERREIRA DA PAZ JÚNIOR1, GUSTAVO OLSZANSKI ACRANI1 (1) Universidade Federal da Fronteira Sul (UFFS), Campus Passo Fundo, BRASIL. Introduction: The relationship between risk factors (RF) for Cardiovascular Diseases (CVD) and patients workplace represents an important environmental factor. The Brazilian high school teacher is part of one of the classes that suffers most from environmental factors, occupying the second category of professionals with the highest presence of occupational diseases by the International Labour Organization (ILO). Objective: To analyze the prevalence of risk factors for cardiovascular disease among teachers of basic education in public schools in the city of Passo Fundo-RS-Brazil as well as the variables related to the outcome. Methods: This is a cross-sectional study, whose data were collected between August 2019 and February 2020 through a questionnaire sent to teachers by email and messages in mobile phone social network groups. Sociodemographic, life, health and employment data were collected. Cardiovascular risk stratification was calculated from the Brazilian Guidelines on Arterial Hypertension published by the Brazilian Society of Cardiology update 2020. Results: The sample consisted of 225 teachers, 91.1% women, 64.9% over 40 years old, 89.3% white, 54.5% with monthly income below R$ 5,000, 52.8% with more than 16 years of career, 73.4% with more than 30 working hours per week. After the analysis, it was found that 32% of those surveyed have a high risk for cardiovascular comorbidities in 10 years. It was possible to verify a statistically significant relationship between the presence of high cardiovascular risk among male teachers (60%, p = 0.005), age over 60 years (71.4%, p = 0.013), obesity (44%, p = 0.001), anxiety (37.5%, p = 0.018), RSI/WRMD (45.6%, p = 0.001) and exhaustion (42.9%, p = 0.044). Conclusions: The present study showed a high number of professors at high risk for cardiovascular disease in 10 years. It also found high rates of associated risk factors that denote negligence in the management of health care programs for teachers. The continuity in the production of studies on the relationship between teaching work and cardiovascular risk is fundamental for the development of measures that are, in fact, effective in combating the disease process, avoiding further complications in the future. 108709 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIELA DE OLIVEIRA SALAZAR1, Glessiane de Oliveira Almeida1, Felipe J Aidar1, Marcos Antonio Almeida-Santos1, Ikaro Daniel de Carvalho Barreto1, Dihogo Gama de Matos1, Cláudia Bispo Martins-Santos1, José Icaro Nunes Cruz1, Joselina Luzia Menezes Oliveira2, Enaldo Vieira de Melo1, José Augusto Soares Barreto Filho2, Antônio Carlos Sobral Sousa2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital Introduction: One of the strategies to control hypertension, recommended by some guidelines, is the self-measurement of blood pressure (BP). However, the benefits of this practice are not yet unanimous. Objective: To assess whether there is an association between non-standardized BP self-measurement and the control of systemic arterial hypertension (SAH), self-medication and emergency department visit. Methodology: Observational, analytical and cross-sectional study. Information was obtained through a standardized questionnaire and medical records of patients seen at cardiology outpatient clinics in Sergipe (Brazil). Adults with SAH were included. BP was considered controlled when systolic and diastolic pressure did not exceed 130 mmHg and 80 mmHg, respectively. The Shapiro Wilk, Chi-square with odds ratio and the Mann-Whitney U tests were performed. A two-tailed p ≤ 0.05 was set for statistical significance. Results: The sample consisted of 1000 patients, with a mean age of 61 ± 12.5 years, 57.1% were women and 52.3% had comorbidities. Most (93.8%) used a digital device for self-measurement and 21.1% were aware of the need for annual calibration of the device, which was performed in 10.7% of the total. The highest frequency of self-measurement in the population was 1 to 3 times/day (35%). The symptom that most induced BP self-measurement was headache (44.4%; p < 0.0001). The most prevalent behaviors after verifying high BP were doing nothing (49.9%) and self-medication (26%), followed by looking for urgency and trying to contact the doctor (22.8% and 1.3%). 99.2% reported that there was no guidance on how to measure BP. There was no association between sex or age with BP self-measurement. Patients with comorbidities perform more self-assessment (p = 0.005), including diabetics, dyslipidemic patients, patients with obstructive arterial disease (p < 0.001) and stroke (p = 0.010). Self-measurement was associated with uncontrolled BP (p < 0.001; OR 1.953; 95% CI 1.707–2.233), emergency visits in the last 12 months for hypertensive causes (p < 0.001; OR 3.410; 95%CI 2.928–3.972) and to self-medication (p < 0.001; OR 1.958; 95% CI 1.703–2.251). Conclusion: Non-guided BP self-measurement, practiced by 93.8% of the sample, was associated with non-control of SAH, self-medication and more visits to the emergency department in the last 12 months. 108711 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AUREA NATHALLIA GOMES DE SOUZA1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá UNIFAP Introduction: In 2012, law nº 12.732/12 was approved in Brazil, which provides for the first treatment of patients with proven malignant neoplasia and establishes a maximum period of 60 days from the diagnosis as the start date. Objective: To analyze the data about the year of diagnosis and year of treatment, observing compliance with law nº 12.732/12 in force in Brazil and other related aspects. Method: Were used the data available in PAINEL-ONCOLOGY, from the Department of Informatics of the Unified Health System (DATA-SUS), for more detail, in the detailed diagnosis category, C-38 (Malignant neoplasm of the heart, mediastinum and pleura), year of treatment and year of diagnosis and for a deeper analysis, the filters Month/Year of diagnosis and Month/Year of treatment were used, combining them and generating tables for analysis. Results: It is important to highlight the absence of data registered in the Information System over the years analyzed. Of the patients who were diagnosed in 2019, there is no information about the year of treatment for about 52.36%. Those who received their diagnosis in 2020 and 2021, the numbers also exceeded 50% of patients, being, respectively, 51.76% and 53.84%, approximately. In addition, some data were filled in incorrectly, stating that the patient started treatment one month before being diagnosed. Using the filter Month/Year of diagnosis and Month/Year of treatment, it can be said that law nº 12,732/12 is complied with in most of the registered cases, but it was observed that in some cases there is a delay in starting treatment, overloading the subsequent months. The annual general data show that 26 people who were diagnosed in 2019 started their treatment in 2020, and when looking at the detailed data per month, only 8 were registered according to the month, of these, 5 in the period governed by the law. And 20 people diagnosed in 2020 started treatment in 2021, but in the detailed data per month, there is information about 4, and 3 started in the period governed by the law. There is no detailed information about the year 2021. Conclusion: The analysis was hampered by the lack of data, which also has an impact on the absence of public policies aimed at the delay in starting the treatment of malignant neoplasms in general, which can have negative consequences for the prognosis of patients. The records show partial compliance with Law nº 12.732/12. 108721 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIELA DE OLIVEIRA SALAZAR1, Glessiane de Oliveira Almeida1, Felipe J Aidar1, Marcos Antonio Almeida-Santos1, Ikaro Daniel de Carvalho Barreto1, Dihogo Gama de Matos1, José Icaro Nunes Cruz1, Cláudia Bispo Martins-Santos1, Joselina Luzia Menezes Oliveira2, José Augusto Soares Barreto Filho2, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital Introduction: The number of hypertensive people in the world has doubled to 1.28 billion since 1990. It is estimated that about 50% of hypertensive people are uncontrolled. Objectives: To assess whether there are differences between the public and private systems in terms of blood pressure control and which factors predict non-control of systemic arterial hypertension (SAH) in these groups. Methods: Observational, cross-sectional and analytical study including hypertensive patients treated at cardiology outpatient clinics of the Unified Health System (SUS) and the Supplementary Network (RS) in Sergipe (Brazil). Information was obtained using a standardized questionnaire and medical records. The Shapiro Wilk, Chi-square, Mann-Whitney U test and binary logistic regression (backward stepwise method) were performed, which considered the model entry a p ≤ 0.25 and permanence ≤ 0.05. Results: Of the total, 50% of the patients were from the UHS and 50% from the SN. UHS users were more diabetic (24.6% vs. 18.6%; p = 0.021) and dyslipidemic (42.6% vs. 25.4% p < 0.001). UHS patients had more uncontrolled SAH, more visits to the emergency room in the last 12 months due to hypertension and fewer regular consultations with the cardiologist, while SN patients performed more self-medication. There was no difference regarding the self-measurement of blood pressure (BP). The predictors of uncontrolled SAH in both groups were emergency visits for hypertension and BP self-measurement. In the UHS, there were other factors, such as female sex and self-medication (Tables 1 and 2). Conclusion: There was a higher prevalence of uncontrolled SAH in the UHS, with female sex and self-medication being predictors of uncontrolled BP in this group, and self-measurement and emergency room visits predictors in both groups. 108727 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PRISCILA SANTOS MARIANO1, Amanda Cyntia Lima Fonseca Rodrigues2, Isabelly Salgado Marin3, Heloisa Belinati Pereira Perez3, Maria Carolina Casagrande3 (1) Universidade Federal de Ouro Preto; (2) Deutsche Hochdruckliga e.V. DHL® Deutsche Gesellschaft für Hypertonie und Prävention; (3) Centro Universitário Ingá Background and Aims: Arterial hypertension (AH) has become a common childhood health condition. When compared to adulthood, screening for childhood AH is complicated and unstable. This stems from the misunderstanding of the effects of AH in childhood, due to the scarcity of information about the long-term consequences of this condition, proof of this is that many pediatric patients have altered blood pressure and are not diagnosed or treated. Based on this evidence, there is a need for in-depth studies about primary hypertension in childhood and how its diagnosis and treatment can act in the prevention of cardiovascular diseases in adult life. Methods: This systematic review used the “PubMed” database to search for the descriptors “Arterial Hypertension” and “Childhood”, associated with articles from the last 10 years, finding 122 articles, which 38 were selected to perform the abstract. Results: The prevalence of AH in the age group from 7 to 17 years remained stable, ranging from 4.4 to 6.4%. However, the risks of developing SAH increased considerably from 6.3% in 1995 to 19.2% in 2014, among the predisposing factors obesity stands out. In other analyses, other possible predisposing factors to childhood obesity, such as longitudinal effects of maternal depression, unhealthy lifestyle, high screen time and low levels of physical activity. In addition, it can also be mentioned that newborns of women with greater weight gain in the first trimester of pregnancy had an increased risk of overweight or obesity, as well as elevations in diastolic blood pressure. Children born to mothers who had gestational diabetes mellitus had significantly higher systolic and diastolic blood pressure. Conclusion: Childhood hypertension is becoming progressively more common in the pediatric population, which demonstrates an enormous public health challenge worldwide. There is a correlation between BMI and AH level in overweight adolescents, and the obese BMI range in this age group is related to an increased risk of incidence of cardiovascular diseases, morbidity (ischemic heart disease and stroke) and mortality in adulthood. Early identification of abnormal AH at young ages is of utmost importance, as it is possible to increase its control and awareness, in addition to reducing progression to higher levels of AH and target organ damage. 108852 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES IGOR MARQUES JORDÃO1, Alana Helen Dos Santos Matos1, Ana Beatriz Cordeiro Prates1, Beatriz Dias Pinheiro1, Andre Barbosa de Andrade1, Isadora Gonçalves Roque1, Lucas Lopes Toledo1, Isabel Pellegrinelli Bacelar1, Fernando Coletti Mazarão1, José Luiz Padilha da Silva2, William Antônio de Magalhães Esteves1, Maria do Carmo Pereira Nunes1 (1) Federal University of Minas Gerais (UFMG); (2) Federal University of Parana (UFPR) Background: Rheumatic heart disease (RHD) is still a major health problem, especially in low- to mid-income countries, leading premature deaths owing to valvular disease (VD). Although left-sided valvular involvement is most commonly seen in RHD, the tricuspid valve can also be affected. However, there is a lack of information about the prognostic value of primary tricuspid valve disease (TVD) in RHD. Objective: This study aimed to determine the impact of TVD on clinical outcome in RHD. Methods: This prospective study enrolled patients with RHD referred to a tertiary center in Brazil for management of VD between 2011 and 2021. Primary rheumatic TVD was defined by echocardiographic features including thickening of leaflets associated with some degree of restricted mobility. Patients with rheumatic TVD were matched to patients with mitral valve disease (MVD) using 1:1 genetic matching algorithm that maximized balance of baseline covariates prior to exploring outcome differences. The main outcome was either need for MV replacement or death. Results: Among 694 patients eligible for the study, age of 47 ± 13 years, 84% female, 39 patients (5.6%) had rheumatic TVD. After excluding patients with incomplete data, 33 patients with TVD were matched to 33 controls based on age, right-sided heart failure, atrial fibrillation, and MV area. Over a median follow-up of 28 months [interquartile range: 8–71], 28 underwent MV replacement, and 6 patients died. Kaplan–Meier survival estimates showed worse event-free survival in patients with TVD compared to matched controls (Figure 1). TVD was predictor of adverse events with a hazard ratio of 3.39 (95% CI: 1.56, 7.35, p = 0.002) in the genetic matched cohort with balance on baseline covariates of interest. Conclusions: In the setting of RHD, rheumatic TVD significantly increased risk of adverse events compared with matched controls. The involvement of TV may express overall disease severity that adversely affects clinical outcome. Our study is the first to assess clinical outcome using genetic matching approach to account for baseline differences in patients characteristics. 108743 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JESSICA SILVA ROCHA1, Gabriel Silva Rocha2, André Moisés de Oliveira Nunes4, Geovanna Vitória da Cruz Xavier Silva1, Júlia Ranielly Oliveira Rios1, Lucas de Lacerda Ramos3 (1) Centro Universitário UniFTC (UniFTC); (2) Hospital Ana Nery (HAN); (3) Universidade Federal da Bahia (UFBA); (4) Escola Bahiana de Medicina e Saúde Pública (EBMSP) Introduction: Takotsubo cardiomyopathy (TCM) is caused by catecholamine surge, which is also observed in COVID-19 disease due to cytokine storm. TCM refers to a transient left ventricular systolic dysfunction, usually with associated chest pain, whose main differential diagnosis is acute coronary syndromes. Although vaccination against SARS-CoV-2 is the primary factor in combating the pandemic, myocarditis has been demonstrated in reports as a side effect of the vaccine. And, despite being rare cases, a new category is being reported, with studies still limited, such as TCM. Objectives: To evaluate the association between the incidence of TCM and vaccination against the COVID-19 virus, in addition to characterizing the rate of post-vaccination TCM hospitalizations. Methods: This is a systematic review, in which the databases available on the platforms Pubmed, Lilacs, Medline were analyzed. For this, studies that address Takotsubo Syndrome related to COVID-19 vaccination were selected, following the PRISMA guidelines. As for the inclusion criteria, it consists of case reports presenting TCM as the only differential diagnosis, published in the last 2 years and available in the electronic version. The excluded studies were those that showed distance from the central objective of the research and duplication. Results: Seven eligible studies were selected for data collection. Of the results obtained, the female sex predominated (85.75%), mean age 62.1 ± 17.8 years, the mean number of days from the vaccine to the event was 3 days and 71.4% of the cases had some comorbidity. Conclusion: Typical epidemiological characteristics of Takotsubo Syndrome were evidenced, such as female gender, advanced age and the presence of comorbidities, in which the event was exclusively related to COVID-19 vaccination, subjectively excluding other causes. Therefore, the need for further studies characterizing TTK, as well as its relationship with mRNA vaccines is important, since there are also previous reports correlating TTK with the influenza vaccine. And from that, to evaluate the safety of mRNA vaccines and optimize patient outcomes. 108748 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GIULIA VITORIA NASCIMENTO DA SILVA1, LARISSA DACIER LOBATO COMESANHA2, TEREZA MARIA MEIRELES FERNANDES DA SILVA1, MARIANA DOS SANTOS GUIMARÃES2, GABRIEL REZENDE NEVES1, MIGUEL CHAMOSO GILSANZ NETO1, PAULO CESAR LOBATO MAGALHÃES2, GABRIELA DE ARRUDA CUSTÓDIO3, HENRIQUE CUSTÓDIO DA SILVA1 (1) Universidade do Estado do Pará (UEPA); (2) Universidade Federal do Pará (UFPA); (3) Centro Universitário Metropolitano da Amazônia (UNIFAMAZ) Introduction: Professional athletes constantly suffer physiological and anatomic alterations in the cardiovascular system in order to balance the intense cardiac activity during exercises. Although women develop less cardiac diseases in their life as a result of healthier habits of life than men, feminine athletes are not exempt from the constant cardiovascular adaptation inherent to high performance sports and its risks. In this regard, changes in the stimulant complex of the heart and in its structure may lead to some severe complications such as arrhythmias and cardiac insufficiency. Objective: To identify electrocardiographic abnormalities in athletes from a professional women’s soccer team. Method: Cross-sectional, analytical-descriptive and quantitative study. Data were obtained through medical records of 20 professional female soccer athletes, in the year 2020, in the city of Belém, Brazil. In addition to electrocardiographic records, information on age, family history, chest pain when practicing physical activity and consumption of alcohol drinks were also analyzed. Chi-square test and Fisher’s exact test were used for statistical analysis of variables. Results: The average age of the athletes were 25.1 ± 3.9 years. In the study population, only 4 participants (20%) had ECG abnormalities, which were 2 right bundle branch conduction delay (50%), 1 left ventricular hypertrophy signs (25%) and 1 right bundle branch block (25%,). There was no statistical significance between alcohol consumption and the presence or absence of ECG changes (p > 0.05). Moreover, there were also no statistically significant differences associated with ECG changes when related to the presence of chest pain during physical activity or the existence of a family history of sudden death or heart disease (p > 0.05). Conclusion: The findings of the present study showed electrocardiogram abnormalities in 20% of the athletes included, but not related to chest pain, family history of sudden death or heart disease, nor to the use of alcohol drinks, being in similarity with other studies available in the literature. Despite this, the importance of continuity of cardiological medical evaluation by athletes for the prevention of cardiovascular diseases and possible sudden death is highlighted. 108756 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION KARINE OLIVEIRA LIMA1, Laura Cordeiro Gomes2, Philip Brainin3, Marliton Vinicius Pedrosa Evangelista1, Isabelle Victória Martins Vieira1, Anna Engell Holm3, Luan Oliveira Matos1, Bianca Vasconcellos Rodrigues Lopes4, Iara Fernanda Vasconcelos de Oliveira e Silva1, Tor Bierning-Sorensen5, Cláudio Romero Farias Marinho2, Odilson Marcos Silvestre5 (1) Multidisciplinary Center, Federal University of Acre, Câmpus Floresta, Cruzeiro do Sul, Acre, Brazil; (2) Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; (3) Department of Cardiology, Herlev-Gentofte University Hospital, Hellerup, Denmark; (4) Health and Sport Science Center, Federal University of Acre, Rio Branco, Acre, Brazil; (5) Faculty of Biomedical Sciences, Copenhagen University, Copenhagen, Denmark Background: The prevalence of hypertension in malaria endemic areas seems to be higher than in malaria-free areas. In these regions, a higher rate of hypertension has been detected in younger people when compared to developed countries. We aimed to investigate the relationship between malaria episodes and hypertension in the Brazilian Amazon. Methods: The study was conducted in the Brazilian Western Amazon from June 2020 to August 2021. Patients with a lifetime history of malaria, but not infected at inclusion had a cross-sectional assessment. However, those who were infected with Plasmodium spp. at enrollment (T0) had a follow-up evaluation (FUP) performed 34 ± 7.2 days later. All of them answered a questionnaire concerning clinical and sociodemographic information and underwent physical examination. Patients were classified as hypertensive through previous medical diagnosis and/or according to the International Society of Hypertension parameters: ≥140 systolic blood pressure (SBP) and/or ≥90 diastolic blood pressure (DBP). The mean between both arms were considered for SBP and DBP. Since variables were non-parametric, Wilcoxon signed-rank test was applied to compare blood pressure betweenT0 and FUP. Logistic regression was performed to assess the risk of hypertension as a function of the age at the first malaria episode. Results: Seventy-six symptomatic malaria patients completed the follow-up evaluation. The median age was 37 years (IQR 26 to 48) and 59% were males. A significant increase in the SBP (median [IQR] T0 119.5 mmHg [111– 129.5] vs FUP 122.5 mmHg [113.75–134], P = 0.015) and DBP (median [IQR] T0 74.5 mmHg [68–82.75] vs FUP 77.5 mmHg [72.5–84], P < 0.001) was found in the follow-up when compared to enrollment. Additionally, a total of 430 patients were included in the cross-sectional study [53% males; median age 39 years (IQR 30 to 47)]. Median age of the first malaria episode was 17 (IQR 12 to 27) years. After adjusting for number of previous malaria episodes, age, abdominal circumference, income, sex, smoking and diabetes, age at first malaria infection was associated with higher risk of hypertension [Odds ratio 1.031 (95% CI 1.006 to 1.057), P = 0.014]. Conclusion: Age at the first malaria episode seems to influence blood pressure levels. Both SBP and DBP increased 34 days after infection. More studies to investigate this association are required to improve health policies on hypertension in communities living in malaria endemic areas. 109436 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JOÃO PEDRO TORRES NEIVA RODRIGUES1, Raquel Vieira Torres1, Pedro Viana Diniz1 (1) Universidade Federal de Juiz de Fora Introduction: Risk factors for stroke onset are well known. However, factors that may act as a trigger still need clarification. Objective: We aimed to review data from the literature to assess the role of physical exertion or emotional distress as triggers of stroke. Methods: Reviewers researched on Pubmed, Embase, and PsycINFO for cohort, case-control, or case-crossover studies with exposition to triggers within 3 months of stroke onset. PRISMA protocol was used. Results: 323 articles were found, and after applying inclusion criteria, 17 studies remained. Combined, there were 1168152 cases of stroke. Physical exertion was defined in most articles as activities that require more than 5 METs to be performed, however, sexual activity, and lifting >23 kg, were also included. Eight studies analyzed this group, and 5 studies reported a correlation between physical exertion and the onset of stroke. 15 studies reported emotional triggers such as anger, psychological distress, startling, positive, and negative emotions were investigated. A correlation between emotions and the onset of stroke was found in 12 studies (Table 1). Different cutoffs were used to determine an emotional outburst, which could increase studies heterogeneity and could cause discordant results between them. Self-reported questionnaires can be prone to memory bias. Conclusion: Physical exertion and emotional triggers were associated with an increased risk of stroke onset in most studies. 108793 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION GUSTAVO SANTOS PORFIRO1, Gustavo Santos Porfiro1, Leonardo Favaro Pereira1, Rosemberg Rodrigues Dal Gobbo1, Fernando Luiz Torres Gomes1 (1) Universidade Federal do Espírito Santo; (2) Universidade Federal do Espírito Santo; (3) Universidade Federal do Espírito Santo; (4) Universidade Federal do Espírito Santo; (5) Universidade Federal do Espírito Santo; (6) Universidade Federal do Espírito Santo Introduction: Heart diseases are the leading cause of death in the world, affecting developed and underdeveloped countries, and have been presenting progressive and alarming growth. Early diagnosis of this type of condition is fundamental to reduce mortality. The main and most common way of diagnosing heart disease is the electrocardiogram (ECG), which is totally dependent on the supervision of a specialized professional. Consequently, it is pertinent to observe the need for preventive diagnosis in order to minimize the high mortality rates of these diseases. Objective: To investigate the selection and prediction of Machine Learning (ML) algorithms to facilitate the process of identifying heart diseases from electrocardiogram signals. Methods: We conducted a literature review, covering the period from January 2021 to January 2022, in the Pubmed, Cochrane and Physionet databases. The descriptors “machine learning”, “heart diseases”, “electrocardiogram”, separated by Boolean operators AND, were searched in the Medical Subject Heading. Among the inclusion criteria were full-text articles published from 2011 onwards. Results: Performance evaluation of an ML algorithm was performed by means of statistical metrics relating overall prediction hit rate, accuracy and the confusion matrix to the treatment of tabular data. It was obtained from algorithms and solutions with open source code a hit rate higher than 80% both in predicting normal ECG exams and in predicting classes that presented heart disorders, such as myocardial infarction, being that in some cases the hit rates were higher than 75% for normal exams and almost 90% in relation to exams with myocardial infarction. Conclusion: The use of intelligent software based on Machine Learning to facilitate the diagnosis of heart diseases is a promising advance, given the alarming growth of these diseases according to data from the World Health Organization. With increasingly advanced technology and improved resources, this software can partially reduce the dependence on analysis by a professional, and can indicate findings that may be overlooked by professionals. It is, then, an ally to the professionals who deal with these types of exams, and not their substitutes. 108798 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING CAROLINA DOS ANJOS BASTOS1, Carolina dos Anjos Bastos1, Eduardo Pires dos Santos1, Anderson Rafael Pesamosca1, Tânia Zaleski1, Wilton Francisco Gomes1 (1) Faculdades Pequeno Príncipe (FPP) Background: Cardiovascular diseases (CVD) remain the leading cause of premature death worldwide. There are several diagnostic methods for its detection, among them coronary computed tomography angiography (CTA), with increasing use in recent years. Defining the most appropriate complementary test, in terms of accuracy for clinical decision making and rationalization of resources, remains a challenge in medical practice. Objective: To analyze the appropriateness of the indications for CTA in a sample from a private clinic in Curitiba, taking as reference the II Guidelines for Cardiovascular Magnetic Resonance and Computed Tomography of the Brazilian Society of Cardiology and Brazilian College of Radiology (IIDRT) and the Roll of the National Agency for Supplementary Health (ANS). As a secondary objective, to analyze the main variables that correlated with the presence of obstructive CAD in this sample. Method: This is an observational and retrospective study, with the analysis of 2595 consecutive CTA exams for the evaluation of CAD, between January 2019 and December 2020. Indications were classified according to IIDRT recommendation classes and ANS Roll recommendations. Multivariate analysis was used for correlation between independent variables and outcome. Results: Regarding the IIDRT indications for the performance of CTA, 1170 (45.1%) individuals fit into recommendation class I, 10 (0.4%) into IIa, 175 (6.7%) into IIb, 550 (21.2%) into III and 690 (26.6%) did not fit into any expected indication. As for the ANS Roll, 1620 (62.4%) individuals fit the indication criteria, while 975 (37.6%) did not. Risk factors such as dyslipidemia, diabetes and family medical history were associated with obstructive CAD in CTA (p < 0.001). Conclusion: 47.8% of patients referred for CTA had a class III recommendation or did not have indications according to the IIDRT. By ANS criteria, over 37.6% of patients had non-predicted indications. This denotes that a critical analysis of current recommendations as well as clinical practice should probably be performed. 108803 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERIOPERATIVE EVALUATION EDUARDO PORTO SANTOS1, RAFAEL FORTES LOCATELI1, ANNA CAROLINA FLORES MARIATH2, VITÓRIA CAROLINA KOHLRAUSCH1, ISABELLA KLAFKE BRIXNER1, GABRIELLE LENZ DE ABREU2, ANÍBAL PEREIRA ABELIN1, DIEGO CHEMELLO1, MATEUS DINIZ MARQUES1 (1) UNIVERSIDADE FEDERAL DE SANTA MARIA; (2) HOSPITAL UNIVERSITÁRIO DE SANTA MARIA Background: Infection is the most frequent non-cardiac complication after cardiac surgery. Its incidence is associated with increased mortality, hospitalization days and costs. Objective: The aim of this study is to describe the incidence of infections in patients undergoing cardiac surgery at the University Hospital of Santa Maria and hospitalization days and mortality associated with. Methods: This is a descriptive, longitudinal and retrospective study analyzing the medical records of patients undergoing cardiac surgery between September 2018 and April 2021. Patients undergoing pacemaker implantation or pericardial surgery or those who died intraoperatively were excluded. The analysis was performed using the software Stata version 15.1, and the results were expressed as percentage, mean and standard deviation. Results: From a database of 200 patients, 194 filled the inclusion criteria. Of these, 60 patients (30.92%) were diagnosed with infection and used antibiotics postoperatively and 16 died (26.67%). The mortality among patients without nfection was 6.0% (8 patients). Infection was associated with death during hospitalization (x² = 16.37; p < 0.001), with 66.67% of deaths occurring among patients with infection. individuals with infection had higher mean hospitalization days than those without infection using the Mann Whitney U test (20 ± 15 days and 9 ± 5 days, respectively, U = 1465.500, Z = –7.099, p < 00.1). Conclusions: The study portrayed the reality of a tertiary public hospital for heart surgery in the countryside, with a high incidence of postoperative infection. The diagnosis of infection was associated with longer hospital stay and higher mortality. The factors associated with higher incidence of infection and management factors need to be addressed in detail in future studies. 108808 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JOSÉ ICARO NUNES CRUZ1, Cláudia Bispo Martins-Santos1, Gabriela de Oliveira Salazar1, Juliana Maria Chianca Lira1, Mayara Evelyn Gomes Lopes1, Ana Luísa Lisboa Prado1, Marília Marques Aquino1, Nathalia Luiza Silva Sobral1, Arthur Leite Lessa1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or – São Lucas Hospital Introduction: Cardiovascular diseases are important causes of morbidity and mortality worldwide. Among these, coronary artery disease (CAD) stands out as a significant cause of death and health costs. Some studies demonstrated that female sex is a protective factor for CAD in the premenopausal period, suggesting a beneficial action of endogenous estrogens on the heart. In this context, it is essential to analyze the risk factors and prevalence of CAD between the sexes. Objectives: To assess the frequency of CAD, as well as its characteristics, according to sex. Methods: This is an observational, cross-sectional, analytical study. The sample included patients who underwent coronary computed tomography angiography (CCTA) in a private hospital. Coronary stenosis ≥ 50% was considered significant on CCTA. Statistical analysis was conducted using Chi-Square test, with significance level set at 5% for comparisons. Odds ratio (OR) and 95% confidence intervals (CI95%) were also described. All analyses were performed using SPSS Statistics software, version 22.0. Results: The sample consisted of 1250 patients, of which 48.3% (604) were female. The mean age was 57.9 ± 12.9 years, with a non-significant difference between the sexes (p > 0.05). Women demonstrated higher frequencies of obesity (27.9% vs. 21.8%; p = 0.027) and sedentary lifestyle (75.3% vs. 65.3%; p = 0.001). Men demonstrated higher rates of smoking (4.6% vs. 3.8%; p = 0.041) and alcohol consumption (41.2% vs. 18.0%; p < 0.001). Male subjects exhibited higher prevalence of CAD (52.6% vs. 38.1%; p < 0.001), as well as higher chance of calcified plaques (OR: 1.95; CI95%: 1.40–2.72; p < 0.001) and non-calcified plaques (OR: 1.75; CI95%: 1.08–2.82; p = 0.021). In addition, men were more likely to have significant coronary stenosis (OR: 2.65; CI95%: 1.82–3.85; p < 0.001), as well as triple vessel CAD (OR: 3.88; CI95%: 2.02–7.46; p < 0.001) and double vessel CAD (OR: 2.76; CI95%: 1.72–4.43; p < 0.001). Conclusions: The prevalence of CAD was lower among women. Men were more likely to have calcified and non-calcified plaques, significant coronary stenosis, double vessel CAD and triple vessel CAD. Thus, it is concluded that females are less affected by CAD. 108817 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GABRIELA DE OLIVEIRA SALAZAR1, José Icaro Nunes Cruz1, Cláudia Bispo Martins-Santos1, Juliana Maria Chianca Lira1, Lucas Villar Shan de Carvalho Cardoso1, Nathalia Luiza Silva Sobral1, Ana Luísa Lisboa Prado1, Giulia Vieira Santos1, Mayara Evelyn Gomes Lopes1, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa2, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Lucas Hospital Introduction: Low cardiorespiratory fitness (CF) is a risk factor for cardiovascular morbidity and mortality. Women have significantly lower CFs than men. Furthermore, alcohol consumption is a known determinant of disease burden, although there is still no consensus on the influence of alcohol on CF. In recent years there has been an increase in alcohol consumption among women. Objective: To identify predictors of low CF in women, with emphasis on the influence of social alcohol consumption on CF. Methods: Observational, cross-sectional, analytical study, with data collection from record of echocardiogram exams under physical stress performed in Sergipe (Brazil). Female patients were included. The CF was classified according to the metabolic equivalent of task (MET) as low (MET < 7.9), intermediate (7.9 ≤ MET < 10.9) and high (MET ≥ 10.9). Statistical analysis included the chi-square test and multinomial logistic regression using SPSS Statistics software. A value of 5% was set for statistical significance. Results: 2202 patients were included, with a mean age of 58.48 ± 10.9 years. Among the risk factors, 54.1% were hypertensive, 47.5% sedentary, 46.4% dyslipidemic, 21.9% obese, 17.1% alcoholic and 4.3% smokers. CF was considered low in 35%, intermediate in 37.5% and high in 27.5% of the population studied. Alcohol consumption was a predictor of low CF in women, compared to those with high and intermediate CF, as well as sedentary lifestyle. Systemic arterial hypertension (SAH) and obesity predicted low CF when compared to the high CF group (Table 1). Conclusion: Alcohol consumption was an independent predictor associated with low CF in women. The same was verified with the presence of sedentary lifestyle, SAH and obesity. 108835 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING JOSÉ ICARO NUNES CRUZ1, Gabriela de Oliveira Salazar1, Cláudia Bispo Martins-Santos1, Lara Teles Alencar Duarte1, Nathalia Luiza Silva Sobral1, Juliana Maria Chianca Lira1, Raisan Almeida Santos2, Myllena Maria Santos Santana1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2, Luiz Flávio Galvão Gonçalves2 (1) Federal University of Sergipe; (2) Rede D’Or – São Lucas Hospital Introduction: The pathophysiology of ventricular dysfunction comprises cardiac remodeling with formation of diffuse cardiac fibrosis, a marker of poor prognosis. Native T1 mapping, performed by cardiac magnetic resonance imaging (CMRI), is a non-invasive method of assessing compromised myocardial areas, such as fibrotic areas, being considered a sensitive marker of cardiovascular morbidity and mortality. Objectives: To associate native T1 mapping with parameters related to left ventricular dysfunction on CMRI. Methods: This is an observational, cross-sectional, analytical study. The sample included patients who underwent CMRI in a Phillips Ingenia 1.5T magnet, from a private hospital located in Sergipe (Brazil), in 2021. Native T1 mapping of the left ventricle was compared with other variables related to ventricular function, which were indexed to body surface when measurements were borderline. Statistical analysis was performed using the Mann-Whitney U test and one-way analysis of variance (ANOVA) adjusted by bootstrapping, followed by Games-Howell post-hoc. All analyses were performed using SPSS Statistics software, version 22.0. Results: Among 415 patients who underwent CMRI in 2021, native T1 mapping was originally obtained for 225 patients (54.2%). The median native T1 mapping in the included sample was 1036 ms (IQR: 1008.0–1067.0 ms). Native T1 mapping values were significantly higher in patients with increased left ventricular (LV) end-diastolic volume (1055.5 vs. 1035.0 ms; p = 0.003), increased LV mass (1066.5 vs. 1034.0 ms; p < 0.001) and increased left atrium (LA) size (1053.0 vs. 1033.0 ms; p < 0.001). The native T1 mapping was also superior in patients with reduced left ventricular ejection fraction (LVEF) compared to patients with preserved LVEF (1143.53 vs. 1032.03 ms; p = 0.001), as well as among patients with LVEF borderline compared to those with preserved LVEF (1077.35 vs. 1032.03 ms; p = 0.007). Conclusions: Elevated native T1 mapping was strongly associated with indicators of cardiac dysfunction, such as greater LV end-diastolic volume, greater LV mass, greater LA dimension and reduced LVEF. Thus, the study confirms the applicability of native T1 mapping on the assessment of heart failure. 108857 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION ANA RACHEL BUCAR CERVASIO1, Clara Avelar Mendes Vasconcellos1, Carlos Augusto Parente Macedo Moura1, Daniel Barreto Kendler1, Deisianny Santos Ferreira1, Elizabeth Silaid Muxfeldt1, Karine da Silva Guimarães1, Gabriela Gama Zagni Jardim1, Lívia Lopes Monteiro de Barros Junqueira1, Rodney Barberá Boghossian1, Tomás de Souza Mello1, Vitória Santa Marinha Flumignan1 (1) IDOMED – Universidade Estácio de Sá, Medicine School, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil – UNESA Background: Obesity is increasing in younger populations, and is associated with a high cardiovascular (CV) risk, however, it is not clear whether metabolically healthy obesity (MHO) may have a lower CV risk or if it is just an earlier stage of the disease. Objective: To evaluate the prevalence and cardiovascular (CV) risk factors associated with metabolically healthy obesity (MHO) in a young population assisted by a Family Health Care unit in a large urban center. Design and Methods: A cross-sectional population study for CV risk assessment in adults aged 20–50 years old from a Family Health Care unit. Demographic, anthropometric data and CV risk factors were recorded. All underwent office blood pressure (BP) measurements, laboratory evaluation (lipid and glycidic profile). Obesity was defined as a body mass index (BMI) > 30 kg/m2 and MHO are those who have less than 3 of the following criteria for metabolic syndrome: office BP higher or equal to 130 × 85 mmHg, hypertension, fasting blood sugar higher or equal to 100 mg/dL, HDL < 40 mg/dL (men) and 50 mg/dL (women), triglycerides >150 mg/dL and waist circumference > 102 cm (men) and > 88 cm (women). Results: A total of 632 individuals were evaluated (60% female; mean age 37 ± 9 years). The prevalence of obesity was 26%, of which 73% were classified as MHO. Obeses are older, with a higher prevalence of physical inactivity (51% vs 41%, p = 0.03), hypertension (44% vs 19%, p < 0.001), dyslipidemia (50% vs 36%, p = 0.002), and diabetes (7% vs 2%, p = 0.001) with higher systolic OBP. MHO compared to unhealthy ones are significantly younger and smoke less. Despite being obese, they have lower BMI (33.6 vs 35.2 kg/m2, p = 0.02) and abdominal circumference (102 vs 110 cm, p = 0.03), with lower diastolic BP. Conclusion: MHO was more prevalent in this young population and seems to have a lower CV risk, however it is not clear whether these younger and less obese individuals are only at an earlier stage of the disease. Perhaps the CV diseases onset is postponed for a few years. Even so, these individuals should not be excluded from public health policies as a form of primary prevention. 108872 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION CLARA AVELAR MENDES DE VASCONCELLOS1, Tomás de Souza Mello1, Karine da Silva Guimarães1, Gabriela Gama Zagni Jardim1, Ana Rachel Bucar Cervasio1, Carlos Augusto Parente Macedo Moura1, Felipe Rey Costa Tostes1, Pedro Henrique dos Santos Lemos1, Clara Maria da Costa Muguet1, Joana Sauerbronn Corrêa da Costa1, Inah Maria Drummond Pecly1, Elizabeth Silaid Muxfeldt1 (1) IDOMED – Universidade Estácio de Sá, School of Medicine, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil. Introduction: The association between kidney lesion, hypertension (HTN) and cardiovascular risk (CV) is well established. Asymptomatic patients with decreased glomerular filtration rate (GFR) and/or increased albuminuria seem to have increased CV risk. The refinement of HTN diagnosis using Home Blood Pressure Monitoring (HBPM) allows to identify individuals with different phenotypes: normotension, sustained HTN, masked HTN and white-coat HTN, increasing diagnostic accuracy. Objective: To assess kidney subclinical lesions among different HTN phenotypes identified by HBPM in an adult population enrolled in a Family Health Strategy unit. Design and Methods: Cross-sectional, populational study with adults between 20–50 years old enrolled in a Family Health Strategy unit in a metropolis. Sociodemographic, anthropometric and CV risk factors were registered. Office blood pressure (BP) was the average of two consecutive measurements, and HBPM followed a 7-day protocol. HBPM < 135 × 85 mmHg and office BP < 140 × 90 mmHg were considered normal, identifying four phenotypes: normotension (controlled office BP and HBPM); white-coat HTN (uncontrolled office BP and controlled HBPM); masked HTN (controlled office BP and uncontrolled HBPM) and sustained HTN (uncontrolled office BP and HBPM). Albuminuria was dosed in an isolated urine sample whereas GFR was calculated by CKD Epi formula using serum creatinine. Results: 483 individuals were evaluated (39% male; mean age: 37.6 ± 8.8 years). More often, individuals with white-coat HTN (9.3%) are male, with greater neck circumference and higher prevalence of metabolic syndrome. Individuals with masked HTN (10%) are more obese, with increased neck and waist circumferences whereas those who present sustained HTN are predominantly male, more obese, with increased neck and waist circumferences and higher prevalence of diabetes and metabolic syndrome. Analyzing four phenotypes, a progressive albuminuria increase was observed among normotensive patients: white-coat HTN 4.0 ± 2.2; masked HTN 5.3 ± 3.4 and sustained HTN 7.1 ± 1.4 mg/g creatinine. We also observed an increased serum creatinine and decreased GFR: white-coat HTN 0.74 ± 0.16/132 ± 34; masked HTN 0.79 ± 0.17/123 ± 28; sustained HTN 0.81 ± 0.32/120 ± 59, respectively, although without statistical significance. Conclusion: The performance of HBPM refined HTN diagnosis and was able to early identify a progressive worsening of kidney function parameters in the different phenotypes. 110530 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIELA FREITAS VALVERDE 1, Gabriel Martins Nogueira1, Bruna Marmori Lima1, João Victor Araújo de Oliveira1, Adriana Santiago de Carvalho Borges1, Lorena de Souza Santos1, Gabriel Von Flach Sarmento1, Alexandra Brito Rocha da Silva1, Jéssica Reis de Jesus1, Ana Flávia de Souza Moura1, Constança Margarida Sampaio Cruz2 (1) Escola Bahiana de Medicina e Saúde Pública (EBMSP); (2) Obras Sociais Irmã Dulce (OSID) Introduction: Resistant Arterial Hypertension (RHTN) is a clinical condition in which blood pressure (BP) remains above target even with the concomitant use of three specific antihypertensive drugs of different classes at maximum doses or within the target using 4 or more of those. The main factor interfering with BP control is drug non-adherence, which can be assessed by the 8-item Morisky Medication Adherence Scale (MMAS-8), an update of the four-item, still not widely found in national studies on RHTN. Thus, this study aims to estimate the rate of medication adherence in patients with apparent RHTN and correlated factors in a teaching-care outpatient clinic. Methods: This is a cross-sectional study carried of patients treated at a cardiology teaching-care outpatient clinic, over 18 years old, diagnosed with hypertension and with at least six months of antihypertensive treatment. Patients with severe psychiatric and/or cognitive limitations, who did not meet RHTN criteria and with secondary hypertension were excluded from the sample. Medication adherence was collected through the application of the MMAS-8 questionnaire in person, in an outpatient clinic and by telepresence, through a virtual form. The score obtained for the therapeutic adherence rate ranges from zero to eight, with eight considered high adherence, ≥ 6 and < 8, medium adherence and < 6, low adherence. Results: A total of 31 patients were included in the study, 23 (74.19%) women and a median age of 60 (IQR 53–65) years. Furthermore, 18 (58.06%) had a family history of hypertension, 20 (64.5%) had no BP control, 25 (80.64%) were sedentary, nine (29.03%) were alcoholics and the most prevalent comorbidity was Diabetes Mellitus (35.4%). The body mass index (BMI) was, on average, 32.37 (IQR 27.10–35.86), the median time of diagnosis of hypertension was 10 (IQR 4–15.5) years and the median number of antihypertensive drugs in use and the median total number of drugs were four (IQR 3–4.5) and seven (IQR 5–8.5), respectively. The median score obtained on the MMAS-8 was 5.75 (IQR 4.75–7.75), considered low adherence. It was found that the no BP control (p = 0.020), a higher BMI (p = 0.043) and higher serum urea values (p = 0.045) are correlated with a lower rate of adherence to antihypertensive drugs. Conclusion: Low outpatient medication adherence to antihypertensive drugs was found. There was a correlation between adherence and the control variables of BP, BMI and serum urea values. 108904 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FRANCISCO JEAN DE MOURA SANTOS FILHO1, Carlos Eduardo Batista de Lima1, André Gonçalves Honório Carvalho1, Maria Clara Lima Almeida2, Paulo Márcio de Sousa Nunes1, Antônio Maycon da Silva Sousa1, Patryck Araújo Dantas1, Mauro Guimarães Albuquerque1, Ginivaldo Victor Ribeiro do Nascimento1, Francisco César de Oliveira Gonçalves1, Arthur Oliveira Nogueira e Lago1, Jardel Silva Santos3 (1) Hospital Universitário da Universidade Federal do Piauí (HU-UFPI) – Empresa Brasileira de Serviços Hospitalares (EBSERH); (2) Instituto do Coração do Hospital das Clínicas da Universidade de São Paulo – InCor HCFMUSP; (3) Universidade Estadual do Piauí (UESPI) – Hospital Getúlio Vargas (HGV) Introduction: Chagas cardiomyopathy is a myocarditis that can progress to dilated non-ischemic cardiomyopathy, heart failure, ventricular arrhythmias and thromboembolic complications. During the chronic phase, an important proportion of patients complain of atypical chest pain. Some studies showed a lower occurrence of chronic coronary disease in Chagas disease, but this fact is not clarified. Objective: Comparative analysis of structural cardiac alterations and the presence of severe obstructive coronary artery disease in patients with and without Chagas disease. Methods: A paired case-control study for atherosclerosis risk factors based on the Framingham risk score (FRS). From November 2014 to November 2019, all patients undergoing coronary angiography at the University Hospital of the Federal University of Piauí, in an elective basis for investigation of coronary disease, were evaluated and chagasic patients were selected, confirmed with two positive serological tests by different methods. The control group was selected consecutively in a 1:3 ratio. For statistical analysis, the chi-square test was used, considering the value of p < 0.05 for statistical significance. Results: Among 1120 patients evaluated, 15 patients with Chagas disease were identified and 45 patients without a diagnosis of Chagas disease were selected. The FRS variables, including age, sex, arterial hypertension, diabetes, physical inactivity, smoking, dyslipidemia and ejection fraction showed no statistical difference between the groups. The occurrence rate of CAD was lower in chagasic patients: 13.3% versus 64.4%, and for severe CAD the percentages were 13.3% and 51.1% (p = 0.003). The presence of structural cardiac alterations was high in both groups, being 80% of the chagasic and 73.3% of the non-chagasic, with no statistical difference (p = 0.606). Conclusion: In this series, the presence of segmental structural alterations of the left ventricle was high in both groups, while in the evaluation of coronary artery disease there was a lower occurrence in the group of patients with Chagas disease. 108910 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING GABRIELA DE OLIVEIRA SALAZAR1, Cláudia Bispo Martins-Santos1, José Icaro Nunes Cruz1, Lara Teles Alencar Duarte1, Nathalia Luiza Silva Sobral1, Juliana Maria Chianca Lira1, Mayara Evelyn Gomes Lopes1, Raisan Almeida Santos2, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa2, Joselina Luzia Menezes Oliveira2, Luiz Flávio Galvão Gonçalves2 (1) Federal University of Sergipe; (2) Rede D’Or São Lucas Hospital Introduction: Risk stratification in patients with chronic infarction is based on left ventricular (LV) morphological and functional parameters, such as left ventricular ejection fraction (LVEF), left ventricular volumes, extent of delayed gadolinium enhancement, among others risk scores. However, native T1 and extracellular volume (ECV), tools for the assessment of interstitial fibrosis, when measured in the myocardium distant from the infarcted territory, present an association of poor prognosis in patients with chronic infarction. Objective: To evaluate the correlation between native T1 and ECV with LV morphological and functional parameters in patients with chronic infarction. Methods: Observational, cross-sectional, analytical study. Patients who underwent cardiac magnetic resonance imaging using a Philips Ingenia 1.5T magnet in 2021 in Sergipe (Brazil) were included. Patients with delayed transmural and/or subendocardial enhancement compatible with myocardial infarction were selected and those with acute infarction or other diagnoses were excluded. T1 measurements were performed in myocardium without delayed enhancement using specific software, and ECV was calculated using native and post-contrast T1 of the myocardium and cavity, in addition to hematocrit. The analysis included the execution of the Shapiro-Wilk tests, Spearman correlation and Mann Whitney U test. The significance level was set at 5%. Results: Of the 75 patients with delayed subendocardial or transmural enhancement, 56 (74.7%) had chronic infarction. The mean age was 68.14 ± 10.24 years. The mean value of native T1 was 1044 ± 43 ms and ECV was 29.8% ± 4.7. There was a negative correlation between T1 and LVEF (ρ-0.374; p = 0.027). The group with increased T1, considering local reference value (>1030 ms), had a higher LV systolic diameter (Md 47.2 vs. 40.2; p = 0.049; TDE-LC 69.4%) and lower LVEF (Md 43 vs. 58; p = 0.038; TDE-LC 70.4%). The group with increased ECV (ECV > 28%) had higher LV mass (Md 96 vs. 120; p = 0.019; TDE-LC 78.4%). Conclusion: The value of native T1 showed an inverse correlation with LVEF. The group with increased T1 had higher LV systolic diameters and lower LVEF. The group with increased ECV had higher LV mass. These findings suggest that the increase in T1 and ECV may be associated with LV remodeling, resulting in diffuse interstitial fibrosis, which may impact the long-term prognosis. 108914 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIELA DA SILVA NASCIMENTO1, Fernanda Oliveira de Carvalho Carlos1, Carlos Filipe dos Santos Pimenta1, Victor Margallo1, João Gabriell Bezerra da Silva1, Bianca Zattar de Mello Barreto1, Carolina de Carvalho Fortes1, Marcus Vinicius Serejo Borges Vale da Silva1, Sofia Luz Coutinho Botelho Lobo1, Arthur Fernandes Cortez1, Bernardo Chedier1, Elizabeth Silaid Muxfeldt1 (1) UFRJ, Universidade Federal do Rio de Janeiro – Faculdade de Medicina, Hospital Universitário Clementino Fraga Filho – ProHart Inflammatory: Biomarkers and Obesity in Resistant Hypertension Fundamental: In the last few years, obesity has acquired pandemic features, being an important public health’s problem and being strongly related to cardiovascular diseases, beyond of being responsible for the difficulty of blood pressure and metabolic control of patients with resistant hypertension (RHT) apparently due to an inflammatory process that underlines this unfavorable context. Objective: To evaluate the relationship between inflammatory biomarkers and obesity in a large cohort of patients with RHT. Methods: This cross-sectional study evaluated 423 subjects with RHT (30.5% male; 63.9 ± 10.8 years old), 215 (50,8%) of whom are obese. In all subjects the inflammatory biomarkers, TNF-α, MCP-1, E-Selectin and PAI-1, were dosed. Socio-demographic characteristics, anthropometric measurements and cardiovascular risk factors were recorded. Variance analysis compared the serum levels of 4 biomarkers inflammatory and the bivariate analysis compared resistant hypertensives with and without obesity. Results: Obese subjects are younger, with a higher prevalence of peripheral obstructive arterial disease. No difference was found concerning to the blood pressure, neither to subclinical lesions. The values of PAI-1 (123 [107–164] vs 113 [89–138] and E-Selectin (53.2 [34.2–68.6] vs 44.6 [20.8–62.0] were significantly higher in patients with obesity. The other biomarkers evaluated did not evidence association with obesity. Conclusion: Among the inflammatory biomarkers evaluated, which was most strongly correlated with obesity was PAI-1 and E-Selectin. According to literature PAI-1 is tightly linked to metabolic disorders, such as insulin resistance and central obesity, while E-Selectin has an important role on atherosclerosis’s development. 108916 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION CAMILA BELLO NEMER1, Bianca Zattar de Mello Barreto1, Carolina de Carvalho Fortes1, Marcus Vinicius Serejo Borges Vale da Silva1, Sofia Luz Coutinho Botelho Lobo1, Aline de Hollanda Cavalcanti1, João Carlos Moreno de Azevedo1, Carlos Filipe dos Santos Pimenta1, Taissa Lorena dos Santos1, Bernardo Chedier1, Elizabeth Silaid Muxfeldt1, Christian Nejm Roderjan1 (1) Universidade Federal do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho – ProHArt Objective: To evaluate the impact of a long-term use of CPAP on arterial stiffness (AS) measured by pulse wave velocity (PWV) in patients with resistant hypertension (RHT) and moderate-severe obstructive sleep apnea (OSA). Design and Methods: An observational prospective study was performed in 121 patients (38% male, mean age 60.7 ± 7.8 years) with RHT and moderate/severe OSA (AHI > 15/hour) divided in 2 groups using CPAP (CPAP group – n = 62) and not using CPAP (control group – n = 59) and followed for at least 12 months. Pulse wave velocityand ABPM were measured before and after follow-up. Primary outcomes were changes in aortic stiffness. Intergroup comparisons of differences in PWV were assessed by a general linear model with allocation group (CPAP or control) as a fixed factor and adjusted for their respective baseline PWV values in addition to gender, age, and 24-hour SBP values. Per-protocol analysis was performed excluding patients with poor CPAP adherence. Results: Patients were followed-up for a median of 68 [49–81] months. They used a median of 5 [3–8] anti-hypertensive drugs and had mean PWV of 8.41 ± 1.52 m/s. CPAP and control groups were similar in their baseline demographic, anthropometric, office and ambulatory BP, PWV, and laboratory characteristics. Control group had a mean increase in PWV of 0.88 m/s (95% CI: 0.52–1.25 m/s, p < 0.001), whereas CPAP group had an average increase in PWV of 1.20 m/s (95% CI: 0.84–1.55 m/s; p < 0.001). After adjustment for initial PWV, gender, age and 24-systolic BP, the mean difference between CPAP and control groups was +0.32 (95%CI: 0.17–0.83; p = 0.20). In a subgroup analysis with patients with higher PWV (>10 m/s) the mean difference adjusted was –0.20 (95%CI: –2.17–1.75, p = 0.83). Conclusions: Long-term treatment with CPAP did not reduce aortic stiffness in RHT with moderate/severe OSA, but it may prevent its worsening in relation to control group in those with baseline increased arterial stiffness. 108917 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERIOPERATIVE EVALUATION RAFAEL FORTES LOCATELI1, Vitória Carolina Kohlrausch1, Eduardo Porto Santos1, Isabella Klafke Brixner1, Anna Carolina Flores Mariath2, Gabrielle Lenz de Abreu2, Aníbal Pereira Abelin1, Diego Chemello1, Mateus Diniz Marques1 (1) Universidade Federal de Santa Maria – UFSM; (2) Hospital Universitário de Santa Maria – HUSM Introduction: Arrhythmias are one of the most frequent cardiac complications in the cardiac surgeries postoperative. Atrial fibrillation (AF) is the most common arrhythmia with 20 to 50% incidence. Most cases occur in the first five postoperative days, with a maximum incidence on the 2nd day. The consequences of AF are increased hospital stay, risk of recurrence and increased mortality. Objective: This study aims to compare mortality and hospital stay among patients who presented AF and those who did not have AF in the postoperative period of cardiac surgery at the University Hospital of Santa Maria from January 2011 to April 2021. Methods: This is a longitudinal, descriptive and retrospective study reviewing physical and electronic medical records of patients undergoing cardiac surgery at public tertiary hospital at Santa Maria, Brazil. Exclusion criteria are age younger than 18 years or a procedure involving only the pericardium or pacemaker implantation. The variables analyzed for this study were the occurrence of AF, period of occurrence and death during hospitalization. Results: From a database of 856 patients, 30,96% patients were diagnosed with AF in the postoperative period (n = 265). The mean number of days for the occurrence was 3,35 and the day with the highest incidence was the 2nd. The total number of deaths in the study was 82 (9,57%). Among those who had AF, the mortality was 7,54% (n = 20) and the odds ratio was 0,6965 (CI 95% 0,41–1,17; p = 0,08). The mean hospitalization days for those who had AF was 14 ± 13 days, and 10 ± 9 days for those without AF. Those without AF had lower hospitalization days (–3.57 days, CI 95% – 5.13 – –2; p < 0.0001). Conclusions: This analysis highlights the reality of a tertiary hospital in southern Brazil. AF occurrence was not associated with overall mortality during hospitalization despite a longer hospital stay. Data continues to be collected and future analyzes will show more accurate results. 108919 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERIOPERATIVE EVALUATION VITÓRIA CAROLINA KOHLRAUSCH1, Vitória Carolina Kohlrausch1, Rafael Fortes Locateli1, Eduardo Porto Santos1, Anna Carolina Flores Mariath2, Gabrielle Lenz de Abreu2, Anibal Pereira Abelin1, Diego Chemello1, Mateus Diniz Marques1 (1) Universidade Federal de Santa Maria – UFSM; (2) Hospital Universitário de Santa Maria – HUSM Introduction: Stroke in the cardiac surgery postoperative is an important neurologic complication. Postoperative stroke is defined as occurring up to 30 days after the procedure, with 56% of them occurring within the first 24 hours. The incidence usually varies between 1.3 to 5% of the procedures and the mortality is from 13 to 41%. Objective: The purpose of this study is to describe the incidence and mortality of stroke in the postoperative period of cardiac surgery at the University Hospital of Santa Maria from January 2011 to April 2021. Methods: The physical and electronic medical records of patients undergoing cardiac procedures at the HUSM were reviewed, between January 2011 and April 2021. This is a retrospective and longitudinal study. and pericardium or only pacemaker implantation procedures were not included. A total of 857 medical records were reviewed and the variables of stroke presentation in the postoperative period, time in days of stroke after surgery and death were evaluated. Data were analyzed using EpiInfo software (version 7.2.5.0.) and comparisons were made based on articles indexed in the PubMed platform. Results: Of the 857 patients included in the study, 3.73% had a stroke in the postoperative period (n = 32; SD = 2.66%–5.22%) – with 46.88% occurred in the first 24 hours after surgery (n = 15; SD = 29.09%–65.26%). The overall mortality was 9.57% (n = 82; SD = 7.78%–11.72%), and higher in the group with perioperative stroke (25%;n = 8; SD = 11.46%–43.40%) than in the group that did not develop this complication (8.97%; n = 74; SD = 7.21–11.11%), with OR = 3.3829, x2 = 9.13, p < 0,005. Conclusions: Despite the low income and economical barriers faced by public hospitals in Brazil we showed that a tertiary public hospital in southern Brazil has similar results than general hospitals around the world regarding neurologic complications in the cardiac surgery postoperative. The database continues to be filled and new studies are being written, analyzing variables such as sex, age and previous diseases. 108926 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION KARINE DA SILVA GUIMARAES1, Gabriela Gama Zagni Jardim1, Ana Rachel Bucar Cervasio1, Clara Avelar Mendes de Vasconcellos1, Tomás de Souza Mello1, Carlos Augusto Parente Macedo Moura1, Mariana Stutz Klen1, Heitor de Oliveira Moraes Rêgo Bandeira1, Diego Alves Calvão1, Ramon Narde Simão1, Ana Cristina Tenório da Costa Fernandes1, Elizabeth Silaid Muxfeldt1 (1) IDOMED – Universidade Estácio de Sá, Medicine School, Campus Vista Carioca, Rio de Janeiro – RJ Background: In primary care it is necessary to identify early cardiovascular risk factors and target organ damage with low complexity procedures. The most available with low cost are Ankle-Brachial Index (ABI), left ventricular hypertrophy by voltage index in ECG and aortic stiffness by pulse pressure (PP) calculation. Objective: To evaluate cardiovascular risk factors and subclinical target organ damage in a young and apparently healthy population assisted in a Primary Healthcare unit. Design and Methods: A cross-sectional population study for cardiovascular risk assessment in adults aged 20–50 years old provided by a Primary Healthcare Unit. A total of 632 individuals were evaluated (40% male; mean age 36 ± 9 years). Sociodemographic, anthropometric data, and traditional cardiovascular risk factors were recorded. All underwent office Blood Pressure (BP) and pulse pressure (PP) (systolic BP minus diastolic BP) was calculated. Ankle-brachial index (ABI) was also calculated after BP measurements in the 4 limbs. The median of the ABI was 1.14 [1.08–1.22], which is the cutoff point used to define early changes. All participants were submitted to ECG to calculate Sokolow-Lyon Index (SLI) and Cornell Voltage Index (CVI) for left ventricular hypertrophy diagnosis. We considered the median of the SLI (20 mm) and the CVI (11 mm) as the cutoff point for early alterations. Results: The prevalence of hypertension was 16%. The median [IQR] office PP was 46 [39–52] mmHg. Elevated Office PP (> 60 mmHg) were identified in 64 participants. High office PP was more frequent in men, obese, with increased neck circumference and lower ABI (1.07 vs 1.16, p < 0.001). It was also associated with higher voltage index: SLI 21.9 vs 19.8, p = 0.04 and CVI 13.4 vs 7.1, p = 0.03. Individuals with decreased ABI are more obese with smaller neck circumference (10 vs 5%, p = 0.03). They also had higher systolic BP (125 vs 119 mmHg, p < 0.001) and PP (49 vs 43 mmHg, p < 0.001). A total of 362 ECGs were performed. Increased SLI was more frequent in men, younger and overweight, in addition to a higher prevalence of hypertension and higher PP. Those with increased CVI are more often male, obese with higher BP levels. Conclusion: In this young population, early changes in subclinical target organ damage already identify a higher cardiovascular risk profile, indicating the importance of implementing primary prevention measures to reduce this risk. 109374 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION BRENDA BARETA SIRONI1, Wendell Arthur Lopes1, Caroline Ferraz Simões1, João Carlos Locatelli1, Higor Barbosa Reck1, Victor Hugo de Souza Mendes1, Gustavo Henrique de Oliveira1, Rogério Toshiro Passos Okawa1 (1) Universidade Estadual de Maringá Introduction: Obesity appears to affect the right ventricle, considered the forgotten chamber in cardiac evaluation. The TAPSE (tricuspid annular plane systolic excursion) have been used for this evaluation, and more recently, new methods of evaluating the right ventricle function have been developed, in which the right free wall Longitudinal Strain, and the FAC (fractional area change), are highlighted. Studies have shown that aerobic training seems to have some effects on the right ventricular structure and function, however little is known about the impact of different types of training on it. Objective: To investigate the effects of 8 weeks of HIIT (high intensity interval training) and MICT (moderate intensity continuous training) on right ventricle by three different methods (TAPSE, FAC and right strain) in obese women. Methods: The sample consisted of 44 women with obesity grade I or II (age: 28.4 ± 4.8 years; BMI: 35.6 ± 3.1 kg/m2), who were randomly allocated to the HIIT group (n = 22) and MICT (n = 22). The training protocols were adjusted so that both promoted similar energy expenditure. Echocardiographic measurements of left and right ventricular function and morphology were evaluated using the Vivid T8 GE ultrasound system. The Vo2 max, was measured during a cardiopulmonary stress testing, with the Cortex device. Body composition was measured using a tetrapolar bioimpedance device. Data were analyzed using the Statistical Package for the Social Sciences (SPSS, IBM®). Differences between the HIIT and MICT groups and moments before and after 8 weeks of training were explored by the ANOVA test for repeated measures. Results: Twenty-five obese women completed 8 weeks of HIIT (n = 11) or MICT (n = 14). Both HIIT and MICT training significantly increased the TAPSE (p = < 0.006; <0.026), FAC (p = 0.002; <0.001) and right free wall GLS (p = <0.003; <0.001). Only HIIT reduced body mass (p = 0.002), body mass index (p = 0.002), body fat percentage (BF% p = <0.001), and significantly increased VO2peak (p = 0.024.). Conclusion: The results obtained with 8 weeks of training on the right ventricle function showed no difference between the two exercise training interventions, both with a positive impact in the right ventricle function parameters. Overall, aerobic training can be considered as a potential intervention for substantial improvements in the right ventricle function of young women with grade I and II obesity, however, HIIT may have additional benefits. 108978 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION CAROLINA DE CARVALHO FORTES1, Carolina de Carvalho Fortes1, Taissa Lorena Dos Santos1, Camila Bello Nemer1, Sofia Luz Coutinho Botelho Lobo1, Gabriela da Silva Nascimento1, Hugo Farah Affonso Alves1, Lucca Hiroshi de Sá Kimura1, Aline de Hollanda Cavalcanti1, João Carlos Moreno de Azevedo1, Bernardo Chedier1, Elizabeth Silaid Muxfeldt1 (1) Universidade Federal Do Rio de Janeiro – Hospital Universitário Clementino Fraga Filho – ProHArt Objective: To assess blood pressure response to long-term continuous positive airway pressure (CPAP) treatment among patients with refractory hypertension (RfHTN) with moderate and severe obstructive sleep apnea (OSA). Design and Methods: An observational prospective study was performed in 26 patients (35.7% male, average age 59.3 ± 7.9 years) with diagnostic of RfHTN and moderate and severe OSA who were divided in 2 groups to use CPAP (CPAP group, n = 13) or not (control group, n = 15). All of them were submitted to clinical follow-up and their anti-hypertensive medication was adjusted according to the assistant physician. 24-hour ABPM was performed both at the beginning and at the end of the study. Primary outcomes were changes in office and ambulatory blood pressure (BP) and BP control. The comparison between groups of BP changes were calculated by a general linear model with group allocation as a fixed factor and adjusted by its respective BP basal values in addition to gender and age. Results: The median follow-up time was 68 months (IQR: 49–81 months). CPAP and control groups were similar in their baseline demographic, anthropometric, laboratory characteristics, and office and ambulatory BP. They had mean systolic and diastolic 24-hour BP of 142 (15) and 83 (14), respectively. At the end of follow-up, refractory patients using CPAP had a reduction of 8.5 and 5.0 mmHg of office systolic and diastolic BP with an intergroup difference (CPAP and control) of 13.5 (–15.2–42.3) mmHg and 10.1 (–7.5–5.4) mmHg, respectively. The most important difference between groups was noticed at nighttime with an intergroup difference of 10.0 (–8.6–28.7) mmHg of systolic BP and 6.7 (–3.9–17.3) mmHg of nighttime diastolic BP. Out of 28 initial refractory patients, 12 are not refractory anymore, being 3 (20%) in control group and 9 (69.2%) in CPAP group with a significant difference between the groups (p = 0.02) Conclusion: Long-term use of CPAP among refractory hypertensive patients with moderate/severe OSA seems to be effective to reduce and control blood pressure measured both in the office BP and 24-hour ABPM, especially lowering nighttime blood pressure. 108960 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION GABRIELA GAMA ZAGNI JARDIM1, Ana Rachel Bucar Cervasio1, Clara Avelar Mendes de Vasconcellos1, Tomás de Souza Mello1, Karine da Silva Guimarães1, Giovanna Francesca Ferreira Maselli1, Michelle Felipe Falcão1, Ivan da Costa Velho Junior1, Raphaella Ferrão1, Inah Maria Drummond Pecly1, Rafael Bica1, Elizabeth Silaid Muxfeldt1 (1) IDOMED – Universidade Estácio de Sá, Medicine School, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil. Objective: To evaluate the relationship between the main CV risk factors and socioeconomic indicators in a population of adults registered in a Family Health Care (FHC) unit in the center of Rio de Janeiro. Design and Methods: Cross-sectional population study that included adults aged between 20 and 50 years living in the area covered by the FHC in Rio de Janeiro. Demographic data (gender and age), socioeconomic data (education level, profession, employment), CV risk factors (smoking, sedentary lifestyle, obesity, hypertension, diabetes, dyslipidemia) were recorded. The metabolic profile is evaluated through laboratory tests. Those who studied up to high school were considered poorly educated. Results: 604 individuals were enrolled [39% male, mean age: 38.8 ± 8,9 years] The median of schooling was 12 years. 288 individuals had high schooling, 44.5% were male. A total of 130 individuals did not study or work. Women with low education had a higher risk of smoking, obesity and hypertension with no difference regarding labor or study activities. Otherwise, men with low education had higher risk of sedentary lifestyle and hypertension. Among men, not working or studying increased the risk of smoking and hypertension. Conclusion: We found an inverse association between socioeconomic conditions and the prevalence of CV risk factors. Women are more affected by low schooling, while men are more affected by their working occupation. The study suggests that socioeconomic factors influence the CV risk, affecting men and women differently, pointing to the need for public policies to reverse this situation. 109353 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GLÓRIA DE MORAES MARCHIORI1, Glória de Moraes Marchiori1, Bráulio Henrique Magnani Branco1, Mauricio Medeiros Lemos1, Daiane Soares de Almeida Ciquinato1, Elen Cristina Bressiano1, Luciana Lozza de Moraes Marchiori1 (1) Universidade UniCesumar UniCesumar Introduction: Dizziness is a common nonspecific sensation of disorientation or impairment in spatial perception and stability. The pathophysiology of post COVID-19 rotatory dizziness is probably similar to that of other viral infections, with some of its manifestations such as hypercoagulability and microthrombus formation, causing significant circulatory disorders possibly affecting its pathogenesis. Cardiorespiratory fitness is one of the significant variables to monitor health, and the gold standard used has been maximal or peak consumption of oxygen (VO2max or VO2peak) through a direct analysis of gas exchange. Thus, VO2max demonstrates the maximum capacity of an individual to absorb, transport, and consume oxygen fitness may be used to identify those at the most significant risk for severe COVID-19 illness. Association between dizziness complaints and cardiorespiratory fitness has not been investigated in individuals with severe forms of COVID-19. Objective: To verify whether there is a correlation between dizziness complaints and cardiorespiratory fitness among people with severe forms of COVID-19. Methods: This study presents a cross-sectional design part of a broader research named the “Post COVID-19 project.” The human research ethics committee approved the project of the institution, with a sample of people post COVID-19 who responded to the Visual-Analog Scale (VAS) for dizziness. To evaluate cardiorespiratory fitness, it used the clinical assessment and Bruce test to measure oxygen consumption directly (via gas analyzer). Results: Of the 133 participants, with a mean age of 49.8 ± 12.2 years old. The prevalence of self-reported dizziness was 51.1% (n = 68); of these, 9.8% (n = 13) were related to dizziness prior to the diagnosis of COVID-19 and 41.4% (n = 55) were related to dizziness during or after COVID-19. The subgroup analysis for age groups, a difference was found in the middle-age (45–64 years) group for the absolute VO2 peak (p = 0.027). Conclusion: There was a statistically significant difference between dizziness complaints and the VO2 peak among the middle-aged population, with the dizziness group having a lower absolute VO2 peak than the non-dizziness group. 109001 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION SARA CRISTINE MARQUES DOS SANTOS1, Luan Tardem Veloso Teixeira1, Ivan Lucas Picone Borges dos Anjos1, Thaís Lemos de Souza Macedo1, João Pedro de Resende Côrtes1, Paula Pitta de Resende Côrtes1, João Carlos de Souza Côrtes Júnior1, Eduardo Tavares de Lima Trajano1, Carlos Eduardo Cardoso1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Pulse wave velocity is currently (PWV) considered a gold standard parameter in the assessment of arterial stiffness, and may be associated with cardiovascular risk in several groups. Stiffness is characterized by the decrease in arterial distensibility, being present in aging, diabetes, atherosclerosis and chronic renal disease1. The objective of the present study was to perform the analysis of central and peripheral hemodynamic values in men and women aged 30 years old or less. Methodology: Observational and cross-sectional study, carried out from May to July 2021 in students aged up to 30 years (in accordance with the first group for pulse wave velocity of the current brazilian hypertension guideline). 49 participants were selected, 32 female (F) and 17 male (M). It was used an anonymous questionnaire and the values provided by the Arteris device through the oscillometric method: PWV; AIX@75; heart rate (HR); peripheral systolic blood pressure (SBP) and central (CSBP); and peripheral diastolic (DBP) and central (CDBP); centre pulse pressure (CPP), cardiac output (CO); vascular age (VA); peripheral vascular resistance (SVR) and cardiac index (CI). For the general group (G) mean, maximum and minimum values were calculated; while for M and F, only the mean was calculated. Results: Mean age G was 23 years (29 ± 20), M 22 and F 23; PWV G of 4.65 m/s (5.4 ± 3.1), M 4.9 and F 4.5; AIX@75 G of 22.6% (41.7 ± 5.3), M 18.4% and F 25%; SBP G of 111 mmHg (137 ± 88), M 120 and F 107; DBP G 74.5 mmHg (96 ± 56), M 77 and F 73; CSPB G 98 mmHg (118 ± 80), M 103 and F 95; CDPB G 75.5 mmHg (98 ± 54), M 79 and F 73.5; HR G 88 bpm (128 ± 60), M 83 and F 90; CPP G 22 mmHg (36 ± 13), M 24.5 and F 20.5; VA G of 22.6 years (32 ± 18), M 25 and F 21; CO G 3.9 l/min (4.6 ± 2.9), M 4.1 and F 3.81; SVR G 1.30 mmHg/ml (1.58 ± 0.97), M 1.29 and F 1.30; CI G 3.9 l/min/m2 (4.6 ± 2.9), M 4.1 and F 3.8. Conclusion: Men presented higher values of PWV, SBP, DBP, CSPB, CDPB, CPP, VA, CO and CI both in relation to the female group and to the general group. Meanwhile, women obtained higher values in SVR, HR and AIX@75. It is worth noting that the values of CSPB and PWV found in the study participants are within the normal range for the population without cardiovascular risk factor (established in the 2020 hypertension guideline of the Brazilian Society of Cardiology) while they are borderline in the European index of CSPB and adequate for PWV. 109005 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION SARA CRISTINE MARQUES DOS SANTOS1, Luan Tardem Veloso Teixeira1, João Pedro de Resende Côrtes1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, Paula Pitta de Resende Côrtes1, João Carlos de Souza Côrtes Júnior1, Eduardo Tavares de Lima Trajano1, Carlos Eduardo Cardoso1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Family history of hypertension (FHH) is a strong risk factor for the development of high blood pressure (HBP), where individuals of normotensive parents would have a lower occurrence of the stiffening process of the arteries1. It is believed that stiffness also has a genetic influence, as well as HBP and age2. The mechanism of pulse wave velocity (PWV) and HPB is not completely established; there is an increase in PWV with increased blood pressure, but it is still not possible to define which is the cause or consequence3. The objective of the present study was to analyze two groups: with positive family history of HBP (PFH) and the group with negative family history of HBP (NFH) through the answers of questionnaires about self-knowledge, life habits, the values provided by the Arteris device of central hemodynamics and arterial stiffness, such as PWV, AIX@75, central systolic pressure (CSP), central diastolic pressure (CDP), central pulse pressure (CPP) and vascular age (VA). Methodology: Observational, cross-sectional study conducted from May to July 2021 in medical students. Done through an anonymous questionnaire and analysis of the pulse wave by the oscillometric method in the Arteris device. The mean was calculated using Excel. Evaluation of sample normality (Shapiro-Wilk), multiple logistic regression, with confidence level of 95% and calculated odds ratio by GraphPad Prism Software version 9.2. Results: Total of 59 participants, mean age of 25.29 (20 ± 42), where 45 belonged to the PFH group and 14 to the NFH group. When analysing the values obtained by the device, the PFH group obtained averages of: PWV 4.82 m/s (6.6 ± 3.1); AIX@75 22.59% (41.67 ± 5.33); CSP 97.47 mmHg (124 ± 11); CDP 77.13 mmHg (106 ± 58); CPP 36.27 mmHg (61 ± 21) and VA 24.84 (45 ± 18). While in the NFH group, means of: PWV 4.76 m/s (5.4 ± 4.2); AIX@75 18.53% (37.67 ± 8.67); CSP 99.86 mmHg (118 ± 81); CDP 75.14 mmHg (98 ± 54); CPP 37.85 mmHg (52 ± 26) and VA 23.36 (32 ± 18). The lower the PWV, the lower the chance of having PFH (OR: 0.4) and the lower the CPP (OR = 0.7), CDP (OR = 0.9) and PWV (OR = 0.3); the lower the probability of being hypertensive. Conclusion: In the PFH group, the values of PWV, AIX@75, CDP and VA were higher than those found in those NFH. Also, it was possible to show statistically that the PFH can influence the increase in the value of PWV, the reduction of the levels of PWV and other hemodynamic parameters are important for prevention of HBP. 109013 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION YURI BARBOSA ARAÚJO1, Glebson Santos Sobral1, Rafael Alexandre Meneguz Moreno1 (1) Universidade Federal de Sergipe Background: For years, the search for clinically useful assessment methods with good accuracy, low cost, non-invasiveness and easy management has been stimulated, aiming to improve the prevention and treatment of atherosclerotic diseases. Objective: To verify the diagnostic potential of the following indices in patients with clinical Atherosclerotic Cardiovascular Disease (ASCVD): Castelli I and II indices, Atherogenic Coefficient, Atherogenic Index of Plasma (AIP) and the variation of the perfusion index between 90 and 120 seconds after reactive hyperemia in the evaluated limb (ΔPI90–120). Methods: A case-control study, of whom 161 patients were included so far, of which 44 (27.3%) were part of the ASCVD group, selected by means of a cinecoronariography, exercise stress test, coronary CTA, cranial CT, carotid or lower limb artery Doppler. To estimate the potential of these atherogenic indices, sensitivity, specificity, positive (PV+) and negative (PV–) predictive value, positive (LR+) and negative (LR-) likelihood ratios were estimated through receiver operating characteristic (ROC) curves. Additionally, multivariate logistic regression was performed to estimate the association of these indices with the outcome. Values of p < 0.05 were considered statistically significant. Results: The number of patients with a history of smoking, hypertension, diabetes mellitus and dyslipidemia was higher in the ASCVD group, as well as mean age and some laboratory parameters: HDL-c, triglycerides, HbA1c and creatinine. The ROC analysis showed better performance with AIP and ΔPI90–120. For the former, a cut-off of (> 0.04) achieved an LR+ and LR- of 4.84 and 0.15, respectively (sensitivity 88.0%; specificity 81.8%; PV+ 64.7%; PV– 94.7%). For the latter, a cut-off of (≤ 56.6) achieved an LR+ and LR- of 5.76 and 0.30, respectively (sensitivity 73.6%; specificity 87.2%; PV+ 68.4%; PV– 89.8%). In multivariate analysis adjusted for age and comorbidities, both AIP and ΔPI90–120 were independently associated with ASCVD: respectively, OR 23.6 (95%CI 4.3–130.9; p < 0.001) and OR 17.8 (95%CI 3.3–97.0; p < 0.001). Conclusions: AIP and ΔPI90–120 were independent predictors of ASCVD. In addition, both have better diagnostic performance for ASCVD compared to the other evaluated indices, making them attractive for future trials and possible contribution to the prevention and treatment of atherosclerotic diseases. 109020 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SARA CRISTINE MARQUES DOS SANTOS1, Thaís Lemos de Souza Macedo1, João Pedro de Resende Côrtes1, Ivan Lucas Picone Borges dos Anjos1, Eduardo Tavares de Lima Trajano1, Carlos Eduardo Cardoso1, João Carlos de Souza Côrtes Júnior1, Eucir Rabello1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: With high mortality rates, the out-of-hospital cardiac arrest (OHCA) is a subject of discussion in the medical field and currently, it is emphasized the inclusion of CPR training for laymen and health professionals, in order to reduce the number of deaths and sequelae in victims. The objective of the present study was to analyze the medical students knowledge regarding the care of victims of CPR and their ability to perform this care. Methodology: This is an observational and cross-sectional study, with quantitative data collection obtained through an anonymous questionnaire, containing questions related to the recognition of a CRA and the CPR maneuver procedures by medical students during the years 2018 to 2021. Results: Of the total 468 students interviewed, 149(31.84%) answered correctly about maximum time limit from which a cardiac arrest can be considered irreversible. Regarding the use of the AED, 222(47.44%) said they knew how to use it, with 158(33.76%) reporting having learned to use it in college. Still on the AED, 265(56.62%) do not know the difference between shockable and non-shockable rhythms, but 307(65.60%) understand that this rhythm will influence in the conduct of care. Regarding the use of AED during CPR, 169(36.11%) answered correctly stating that it should be used as soon as possible. In pediatric CPR, 51(10.90%) could correctly answer regarding the depth of compressions. In the victim’s pulse reassessment, 203(43.38%) did not know the correct periodicity. At the end of the questionnaire, 206(44.02%) considered themselves able to perform a CPR. Conclusion: It was possible to observe from the present study that more than half of the students do not know the ideal time to help the victim of CRA, the time of pulse reassessment during the assistance and about 90% of them, would not do the pediatric compression effectively. Regarding the AED, about 53% do not know, also not knowing the difference of the rhythm found and the need for its immediate use in a CRA. Less than half of the interviewees consider themselves capable of performing CPR. The need to implement courses in hands-on modality is emphasized, where students can realistically train the care and thus, fix it in their professional life, thus bringing benefits in post-CPR survival rates. 109023 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SARA CRISTINE MARQUES DOS SANTOS1, Thaís Lemos de Souza Macedo1, Ivan Lucas Picone Borges dos Anjos1, João Pedro de Resende Côrtes1, Eduardo Tavares de Lima Trajano1, Carlos Eduardo Cardoso1, Eucir Rabello1, Esmeralci Ferreira2, João Carlos de Souza Côrtes Júnior1, Paula Pitta de Resende Côrtes1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras; (2) Universidade do Estado do Rio de Janeiro Introduction: Cardiac arrest (CA) is defined as the sudden interruption of necessary mechanical ventricular activity and breathing. Clinically, pulse, breath, and consciousness are absent. Biologically, there is still a viable brain and other biological functions. (1) In Brazil, approximately 200 thousand individuals are victims per year of CA, 50% of them occur in extra-hospital environments such as homes, shopping centers, stadiums, airports, sports centers, among other spaces (2). In the United States of America, approximately 350,000 people die annually from CA, and, currently, the prevalence of training in cardiopulmonary resuscitation is low (3). Chest compressions associated with the use of an automatic external defibrillator (AED) are essential for survival and reducing post-CA sequelae. The present study aimed to analyze the knowledge of medical students about the use of DEA. Methods: Observational and cross-sectional, quantitative, and qualitative study between 2018 and 2019, through an anonymous survey, distributed after approval by the Research Ethics Council, addressing questions related to the knowledge of medical students about handling an AED during a CA. Results: Of 291 students interviewed, 162 (55.67%) knew how to handle an AED. Of these, 115 (39.52%) learned at college, 45 on courses (15.46%), 3 (1.03%) over the internet, 1 at work (0.34%), and 127 (43.65) did not inform or not applied. A total of 115 (39.52%) knew the difference between shockable and non-shockable rhythms. However, 190 (65.29%) reported knowing that the cardiac rhythm influences the conduct in the CA, 88 (30.24%) did not know and 13 (4.46%) did not inform. In assisting the victim of CA, 92 (31.62%) answered correctly that the AED should be used as soon as possible, 94 (32.3%) said that the use should be made later, 56 (19.24%). When asked about the locations that could identify the presence of the DEA, 92 (31.62%) mentioned shopping centers, 92 (31.62%) in universities and schools, 45 (15.46%) in gyms, 3 (1, 03%) in cinemas and 147 (50.52%) did not know. Conclusion: It was observed that slightly more than half of the students consider themselves capable of using an AED in an emergency and less than half know the difference between heart rhythms. However, they know that it will influence the conduct of the CPR. Less than a third knew the correct moment to use the AED. It shows that is necessary to implement practical workshops on CA management through universities, targ. 109024 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ISRAEL FIGUEIRA LEMOS1, Israel Figueira Lemos1, Luiz Carlos Figueiredo Filho1, Maria Eduarda Dantas da Veiga1, Mariana Lassance Maya Palheta1, Juliana de Sousa Tavares1, Ivan Cuoco Sampaio1, Rafael Augusto Silva Cabeça1, João Lucas Silva Sales1, Luma Maria Favacho Bordalo1, Ingrid Jade Muniz Wanderley1, Paula Larissa Baía Lima1 (1) Universidade do Estado do Pará Introduction: Extracorporeal circulation, especially associated with children undergoing cardiac surgeries, is related to situations of decline in serum levels of thyroid hormones. Studies show that the rates of improvement in prognosis in surgical outcome situation in these patients can be optimized by supplementation of these hormones, due to their importance in vital functions, becoming essential in the improvement in operative outcome. Objectives: To evaluate the effectiveness of thyroid hormonal supplementation in pediatric cardiac surgeries for improvement of the operative prognosis. Methods: Systematic review using the PICO strategy and guiding question: “Does the supplementation of thyroidal hormones influence the recovery from cardiac surgery in pediatric patients?”. Inclusion criteria: randomized clinical trials and articles that focus on the effect of thyroidal hormones supplementation in pediatric patients undergoing cardiac surgeries. Exclusion criteria: articles with methodological approach focused on surgical techniques rather than the supplementation of these hormones. The inclusion of articles followed the filtering protocol of the PRISMA diagram. The articles were selected from PubMed and VHL databases with the temporal delineation from 2000 to 2022. To reduce the risk of bias, Rob 2 software guidelines were used to analyze the articles. Furthermore, the descriptors used were: “thyroid hormones”, “heart surgeries” and “children”. Results: At first, 8 articles were found in the databases by means of the descriptors, of these, 5 fit the selective criteria. In the postoperative period, with the administration of T4, there was no improvement in hemodynamic performance or less use of mechanical ventilation in patients. Moreover, 4 studies reported in common the role of improvement in myocardial function by T3 administration, highlighting: improvement in cardiac output, reduction of systemic vascular resistance and ischemia-reperfusion lesions. In pharmacokinetic terms, doses between 0.4–0.8 μg/kg are safe and effective. Conclusion: Efficacy of thyroid hormone supplementation was evidenced in the prognostic evaluation of surgical pediatric conditions, which allowed for an improvement of the condition. A limitation of this study was the scarcity of studies, that still absent in the literature, for the comparison of the longer surgical evaluation time with the supplementation of thyroidal hormones. 109462 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY PEDRO AUGUSTO MOREIRA ZAPPA1, Sérgio Lima de Almeida1, Fernando Graça Aranha1, Luis Enrique Portugal1, Marli Annes1, Giani Osni Alves1, Giovanna Grunewald Vietta2, Portiuncola Gorini1, Adriana Ferraz Mrtins1, José Fernando Arruda1, Roberta Luiza Salum1 (1) Hospital SOS Cárdio; (2) Universidade do Sul de Santa Catarina Background: The high prevalence of patients with aortic stenosis has led to the development of new surgical treatment options, such as rapid implant valves. These can reduce transoperative times, which are crucial for reducing complications for patients benefiting from this type of prosthesis. Objective: To evaluate the difference between the clinical, surgical and echocardiographic profile of patients undergoing aortic valve replacement procedure using rapid implant prosthesis and conventional bioprosthesis. Method: Case-control study, carried out at Hospital SOS-Cárdio in Florianópolis; population of 60 cases and 119 controls, the controls were selected at random. The variables studied were demographic, clinical, surgical and echocardiographic factors, in order to identify differences between the times of aortic clamping and extracorporeal circulation. Results: 73.3% of patients in the case group were at high risk, compared to 39.5% of controls (p < 0.001). Mortality was similar between groups (p > 0.999). The CPB time was on average shorter for the case group (72.35 ± 18.459 minutes), as well as the Clamp time (62.87 ± 16.802) (p < 0.001). The mean transvalvular gradient differed between groups: 7.95 ± 2.74 mmHg for cases, and 13.19 ± 13.142 mmHg for controls (p < 0.001). Conclusion: The study demonstrated a reduction in cardiopulmonary bypass times and aortic clamping in the case group, as well as, this same group showed better hemodynamic performance in the prosthesis implanted in it. There was no difference in mortality between the groups, which demonstrated a better result when using the rapid implant prosthesis, as it presented significantly increased risks. 109032 Modality: E-Poster Scientific Initiation – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE SARA CRISTINE MARQUES DOS SANTOS1, Ivan Lucas Picone Borges dos Anjos1, Lívia Liberata Barbosa Bandeira1, Thaís Lemos de Souza Macedo1, Eucir Rabello1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Promoting the approach of medical students to patients aims to encourage the implementation of humanization in the doctor-patient relationship1. In a study carried out in 2008, a total of 82.5% of respondents who were under hospitalization, considered themselves stressed3. Because of the stress observed during hospitalization and the benefit that humanization of care can bring to patients, the objective was to bring first-time medical students closer to patients admitted to a teaching hospital with a humanized focus and to evaluate through questionnaires anonymously. Methodology: Prospective, observational, and cross-sectional study, with the opinion of the Research Ethics Council (CEP) No. 1,963,944 and carried out from 2017–2019 under the guidance of teachers to first-time medical students in accompanying hospitalized patients with vision humanized as a person. An anonymous questionnaire of quick responses to patients was administered on the participation of students during hospitalization. Results: A total of 1732 hospitalized patients were interviewed and 1156 (66.7%) considered the participation of students with a humanized focus as excellent; 492 (28.4%) as good; 69 (4%) as regular and only 09 (0.5%) as poor, with 06 (0.3%) not responding. They considered that the student’s participation during hospitalization was positive, bringing more comfort and tranquility to patients, according to the reports of the questionnaires, during hospitalization, seeking to supply problems reported by patients in various areas reported. Of the total group, 835 (48.2%) of them reported that their hospitalization was responsible for the occurrence of disorders in their routine, 351 (42%) being concerned about the disease, 235 (28.1%) reporting communication problems with family members, 232 (27.8%) with work, financial 151 (8.7%), 24 (1.4%) problems with personal hygiene and 48 (2.7%) did not report. Conclusion: Humanization in hospitalized patients stands out as an important tool in the treatment because of the reduction of stress generated by the disease and hospitalization. The present study demonstrated that 95.15% of the patients perceived the student’s participation as positive from the humanized point of view. Humanized treatment should be encouraged by the doctor’s initial training, contributing to stress reduction, optimizing treatment. 109043 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION CLÁUDIA BISPO MARTINS SANTOS1, Gabriela de Oliveira Salazar1, José Icaro Nunes Cruz1, Lucas Villar Shan de Carvalho Cardoso1, Ana Luísa Lisboa Prado1, Giulia Vieira Santos1, Letícia Luiza Gomes Marques1, Arthur Leite Lessa1, Edvaldo Victor Gois Oliveira1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Federal University of Sergipe Introduction: Physical exercise leads to cardiovascular and autonomic adaptations essential for most favorable prognosis of chronic coronary disease (CCD), increasing aerobic capacity expressed by maximal oxygen consumption (VO2MAX). Objective: To analyze the behavior of cardiorespiratory capacity in patients with CCD undergoing Exercise Stress Echocardiography (ESE). Methods: Cross-sectional study between January/2000 and January/2022 of patients with CCD who underwent ESE at a cardiology referral service in Sergipe (Brazil). A total of 2000 patients (61.04 ± 10.17 years) were categorized as sedentary or active according to the definitions of the World Health Organization. Student’s t test or Mann-Whitney test and chi-square test was used. The correlation of VO2MAX with clinical variables was evaluated using Pearson’s or Spearman’s tests. A significance level of 5% was adopted. Analyzes were performed using SPSS Statistics software. Results: A total of 1033 (51.66%) sedentary and 967 (48.34%) active individuals were evaluated. The active patients reached higher VO2MAX (35.41 vs. 30.48 mg/kg.min; p < 0.001) and, the sedentary, higher diastolic pressures after exercise (85.05 vs. 83.99 mmHg; p = 0.003). Sedentary lifestyle was associated with the female sex (45.5% vs. 34.7%; p < 0.001). VO2MAX was negatively correlated with resting systolic blood pressure, wall motion score index after exercise and age – being those relationships stronger in the active group; resting diastolic blood pressure was positively correlated with VO2MAX only in sedentary individuals – Table 1. Conclusions: Practicing physical exercise permitted the increase of aerobic capacity, reducing systolic pressure levels at rest and correlating with lower wall motion score index after exercise. 109048 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CLÁUDIA BISPO MARTINS-SANTOS1, José Icaro Nunes Cruz1, Gabriela de Oliveira Salazar1, Juliana Maria Chianca Lira1, Lucas Villar Shan de Carvalho Cardoso1, Mayara Evelyn Gomes Lopes1, Marília Marques Aquino1, Octavio Morais Veloso1, Giulia Vieira Santos1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Federal University of Sergipe Introduction: Despite the clear association between smoking and atherosclerotic diseases, the discussion about the smoker’s paradox persists, given the low reactivity of platelets induced by smoking. Objective: To evaluate the occurrence of smoking-related myocardial ischemia (MI) in patients with previous Acute Myocardial Infarction (AMI) submitted to Exercise Stress Echocardiography (ESE). Methods: Cross-sectional study between January/2000 and January/2022 with individuals presenting a positive history for AMI who underwent ESE at a cardiology referral service in Sergipe (Brazil). A total of 737 patients (61.01 ± 10.78 years) were categorized according to tobacco exposure: smokers, former smokers and non-smokers. Analysis of variance (ANOVA) was used for multiple comparisons with post-hoc Bonferroni and the chi-square test were applied. A significance level of 5% was adopted. Binary logistic regression was performed to identify whether smoking was independently associated with MI. To enter the model, the significance level was p < 0.10 and, to remain, p < 0.05. Analyzes were performed using SPSS Statistics software. Results: Fifty-four (7.33%) smokers, 581 (78.83%) ex-smokers and 102 (13.84%) non-smokers were evaluated, which did not presented differences in the distribution of sex, systemic arterial hypertension, family history and systolic and diastolic blood pressures at rest (p > 0.05). The age was higher on the non-smoker group when compared to smokers (63 vs. 58 years; p = 0.022). Smokers were more likely to be diabetics (p = 0.0037), and ex-smokers, non-diabetics (p = 0.0010). Former smokers were generally non-dyslipidemic (p = 0.0051), while non-smokers were dyslipidemic (p < 0.0001). The chance of having MI was higher among smokers (OR = 2.44; 95%CI 1.14–5.22; p = 0.022) and former smokers (OR = 2.24, 95%CI 1.42–3.53, p = 0.001). Conclusions: When evaluating post-AMI patients, being a former smoker was independently associated with MI. The predictive model also showed that the chance of having MI was even greater among those who continued to smoke. 109050 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CLÁUDIA BISPO MARTINS-SANTOS1, José Icaro Nunes Cruz1, Gabriela de Oliveira Salazar1, Ullany Maria Lima Amorim Coelho de Albuquerque1, Myllena Maria Santos Santana1, Lucas Villar Shan de Carvalho Cardoso1, Marília Marques Aquino1, Nathalia Luiza Silva Sobral1, Lara Teles Alencar Duarte1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Federal University of Sergipe Introduction: When myocardial ischemia (MI) is documented and there is no obstruction of coronary arteries, the diagnosis of ischemia with no obstructive coronary artery disease (INOCA) is established, which has an adverse prognosis. Objective: To analyze the factors associated with the absence of obstructive coronary artery disease (CAD) in patients with MI. Methods: Cross-sectional study between January/2000 and January/2022 with individuals presenting MI on Exercise Stress Echocardiography carried out at a cardiology referral service in Sergipe (Brazil). A total of 1631 patients (59.80 ± 10.56 years) were analyzed, divided into patients with INOCA – defined by coronary angiography – and with obstructive CAD. Mann-Whitney and chi-square tests were applied. A significance level of 5% was adopted. Analyzes were performed using SPSS Statistics software. Results: 500 (30.66%) individuals had INOCA and 1131 (69.34%) had CAD. Patients with INOCA were more likely to be female (56.6% vs. 35.6%; p < 0.0001). The INOCA group showed a lower tendency to have dyslipidemia (57.0% vs. 70.4%; p < 0.0001) and a positive family history of cardiovascular diseases (54.6% vs. 64.2%; p < 0.0001). There was no association between absence of CAD on MI and the factors: hypertension, diabetes mellitus and obesity (p > 0.05). Patients with INOCA tended to be younger (58 vs. 60 years; p < 0.0001) and the analyses of wall motion score index at rest and after exercise dit not presented significant differences (p > 0.05) between the groups. Systolic blood pressure levels at rest and after exercise did not differ between INOCA and CAD (p > 0.05). Conclusions: Female sex, absence of dyslipidemia and negative family history for cardiovascular events were factors associated with INOCA. There was no difference in the involvement of left ventricular motility, which suggests that ischemia in patients with CAD and INOCA has a similar degree. 109052 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING CLÁUDIA BISPO MARTINS-SANTOS1, Gabriela de Oliveira Salazar1, José Icaro Nunes Cruz1, Nathalia Luiza Silva Sobral1, Juliana Maria Chianca Lira1, Lara Teles Alencar Duarte1, Raisan Almeida Santos2, Giulia Vieira Santos1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1, Luiz Flávio Galvão Gonçalves2 (1) Federal University of Sergipe; (2) Hospital São Lucas Introduction: Cardiovascular magnetic resonance (CMR), through the myocardial T1 time mapping technique, provides the tissue characterization of myocardial fibrosis, both localized and diffuse, in a quantitative manner, in order to overcome some limitations of the evaluation of late enhancement. Objective: To analyze CMR parameters in relation to the increase in native T1 map levels in patients without delayed enhancement, in order to demonstrate its role as a marker of advanced heart disease. Methods: Cross-sectional study between January and December 2021 with individuals undergoing CMR who did not present late enhancement at a cardiology referral service in Sergipe (Brazil). A total of 131 patients (54.54 ± 16.96 years) were analyzed, divided into two groups according to the increase (≥1030 ms) or not (<1030 ms) in the levels of native T1 acquired by the modified look locker method (MOLLI) in a magnetic field of 1.5 Tesla. Student’s t, Mann-Whitney and chi-square tests were applied. A significance level of 5% was adopted. Analyzes were performed using SPSS Statistics software. Results: 47 (35.88%) individuals had increased levels of native T1 and 84 (64.12%) presented levels below 1030 ms. The group with increased T1 had more females than the group without increased T1 (72.3% vs. 32.1%; p < 0.0001). Patients with increased native T1 levels had lower left ventricular ejection fraction values when compared to those with normal T1 levels (56.32% vs. 62.35%; p = 0.014). The group with high native T1 had even lower values of left ventricular mass (95.34 g vs. 105.41 g; p = 0.027), right ventricular end-diastolic volume (108.89 ml vs. 129.52 ml; p = 0.007) and right ventricular measurements at short axis orientation (3.66 cm vs. 3.99 cm; p = 0.001). Conclusions: Elevated levels of native T1 mapping were associated with female sex, as well with lower left ventricular mass and ejection fraction values in those patients without late enhancement. Therefore, the role of CMR in the assessment of ventricular function is reiterated and its application in patients without late enhancement is demonstrated. 109069 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS FILIPE ALVES RAMOS 1, Augusto Dê Marco Martins2 (1) Catholic University of Brasília; (2) Biocárdio Institute Introduction: Acute myocardial infarction is one of the main causes of death in Brazil. According to the Ministry of Health, risk factors such as (1) smoking, (2) dyslipidemia, (3) hypertension, (4) obesity, (5) diabetes, and (6) stress have contributed to an increase in the number of deaths associated with infarction, and thus, generating social and economic impacts on the Brazilian society. Objective: To investigate the number of deaths associated with Acute Myocardial Infarction in Brazil between 1999 and 2019. Methods: This is a cross-sectional epidemiological study carried out between 1999 and 2019 on the number of deaths from acute myocardial infarction reported in Brazil. The analysis of information had been extracted from the Department of Informatics of the Brazilian Unified National Health System (DATASUS). Results: In the analyzed period, 1,610,781 deaths from acute myocardial infarction were reported in Brazil. There was a 28.42% increase in the mortality rate in 2019 when compared with the rate reported in 1999. The number of deaths in Brazil in 2019 (95,557 deaths) was 65.00% higher than in 1999 (57,913 deaths). The male sex was the most afflicted and corresponded to 59.03% of the deaths reported in that period. Regarding the deaths by race, it was observed that white people (57.03%) and pardo people (29.48%) were the most affected. When grouped by age, it was observed a higher frequency of mortality rate between 70 to 79 years old: 411,161 (25.53%); followed by 80-year-olds: 380,371 (23.61%); 60 to 69 years: 378,933 (23.52%); 50 to 59 years: 263,790 (16.38%); 40 to 49 years: 126,090 (7.83%); 30 to 39 years: 37,176 (2.31%) and under 30 years old: 11,244 (0.70%). In 2019, the mortality rate per 100,000 people in Brazil was 45.36. Regarding the numbers by region, it was noted that the southeast region was responsible for 48.45% of deaths reported in Brazil in the analyzed period. Conclusions: In the light of the results obtained, it was verified that the mortality rate and the number of deaths due to acute myocardial infarction increased over the years. In this study, it was observed a higher frequency of deaths from acute myocardial infarction among white men over the age of 50. Therefore, it is essential to promote health campaigns against stress, sedentary lifestyle, and poor diet, which are aimed at the prevention of coronary artery disease. 109074 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARYANA HELENA DE SOUZA MENDONÇA RIBEIRO1, Jeanne du Fay de Lavalla2, Luciene Ferreira Oliveira Mota3, Annabelle Santos Volgman4 (1) Medicine College of Itajubá, Itajubá, MG, Brazil; (2) Department of Internal Medicine, Rush University Medical Center, Chicago, IL, USA.; (3) Department of Cardiology, Medicine College of Itajubá, Itajubá, MG, Brazil; (4) Division of Cardiology, Rush University Medical Center, Chicago, IL USA Introduction: While a diverse workforce improves the care of patients, encourages different views, and reduces bias in the working environment, female cardiologists continue to be <30% of the workforce in the United States and Brazil. Therefore, including women’s recruitment as a priority for the cardiology community through understanding and discovering what inspires women to follow a cardiology career is essential. We conducted a survey of students from a Brazilian medical school to ascertain their motivations to consider cardiology and sought to find sex differences among the students. Objective: Identify factors which affect medical students’ interest in cardiology and assess any sex differences. Methods: Medical students answered a digital survey based on the science interest survey, validated by the Rasch scale and modified to address cardiology interest questions. The survey consisted of 20 questions, answered in the Likert scale and divided into five categories: class, family, friends, informal (learning outside the classroom), and professors. Altogether, 100 medical students (28 men/72 women) from the first to sixth year answered this survey. p < 0.05 was considered significant. Results: In both sexes the influence of professors had higher median ratings compared to the classes, family, friends, and informal. Friends had the least influence on students’ interest in cardiology. There were no significant sex differences. Conclusion: In this cohort of Brazilian medical students, professors had the most influence on encouraging medical students into pursuing cardiology. In the same way, practical classes in cardiology wards and visits to specialized centers appeared to be a good alternative to inspire men and women on becoming cardiologists. 109075 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA LUISA SOARES CHIARETTI1, Bianca de Almeida Nunes2, Livia Brito Oliveira2, Glicia Gleide Gonçalves Gama2, Fábio Figueiredo Costa2, Adriana Lopes Latado2 (1) Faculdade de Medicina da Bahia da Universidade Federal da Bahia – FMB-UFBA; (2) Hospital Universitário Professor Edgar Santos da Universidade Federal da Bahia/Empresa Brasileira de Serviços Hospitalares – HUPES-UFBA/EBSERH Introduction: The rapid development of effective and safe vaccines was set as a priority due to the COVID-19 pandemic. This acceleration generated an infodemic, raising questions about the safety of the vaccines between the population, including cardiac patients. Objectives: Evaluate the safety of COVID-19 vaccination in patients with cardiopathies. Methodology: Unicentric prospective observational cohort that evaluated vaccination cover, frequency and severity of adverse events (AE) associated with the COVID-19 vaccines between patients with cardiopathies followed up in a cardiology referral service, in Salvador, Bahia, Brazil, between 2021 and 2022. AE were defined as any unwanted medical occurrence after the vaccination, which were categorized as severe (SE) or non-severe (NSE) events. Descriptive analysis of the data were performed and the inferential analyses were exploratory. Significance level adopted was 5%. Results: 259 patients were included, 65,4% were women, 83,3% were black or brown. The average age was 62.4 (+15.8) years). Among morbidities, 81,4% had arterial hypertension, 44,6% had diabetes mellitus, 23% had chronic kidney disease, 78,7% had dyslipidemia, 16,3% had atrial fibrillation, 11,2% had previous myocardial infarction and 36,6% were diagnosed with heart failure. With regard to vaccination, 84,3% (204) had received at least two doses of vaccine, of which 38,0% received the CoronaVac, 30,2% (78) received the AstraZeneca, and 15,1% received Pfizer. 1,9% received a single dose of Janssen and 14,7% (38) did not know about vaccine type. By the end of the study, 35,6% had received a third dose of vaccine, 97,5% (81) were given Pfizer and 2,5% were given CoronaVac. Of total vaccinated, 30% reported NSE (local soreness, fever, myalgia or headache, during less than 3 days) and no SE (life-threatening events, requirement of hospitalization or causes of permanent sequelae) were reported. Patients who reported AE were younger (56,5 + 15,1 vs 65,5 + 14,9 years; p < 0,01), mostly women (36,8% vs 18,3% p = 0,01) and without hypertension (40,6% vs 34%, p = 0,04). The AE were more frequently reported after vaccination with adenovirus carrier or RNA vaccines (AstraZeneca, Janssen and Pfizer) than with Coronavac (p < 0,01). Conclusion: The COVID-19 vaccines available in Brazil are safe for application in patients with cardiopathies. AE were frequent but without gravity, being more frequent in women and after vaccination with adenovirus carrier or RNA immunizers. 109122 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LEO CHRISTYAN ALVES DE LIMA1, Hildeman Dias da Costa2, Laura Jane França Lacerda1, Matheus Akira Suzuki de Oliveira2, Leticia Luana Alves Ferreira1, Emanuelle Toneto Souza Silva1, Andressa Flores da Costa1, Carla Cibelly Mesquita Almeida1, João Paulo Lucena1 (1) Centro Universitário São Lucas (UniSL); (2) Universidade Federal de Rondônia (UNIR) Introduction: Chronic rheumatic heart disease (CRHD) is a non-suppurative complication of pharyngotonsillitis caused by group A beta-hemolytic streptococcus and results from a delayed immune response in genetically predisposed populations. It is a leading cause of cardiovascular disease in developing countries, accounting for approximately 15 percent of all heart failure patients in endemic countries. Objectives: To describe the epidemiological profile of hospitalizations for Chronic Rheumatic Heart Disease in adults between the years 2009 and 2019. Methodology: Epidemiological, descriptive, retrospective study. Data were obtained from the IT department of the Unified Health System – DATASUS, from January 2009 to December 2019, with individuals aged 20 to 79 years, evaluating hospitalization rates, mortality and pattern of carriers: range age, race and sex. Results: In the analyzed period, 3830 hospitalizations were observed, 2052 were female and 1778 were male. Regarding race, there were 2089 occurrences in pardos, 272 in whites, 76 in blacks, 20 in yellows, 7 in indigenous people. The age group with the highest number of cases was between 40 and 49 years, with 777 individuals. The average total hospital stay was 14 days, representing a total expense of R$31,020,178.05, with 2017 being the year with the highest number of hospitalizations (534) and 2010 the lowest number (295). The total mortality rate was 9.63, with the state of Amapá having the highest rate (18.37), followed by Rondônia (12.27), with a peak in 2014 (17.49). The total number of deaths corresponded to 369 deaths, with the state of Pará holding the highest number (143), followed by Amazonas (87). Conclusion: CRHD is still a major public health problem, especially in developing countries, resulting in huge health expenditures. The present study demonstrated a large number of cases of the disease in the northern region of Brazil. Between the years 2009 to 2019, the epidemiological profile was characterized by females, brown, aged between 40 and 49 years. The state with the highest number of cases was Pará and the highest mortality rate found was in Amapá. The description of these sociodemographic and epidemiological characteristics guide the planning of health actions in order to improve the population’s quality of life. 109124 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DOMINGOS ALVES DE SANTANA NETO1, Anna Mayse Feitosa da Silva1, Mariana Evaristo Leite2, Sarah Mariani Rocha Oliveira1, Ana Beatriz Vaz de Araújo1, Katia Vergetti Bloch1 (1) Universidade Federal do Rio de Janeiro (UFRJ); (2) Universidade Federal de São João del-Rei (UFSJ) Introduction: Hypertension (HTN) is one of the main risk factors for cardiovascular diseases. Hypertensive adolescents are at higher risk of becoming hypertensive adults. Heart rate (HR) is a simple measure associated with sympathetic activity and can be influenced by numerous environmental factors. Elevated resting HR in adolescents can be a predictor of HTN in adults. Objective: To explore the association of Hypertension with Heart rate in Brazilian adolescents. We also aimed to investigate the influence (confounding or effect modification) of sex, age, physical activity, and obesity in this association. Methods: The Study of Cardiovascular Risks in Adolescents (ERICA) was a national, school-based, cross-sectional study. The sample has a complex design, being composed of adolescents aged between 12 and 17 years old from public and private schools located in Brazilian municipalities with more than 100,000 inhabitants. Data were collected using a self-administered questionnaire on an electronic device. Blood pressure and heart rate at rest were measured with an oscillometric device. Weight and height were measured to calculate the body mass index (weight/height2), classified according to WHO reference curves by sex and age. Physical activity was evaluated using a list of 24 modalities, including information on frequency (days) and time (hours and minutes) of practice during the last week. Adolescents who accumulated more than 300 min/week of physical activity were classified as active. Raw and adjusted linear regression models were performed in Stata 15.0. Results: Age and obesity were confounding variables, while sex and physical activity were effect modifiers for the association between Hypertension and Heart rate (p < 0.001 for interaction). Girls and hypertensive adolescents had the highest Heart rate levels (88.5 bpm; 95%CI 87.0 to 90.0), while boys and non-hypertensive adolescents had the lowest (78.1 bpm; 95%CI 77.6 to 78.5) adjusted for age and obesity. The Heart rate of hypertensive and inactive adolescents was 87.6 bpm (95%CI 86.0 to 89.1), and non-hypertensive and active adolescents was 79.1 bpm (95%CI 78.6 to 79.5). Conclusion: The observed association of Hypertension with Heart rate was independent of age and obesity. The effects of sex of physical activity on this association should be considered in longitudinal studies to estimate the prediction. 109217 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION RAYANNE ALESSANDRA DA SILVA BARRETO1, Anderson Brasileiro de Araújo1, Ana lívia Gadelha Xavier da Nóbrega1, Beatriz Gadelha e Xavier1, Bianca Vasconcelos Braga Cavalcante1, Letícia Lacerda Burity1, Vinicius Vieira Leandro da Silva1 (1) Faculdade de medicina Nova Esperança-FAMENE Introduction: Preeclampsia is defined as hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg), arising after 20 weeks of gestational age with proteinuria (300 mg) or other signs of end-organ damage and is an important cause of maternal and perinatal morbimortality, particularly when of early onset associated with deficient intravascular production of prostacyclin, a vasodilator, and excessive production of thromboxane, a vasoconstrictor and stimulant of platelet aggregation. These observations led to the hypotheses that doses greater than 80 mg/day of acetylsalicylic acid, might delay or prevent development of preeclampsia. Prophylaxis is recommended in women at high risk of this disease that includes history of preeclampsia, chronic hypertension, family history of preeclampsia, renal disease, a body mass index greater than 30, multifetal gestation, and sociodemographic characteristics. Should be initiated between 12 weeks and 28 weeks of gestation, optimally before 16 weeks, and continued daily until delivery. Objective: To assess the effectiveness and safety of doses greater than 80 mg/day of acetylsalicylic acid (ASA), in early pregnancy with increased risk of preeclampsia with the objective of delaying or preventing this disease. Methods: Keywords were combined for electronic databases search. Relevant citations were extracted from PubMed, Elsevier and web science from 2007 to 2021. This systematic review includes only randomized, controlled trials. The population in the studies involved pregnant women at risk of preeclampsia treated with aspirin low-dose initiated at or before 16 weeks of gestation. Results: 17 trials (n = 15.908) to assess maternal and perinatal health outcomes and 15 trials (n = 15.767; 10 good-quality) to assess prevention of preeclampsia. All trials were placebo-controlled. ASA treatment beginning in early gestation was associated with a greater reduction in the incidence of preeclampsia than treatment beginning in late gestation. Conclusions: Daily low-dose acetylsalicylic acid use in pregnancy is considered safe. Treatment with this drug initiated early in pregnancy is an efficient method of reducing the incidence of preeclampsia and is associated with a low likelihood of serious maternal, or fetal complications, or both, related to use. 109129 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARINA DE ANDRADE BATISTA1, Ana Verena Silva Santos1, Daiane Lima dos Santos2 (1) Cetro universitário UniFTC; (2) Universidade Estadual da Bahia Introduction: According to the Brazilian Society of Cardiology (2015), there are about 350 thousand new cases of Acute Myocardial Infarction (AMI) per year and 40 to 65% of them evolve to death in the first hours. Among cardiovascular diseases, AMI has one of the highest rates of morbidity and mortality. In the period of the COVID-19 pandemic, given the restrictions to seek health services, the possibility of underreporting of AMI cases in the country is raised. In addition, from 2019 to 2020, there was a decline of 89.97% in patients who sought the cardiology outpatient clinic. Therefore, given this public health problem, there is an urgent need to analyze the registration of these cases in regional units in Brazil. Objective: To analyze the number of confirmed cases of acute myocardial infarction, before and during the Covid-19 pandemic, among the Regional Units of the Brazilian territory, comparing the periods from 2017 to 2019 and 2019 to 2022. Method: This is a retrospective epidemiological study of an analytical nature, carried out using data published on the Ministry of Health portal from 2017 to 2022. For this study, confirmed cases of AMI were analyzed by Regional Unit from 2017 to 2022. Appraisal by the Research Ethics Committee is unnecessary because this research used public data, without identification of the participants. Results: In the analyzed period, 738,196 cases of AMI were confirmed in Brazil, with 362,649 in the pre-pandemic period (1) 2017 to 02/2019 and 370,547 in the pandemic period (2) from 03/2019 to January 2022. Studying the regional units in period 1, the following stand out: Southeast with 49.3% (n = 178,942), Northeast with 19.7% (n = 71,509) and South with 19.3% (n = 70,021) of cases. In period 2, regional highlights were in charge of the same regional units, respectively, with 48.8% (n = 180,945) of cases, 19.8% (n = 73,724) and 18.8% (n = 69,868). Therefore, during the pandemic, there was an increase of 2.1% (n = 7,898) compared to the pre-pandemic period. Conclusion: While access to health services has declined during the pandemic, AMI records have increased. However, given the possible underreporting, it is inferred that the increase was even more significant. Since AMI has high morbidity and mortality, many individuals were left without proper assistance during this period, increasing their susceptibility to AMI and its complications. 109131 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ANA CLARA DE PAULA CALDAS1, Ana Clara de Paula Caldas1, Juliana Rodrigues Soares2, Vinicius Tostes Carvalho2, Lucas Lodi-Junqueira2, Guilherme Rafael Sant’Anna Athayde2, Paulo Henrique Melo1, Fernando Coutinho1, Lucas Posoli1, Pedro Emanuel Carvalho1, William Antonio M. Esteves1, Maria Carmo Pereira Nunes2 (1) Universidade Federal de Minas Gerais (UFMG); (2) Hospital das Clínicas da Universidade Federal de Minas Gerais (HC – UFMG) Background: Rheumatic mitral stenosis (MS) results in elevated left atrial (LA) pressures and subsequent atrial remodeling, atrial fibrillation (AF), and thrombus formation. Progressive LA enlargement can occur, despite the relief of valvular obstruction with valve intervention. This study evaluates LA function by three-dimensional echocardiography (3DE) in rheumatic MS patients undergoing percutaneous mitral valvuloplasty (PMV) to assess the impact of AF on LA function. Methods: A total of 160 MS patients referred to PMV were prospectively enrolled. LA volumes and function were measured by 3DE pre, immediately after PMV, and at 1 year follow-up. Results: Mean age was 44 ± 12 years, and 134 (84%) patients were women. Pre PMV, 44 (28%) were in AF, and 66 (41%) were in NYHA functional class III or IV. LA maximum volume decreased from 55.5 ± 23.1 ml/m² to 53.5 ± 29.7 ml/m² 48 hours after the procedure (p = 0.147), and to 50.5 ± 24.6 ml/m² at 1 year follow-up (p = 0.011). LA emptying fraction increased from 20.4 ± 10.1 ml/m², to 28.7 ± 11.4 ml/m² after 48 hours of procedure (p < 0.001), and to 32.6 ± 13.3 ml/m² at 1 year follow-up (p = 0.003). In AF patients, LA emptying fraction was 13.8 ± 7.5% at baseline, 21 ± 9.3% 48 hours after the procedure (p = 0.039) and 20.8 ± 8.7% at follow-up (p = 0.946) (Figure 1). Age, initial presence of AF, and postprocedural mean gradient were identified as the most significant predictors of late LA function. Conclusions: LA presents reverse remodeling after PMV. This remodeling is higher immediately after the procedure, but continues to occur in the following months. In patients with MS and AF, volumes remained unchanged after PMV, either at 48 hours and at follow up, and only the emptying fraction improves at 48 hours, without further improvement at follow up. 109143 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ERICK BRODER BICHARA1, Victor Arthur Soares Costa Araújo1, Diego Moreira Arruda1 (1) Escola Bahiana de Medicina e Saúde Pública Background: Pulmonary embolism (PE) is one of the most frequent underdiagnosed acute cardiovascular diseases. In the COVID-19 first wave, especially, there were many disorders with similar symptoms. The pandemic has created an atmosphere of uncertainty among healthcare professionals and this fear leaded to a pulmonary embolism diagnosis even more underdiagnosed within the focus on COVID cases. Increased D-dimer works as a predictor for the presence of COVID. This indicator is still an essential factor for diagnosis PE, and it’s increase in patients with COVID can result in prothrombotic state. Methods: Quantitative, descriptive study, carried out using secondary data from the Hospital Information System of DataSUS, about the monthly frequencies of hospitalizations and deaths related to pulmonary embolism. Objective: Describe the epidemiological profile of hospitalizations and deaths resulting of pulmonary embolism in periods pre, during and post-COVID-19 pandemics in Brazil. Results: In pre-pandemic period (Apr/19–Jan/20), there were 7915 hospitalizations in the country, 1948 in capitals. 2979 hospitalizations occurred in men and 4936 in women. Age group 60–69 years (1496) led. As for deaths, there were 1387 in country, 344 in capitals. 560 deaths in men and 827 in women. The age group 80 years and over (358) led. In 1st wave period (Feb/20–Nov/20), there were 7173 hospitalizations in country, 1777 in capitals. An increasing pattern was observed between Dec/19–Feb/20 (28, 141 and 222) – pandemic beginning – and a decreasing between Oct/20–Nov/20 (187 and 79) – end of the first wave. 2803 hospitalizations occurred in men and 4370 in women. Age group 60–69 years (1406) led. As for deaths, there were 1289 in country, 351 in capitals. 527 deaths in men and 762 in women. Age 80 and over (333) led. During vaccine period (Jan/21–Oct 2021), there were 8087 hospitalizations in country, 1936 in capitals. This period had the highest number among the three periods (1042 in Jul/21). There was an increasing pattern from Aug/20–Apr/21 (4, 6, 23, 47, 109, 178, 182, 185, 232) and a decreasing from Jun/2021–Oct/21 (256, 253, 196, 151, 97). 3440 hospitalizations occurred in men and 4647 in women. Age group from 60–69 years (1565) led the cases. As for deaths, there were 1585 in country and 387 in capitals. 692 deaths in men and 893 in women. Age 80 and over (402) led. Conclusion: Both cases and deaths decreased during 1st wave period and increased during vaccine period. 109708 Modality: E-Poster Scientific Initiation – Non-case Report Category: PSYCHOLOGY VINICIUS VIEIRA LEANDRO DA SILVA1, Leticia Lacerda Burity1, Beatriz Gadelha e Xavier1, Bianca Vasconcelos Braga Cavalcante1, Ana Lívia Gadelha Xavier da Nóbrega Pereira1, Rayanne Alessandra da Silva Barreto1, Giovanna Alves de Souza2, Eduardo Franco Correia Cruz Filho3, Salmo Vasser Paranhos de Oliveira4, Beatriz Calsolari Ranha5, Idemar Luis Cover Filho6, Luís Fernando Brito Ferreira7 (1) Faculdade de Medicina Nova Esperança (FAMENE); (2) Universidade Cidade de São Paulo (UNICID); (3) Centro Universitário de João Pessoa (UNIPE); (4) Universidade: Instituto de Ensino Metropolitano de Ensino Superior (IMES – Univaço); (5) Universidade do Estado do Rio de Janeiro (UERJ); (6) Faculdade Ciências Médicas (FCM); (7) Centro Universitario Facisa (FACISA) Introduction: Cardiovascular diseases (CVDs) are the leading cause of death in the world, bringing concern to the main global health agencies. Among the risk factors for CVDs, emotional aspects are increasingly being associated with increased cardiovascular risk. In the clinical practice of cardiac surgical patients, it is observed that psychological factors such as depressed mood, anxiety and fear of dying influence the patient’s recovery. Objective: This abstract aims to show the benefits of psychotherapy in patients after cardiac surgery, and its relationship with cardiovascular disease. Methods: This is an integrative review based on relevant publications on databases of the Latin American and Caribbean Literature on Health Sciences Information (LILACS), Scientific Electronic Library Online (SciELO), Virtual Health Library (BVS) and PubMed, in English and Portuguese from 2007 to 2020. Results: It is known that the frequency of psychiatric disorders in hospitalized patients after cardiac surgery is between 20 and 60%, where anxious and depressive disorders due to fear of loss of autonomy or death stand out. Psychotherapy has brought several benefits to hospitalized patients, where there has been a reduction in the symptoms of psychological disorders, greater adherence to medication and diet, and an improvement in the acceptance of the procedures performed, leading to a reduction in the number of hospitalization days, helping in a better patient recovery. Conclusions: Therefore, we can conclude that the support of psychotherapy in patients who have undergone cardiovascular surgery has brought relevant results, causing a faster and more comfortable recovery for hospitalized patients. 109182 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GUILHERME OLIVEIRA MAGALHÃES COSTA1, Bruno Oneto Y Viana Pintos2, Maurício Pimentel2, Anderson Donelli da Silveira2 (1) Universidade Federal do Rio Grande do Sul (UFRGS); (2) Hospital de Clínicas de Porto Alegre (HCPA) Introduction: Atrial fibrillation (AF) is associated with a worse prognosis in patients with heart failure (HF). A blunted chronotropic response seems to be related to worse prognosis and functional capacity in those patients, independently of beta-blocker use. Objectives: To evaluate heart rate response during cardiopulmonary exercise testing (CPET) in patients with both HF and AF and its association with cardiovascular events. Methods: Retrospective cohort study including patients with HF and AF assessed by CPET, followed-up in an outpatient clinic at a public university hospital. The primary endpoint was death or need for heart transplant. T-Test for independent samples or Mann-Whitney U test were used when appropriate. The best cut-off point for the chronotropic index was obtained by a ROC curve analysis. Univariate and multivariate survival analysis was performed using Cox regression and Kaplan-Meier curve. Results: A total of 1083 patients with HF comprised the entire cohort, 208 of those with coexisting AF. Mean age was 58 (±11) years and average follow-up time was 42 (±20) months. 31% were female, 19% ischemic, 15% had HFpEF, 85% had HFrEF and 93% were receiving beta-blockers. Primary outcome occurred in 21.4% of patients. It was more frequent in patients who had lower peak HR (123 vs 141 bpm, P < 0.001), ΔHR (47 vs 58 bpm, P = 0.004), chronotropic index (0,57 vs 0,78, P < 0.001), peak VO2 (15.4 vs 16.9 ml/kg/min, P = 0.03) and OUES (1.18 vs 1.39, P = 0.002). Univariate survival analysis showed that the same variables remained as outcome predictors. A chronotropic index <0.6 was associated with an HR 2.7 (IC 95% 1.5 –4.7) to the primary endpoint in the univariate analysis and an HR 3,1 (IC 95% 1,2–7,8) in the multivariate adjusted for sex, age, CKD, DM and use of beta-blockers, remaing as the strongest risk predictor in our sample. Conclusion: A blunted HR response in patients with HF and AF, evidenced by lower chronotropic index, ΔHR and HR peak, was associated with an increased risk of death or need for heart transplant in our sample. These findings question the real benefit of beta-blocker use in this scenario. Further studies evaluating the reduction or withdrawal of these medications could help to answer our question. 109185 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY NICOLE DE OLIVEIRA SANTOS1, Giovana Rezende Fernandes Costa1, Jennifer Klassen Boeing1, Luís Antonio Zorzi Santin1, Adriana Saito Jasper2 (1) Universidade Positivo (UP); (2) Associação Hospitalar de Proteção à Infância Dr. Raul Carneiro/Hospital Pequeno Príncipe (HPP) Introduction: Critical congenital heart diseases (CCHD) require intervention as early as possible. The delay in its diagnosis increases the risk of morbidity and mortality in newborns (NB). Prenatal diagnosis of CCHDs is at first done using morphological ultrasound, and then more thoroughly by fetal echocardiography, allowing for a more deliberate management planning of birth conditions with the family and health-care providers. However, these exams are not offered to all pregnant women by the Healthcare System in Brazil. Objective: To correlate the antenatal diagnosis with the origin of newborns admitted for critical congenital heart defects in a neonatal intensive care unit of a tertiary hospital in Curitiba, Paraná, Brazil. Methods: This was a retrospective study reviewing medical records covering the period from January 1st 2015 to December 31st 2019 of patients diagnosed with critical congenital heart disease who were admitted to the Neonatal Intensive Care Unit. A descriptive analysis of the collected data was performed. Results: The study included 127 patients. The CCHD type incidence found in our sample was: coarctation of the Aorta (20.5%), transposition of great arteries (15.7%), pulmonary atresia (13.4%), hypoplastic left heart syndrome (11.8%), tetralogy of Fallot (3.1%), anomalous pulmonary vein drainage (3.1%), Ebstein anomaly (3.1%), tricuspid atresia (2.4%), truncus arteriosus (2.4%), double outlet right ventricle (1.6%), aortic stenosis (0.8%) and two or more heart defects combined (22%). Prenatal diagnosis, performed on 41.7% of the patients, showed statistically significant relation with the origin of the NB (p < 0,0001). Curitiba, capital of the state of Paraná, had the highest proportion of prenatal diagnosis (69%) when compared to the Greater Curitiba region (25.9%), the countryside region of Paraná (25%) and other Brazilian states (50%). Conclusion: Most patients with CCHD weren’t diagnosed prenatally and, amongst the NB with a prenatal diagnosis, most were from Curitiba – PR. Due to the available infrastructure in the capital, Curitiba has the necessary technology and professionals qualified to perform diagnosis, scanning protocols and practice related to prenatal screening, suggesting an important role in their implementation. 109188 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES ALEXANDRE NEGRÃO PANTALEÃO1, Nayana FA Gomes1, Vicente Rezende Silva1, William AM Esteves1, Marildes Luiza de Castro1, Lucas Chaves Diamante1, Fernando Cunha Ruffo1, Matheus de Oliveira Ferreira1, Luís Felipe Rezende de Almeida1, Maria Carmo P Nunes1 (1) School of Medicine, Federal University of Minas Gerais (UFMG) Introduction: Rheumatic heart disease (RHD) is the leading cause of cardiovascular death in children and young adults, and disproportionally affects low- and middle-income countries. Mitral regurgitation (MR) is the most common valve abnormality in RHD and it is often associated with atrial fibrillation (AF). AF is the most prevalent sustained arrhythmia in RHD and it is a marker of disease severity with poor prognosis. Objective: This study aims to investigate the association between AF and MR progression in patients with RHD. Methods: Consecutive RHD patients with non-severe MR associated with any degree of mitral stenosis were selected between 2011 and 2021. Primary endpoint was progression of MR, which was defined as an increase of one grade in MR severity from baseline to the last follow-up echocardiogram. AF diagnosis was based on a history of permanent AF supported by past 12-lead electrocardiogram (ECG). Diagnosis of new-onset AF in patients with sinus rhythm at baseline was confirmed by 12-lead ECG. Results: 539 patients were included, aged 46.2 ± 12 years, and 83% were women. At a mean follow-up time of 4.2 years (IQR: 1.2 to 6.9 years), 54 patients (10%) displayed MR progression with an overall incidence of 2.4 per 100 patient-years. Patients who had new-onset AF during the follow-up were at risk for progression (adjusted hazard ratio [HR] 2.447, 95% CI 1.035–5.788) and patients with permanent AF were at the highest risk (HR 4.459, 95% CI 2.148–9.631) compared with patients in sinus rhythm. Conclusions: In RHD patients with a full spectrum of MR severity, progression of MR occurs over time, associated with the presence of AF. AF burden either permanent or new-onset AF led to an increased risk of progression of MR. 109194 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FILIPE ALVES RAMOS 1, ROSANGELES KONRAD2 (1) Catholic University of Brasilia; (2) Hospital Anchieta – Cárddio Clinic Introduction: Heart failure is a cardiovascular pathology that presents a high mortality rate. According to the World Health Organization (OMS), 23 million people live with this disease in the world. Hypertension, as well as diabetes and coronary artery disease are the main risk factors for the development of heart failure and are related to their worst prognosis. Objective: Analyzing the evolution of the number of deaths from heart failure in Brazil between 1999 and 2018. Methods: This is a descriptive and quantitative cross sectional study conducted between 1999 and 2018 on the number of deaths from heart failure documented in Brazil. The analyzed data were obtained through the Department of Informatics of the Brazilian Unified National Health System (DATASUS). Results: From 1999 to 2018, 550,969 deaths from heart failure were recorded in Brazil. The mortality rate in 2018 decreased 29.71% compared to 1999. The number of deaths in Brazil in 2018 (26.482 deaths) was 10.32% lower than the recorded in 1999 (29.531 deaths). In relation to race/skin color deaths, it was found that white people 297.198 (53.94%) and pardo people 157.282 (28.55%) were the most affected. In the distribution by age groups there was a higher prevalence of mortality over 80 years: 233.283 (42.34%); followed by 70 to 79 years: 144.164 (26.16%); 60 to 69 years: 89.072 (16.17%); 50 to 59 years: 46.643 (8.46%); 40 to 49 years: 20.871 (3.79%); 30 to 39 years: 8.589 (1.56%) and those under 30: 7.747 (1.41%). There was a 29.71% reduction in the mortality rate for every 100,000 people, when compared to the one registered in 2018 (12.66%) with that documented in 1999 (18.01%). Conclusions: Through the results presented, it is noted that the mortality rate, as well as the number of deaths from heart failure has decreased in Brazil over the years. This reduction is justified by the greater use of beta-blockers and angiotensin-converting enzyme, which reduce the morbidity and mortality associated with heart failure. Add to this, the reduced selection of heart failure as a basic cause of death in the face of other cardiovascular diseases, such as hypertension. In the present study, there was a higher frequency of heart failure deaths among white men over the age 60. 109196 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LARISSA SILVA FERREIRA1, Larissa Silva Ferreira1, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá (UNIFAP) Introduction: Torsades de pointes is a rare type of ventricular cardiac arrhythmia that is characterized by an increase in the QT interval on the electrocardiogram (ECG). After the beginning of the COVID-19 pandemic, the use of chloroquine and hydroxychloroquine (HCQ) became part of treatment protocols. However, later studies demonstrated the low efficacy of these drugs for the treatment and the increase of cardiovascular risks such as angina, arrhythmias and heart failure. Objective: To analyze the mortality trend due to cardiac arrhythmias in the northern region between the periods of 2020 and 2022. Methods: Epidemiological study with a cross-sectional design of mortality and arrhythmia in Brazil between 2020 and 2022. The investigated data were extracted from the Department of Informatics of the Unified Health System (DATASUS). Results: In the period investigated, 18,741 deaths from arrhythmias were documented in Brazil. The growth in the mortality rate was from 11.64% in January 2020 to 15.62% in January 2022. It was observed that the highest mortality rate occurred in the Midwest region (35.04%). Furthermore, the number of hospitalizations for arrhythmias in this period was 27,887, being higher in the Southeast region, which represents 47.7% of hospitalizations. Regarding deaths by sex, it was found that males were the most affected (15.43%) as well as in the number of hospitalizations (56.03%). In the distribution by age groups, higher mortality was observed in individuals aged 80 years or older (16.83%) compared to those aged 55 to 59 years (14.80%). It was also noted an increase in cases of hospitalization in this period in the age group from 65 to 69 years, representing 53.3% of the total number. Conclusions: Based on the results obtained, there was an increase in cases of hospitalizations and deaths due to arrhythmias in the period studied. This mortality was higher in males and in the age group from 65 years old. Thus, HCQ can prolong the ECG QT interval and can trigger an arrhythmia called torsades de pointes. Thus, the use of this drug during the COVID-19 pandemic may, indirectly, have been one of the aggravating factors in this arrhythmia mortality rate. 109241 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY LEONARDO YOHAN SANTOS CHUCRE DE LIMA1, Elizama Raquel de Souza Silva1, Bruna Lisboa Nunes1, Isabela Ferreira de Freitas1, Luiz Felipe Leão Lima1, Micandria Yanka Fender Lobato1, Luma Maria Favacho Bordalo1, Fernando Gabriel dos Santos Santiago1, Caio Vinicius Botelho Brito1 (1) Laboratório de Cirurgia Experimental da Universidade do Estado do Pará Introduction: Noncommunicable diseases (NCDs) are responsible for 70% of global deaths and about 45% of those are of cardiovascular origin. In Brazil, 72% of deaths result from NCDs and 30% are by cardiovascular diseases (CVDs). Thus, the importance of finding therapeutical strategies and new pharmacological models is evident. In this context, a great number of traditional herbs which have high anti-oxidative and anti-inflammatory capacities have been referred to as effective and might prove beneficial against CVDs. Objective: To assess the existence of cardioprotective effects in medicinal plants. Methods: This study is a systematic review with meta-analysis through the PRISMA method. Searches were conducted in the MedLine and LILACS databases, through the “Medicinal plants” and “Cardiovascular diseases” descriptors and their synonyms indexed in DeCS/MeSH. Randomised studies, case reports and observational studies published in the last 5 years in english and portuguese were included. Integrative reviews and articles without free full text available were excluded. Research resulted in 290 articles that were filtered for analysis by 4 reviewers. Results: The primary outcomes showed important effects of different medicinal plants over congenital cardiopathies’ treatment, highlighting its antiatherogenic potencial and reduction of cardiovascular risk factors, especially through lowering body mass index (BMI), waist circumference and cholesterol levels. Furthermore, as secondary outcomes, it was possible to observe a hypotensive action of Acacia longifolia, Urtica Dioica and Viola Odorata extracts, and the regularization of biomarkers and inflammatory response as a result of citrus flavonoids and polyphenols combination, which improved both lipid panel and flow-mediated dilation in studied patients. Moreover, the use of oils, such as Brazil nuts and coconut combined with epigallocatechin gallate, is effective against lipid peroxidation and decreases risk of CVDs. Conclusions: Medicinal plants are a great alternative for treating and preventing CVDs and might be used to develop future studies for promising medicines. 112393 Modality: E-Poster Scientific Initiation – Non-case Report Category: PSYCHOLOGY SUELLEN KEYZE ALMEIDA LIMA1, Dra. Antoinette Oliveira Blackman1, Beatriz Montenegro Oliveira1, Dra. Solange Lopes da Silva1, Adda Cecília Batista de Carvalho Vieira1, David Ricardo Bernal Lima Hernandez1, Pedro Henrique Alves Miranda1, Patrícia Brito de Almeida Borges1 (1) Centro Universitário do Planalto Central Apparecido dos Santos Introduction: Remarkable changes in the work situation have led to the increasing importance of psychomental and socioemotionally demanding conditions at work. Psychic distress at work can encompass stressful situations and numerous consequences that compromise the worker’s lifestyle, including cardiovascular risk. Objective: To understand the main triggers of uneasiness at work and relationships between psychic suffering in public security personnel and the risk of cardiovascular diseases among these professionals. Method: An integrative literature review was carried out in the databases BVS-PSI, Scientific Eletronic Library Online (SCIELO), PubMed/MEDLINE, Google Scholar, UptoDate, PePSIC. Results: This study analyzed the association between work-related behavioral characteristics and the main triggers related to illness, such as load, pace, schedule, work environment, among others. Final Considerations: We present reflections about behaviors adopted by the servers, as tools to their process of getting sick; we also point out aspects to be highlighted in prevention and assistance actions. And it links an immediate alert system for anticipation of care to the health of these servers, because they are getting sick. 109224 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JULIANA DE ALMEIDA SILVEIRA1, Sara Cristine Marques dos Santos1, Anna Júlia Tamiozzo Reis1, Girley Cordeiro de Sousa1, Juliana Alves Costa1 (1) Universidade de Vassouras Introduction: Among the congenital abnormalities, cardiac malformations are the most responsible for death, being the main cause of infant death in first world countries. Tetralogy of Fallot (TF) is the most frequent cyanotic congenital heart disease, with an incidence of 10% of congenital heart defects1. TF consists of four concomitant anomalies, namely: right ventricular outflow tract obstruction (RVOT), sub-aortic interventricular communication (VSD), aortic dextroposition associated with overriding over the interventricular septum and right ventricular hypertrophy. The importance of TF lies not only in its incidence, but also in the severity of symptoms and the effectiveness of surgical correction, especially when performed in childhood. Objective: To analyze the current panorama of Tetralogy of Fallot procedures and variants in children and adolescents performed in Brazil for 10 years and to correlate the current epidemiology with the results obtained. Methods: A systematic review of the literature and an observational, descriptive and cross-sectional collection of data on correction of Tetralogy of Fallot and variants in children and adolescents, available in DATASUS – Hospital Information System of the SUS (SIH/SUS) were carried out for a ten-year period – December 2011 to December 2021 – evaluating the value of public spending, complexity, mortality rate, deaths, permanence and character of care and articles available in Scielo and PubMed. Results: In the analyzed period, 2,777 hospitalizations were observed for the performance of correction procedures for Tetralogy of Fallot and variants in children and adolescents, representing a total expense of R$ 65,165,447.56. The total mortality rate in the 10 years studied was 10.44, corresponding to 290 deaths, with 2011 being the year with the highest mortality rate, 12.99, while 2018 had the lowest rate, 8.16. The Brazilian region with the highest number of hospitalizations was the Southeast with 1,265 hospitalizations, followed by the Northeast region with 627. The Midwest region had the highest mortality rate 20.29, while the Southeast region had the lowest rate, with a value of 7.67. Conclusions: The present study identified that the rate dropped from 8.89 in 2009 to 8.76 in 2019. It is worth noting that the Southeast region, despite having the highest number of hospitalizations, has the lowest mortality rate, evidencing the effectiveness of the services performed. 109230 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION CECÍLIA RODRIGUES VIANA 1, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Larissa Silva Ferreira1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) UNIVERSIDADE FEDERAL DO AMAPÁ (UNIFAP) Introduction: Systemic arterial hypertension (SAH) consists of a chronic increase in blood pressure to values greater than or equal to 140 mmHg for systolic pressure and 90 mmHg for diastolic pressure. It is considered a multifactorial condition, which presents important risk factors, such as obesity and sedentary lifestyle. It represents one of the most relevant conditions for the incidence of cerebrovascular diseases. Objective: To analyze the epidemiological aspects related to mortality from hypertensive diseases in Brazil, by region, from 2009 to 2019. Methodology: Cross-sectional epidemiological study of mortality from hypertensive diseases by Brazilian region, during the years 2009 to 2019, from the data analysis from the SUS Hospital Information System (DATASUS). Results: In the analyzed period, Brazil recorded 530,382 deaths from hypertensive diseases, with 29,149 in the North region (5.49%), 165,717 in the Northeast (31.24%), 228,553 in the Southeast (43.09%), 73,711 in the South (13.89%) and 33,252 in the Midwest (6.26%). In relation to deaths by race/color, white individuals were the most affected in Brazil (n = 250,330), however, when analyzing by region, changes in this situation are noted, since in the North region, the predominance was of pardos (66.45%), as well as in the Northeast region (62.2%) and in the Center-West region (48.07%). The Southeast and South regions followed the trend of Brazil with the predominance of the white population, corresponding to 57.39% and 84.85%, respectively. Regarding gender, in Brazil, women were the most affected (53.05%), however, when analyzing by region, there are differences in the North and Center-West regions, in which the male population had the highest rates., 52.83% and 51.56%, respectively. The Northeast, Southeast and South regions maintained the Brazilian trend and their female population was the most affected, representing 53.08%; 53.95% and 54.63% of the total, respectively. Regarding the age group, the population aged 80 years and over is the one that dies the most from hypertensive diseases (n = 222,829) in the country. This holds true in all regions, with the Southeast having the highest number of cases (39.17%), followed by the Northeast (35.44%), South (14.68%), Central-West (5.39%) and North (5.29%). Conclusion: It’s concluded that SAH mortality in Brazil presents a heterogeneous aspect when analyzed by region, since, generally, Brazilian prevalence trends were not maintained for all. 109231 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARIANA EVARISTO LEITE1, Domingos Alves de Santana Neto2, Sarah Mariani Rocha Oliveira2, Anna Mayse Feitosa da Silva2, Ana Beatriz Vaz de Araújo2, Katia Vergetti Bloch2 (1) Universidade Federal de São João del-Rei (UFSJ); (2) Universidade Federal do Rio de Janeiro (UFRJ) Introduction: Physical activity is defined as any bodily movement generated by the skeletal muscles that increase energy expenditure. During physical activity, the autonomic nervous system promotes changes in the cardiovascular system to compensate for the increased metabolic demand required during the effort, including increased heart rate and stroke volume. Resting heart rate usually decreases with physical conditioning, and the stroke volume increases. Therefore, heart rate can be a marker of physical conditioning that accurately assesses physical activity levels. Objective: The study aimed to analyze the association of resting heart rate with physical activity. We also tested the effect of sex and obesity on this association in Brazilian adolescents. Methods: The Study of Cardiovascular Risks in Adolescents was a cross-sectional, national, school-based study with adolescents aged 12 to 17 years old from public and private schools in municipalities with more than 100 inhabitants. An oscillometric blood pressure monitor assessed heart rate. Leisure-time physical activity was categorized according to the week volume (active ≥ 300 min and inactive = zero min). Obesity was classified by body mass index (BMI = weight/height2) according to age and sex. Data analysis was performed using Stata 15.0, estimating raw and adjusted linear regression models. Results: A total of 73,399 adolescents were evaluated, of which 49.6% were considered inactive (60.2% were girls and 36.9% were boys). The mean heart rate of the inactive group was 83.4 beats per minute, and that of active adolescents was 79.7 beats per minute, p < 0.001. Adolescents with obesity had higher heart rate than those without obesity (83.7 vs. 81.2 beats per minute, respectively; p < 0.001). Adjusting for age, sex and obesity modified the association of physical activity and heart rate (p < 0.001 and p = 0.03 for interaction, respectively). Conclusion: Active adolescents have lower resting heart rate than inactive ones, indicating a predominance of parasympathetic modulation instead of sympathetic modulation. These results suggest that the physical activity classification adopted in the study reflects the physical conditioning of adolescents and that sex and obesity modify the effect of the relationship between physical activity and heart rate. 109247 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM DANIELLA NUNES PEREIRA1, Maíra Viana Rego Souza-Silva1, Magda Carvalho Pires1, Lucas Emanuel Pereira Ramos1, Maria Aparecida Camargos Bicalho2, Fernando Anschau3, Yara Cristina Neves Marques Barbosa Ribeiro4, Milton Henriques Guimarães Júnior5, Luanna Silva Monteiro Menezes6, Neimy Ramos de Oliveira7, José Miguel Chatkin8, Milena Soriano Marcolino1 (1) Universidade Federal de Minas Gerais; (2) Fundação Hospitalar do Estado de Minas Gerais; (3) Grupo Hospitalar Conceição; (4) Hospital Metropolitano Dr. Célio de Castro; (5) Hospital Márcio Cunha; (6) Hospital Metropolitano Odilon Behrens; (7) Hospital Eduardo de Menezes; (8) Hospital São Lucas da PUCRS Introduction: Despite the evidence of lack of benefit, chloroquine has still been prescribed by some doctors to treat coronavirus disease 2019 (COVID-19) patients. Objectives: To describe and analyze the side effects and outcomes associated with the use of chloroquine and hydroxychloroquine in COVID-19 therapy. Methods: This retrospective cohort of a substudy of the multicentric cohort Brazilian COVID-19 Registry, which included consecutive COVID-19 patients, admitted to 37 hospitals in 17 Brazilian cities, from March to September 2020. A propensity score was used to select the sample, matched by age, sex, cardiovascular comorbidities and hospital. Chi-square or Fisher tests were used to compare categorical variables and the Wilcoxon test for numerical variables. Results: From 7,732 COVID-19 patients, 280 (89.7%) were treated with hydroxychloroquine and 32 with chloroquine (10.3%) and 515 were randomly selected as controls. Demographic data and comorbidities are shown in Table 1. During hospitalization, 3.2% of the chloroquine patients presented side-effects to the medication and 40.0% of them required therapy interruption. Electrocardiography abnormalities were more prevalent in patients using chloroquine than controls (36.5% vs 26.9%). With regards to patient outcomes, patients who used chloroquine/hydroxychloroquine had a higher frequency of admission in intensive care units (45.2 vs. 34.0%, p = 0.002) and mechanical ventilation (34.6 vs. 25.5% p = 0.008). Mortality was not significantly different between groups. Conclusion: Patients undergoing COVID-19 treatment with chloroquine/hydroxychloroquine presented higher frequency of intensive care unit and mechanical ventilation requirement than matched controls who did not use the medications. In-hospital death rates were similar. 109250 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY GIOVANA REZENDE FERNANDES COSTA 1, Jennifer Klassen Boeing1, Luís Antonio Zorzi Santin1, Nicole de Oliveira Santos1, Adriana Saito Jasper2 (1) Universidade Positivo (UP); (2) Associação Hospitalar de Proteção à Infância Dr. Raul Carneiro/Hospital Pequeno Príncipe (HPP) Introduction: Congenital heart disease (CHD) is the most common congenital disease among newborns (NBs). CHDs defined as critical, generally dependent on the ductus arteriosus’ permeability, account for around 20% to 25% of heart diseases. The definition of the clinical-epidemiological profile of these patients can help in proposals and optimization of treatments. Objective: Perform an epidemiological analysis of critical congenital heart diseases (CCHDs) in NBs admitted to the Neonatal Intensive Care Unit of a tertiary care hospital in Curitiba/PR. Methods: This was a retrospective study reviewing medical records covering the period from January 1st 2015 to December 31st 2019 of patients diagnosed with CCHDs who were admitted to the Neonatal Intensive Care Unit. Results: The sample consisted of 127 patients. The incidence of diagnoses found is represented in the graph. Sixty-five (51.2%) male and 62 (48.8%) female patients were identified. Regarding gestational age, 0.8% of NBs were born at less than 30 weeks, 7.1% between 30 and 33 weeks, 13.4% between 34 and 36 weeks, 47.2% 37 to 38 weeks and 31.5% over 39 weeks. Considering the clinical outcomes, 38.6% died, of which 73.5% of deaths were due to cardiogenic shock, 24.5% due to septic shock and 2% due to other causes. When relating the type of CCHD with the number of deaths, the mortality rate was higher in cases of hypoplastic left heart syndrome (p < 0.017) and tricuspid atresia (p < 0.027). Conclusion: Within our sample, the main diagnoses were hypoplastic left heart syndrome, coarctation of the aorta, transposition of the great arteries, tetralogy of Fallot, and pulmonary atresia, following what is detailed in literature. No level of significant correlation was found between the type of CCHD and gender and between the type of CCHD and gestational age at birth. There was a statistical significance in the correlation between mortality and the two heart diseases: hypoplastic left heart syndrome and tricuspid atresia, in most cases resulting in death. These data should be evaluated as opportunities to improve care for heart disease. 109256 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY CECÍLIA RODRIGUES VIANA 1, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Larissa Silva Ferreira1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) UNIVERSIDADE FEDERAL DO AMAPÁ (UNIFAP) Introduction: Congenital heart diseases (CHD) consist of macroscopic abnormalities of the heart or great vessels and constitute an important cause of infant death, death occurring in live-born children from the moment of birth to an incomplete year of age. These malformations can occur in the first eight weeks of pregnancy, in which the cardiac system begins its formation. The patient may have respiratory and circulatory failure as the main complications resulting from this pathology, which are decisive for the compromise of the child’s quality of life. Objective: To analyze the epidemiological aspects regarding infant death due to congenital heart disease in the North region, from 2016 to 2019. Methods: Cross-sectional epidemiological study of infant mortality due to congenital heart disease in the North region between 2016 and 2019, based on the analysis of data from the Hospital Information System (SIH) from the DATASUS database. Results: During the study period, 1,342 infant deaths from congenital heart disease were reported in the North region, which corresponds to 11.9% of the total number of deaths from this cause in Brazil (11,277 deaths). In 2016, the number of deaths was 301 children, in 2019 this figure was 349, which represents an increase of 15.95% during the years analyzed. Regarding deaths by color/race, it was noted that brown individuals were the most affected: 830 (61.85%), followed by declared whites: 354 (26.37%). With regard to maternal schooling, it was observed that mothers with schooling between 8 and 11 years are the most affected (37.77%) by this fatality. In terms of gender, males had a higher number of cases: 705 (52.53%) versus 630 (46.94%) throughout the analyzed period. Conclusions: Based on the data obtained, it can be concluded that the highest incidence of infant deaths from congenital heart disease in the North region occurs in mixed-race male individuals whose mother has between 8 and 11 years of schooling. There was also an increase in the number of cases between 2016 and 2019. 109257 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM EDUARDO BARROS SCHAUSTZ1, João Dario Martins de Mattos1, Andréa Silvestre de Sousa3, Emiliano Horacio Medei2, Denílson C. de Albuquerque1, Olga Ferreira de Souza1, Fernando A. Bozza1, Gabriel C Camargo1, Paulo Henrique Rosado de Castro1, Ronir Raggio Luiz2, Renata Moll-Bernardes1 (1) D’Or Institute for Research and Education (IDOR); (2) Federal University of Rio de Janeiro (UFRJ); (3) Evandro Chagas National Institute of Infectious Diseases (INI) Background: Myocardial injury has been associated with adverse outcomes and mortality in patients with Coronavirus disease 2019 (COVID-19); however, there are still important knowledge gaps regarding its pathogenesis and clinical implications. Aim: To assess the prevalence and mortality associated with myocardial injury in a large population of hospitalized patients with COVID-19. Methods: Consecutive adult patients hospitalized with COVID-19 were prospectively included in a multicenter registry (n = 3246). Troponin levels were measured in the first week of hospitalization (n = 1476) and normalized according to the 99th percentile upper reference limit (URL). Demographic, clinical, and laboratory data were collected, and a logistic regression model was used to quantify the association with the primary outcome. Results: Myocardial injury (troponin levels > URL) was detected in 353 cases (23.9%). Total in-hospital mortality was 10.9% (IC95%: 9,8% – 12,0%). The mortality rate was higher among patients with cardiac injury (22,7% vs 5.5%) and increased for each level of troponin (Figure 1). In-hospital mortality and myocardial injury were associated with older age, reduced oxygen saturation, diabetes, hypertension, heart failure, coronary artery disease, and chronic renal disease, but were not associated with sex. After adjusting for age and comorbidities, a multivariable logistic model showed a higher risk of death in patients with cardiac injury with an odds ratio of 2.99 (95% CI: 2,06–4,36). According to the model, age, oxygen saturation ≤93% and diabetes were also independent risk factors for mortality with COVID-19. Conclusions: Myocardial injury was detected in 23.9% of the patients hospitalized with COVID-19 and was an independent predictor of in-hospital mortality. Mortality rate increased for each higher troponin category level. Myocardial injury and mortality were also associated with age, reduced oxygen saturation and cardiovascular comorbidities. 109266 Modality: E-Poster Scientific Initiation – Non-case Report Category: ANTICOAGULATION BEATRIZ ROCHA DARZÉ1, Carolina Costa da Silva Souza1, João Victor Santos Pereira Ramos1, Queila Borges de Oliveira2, Mateus Santos Viana2, Eduardo Sahade Darzé2, Luiz Eduardo Fonteles Ritt2 (1) Escola Bahiana de Medicina e Saúde Pública; (2) Instituto Dor de Pesquisa e Ensino, Hospital Cárdio Pulmonar Introduction/Background: Fondaparinux is an effective and safe anticoagulant in the treatment of non-ST segment elevation acute coronary syndromes (NSTE-ACS), when compared to enoxaparin. However, due to the underrepresentation of the obese population in clinical trials, the application of these results to obese patients is still uncertain. Purpose: To compare fondaparinux to enoxaparin in the treatment of obese patients with NSTE-ACS. Methods: This is a retrospective cohort study including obese patients (BMI ≥ 30 kg/m2) admitted with a diagnosis of non-ST segment elevation myocardial infarction (NSTEMI) or unstable angina (UA) and treated with fondaparinux or enoxaparin at a tertiary hospital, between 2010 and 2017. The study compared clinical and laboratory characteristics between the fondaparinux and enoxaparin groups using the Chi-square and Mann Whitney tests, when appropriate. The incidence of the primary outcome (death, reinfarction, stroke or major bleeding) was compared between groups and a p-value < 0.05 was used for all analyses. The study complied with the Declaration of Helsinki and was approved by the institutional review board. Results: A total of 335 patients with obesity and NSTE-ACS were included, of which, 238 used fondaparinux and 97, enoxaparin. The mean age was 64 ± 12 and 52.5% were male. The prevalence of diabetes, hypertension, dyslipidemia, previous diagnosis of coronary artery disease, previous stroke and use of an invasive strategy were similar between fondaparinux and enoxaparin groups. The incidence of the primary outcome was 4.2% in the fondaparinux group and 5.2% in the enoxaparin group (p = 0.702). There were no significant differences between groups when the primary outcome components were analyzed separately. The incidence of death, stroke, myocardial infarction, and major bleeding in the fondaparinux and the enoxaparin groups were, respectively, 1.3% vs 3.1% (p = 0.251), 0% vs 1% (p = 0.117), 1.3% vs 3.1% (p = 0.251), 2.1% vs 0% (p = 0.355). Conclusion: In a sample of patients with NSTE-ACS and obesity, there was no difference in the occurrence of the composite outcome (death, stroke, reinfarction and major bleeding) between patients using fondaparinux or enoxaparin. Randomized trials are needed to confirm these findings. 109267 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JULIANA DE ALMEIDA SILVEIRA1, Sara Cristine Marques dos Santos1, Girley Cordeiro de Sousa1, Anna Júlia Tamiozzo Reis1, Yasmin Monteiro Mandu1 (1) Universidade de Vassouras Introduction: Interventricular communication (IVC) is defined as the absence of septal tissue, which allows communication between the ventricles. Among the congenital heart diseases, it is the most frequent, corresponding to about 35%. In Brazil, data indicate an incidence variation of 0.6/1000 live births and it is estimated that 28,000 new cases will appear per year, of which 23,000 will require surgical intervention. In view of the above, a more in-depth knowledge about this anomaly becomes, therefore, a subject of great interest not only for its high frequency, high number of hospitalizations and expenses for the health system, but also serves as an alert for the need of more comprehensive research in order to know the possible risk factors and make an early diagnosis. Objective: To analyze the current panorama of CIV Correction procedures performed in Brazil for 10 years. Methods: A systematic literature review and observational, descriptive and cross-sectional collection of IVC Correction data, available in DATASUS – SUS Hospital Information System (SIH/SUS) for a period of ten years – December 2011 were carried out. to December 2021 – evaluating the value of public spending, complexity, mortality rate, deaths, permanence and character of care and articles available in Scielo and PubMed. Results: In the analyzed period, there were 2,951 hospitalizations for IVC Correction procedures, representing a total expense of R$55,801,572.62. The Brazilian region with the highest number of hospitalizations was the Southeast with 1,228 hospitalizations. The region with the highest number of deaths was the Southeast with 79 cases, while the North region had the lowest number, with 6 deaths recorded. The North region had the highest mortality rate (8.96). The Northeast region had the lowest rate, with a value of 5.58. Conclusions: In view of the data, there is a higher prevalence of hospitalizations in the three most industrialized Brazilian regions (Southeast, Northeast and South). On the other hand, emergency procedures have the highest mortality rate. Furthermore, the highest mortality rate occurs in the regions with the lowest prevalence, the North and Midwest regions, and the deaths are higher in the Southeast and Northeast regions. Thus, there is a need to expand data collection in order to improve the current epidemiological analysis. 109272 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DANIEL DE OLIVEIRA MEIRELES 1, Sara Cristine Marques dos Santos1, Thaís Lemos de Souza Macêdo1, Ivan Lucas Picone Borges dos Anjos1, Patrícia Rangel Sobral Dantas1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: High blood pressure (HTN) and diabetes mellitus (DM) are two common diseases today. Complications of DM and HTN can include chronic kidney disease, cardiovascular, or cerebrovascular disease. The association of these diseases increases cardiovascular risk and morbidity and mortality1. The present study aimed to analyze the current panorama of cases of hypertensive and also diabetic patients in the state of Paraíba for 10 years and to correlate the current epidemiology with the results obtained. Methods: A systematic review of the literature and an observational, descriptive and cross-sectional collection of data on HTN and DM, available at DATASUS – System for registration and monitoring of (HYPERDIA) for a period of ten years – December 2002 to December 2012 – and articles available in Scielo, Lilacs, and PubMed. Results: In the analyzed period, 55,993 follow-up records of patients with HTN and DM were observed, of which 38,103 were female and 17,890 were male. Of the 55,993, 27,486 are considered sedentary, 19,008 of whom are women. Overweight in 25,635 cases. According to the age group, there is a greater number of cases from 55 to 69 years old, with an average of 7,461 cases (obtained through the arithmetic mean of the values shown by DATASUS). There are 14,569 smokers out of the total. Among the total cases, 5,222 patients with previous acute myocardial infarction and 6,513 with stroke were identified. 3,026 cases of diabetic foot were recorded. Counting 1,859 cases of amputation due to DM. Of 55,993 patients, 3,344 have chronic kidney disease. As for risk, 27,784 are considered high risk, 10,217 are very high risk and 17,992 have no calculated risk. Conclusions: From the present study, the prevalence of SAH and DM in women is higher than in men. It is worth emphasizing the need to invest in primary care to control and treat these patients, also preventing new ones. Besides, there is a need for correct notification of procedures, to improve the current epidemiological analysis. 109273 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LARA ALÍPIO PEDROSA1, Ariana Lacerda Garcia1, Beatriz Ribeiro Coutinho de Mendonça Furtado1, Carolina Gonçalves da Cunha Lima1, Gabriella de Figueiredo Falcão1, Laís Nóbrega Diniz1 (1) Faculdade de Medicina Nova Esperança (FAMENE) Introduction: Peripartum cardiomyopathy is a form of dilated cardiomyopathy of uncertain etiology, characterized by reduced left ventricular ejection fraction, which affects women without preexisting heart disease during the last month of gestation or until the fifth month of puerperium. The factors identified as the greatest risk for its development include advanced maternal age, black race, gestational hypertension and family history of cardiomyopathies. Clinical presentation includes signs and symptoms of congestive heart failure with tachycardia, tachydyspnea, edema, orthopnea, swollen jugular veins, and nocturnal cough. Objectives: To discuss the clinical manifestations of peripartum cardiomyopathy, in addition to the link between prolactin levels in the genesis of this cardiomyopathic condition. Methods: This bibliographic review had a qualitative character based on exploratory and selective readings of articles and scientific websites (Sciello, PubMed, UpToDate) referring to the topic in question. Results: The hemodynamic stress of pregnancy on the heart can trigger the clinical manifestation of genetic forms of previously silent cardiomyopathy. Studies show identified evidence of MCP carriers with a family history of cardiomyopathy and a subgroup of patients with mutations in genes related to familial forms of cardiomyopathy. New pathophysiological concepts were recently presented in the literature, involving oxidative stress and prolactin levels in the genesis of MCP. Oxidative stress activates cathepsin D in cardiomyocytes, an enzyme that promotes proteolytic cleavage of prolactin resulting in the 16 KDa fragment, which is a potent factor with pro-inflammatory, antiangiogenic, pro-apoptotic, vasoconstrictor and cardiomyocyte depressant characteristics. In agreement with these findings, inhibition of prolactin secretion with bromocriptine, a dopamine receptor agonist, prevents the development of MCP. Conclusion: Peripartum cardiomyopathy is a condition with several risk factors and without a defined cause. Treatment uses drugs for heart failure in general and the prognosis is still poor. With therapeutic advances, the mortality rate of peripartum cardiomyopathy has decreased, among these, those related to the study of the association of prolactin. Research with a view to clarifying the etiopathogenesis of this disease should be implemented, which may facilitate preventive measures. 109288 Modality: E-Poster Scientific Initiation – Non-case Report Category: ANTICOAGULATION BEATRIZ ROCHA DARZÉ1, Queila Borges de Oliveira2, Mateus Santos Viana2, Eduardo Sahade Darzé2, Luiz Eduardo Fonteles Ritt2 (1) Escola Bahiana de Medicina e Saúde Pública; (2) Instituto Dor de Pesquisa e Ensino, Hospital Cárdio Pulmonar Background/Introduction: The prevalence of obesity has increased in recent decades and, considering its association with a higher risk of Venous Thromboembolism (VTE) and Acute Coronary Syndromes (ACS), it is necessary to establish an anticoagulation regimen suitable for this group of patients. The obese population is historically underrepresented in clinical trials, so the effect of obesity on the efficacy and safety of antithrombotic treatment is still uncertain. Purpose: To evaluate the influence of obesity on the safety and efficacy of antithrombotic therapy in patients with ACS or VTE. Methods: This is a systematic review and meta-analysis, conducted according to the PRISMA methodology, using the MEDLINE/PubMed, Scielo, Cochrane, EMBASE and Lilacs databases. Articles characterized as randomized, cohort or case-control studies that compared the occurrence of clinical outcomes (mortality or bleeding) between obese and non-obese patients using parenteral anticoagulants for the treatment of ACS or VTE were selected. To assess the risk of bias, the Cochrane Risk of Bias Tool and the Newcastle-Ottawa Scale were used. Statistical analysis was performed using Revman 5.4 software with relative risk and 95% CI as analytical parameters. Results: Six articles, with a total of 40.939 patients, were eligible for this review, being 3 randomized clinical trials and 3 retrospective cohorts. Of the patients, 87.7% had ACS. The mortality rate was 2.6% (95% CI 2.2% –3.0%) in obese patients and 3.6% (95% CI 3.3% –4.0%) in non-obese patients, while the rate of major bleeding was 6.0% (95% CI 5.6% –6.4%) in the obese group and 6.2% (95% CI 5.9% –6.5%) in the non-obese group. The incidence of major bleeding was similar between groups with RR: 0.90, 95% CI: 0.77–1.04, p = 0.14. The outcome incidence remained comparable when studies were analyzed separately by anticoagulant: enoxaparin (RR: 0.87, 95% CI, 0.70–1.08, p = 0.21) or UFH (RR: 0.96, 95% CI, 0.79–1.17, p = 0.67). The mortality rate, measured by only 2 studies, was lower in obese patients, with RR: 0.71, 95% CI 0.59–0.87, p = 0.0007. Conclusion: In patients treated for VTE or ACS, rates of thrombotic complications, death and bleeding were comparable between obese and non-obese patients, regardless of the antithrombotic used. 109317 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY ANA LÍVIA GADELHA XAVIER DA NÓBREGA PEREIRA1, Beatriz Gadelha e Xavier1, Bianca Vasconcelos Braga Cavalvante1, Rayanne Alessandra da Silva Barreto1, Leticia Lacerda Burity1, Vinicius Vieira Leandro da Silva1 (1) Faculdade de Medicina Nova Esperança (FAMENE) Introduction: Hypertrophic cardiomyopathy (HCM) is a heart disease that causes anatomical changes in the cardiac structure, the main one being left ventricular hypertrophy. Mavacamten is a molecule capable of inhibiting myosin ATPase and aims to assist in the treatment of hypertrophic heart disease, reducing contractility, improving diastolic function and promoting a better energetic response in the myocardium. The drug was tested in preclinical studies, demonstrating safety and reduction of obstructive symptoms, in addition to improvement in left ventricular filling pressures. Objectives: This article aims to systematically review the existing literature with the results of the use of the drug mavacamtem for the treatment of hypertrophic cardiomyopathy. Methods: This is a systematic review of the literature indexed between 2018 and 2021, published in the Scientific Electronic Library and PubMed databases. Unfinished and inaccessible articles for free were excluded. Results: The clinical presentation of HCM can vary from asymptomatic to advanced heart failure. Its diagnosis is confirmed by echocardiography and/or vascular magnetic resonance imaging with the observation of left ventricular hypertrophy. Currently, HCM therapy is based on the use of beta-blockers as a first line in symptomatic patients and disopyramide as a second option. Mavacamtem was recently tested in phase three of a randomized double-blind placebo-controlled trial with 251 patients enrolled in the program, where 123 were selected for use of the drug. It was evidenced that 45 patients using mavacamten reached the primary goal against the 22 who reached the use of placebo. Patients using the medication had significant reductions in left ventricular outflow tract obstruction after exercise, a greater increase in pVO2, and improvement in symptoms and quality of life compared to those who did not use the drug. In addition, there was a NYHA class reduction in at least 80 patients. In addition, the drug proved to be tolerable and without important adverse manifestations, being similar to placebo. Conclusion: Currently, the use of macavamten must be associated with previously established pharmacological therapy, in an effective, safe and tolerable way, in order to act directly in the pathophysiology of the disease and to avoid or postpone the need for invasive processes in patients. However, additional studies are needed to determine the best therapy for patients with HCM. 109302 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS CAROLINA COSTA DA SILVA SOUZA1, Cláudio Marcelo Bittencourt das Virgens1, Mateus dos Santos Viana1 (1) Escola Bahiana de Medicina e Saúde Pública (EBMSP) Background: Faced with a patient with acute chest pain, the need to rule out the etiology of coronary artery disease (CAD) is substantial due to the high associated mortality. In a scenario of abundant and often unnecessary expenses in the medical field, the need for an initial reasoning to determine which patients need confirmatory tests is highlighted. Because of this, health professionals seek support in the specific characteristics of acute chest pain through the gestalt to determine the pre-test probability of each patient. In view of the wide applicability of this methodology, its validation becomes essential for medical practice. Purpose: To assess the accuracy of clinical judgment in interpreting specific characteristics of acute chest pain for the diagnostic prediction of CAD. Methods: A systematic literature search was carried out through PUBMED, EMBASE, LILACS, SCIELO and CENTRAL databases and manual search. The selected articles were evaluated using the QUADAS-2 tool, and the collected data were used for a qualitative and quantitative analysis of the data, with meta-analysis built according to the fixed effects model, considering the measures of sensitivity, specificity, odds ratio, and diagnostic odds ratio (DOR). The present research was formulated according to PRISMA methodology. Results: Two studies with low risk of bias and good applicability for this systematic review were selected, with a total of 487 patients evaluated in terms of coronary obstruction in an emergency setting. Studies’ results were comparable, with values of sensibility and specificity ranging from 0,49 to 0,47 and 0,52 to 0,66, respectively. The calculated DOR was 1.47 (95%CI, 1.02–2.11), p = 0.04. The McNemar test for heterogeneity was 6.91 (p = 0.009). Conclusion: The gestalt of acute chest pain in an emergency context is not a good accuracy method for the prediction of coronary artery disease. New research is necessary to reinforce these findings. 109346 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOGERIATRICS ELLEN BEATRIZ SOBRAL2, Carolina Jeronimo Magalhaes3, Maria Eduarda Borges Matias2, Sabrina Barreto Braga Pires2, Carolina de Carvalho Moury Fernandes2, Matheus Dantas Soeiro2, Jessica Myrian de Amorim Garcia1, Francisco Bandeira3 (1) Hospital Agamenon Magalhães (HAM); (2) Faculdade Pernambucana de Saúde (FPS); (3) Universidade de Pernambuco (UPE) Introduction: The reduction of muscle strength, mass and function, known as sarcopenia, has a significant association with physiologic aging process. As the world population has aged, multiple comorbidities such as heart failure (HF) and chronic kidney disease (CKD) are frequent in the elderly, which has correlation with inflammatory factors and sarcopenia. Sarcopenia influences quality of life, morbidity, and mortality. Objectives: Evaluate the screening of high risk for sarcopenia in elderly in-patients with CKD and HF. Methods: Cross sectional observational study from August 2020 to December 2021 in a tertiary cardiology center with in-patients ≥65 years. Diagnosis and classification (stage 1 to 5) of CKD was based on CKD epidemiology collaboration equation to estimate glomerular filtration rate (GFR). For this study we established a considered renal disfunction patients with GFR < 60 mL/min/1.73 m2. Sarcopenia screening was performed with Sarc-f and patients with score >5 were defined in a high risk for sarcopenia. Results: A total of 190 patients (53,6% female) were studied. Mean age of 72.9 ± 5.9 years. A total of 49,73% elderly patients had renal disfunction. Stage 3 CKD elderly patients were 31,6% of the population study; 8,9% stage 4; 8,4% stage 5. In elderly patients with both renal disfunction and HF, 36,8% had positive screening for sarcopenia with Sarc-f > 5. The presence of CKD (stages 3 to 5) and HF had no association with the prevalence of high risk for sarcopenia. Conclusions: The screening with Sarc-f in elderly patients with CKD and HF in this study has not shown a significant high risk for sarcopenia. However, screening Sarc-f has providing identification of in-patients at risk of developing sarcopenia, in addition to practicality and specificity in medical practice, which shows the importance of more studies and further analysis. 109543 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY GABRIEL FONSECA GOMIDE1, Jaqueline S Da Silva1, Bruna Rocha1, Tadeu Montagnoli1, Bruno Eduardo Dematté1, Rodolfo Maia1, Eliezer J Barreiro1, Gisele Zapata-Sudo1 (1) Universidade Federal do Rio de Janeiro UFRJ Introduction: Pulmonary hypertension (PH) is a chronic progressive disease characterized by extensive pulmonary vascular remodeling, leading to right ventricle (RV) hypertrophy and failure. Activation of adenosine receptor A2A (A2A-AR) can limit the progression of PH by attenuating vascular and cardiac inflammation and remodeling. This work aims to evaluate the effects of a new A2A-AR agonist, LASBio-1900, in cardiac and vascular dysfunctions on monocrotaline (MCT)-induced PH in male Wistar rats. Methods: After a single injection of MCT (60 mg/kg i.p.), twelve rats were randomly divided in groups and treated orally with either vehicle or LASSBio-1900. Hemodynamic parameters were obtained by Doppler echocardiography and intraventricular pressure measurement. Pulmonary arteries (PA) muscularization was determined through the percentage of the wall portion positively stained with alpha smooth muscle actin (α-SMA) relative to the total transversal area. Perivascular collagen in PA was evaluated using picrosirius red staining and interstitial fibrosis was measured by obtaining the total collagen area per arteriole area. Evaluation of inflammatory process in perivascular PA was performed by immunohistochemistry to iNOS content in as the ratio of stained area to the arteriole area. Results: PH promotes increased PA vascular resistance, since the ratio of pulmonary acceleration time to total ejection time (PAT/TET) changed from 34.35 ± 0.85 in control rats to 23.17 ± 1.51 in MCT and improved in MCT-LASSBio-1900 to 30.86 ± 2.74 (p < 0.05). LASSBio-1900 reduced RV hypertrophy, as it reduced from 55.4 ± 2.3 to 35.3 ± 5.9% (p < 0.05). PH increased RV systolic and diastolic pressures, 19.21 ± 2.04 and 4.73 ± 1.04 mmHg in control to 51.5 ± 5.2 and 11.9 ± 1.3 mmHg, respectively. LASSBio-1900 recovered RV function reducing systolic and diastolic pressures to 28.4 ± 4.0 and 6.51 ± 0.84 mmHg. The increase in wall thickness observed in MCT 61.70 ± 1.11 to 83.24 ± 1.82% was improved by LASSBio-1900 to 75.24 ± 3.46% (p < 0.05). Perivascular fibrosis in PH rats was 15.06 ± 1.34% and LASSBio-1900 reduced the PA collagen deposition to 8.7 ± 1.4% (p < 0.05). iNOS expression evaluated in control of 4.5 ± 0.3 and MCT of 19.0 ± 0.9% (p < 0.05) in iNOS protein content were observed in PH and LASSBio-1900 reduced to 8.7 ± 1.4% (p < 0.05). Conclusion: A2A-AR activation by LASSBio-1900 improved RV hemodynamics and pulmonary vascular structure in PH, indicating a new alternative treatment. 109378 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY BERNARDO BALSAN CAMILLO 1, Carolina Martinez Teixeira1, Rodolfo Balsan Camillo2, Bruna Rocha Mezzomo1, Giuseppe Morales Gentilini1, Joel Soares Dahne1 (1) Universidade Católica de Pelotas; (2) Santa casa de misericórdia de Porto Alegre Introduction: Among the causes of global morbidity and mortality, cardiovascular diseases are among the most prevalent. For the purpose of reducing cardiovascular risk, the use of cholesterol-lowering medications, such as Anacetrapib, has gained prominence in relation to other therapeutic options. Objective: Description of the importance of Anacetrapib in the reduction of cardiovascular events associated with dyslipidemia and atherosclerosis. Method: Systematic review (PRISMA) based on scientific literature from 2011 to 2019, published in PubMed databases. The descriptor used was “Anacetrapib”. Articles that show information about the choice of their choice were removed, and those whose content was not related to the proposed topic were removed. 249 titles were found. Of these, 15 were selected, 7 abstracts were read and 7 papers were selected for this study. Result: The results demonstrate that dyslipidemic patients undergoing cholesterol ester transfer protein (CETP) inhibitors, a key protein involved in reverse cholesterol transport, especially Anacetrapib, had a reduction in mortality associated with atherosclerosis and dyslipidemia. The reduction in mortality is due to the significant reduction in LDL, triglyceride and HDL levels in patients who used the drug as a single therapy. Furthermore, in patients on dual therapy, associated with statins, they have a greater ability to reduce cardiovascular events. Although other CEPTs have been analyzed, the only one that has so far not demonstrated adverse effects such as death from cardiovascular and non-cardiovascular causes was Anacetrapib. In addition to being able to be used in replacement therapy for statins, this medication can be used both in patients who do not reach LDL levels adequate for their risk, and in patients who do not tolerate statin therapy. Conclusion: Despite the occurrence of drugs that there are more studies on the subject, and ignoring the high cost factor, Anacetrapib is a drug that seems to reduce death from cardiovascular causes in patients with dyslipidemia or atherosclerosis, without causing significant adverse effects. 109369 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JOÃO PEDRO COSTA DOS SANTOS1, João Pedro Costa dos Santos1, Mariana Ranucci da Cunha1, Lucas Narciso Balchiunas1, Pedro Henrique Yukio Miyaji1, Henrique Thadeu Periard Mussi1 (1) Universidade Federal Fluminense Introduction: COVID-19 is a respitarory disease that has a physiopathology highly associated with thromboembolic disorders and complications. Some studies suggest that elevated d-dimer can predict poor prognosis in this patients, although cut-off levels are not consensual yet. Objetives: This study aims to correlate admissional d-dimer levels with the need for endotracheal intubation and mortality in COVID-19 patients. Methods: Retrospective cohort study with 142 patients with laboratorial diagnosis of COVID-19 admited at Hospital Universitário Antônio Pedro. We compared mean serum levels of d-dimer between patients submitted to mechanical ventilation or not and with mortality in this group, searching for the association of this variables, using the t-test for this purpose. Statistical analysis was performed in Microsoft Office Excel 2016 and Jamovi 1.6.23. Results: We enrolled 142 patients with laboratorial diagnosis of COVID-19, 53,5% were male, the mean age was 60,3 years, 62,6% were diagnosed with arterial hypertension and 37,3% with diabetes. The mean admissional d-dimer level in the whole population was 2289 ng/ml. In the patients that needed endotracheal intubation the admissional mean levels were 3275 ng/ml and 1809 ng/ml in those who were not intubated. Besides that, the mean admissional levels of d-dimer in the patients that died were 3556 ng/ml and 1834 ng/ml in those that did not have the same outcome. According to the t-test results, high d-dimer admissional serum levels were associated with the need of endotracheal intubation (Statistic = –3,52; p < 0,01) and with mortality (Statistic = –3,96; p < 0,01). Conclusions: This studie concludes that high admissional levels of the d-dimer showed association with poor prognosis in patiets diagnosed with COVID-19 in Hospital Universitário Antonio Pedro. More studies are needed to define a cut-off point and confirm the association between these variables. 109377 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GLÓRIA DE MORAES MARCHIORI1, Glória de Moraes Marchiori1, Daiane Soares de Almeida Ciquinato1, Braulio Henrique Magnani Branco1, Luciana Lozza de Moraes Marchiori1 (1) Universidade UniCesumar UniCesumar Introduction: Obesity and overweight are an important public health problem in society, due to their growth in all age groups and their association with various chronic diseases, especially arterial hypertension. The school constitutes an important environment in the configuration of the teaching staff’s reality of life and aspects related to the conditions and organization of the teacher’s work, which have repercussions on the health-disease process. The working conditions of teachers can contribute to the development of lifestyle-related diseases, including obesity and high blood pressure. High sedentary behavior has been associated with abdominal obesity, and high sedentary leisure breaks have been associated with lower chances of hypertension among public school teachers. Objective: The objective of the present research was to compare hypertension with the body mass index (BMI) in a sample of teachers. Methods: This is a cross-sectional study, part of a thematic project, part of a broader research called PRO-MESTRE. The project was approved by the Humana Research Ethics Committee, in which all participants were previously informed about its purpose and procedures and signed the Free and Informed Consent Form. Inclusion criteria included being elementary and high school teachers, being actively teaching and being in charge of a subject in class, working in the classroom for more than 12 months, in addition to not having had a leave of absence for >30 days in the 12 months previous. The sample of elementary and high school teachers, who answered the questionnaire with objective questions to verify hypertension. BMI (kg/m2) was calculated based on self-reported body weight (in kilograms) divided by height (in square meters), with the following classification: <18.5: low weight; 18.5 to 24.99: normal or eutrophic; 25.0 to 29.99: overweight; and 30.0: obese. The BMI calculation and classification followed the National Center for the Prevention of Chronic Diseases. Appropriate statistical tests were applied adopting a significance level lower than 0.05%. Conclusion: In the present study, there was a significant association between hypertension and BMI in the teacher population. These observations can help health professionals in the diagnosis of arterial hypertension in teachers and in the approach of anthropometric factors and other comorbidities in the clinical evaluation of these professionals, considering the lifestyle. 109393 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JOSÉ GUILHERME MELO SILVA1, Eduardo Fernandes de Oliveira1, Newton Pessoa de Oliveira Neto1, Ana Beatriz Estrela de Sá1, Giordana do Nascimento Nunes1, Victor Matheus Costa Cardoso1 (1) Centro Universitário Tocantinense Presidente Antônio Carlos- UNITPAC Objective: Congenital heart disease is considered the most common malformation and the leading cause of death among birth defects, despite being underdiagnosed in Brazil. The aim of this study was to describe the epidemiological profile of deaths that occurred due to congenital heart septal malformation from 2014 to 2019. Methods: This is a cross-sectional, descriptive and quantitative epidemiological study using data of deaths due to congenital heart septal malformation in the Northern region in the period 2014–2019, which were obtained through the computer department of the single health system (DATASUS). Discussion and Results: According to the age group, most cases of death due to cardiac septal malformation occurred in the first year of life, especially in the first month. Comparing the genders, females were slightly more prevalent than males. Comparing the races/colors, the prevalence occurred mainly in those who considered themselves as brown, followed by white, and thus, gathering more than 90% of the cases of deaths due to congenital malformation of the heart septum. Conclusion: It is evident, therefore, that the research findings point to the importance of an active search for congenital heart diseases in the neonatal period and a greater attention to socioeconomic groups that historically are more exposed. Considering that this is one of the main causes of death in the first year of life. In addition, more comprehensive and detailed research is needed to understand the risk factors and develop preventive plans for some of these disabilities. 109411 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY BRUNO CARDOSO SCHMOELLER1, Leonardo Sinnott Silva1, Italo Mattos Rinaldi1, Gabriel Fernando Elias dos Reis1 (1) Universidade do Sul de Santa Catarina (UNISUL) Background: Primary percutaneous coronary intervention (PCI) is currently the therapy of choice for the treatment of acute ST-segment elevation myocardial infarction (STEMI). However, randomized clinical trials have shown that when the door-to-balloon time exceeds 100 minutes, the benefits of PCI over fibrinolytic therapy is drastically reduced or even eliminated. Objective: To verify the association of door-to-balloon time with patients admitted in business hours and non-business hours. Material & Methods: 57 STEMI patients who underwent primary PCI between April and August 2021 were included. Results: The average door-to-balloon time (D2B) found was 132.16 ± 68.96 minutes. For better characterization, the D2B time was converted into a categorical variable and separated into two groups: below 90 minutes (26.30%) and above 90 minutes (73.70%). Patients undergoing primary PCI outside of business hours, at night and on weekends, had a higher prevalence of D2B time over 90 minutes (66.60%), with a statistical difference compared to patients admitted during business hours (33.40%) (p = 0,027). It is easily noticeable that the longer D2B time at night and on weekends are related to logistical issues. Conclusion: The average D2B time found is above what is recommended. It was evidenced that the delayed PCIs are mostly concentrated in non-business hours, which negatively affects the prognosis of treated patients. 109478 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH FRANCISCO ELTON COELHO DA SILVA FILHO1, Giuseppe Marques Alencar1, Lidia Lillian Santos Barbosa2, Antonio Maycon da Silva Sousa1, Carlos Eduardo Batista De Lima1 (1) Universidade Federal do Piauí – UFPI; (2) Centro Universitário UniFacid – UniFacid Objective: To estimate the total cost of Acute Coronary Syndrome (ACS) in the State of Piauí, between January 2011 and December 2020. Methodology: For the calculation of direct adirect cost we obtained the number and cost of hospitalizations for Acute Myocardial Infarction and Unstable Angina in Piauí, for the Unified Health System (SUS) through DATASUS/TABNET as well as the values related to angioplasties and revascularization surgeries. From this, data for the Supplementary Health System (SSS) were estimated based on specific annual coverage rates. To estimate indirect costs they were incorporated due to loss of productivity associated with early mortality and absenteeism in patients belonging to the occupied population. Results: The sum of the amounts of hospitalizations by ACS and the procedures performed in the period (total direct cost) was R$ 178,943,799.06 (U$ 35,189,136.92). The sum (total indirect cost) of the estimated costs for absenteeism and early mortality was R$ 1,176,271,322.60 (U$ 231,312,696.18). Therefore, the total estimated cost was R$ 1,355,215,121.66 (U$ 266,501,833.10). Conclusion: The estimated average annual cost of ACS in Piauí was R$ 135,521,512.16 (U$ 26,650,183.31) and showed a downward trend in the last five years of the period. 109414 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JARBAS RYGOLL DE OLIVEIRA FILHO1, EVA BRENDA SANTOS SILVA1, THIAGO EMANUEL RODRIGUES NOVAES1, TASSO KFURI ARAÚJO MAFRA1, JOSSIMARA POLETTINI1, GUSTAVO OLSZANSKI ACRANI1, SHANA GINAR DA SILVA1, RENATA DOS SANTOS RABELLO1, IVANA LORAINE LINDEMANN1 (1) Universidade Federal da Fronteira Sul (UFFS), Campus Passo Fundo, BRASIL. Introduction: The Coronavirus Disease 2019 (COVID-19) still represents a global health problem. It is known that such an infection, caused by Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV2), can progress to complex clinical conditions, including Severe Acute Respiratory Syndrome (SARS), requiring hospitalization in the Intensive Care Unit (ICU) and use of ventilatory support. It is also clear that patients with previous cardiovascular diseases, as well as other comorbidities, are at greater risk of developing severe symptoms and progressing to death. Objectives: To identify the prevalence of hospitalization in the Intensive Care Unit (ICU), use of ventilatory support and case evolution in adult patients diagnosed with COVID-19, carriers and non-carriers of chronic cardiovascular diseases (CVD). Methods: Observational study based on data analysis from the Influenza Epidemiological Surveillance Information System (SIVEP-Gripe) in Passo Fundo, RS. The study population consisted of SARS cases due to COVID-19, confirmed and reported from January 1 to December 31, 2020. Sociodemographic and health data were collected from individuals aged between 20 and 59 years. The use of ventilatory support, admission to the ICU and the evolution of the case (death/cure) were used as outcomes of interest, and the exposure analyzed was the diagnosis of CVD. The prevalence was verified with a 95% confidence interval (95%CI) of the outcomes and their distribution according to the predictor variable (chi-square test, admitting an α error of 5%). Results: The sample consisted of 574 participants, with a predominance of men (58.0%) and aged between 41 and 59 years (69.0%). The diagnosis of CVD was observed in 25.3%. The prevalence of ICU admission was 21.0% (CI95 18–24), use of ventilatory support was 65.1% (CI95 61–69) and 12.2% (CI95 9–12) died. A statistically significant relationship was found between CVD and ICU admission (29.0%, p = 0.002) and use of ventilatory support (77.4%, p < 0.001), but not between CVD and deaths (15.6%, p = 0.051). Conclusions: There was a higher prevalence of ICU admission and use of ventilatory support in adults with CVD, a fact that attests to the greater complexity of managing the COVID-19 patient with CVD. The relationship of angiotensin-converting enzyme 2 – the main gateway of SARS-CoV2 – which is also related to CVD, is evaluated. As for lethality, no statistically significant relationship was identified. 109446 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GUSTAVO FIALHO COELHO1, Mariana Moreira Vannier2, Tamara Tâmara de Souza1, Caroline Melo Jordão Reis2, Karla Santa Cruz Coelho1 (1) Universidade Federal do Rio de Janeiro campus Macaé; (2) Centro Universitário Serra dos Órgãos Introduction: The first confirmed death by COVID-19 in Brazil was on 12th March 2020, in the city of São Paulo. From then on, the country’s health system was affected by the pandemic with the occurrence of direct and indirect deaths. Therefore, an analysis of the pandemic’s impacts on other health aspects such as deaths from cardiovascular causes is necessary. Objectives: Analyze the pandemic’s impact on mortality from cardiovascular causes in Brazil, as a whole and at home, using death data from the Civil Registry in 2019, 2020 and 2021. Methods: The Civil Registry Portal was accessed on March 28th 2022 and death data from unspecific (UCC) and specific cardiovascular causes – acute myocardial infarction and cerebrovascular accident (CVA), in the years 2019, 2020 and 2021, referring respectively to the pre-pandemic, 1st year of pandemic and 2nd year of pandemic were collected. Results: When comparing the sequence of years there was a single reduction in deaths from infarction from 2019 to 2020 (–4.28%) with an increase in all other analyzed aspects. From 2019 to 2020 there was an increase in deaths from CVA (0.97%) and UCC (31.17%). While from 2020 to 2021 there was an increase in deaths from infarction (6.40%), CVA (3.55%) and UCC (15.94%). When analyzing deaths at home, there was an increase in all death causes; infarction (4.35%), CVA (26.67%) and UCC (69.89%) from 2019 to 2020 and infarction (4.16%), CVA (4.06%) and UCC (14.65%) from 2020 to 2021. There was also a more accentuated growth in deaths from unspecific than specific cardiovascular causes, especially in deaths at home. Conclusion: Deaths from different cardiovascular causes increased when comparing the pre-pandemic period to the pandemic period and persisted from the first year of the pandemic to the second, almost in its entirety. Deaths from unspecific cardiovascular causes and at home had a significant increase, showing a probable influence of the COVID-19 pandemic on the Brazilian health system. Thus, the pandemic’s evolution in different phases and the adaptation of the health system to it, demonstrated an influence on the number of deaths from cardiovascular diseases. New studies should be carried out, analyzing the deaths registered in registry offices and the hospital admissions data. 109430 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIELA GAMA ZAGNI JARDIM1, Antonio Carlos Eberienos Assad Filho2, Julia Resende de Oliveira2, David Ferreira de Lima Duarte2, Maria Gabriela Pimenta dos Santos2, Paola Pugian Jardim3, Andréa Vaospasse Cocco Faria3, Lílian Soares da Costa4 (1) IDOMED – Universidade Estácio de Sá, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil/Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (2) IDOMED – Universidade Estácio de Sá, Campus Cittá, Rio de Janeiro, RJ, Brasil/Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (3) Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (4) IDOMED – Universidade Estácio de Sá, Campus Vista Carioca, Rio de Janeiro, RJ, Brasil/IDOMED – Universidade Estácio de Sá, Campus Cittá, Rio de Janeiro, RJ, Brasil/Instituto Estadual de Cardiologia Aloysio de Castro/IECAC Background: It is estimated that the prevalence of resistant arterial hypertension (RAH) is 200 million individuals worldwide. Compared to controlled hypertensives, a higher arterial stiffness is observed, and the measurement of pulse wave velocity (PWV) has been, in addition to being a predictor of vascular stiffness, a prognostic marker and increased cardiovascular (CV) risk, especially in low/medium risk populations. Objective: Descriptively analyze the demographics and PWV findings of an RAH cohort. Materials and Methods: Prospective observational study of a consecutive cohort of RAH, who are part of a cohort of individuals at high CV risk (n 163), followed up at a tertiary cardiology unit in Rio de Janeiro. A questionnaire was applied containing sociodemographic data, CV risk factors, comorbidities, medications in use and therapeutic optimization, in addition to reviewing medical records to collect data from complementary exams performed within 12 months. In this group, casual systolic (SBP) and diastolic blood pressure (DBP), central aortic pressure, amplification index and PWV were measured. Results: Fifty-two individuals with RAH were evaluated. The mean age was 65.3 years (42–85 years) and 53.85% (n 28) were female. The most relevant data found were: 75% dyslipidemia, 75% confirmed coronary disease, 55.7% diabetes mellitus, 5.8% chronic kidney disease, 63.8% reported snoring with 25% untreated sleep apnea. In oscillometric analysis, the mean BP was 130.2 × 82.8 mmHg (SBP 91–220 and DBP 68 × 108 mmHg) and PWV 9.45 m/s (6.3–13.3 m/s). Clinical evaluation showed that blood pressure values were elevated by 28.8% for SBP and/or DBP (n 15) and 55.8% for PWV (n 29), using median values as a standard after adjustment for sex, age, and presence of additional risk factors. Conclusion: The data indicate PWV values similar to those of studies with a RAH population of the same age group, emphasizing that a statistically significant percentage presented high PWV values, even in patients with RAH and controlled BP in oscillometric measurements. Additional studies are needed to demonstrate that PWV can be an independent prognostic marker also in high CV risk individuals, which may contribute to the management and pharmacological adjustment of this RAH population. 109454 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY MARCIO CÉSAR RIBEIRO MARVAO1, Márcio César Ribeiro Marvão1, Mariana Kondo Obara2, Alice Barroso Guimarães2, Lucas Campos Maia3, Teresa Giovanna do Carmo Alves4, Danillo Monteiro Porfírio2, Maria Clara Hollanda Cecim4 (1) Instituto Evandro Chagas (IEC); (2) Universidade Federal do Pará (UFPA); (3) Universidade do Estado do Pará (UEPA); (4) Centro Universitário do Pará (CESUPA) Introduction: Among cardiovascular diseases, coronary artery disease (CAD) has been the leading cause of death in Brazil for half a century. Myocardial revascularization (RM) is the most performed cardiac surgery in the country, and aims to improve the blood supply of coronary arteries that have a narrowed or blocked portion, through the implantation of grafts, which can be performed with or without extracorporeal circulation (ECC). Objectives: To quantitatively analyze data on MR using ECC performed in Brazil from 2018 to 2021 and analyze the percentage rate of surgeries by region and national and regional morbidity and mortality rates. Methodology: Cross-sectional, observational and descriptive study, with data from the Hospital Information System of the SUS IT department (SIH/DATASUS) about MR with the use of ECC, including those in which 2 or more grafts were used, performed between 2018 and 2021. Results: There were 67,122 hospitalizations for the procedure studied, with a peak in 2019 (28.7%) and concentrated in the Southeast (42.9%) and South (31.9%) regions. The North region had the lowest value (3.9%). There were 4,003 deaths, with the Southeast region presenting the most expressive amount (39.5%), followed by the South region (32%). The North region presented the lowest numbers (5.5%). The national mortality rate was 5.64 in 2018 and 5.53 in 2019 (a drop of 1.9%), however, during the period of the pandemic, there was a recorded value of 6.51 in 2020 (an increase of 18% compared to 2019). Taking into account the relative mortality rate in the period, the Central-West and North regions had the highest indicators (10.6 and 8.48, respectively). Conclusion: There were 67,122 hospitalizations for coronary revascularization surgery, mainly in the Southeast and South regions, with the lowest rates in the North region. These data may be related to the availability of resources and the greater number of specialized centers in the richest regions. A total of 4,003 deaths were recorded, with the Southeast and South presenting the highest values, which may reflect the number of procedures being proportional to the number of deaths. However, when analyzing relative mortality, the Midwest and North regions led, which implies that there are other factors that increase mortality in these places. During the COVID-19 pandemic, there was an 18% increase in the death rate, suggesting that there may be a relationship between Sars-Cov-2 and CAD complications. 109492 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT IVAN CUOCO SAMPAIO1, Israel Figueira Lemos1, Ingrid Jade Muniz Wanderley1, João Lucas Silva Sales1, Juliana de Sousa Tavares1, Luiz Carlos Figueiredo Filho1, Luma Maria Favacho Bordalo1, Maria Eduarda Dantas da Veiga1, Mariana Lassance Maya Palheta1, Paula Larissa Baía Lima1, Rafael Augusto Silva Cabeça1, Ivan Cuoco Sampaio1 (1) Universidade do Estado do Pará Introduction: Cardiac insufficiency, also known as heart failure, occurs when the heart cannot pump strongly enough to make sufficient blood reach the entire body, causing several secondary effects on patients, including death. Recently, an increase in the number of cases and mortality related to this pathology has been observed, especially amongst the elderly, due to their vulnerability to this disease because of their age. Objective: Carry out a comparative analysis of death and hospital admission numbers on elderly people, caused by Cardiac Insufficiency in Brazil’s regions. Method: This is a quantitative-focused, retrospective cross-sectional study which uses the Brazilian public health system hospital database (“Sistema de Informações Hospitalares do SUS – SIH/SUS”), alongside ICD-10: Cardiac Insufficiency. The data searched refers to elder patients – 60 years or more – who were hospitalized or died between the years 2009 and 2019 in the five Brazilian regions. Results: Regarding the data collected, there was a slightly higher frequency of female hospitalization (50.5%) compared to male (49.5%). The Southeastern Region concentrated the highest number of hospitalizations (41.5%), followed by the South (23.6%), Northeast (22.7%), Midwest (7%) and North (5%). The age group that accumulates the highest number of hospitalizations is 70 to 79 years (36.9%), followed by 60 to 69 years (32.8%), finally, 80 years and more (30.1%). In total, 259.416 patients died due to Cardiac Insufficiency. The Brazilian region with most death rates was Southeast (47.1%), followed by Northeast (24.1%), South (18.1%), Midwest (5.9%) and North (4.8%) regions. The age group that concentrated the most mortality was 80 years or more (52.8%), followed by 70 to 79 years (29.2%) and 60 to 69 years (18.0%). Conclusion: According to the data collected, Southeast region has the highest number of admissions and deaths regarding Cardiac insufficiency, this happens due that region’s population density. Also, as patients grow older, more female patients suffer from this disease. It has been shown that even though the South has the 2nd highest number of hospital admissions, the Northeast possesses the 2nd highest mortality, possibly due to demographic density or poor infrastructure of the public health system, when compared to the South region. 109472 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LAURA MINELLI CANTOIA1, Isabella Molina Silva1, Cláudia Helena Cury Domingos1 (1) Universidade de Ribeirão Preto- UNAERP Introduction: Cardiovascular Diseases (CVD) are the leading cause of death in Brazil and worldwide. In Brazil, CVD continue to be the main cause of death and hospitalizations in the country, reaching the mark of 31% of deaths in the adult population. Risk factors for CVD have been in previous work from the Framingham Heart Study. In Brazil, the prevalence of these cardiovascular risk factors (CV) shows regional variation and an inverse relationship between socioeconomic status and cardiovascular mortality. Objectives: Due to the impact of CVD on public health, nationally and internationally, this study aims to know the variables that make up the CV risk of patients, in order to stratify them into risk groups, identifying the most vulnerable patients and those with greater need for care attention. Methodology: This is an observational, descriptive and transversal study. Data from medical records of patients being followed up at cardiology outpatient clinic of a secondary hospital in Ribeirão Preto, São Paulo, between 2019 and 2022 were included. Individuals aged between 49 and 95 years were included in the study. For CV risk stratification, the “Cardiovascular Risk Stratification Calculator” of the Brazilian Society of Cardiology was used, which allows inferring the patient’s prognosis over the next 10 years. Results: A total of 182 medical records of patients undergoing follow-up at the outpatient clinic were studied. For CV risk stratification, the following variables were used: Presence of Clinical or Subclinical Atherosclerotic Disease, Type 1 or Type 2 Diabetes Mellitus, Gender, Age, Arterial Hypertension, Smoking and Dyslipidemia. Of the study patients, 40 of them (22%) were classified as very high CV risk, 124 (68.1%) as high CV risk, 13 (7.1%) as medium CV risk and 5 (2.7%) as low CV risk. Conclusion: Cardiovascular risk stratification allows establishing a more assertive and individualized clinical approach. When analyzing the clinical profile of these patients, it is observed that most are at high risk for cardiovascular events and, therefore, will need a multiprofessional approach for adherence to lifestyle changes and prescription of adequate drugs, in order to reduce the chances of CV events in the next 10 years. 109490 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ÁDRIA RAYANE LIMA CASCAES1, Ana Josefina Gonçalves Salomâo2, Antônia Evelyn Albuquerque Costa1, Fernando Maia Coutinho3, Vinícius Queiroz Silva1 (1) Universidade do Estado do Pará UEPA; (2) Centro Universitário do Estado do Pará CESUPA; (3) Universidade Federal do Pará UFPA Background: Cardiovascular diseases are one of the main causes for mortality and hospitalizations worldwide. In Brazil this tendency started in the decade of 1960 and persists until nowadays. The increasing incidence of admissions by heart failure from 2008 to 2018 shows the importance of analyzing the scenario of morbidity in the north of Brazil. Objective: Review the epidemiological profile of heart failure morbidity between 2017–2021 in the north of brazil. Methods: Epidemiological and Ecological descriptive study. Data extracted from the Department of Informatics of the Unified Health System (DataSUS) through health information (TABNET), considering age, sex, race and year the patients receive care. Results: From 2017 to 2021, there were 50,264 hospitalizations for heart failure in the northern region of Brazil. The highest number occurred in 2018 (11,255), followed by 2019 (11,078) and 2017 (10,961), the lowest occurred in 2021 (8,176). Analysing distribution by sex, the predominance of admissions was among men with a range of 29,449 (58%), compared to women who total around 20,815 (41%), demostraiting that heart failure affects more in men’s health. The prevalence of the disease can be considered of great importance in the state of Pará, where the number of hospitalizations was the highest in the North, totaling 22,161 during the study period. Pará was not the one with the lowest mortality rate, obtaining about 10%, in comparison, the state of Acre had a much lower number of admissions (1,655) and the highest mortality rate among Brazilian states (17.95%). Ranging from younger than 1 to older than 80 years, elderly between 70 and 79 years old, followed by those between 60 and 69 years old, showed the higher incidence of heart failure hospitalizations. In opposition, the younger ones have the lowest values, and it was observed that hospitalizations begin to decline considerably from those under 30 years of age. In relation to color/race, the prevalence occurs in people self-declared to be brown (30,915) and to a lesser extent in indigenous people. Conclusions: The epidemiological profile identified a higher alarm on the populations of men, self-declared to be brown, elderly between 70 and 79 years and residents of the state of Pará. Therefore, these groups need to be a distinct focus of the health system attention to the prevention of this morbidity, for a more effective management of resources and better care for patients with their specificities. 109494 Modality: E-Poster Scientific Initiation – Non-case Report Category: ANTICOAGULATION LAURA CAETANO DE SÁ 1, Letícia Santos Dias Norberto Ferreira1, Alair Junio Rocha Arantes2, Hebatullah Abdulazeem3, Ishanka Weerasekara4, Raissa Carolina Fonseca Cândido2, Rodrigo Lanna de Almeida5, Samuel Rosa Ferreira2, Tati Guerra Pezzini Assis2, Thais Marques2, Isabela Muzzi1, Milena Soriano Marcolino1 (1) Telehealth Center, University Hospital, Universidade Federal de Minas Gerais; (2) Medical School and University Hospital, Universidade Federal de Minas Gerais; (3) Department of sport and health sciences, Technical University of Minich, Germany; (4) School of Health Sciences, College of Health, Medicine and Wellbeing, The University of Newcastle, Australia; (5) Laboratory of Respiratory Physiology, University of Brasilia Oral anticoagulation is the cornerstone treatment of several cardiovascular diseases, and due to its large applications, different e-health strategies have been proposed and implemented to support the management of such treatment. However, data on the effect of e-health-based anticoagulation management on clinical outcomes are very scarce. Therefore a summary of the best available evidence on the topic is warranted and very appropriate for the current moment, due to the explosion in adoptance of telehealth strategies during COVID-19 pandemic. Our goal was to systematically review the evidence on the impact of telemedicine-based management of oral anticoagulation therapy (OAT) compared to usual care (UC) on thromboembolic and bleeding events. Medline, EMBASE, Cochrane library, LILACS and Google scholar databases were searched from inception to September 2021 for randomized controlled trials (RCTs) comparing telemedicine to UC for OAT management in adult outpatients and reporting any of our outcomes of interest: total thromboembolic events (TTE), major bleeding (MB), mortality or time in therapeutic range (TTR). Risk of bias of included studies was assessed using the Cochrane risk-of-bias tool. Dichotomous outcomes were pooled as relative risks (RR) and continuous outcomes as mean differences (MD), using random-effects models. Regarding the results, twenty-seven RCTs were included, comprising 27 758 patients. Atrial fibrillation and venous thromboembolism were the most common indications for anticoagulation. Different telemedicine strategies were evaluated, such as electronic algorithms for anticoagulant dose adjustment, self-testing and self-management of anticoagulation with remote support, mobile applications for OAT management, among others. Telemedicine resulted in similar rates of TTE (9 studies, RR: 0.88 95% CI: 0.73, 1.06; I²: 0%), MB (9 studies, RR: 0.94, 95% CI 0.82, 1.07; I²: 0%) and mortality (10 studies, RR: 0.98, 95% CI 0.83, 1.17; I²: 0%), when compared to UC, and in slightly improved TTR (15 studies, MD: 2.76, 95% CI 0.37, 5.15; I²: 91%). Concluding, telemedicine-based management of OAT appears to be at least as good as UC when it concerns clinical outcomes, and may be slightly better in regard to quality of anticoagulation, although the confidence in those estimates was moderate or low. 109509 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ADRIANO LEITÃO DE ALMEIDA1, Ana Carolina de Sousa2, Carlos Eduardo Alexandre Silva2, Vivian de Lima Brabo1, Larissa da Silva Cambraia1 (1) Universidade do Estado do Pará; (2) Universidade Federal do Pará Introduction: Acute myocardial infarction (AMI) is defined as the presence of acute myocardial injury associated with the clinical manifestation of ischemia. In Brazil, from 1996 to 2016, the North region was the second region with the highest growth in the mortality rate from AMI. Objective: To describe the epidemiological data regarding acute myocardial infarction hospitalizations in northern region of Brazil between 2012 and 2021. Method: This research is a cross-sectional descriptive study, carried out under a quantitative approach. Data was collected on the website of Hospitalar information system (SIH), from Brazil’s healthcare information department, including the hospitalizations on patients living in north region from 2012 to 2021. This study evaluated the number of hospitalizations and its mean length of stay, which were compared by state, gender, age group, race/ethnicity and character of care. The data was analyzed using Microsoft Office Excel 2017 software. Results: During the period studied, there were a total of 45890 cases of hospitalizations for AMI, with emphasis on the year 2021, which represented about 13.23% of all hospitalizations, with a mean length of 8.2 days. The state of Pará held the higher hospitalization rates, with 19075 cases (41.57%). The highest number of hospitalizations for AMI occurred between 60 and 64 years old, with 7170 cases (15.62%), however, the longest mean length of stay was in the 65–69 age group, lasting 8.8 days. Men had more hospitalizations due to AMI than women, with 69% of cases, as well as a longer permanence time on hospital, with 8.3 days. In people of brownish color, representing 65.79% of hospitalizations for this disease. Regarding the character of care, urgency was the predominant one, with 86.77% of the total hospitalizations. Conclusion: Thus, this study found higher hospitalizations of AMI on brown men above 60 years old who lived in State of Pará, admitted on urgency care. The longer permanence time among advanced age men might suggest a higher morbidity on this group. During the pandemic, the number of hospitalizations was initially stable, however had a significant increase in 2021, which differ from other studies that reported a decrease on hospitalizations due to bigger demand on COVID-19 management, so that could be explained by the allocation of beds initially created for COVID when the incidence decreased, although this relation can be better evaluated on future studies. 109514 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIELA SILVA SANTOS1, Gabriel Barbosa Figueira dos Santos1, Rafael Alexandre Meneguz-Moreno1, Gilberto Andrade Tavares1, Viviane Correia Campos1 (1) Universidade Federal de Sergipe (UFS) – Campus Lagarto Introduction: Patients hospitalized with COVID-19 and heart failure (HF) have higher in-hospital mortality and complications. Besides, HF as a risk factor for severe SARS-CoV-2 infection could be a complication triggered by COVID-19. OBJECTIVE To analyze the complications developed during hospitalization of patients with COVID-19 and HF. Materials and Methods: It was a retrospective study based on review of electronic medical records of adult patients admitted to the Respiratory Disease Unit (RDU) of a unique hospital from northeast Brazil with RT-PCR reagent for SARS-COV2 and HF diagnosis according to the Framingham criteria. Patients were divided into two groups, one with acute heart failure (AHF) and the other with acute chronic heart failure (ACHF), regarding if they presented with HF for the first time or if they already had this diagnosis, respectively. We have analyzed the comorbidities, complications and mortality between groups and complications according to New York Heart Association (NYHA) functional class and mortality as a hard outcome. Results: The sample consisted of 77 participants, 59.7% (n = 46) male, age 67.1 ± 16.4 years, 33.8% (n = 26) with AHF and 66.2% (n = 51), ACHF. The AHF group had as previous comorbidities mostly diabetes (pdee = 0.019) and chronic lung disease (p = 0.017). There was no difference regarding complications and mortality among the groups. Those with NYHA classification IV and positive SARS-COV2 had an increased risk of developing shock (p = 0.001), cardiopulmonary arrest (CA) (p = 0.01), acute respiratory distress syndrome (ARDS) (p < 0.0001), bacteremia (p = 0.008), hemorrhage (p = 0.04), and liver damage (p = 0.04). In-hospital mortality rate was 39% (n = 30). Mortality was higher in patients who presented as complications: shock (p < 0.0001), CA (p < 0.0001), pneumonia (p = 0.009), ARDS (p < 0.0001), bacteremia (p = 0.0003), hemorrhage (p = 0.02), anemia (p = 0.02), cardiac arrhythmia (p = 0.03), and liver damage (p = 0.04). Conclusion: NYHA functional class IV HF and COVID-19 have a higher risk of developing complications and death during hospitalization, especially when they develop pneumonia, ARDS, shock, CA, anemia, hemorrhage, arrhythmia, liver damage, and/or bacteremia. 109698 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LARISSA DE OLIVEIRA BELTRÃO1, Beatriz Ximenes Bandeira de Morais1, Maria Letícia Garrote Bulhões2, Leticia Ferreira Leal3, Catiane Kelly Schaefer4, Isabela Torres Castro1, Vauma Garrote Bulhões Barros5, Rafael José Coelho Maia6, Maria de Fatima Nunes de Oliveira Mesquita6 (1) Faculdade Pernambucana de Saúde; (2) Universidade Estácio de Sá; (3) Universidade de Pernambuco; (4) Universidade de Santa Cruz do Sul; (5) Hospital do Coração de Alagoas; (6) Hospital Agamenon Magalhães Background: Acute Myocardial Infarction (AMI), is the major cause of death worldwide, having Coronary Artery Bypass Graft (CABG) Surgery as a therapeutic option. In 2020, with the outbreak of the COVID-19 pandemic, there was a decrease in the number of patients admitted for AMI symptoms, as well as in the number of CABGs. Objective: This study aims to demonstrate the impact of the COVID-19 pandemic changed the main indicators of hospitalizations of patients with AMI in hospitals of the Unified Health System (SUS) in Brazil, their length of stay, mortality rate, number of CABGs, correlation with sex, age and condition of the procedure and hospital stay, and if there is a tendency for return to normality. Methods: This is a descriptive, cross-sectional study conducted using data from SUS Data Transfer Service, about hospital admissions for AMI and CABGs performed between 2019 and 2021. We analyzed the numbers of admissions and procedures undertaken, as well as, percentage reductions in each year across subgroups. Results: 132,173 patients with AMI were admitted to SUS hospitals in 2019, decreasing by 23,95% in 2020, totaling 129,637. The following year it reduced by 13,25%, amounting to 125,916 patients (Tab1). This decrease was proportional between genders and was more prevalent between 50 and 70 years of age. Additionally, the amount of CABG decreased by 1.92% from 2019 to 2020 and 2.87% in 2021. It was accompanied by a decline in the length of hospital stay in 2020 (11,3 days), when compared to 2019 (11,85 days), followed by an increase in 2021 (11,6 days). Furthermore, there was a slight increase in the mortality rate of patients undergoing CABG, both elective and emergency. In 2019, this rate was 5.95, increasing to 7.11 in 2020 and decreasing to 6.84 in 2021. Conclusions: Hospitalizations for AMI continue to decrease at lower intensity, showing a reduction of almost 50% in the trend of previous year, which leads to belief in a full recovery, if this trend continues, in 2022 or 2023. In CABG, we have a continued decrease, however with signs of an imminent recovery in mortality rate and days of hospitalization. 109516 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY PEDRO HENRIQUE TORRES TIETZ1, Filipe Cirne1, Márcia Moura Schmidt1, Cristiano de Oliveira Cardoso1, Alexandre Schaan de Quadros1 (1) Instituto de Cardiologia do Rio Grande do Sul (IC-FUC RS) Introduction: The gold standard treatment for ST elevation myocardial infarction (STEMI) is primary percutaneous coronary intervention (pPCI), but the clinical impact of performing pPCI off-hours is controversial. Objectives: To compare characteristics and major adverse cardiovascular events (MACE) of STEMI patients submitted to pPCI performed off-hours versus on-hours in a reference cardiology center. Methods: Prospective cohort of patients who underwent pPCI for STEMI from 2009 to 2019 in our institution. Patients treated on- and off-hours were compared as to baseline characteristics and short and long-term events. Clinical events were assessed by Kaplan Meier survival curves and Cox regression analysis. A p value <0.05 indicated statistical significance. Results: 2560 patients were treated off-hours and 1876 patients treated on-hours. Most of the baseline characteristics were well balanced between the groups, including similar door-to-balloon times (70min. × 69min; p = 0.15). Patients treated off-hours had a higher thrombus burden (49.6% × 45.5%; p < 0.01). Radial access was more frequently used off-hours (62.1% × 57.6%; p = 0.01). Procedural success (95.7% × 96.4%; p = 0.21) and peri-procedural complications were similar between the groups(p = 0,97). The off-hours groups had higher rates of MACE at 30 days (10.2% × 8.5%; p = 0.04) and 1 year(15.4% × 13.1%; p = 0.03), as well as higher rates of death at 30 days(7.8% × 6.1%; p = 0.03) and 1 year(11.1% × 9.0%; p = 0.02). No statistically significant differences in ischemic events were seen. Conclusion: In our institution, clinical characteristics, door-to-balloon time, pPCI results and short and long-term ischemic events of STEMI patients treated on and off-hours were similar, but off-hours patients presented higher mortality. 109521 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO GUILHERME CUNHA DE ALMEIDA1, Bráulio Cruz Melo1, Emerson de Santana Santos1, João Victor Andrade Pimentel1, Beatriz Luduvice Soares1, Júlia Souza Diniz1, João Paulo Dias Costa1, Giovanna Medeiros Resende2, Irlaneide da Silva Tavares1, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa1, Joselina Luzia Menezes de Oliveira1 (1) Universidade Federal de Sergipe; (2) Universidade Tiradentes Introduction: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disease in the world. The diagnosis is made by echocardiography and cardiac magnetic resonance (CMR), and its genetic etiology is defined as pathogenic variations in sarcomeric genes causing left ventricular hypertrophy without any other plausible etiology. Methods: A prospective study was conducted with patients referred to our cardiogenetic outpatient service between January 2021 and March 2022. Patients who met the American Heart Association hypertrophic cardiomyopathy diagnostic criteria had DNA sequenced using Next-Generation Sequencing technique. Statistical analysis was performed using R software version 4.0.1. Objective: To describe the population of patients under investigation for HCM. Results: A total of 74 patients were referred and 52 (70.2%) met HCM diagnostic criteria and had a mean age of 49.7 ± 18.9 years and 71.2% were men. A family history of sudden cardiac death was present in 59.6% patients. Beta-blocking agents were the most commonly used medications (44.2%); 7.7% of patients reported having an implantable cardioverter-defibrillator (ICD). Palpitations were the most reported symptom (69.2%). Rest echocardiogram showed a mean septum wall thickness of 16.6 ± 6.6 millimeters. CMR showed a maximal wall thickness of 17.4 ± 7.4 millimeters. 38.4% of patients had a pathogenic variant in gene panel, 70% of those presented pathogenic sarcomeric variants and the 30% presented a pathogenic variant of a phenocopy. Conclusion: The present study corroborates previous findings. Genetic testing has enabled the distinction between HCM and its phenocopies and accurate genetic counseling for the patients’ families. 109522 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY JULIANA DE ALMEIDA SILVEIRA1, Sara Cristine Marques dos Santos1, Daniel de Oliveira Meireles1, Júlia Bardela de Oliveira1, Thaís Lemos de Souza Macêdo1, Ivan Lucas Picone Borges dos Anjos1, Isabela Costa Linhares1, João Paulo Norá Muñoz1, Anna Júlia Tamiozzo Reis1, Anderlúcia Côrrea Guedes1, Girley Cordeiro de Sousa1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Coronary angioplasty is a procedure performed by percutaneous coronary artery revascularization, decreasing myocardial ischemia. Technically, access is via a percutaneous artery, without the need for a thoracotomy. Stent device implantation was used most of the time. The present study aimed to analyze the current panorama of coronary angioplasty procedures with stent implantation performed in Vassouras – Rio de Janeiro, for 10 years and to correlate the current epidemiology with the results obtained. Methods: A systematic literature review and an observational, descriptive and cross-sectional collection of data on coronary angioplasty with stent implantation, available at DATASUS – SUS Hospital Information System (SIH/SUS) for a period of ten years – December 2008 to December 2018 – evaluating the value of public spending, complexity, mortality rate, deaths, permanence, and character of care and articles available in Scielo, Lilacs, and PubMed. Results: In the analyzed period, 1,675 hospitalizations were identified for the performance of coronary angioplasty procedures with Stent implantation, representing a total expenditure of R $ 8,086,346.37, with 2018 being the year with the highest number of hospitalizations (263) and the year of 2008 with the lowest number of hospitalizations (6). With 2018 as the year responsible for the largest amount spent during the period (R $ 1,173,571.59). Of these admissions, 1,025 were in private. Of the total procedures, 1,112 were performed on an elective basis and 563 on an urgent basis, with 1,675 being considered highly complex. The total mortality rate in the 10 years studied was 1.73, corresponding to 29 deaths, with 2011 being the year with the highest mortality rate, 4.17, with 8 deaths while 2015 had the lowest rate, 0,72, with 1 death. The mortality rate for elective procedures was 1.08 compared to 3.02 for urgent procedures. The average total hospital stay was 2.3 days. Conclusions: It can be observed, from the present study, that there was an increase in the performance of the coronary angioplasty procedure with Stent implantation in the period of 10 years, starting in 2008, accompanied by a reduction in the number of deaths, showing the treatment success. Besides, there is a need for the correct notification of procedures, aiming to improve the current epidemiological analysis. 109529 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY WALDEMIR FERRARI JUNIOR1, Leonardo Nunes Sanson1, Mariana Castro Pires1, Capitulino Camargo Junior1, Vitor Reis de Souza1, Rafael Fabiano Machado Rosa1, Júlio Pasquali Andrade1, Mirian Francine Favero1, Laura Peroni Baldino1, Diego Seibel Júnior1, Pedro Enrico Ventura1, Maiquel André Teixera1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre Introduction: Congenital heart diseases (CHDs) represent an important public health problem. Although its etiology is not well understood, the 22q11 deletion syndrome (22q11DS) stands out among its known causes, also known as DiGeorge syndrome/Velocardiofacial syndrome. Objective: The aim of this study was to verify the frequency of 22q11DS among patients with a conotruncal CHD. Methods: The sample consisted of a prospective and consecutive cohort of patients in their first hospitalization in a cardiology intensive care unit (ICU) of a pediatric hospital, during a period of 1 year. An evaluation form was filled out for each patient with the collection of clinical data. ALL of them also underwent high resolution karyotype and 22q11 microdeletion investigation using the fluorescent in situ hybridization (FISH) technique. The classification into conotruncal defect was performed by a collaborating cardiologist based on the results of echocardiograms and catheterizations, and also the surgical descriptions Results: Of all patients with CHD, 52 (25.1%) had a conotruncal defect. Thirty-two were male and their ages ranged from 1 day to 10 years (48% <1 month). The main reason for hospitalization was cardiac surgery (76.9%). The most observed CHD was tetralogy of Fallot (TOF) (40.4%). There were no cases of interruption of the aortic arch. Karyotypic abnormalities were observed in 5 patients (9.6%); however, none of them had 22q11DS. The analysis by the FISH technique could be performed successfully in 51 patients, and the 22q11 microdeletion was identified in 2 cases (3.9%) (both with TOF). Conclusions: The frequency of 22q11DS verified in our study was similar to studies that found values ranging from 4 to 15% and different from others that detected rates between 17 and 48%. These differences seem to be related, especially, with the method of patient selection adopted in the studies. Patients with 22q11DS often have complex heart defects and extracardiac malformations, which can have direct implications in their treatment (especially the surgical one) and prognosis. 109536 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LUÍS FILIPE RIBAS SOUSA1, Brenno Laerth Neves Sousa1, Gabriel Lino Ribas Sousa1, Rafaela França da Silva1, Yana Mara de Oliveira Coelho Mendes1, Elyanne dos Santos Gomes1, Marcus Vinicius Moreira Barbosa1 (1) Instituto Tocantinense Presidente Antônio Carlos de Porto Nacional – ITPAC Porto In December 2019, a city of China called Wuhan onset a new coronavirus infection, named Coronavirus Desease 19 (Covid-19), caused by Severe Acute Respiratory Syndrome-2 (SARS-CoV-2). It has affected more than 457 million people wordwide. The transmission occours person-to-person through close- range contact to respiratory particles. Although SARS-CoV-2 is willing to cause respiratory síndromes, It may cause cardiovascular complications, even in those who do not have past medical history. Thereby, it is intended to do a systematic review, using the PRISMA methodology to identify cardiovascular outcomes in pacientes infected with Covid-19. Scielo and Pubmed are the bases used in this review. Free access articles published in English and Portuguese were included, according to the purpose of the study. It was analyzed 1.817 pacientes infected by Covid-19, where 62.5% male with mean age of 58,8 years-old. According to the database, cardiac manifestations of Covid-19 happened in those who had previous comorbidy, like essential hypertension, diabetes mellitus, dyslipidemia or smoking and alchol using. Miocardical injury are the most common complications, ranging from 8.1% to 36.6%. Arrhythimias were found in 12.7% to 17,6% and myocardical infaction in 0,4% to 9,9%. From this, the studies were able to asses point of care susceptible to cardiovascular complications in people with Covid-19. Among them, men aged over 60 years-old, who chronicle cardiac deasease and white blood cells (WBC) contage ≤1,1 × 10^9 cells/L. It was pointed out that advanced age, especially in men, with risk factons, such as: smoking, diabetes mellitus, hypertension, dyslipdemia, chronic heart desease, chronic kidney desease with glomerular filtration rate <90l/day and WBC ≤1,1 × 10^9 cells/L. It all worsens patient’s prognosis. 109548 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES LUCAS LUCENA BEZERRA1, Guilherme Aragão Melo1, Bruna Ferreira Apolinário Costa1, Maria Júlia Pimentel de Albuquerque1, Cícero Samuel Tavares de Souza1, Lara Guedes Bezerra1, Mariana Diniz de Souza1, Natália Lisboa de Carvalho Wanderley Cavalcanti1, Michelle Alves de Farias1, Antonia Raiane Silva Claudino1, Isabella Cristina Oliveira Pacheco1, Diego Felipe Ferrão Pereira de Andrade Barros1 (1) FACULDADE DE MEDICINA DE OLINDA- FMO Introduction: In-hospital cardiac arrest (IHCA) is an emergency health issue that can lead to morbidity and mortality in a varied number of patients. In Brazil, there is not much data on IHCA found in the literature, but it is known that cases have a bad prognosis and low survival rates. For a better outcome for patients who need a good resuscitation at the IHCA, undergraduate students must go through adequate training to improve their knowledge in Advanced Cardiac Life Support (ACLS) when dealing with cardiac arrest. Objective: The main goal was to review and synthesize evidence on the following question: “What is the scientific evidence on the benefits of educational simulation interventions in advanced cardiology life support training in undergraduate students?”. Methods: This systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The literature research that was performed covered 3 databases (PUBMED, EBSCOHOST, AND ScienceDirect). The period of publication of the articles was from 2018 to 2022. Results: Among 350 articles retrieved, 9 papers were included and totalizes 1152 undergraduate students. Most studies used multiple-choice questionnaires to evaluate knowledge retention and cardiac arrest simulation or other skills tests to evaluate skills retention. The interventions, which were delivered in a variety of ways, have seen improvements such as: better patient outcomes, improved skills, and increased teamwork performance. In one of the articles, the pre-test score and post-test for ACLS knowledge had an 88% improvement. The performance of ACLS maneuvers that most follows the guidelines of the main world institutions, were in the high-fidelity simulation training scenarios. Conclusions: Simulation technology for resuscitation training as an innovative strategy for students, has brought great benefits to the ACLS learning process, helping bring better results in death prevention. However, more high-quality studies are needed, especially in Brazil. 109732 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIEL SOUSA SANTOS1, Danielli Oliveira da Costa Lino2, Ane Karoline Medina Néri3, Carlos José Mota de Lima2, Glauber Gean de Vasconcelos2 (1) Universidade federal do Ceará, School of Medicine.; (2) Hospital de Messejana Dr. Carlos Alberto Studart Gomes.; (3) Universidade de Fortaleza, Postgraduate Program in Collective Health Heart transplantation is a surgical alternative used to treat heart failure unresponsive to clinical treatment, being responsible for improving the expectation and quality of life of patients who present such a condition. After a cardiovascular event, patients and their families deal with numerous changes, including the consequences of the disease or its treatment on everyday functioning. Individuals assume behaviors for which they are not prepared, such as initiating sexual intercourse early, often due to anxiety about living quickly and intensely. The onset of early sexual activity increases vulnerability to sexually transmitted diseases, which interferes with post-transplant survival. Sexual activity is an aspect of quality of life that is important to many patients and partners who may be adversely affected by a cardiac event. Although healthy sexual activity integrates quality of life, few studies addressing this theme have been developed in heart transplant patients. To assess risky behaviors in sexual activity in individuals undergoing heart transplantation. Descriptive research with a qualitative approach, with the interview of 30 heart transplant patients treated at the outpatient clinic specializing in Cardiac Transplantation in 2018. It was observed that 25 patients (83.3%) did not receive guidance on sexual activity. In 66.7% of respondents, risky behaviors were present, such as not using condoms reported by 50% of respondents, having multiple partners reported by 17.9% and early return to sexual activity, contrary to the recommendation of the cardiac transplant team, by 7.1% of the evaluated individuals. We evaluated the presence of risky sexual behaviors among individuals undergoing heart transplantation and observed that most participants had not received guidance regarding sexual activity in the post-transplant period and that most individuals practiced risky sexual behaviors. The contribution of the transplant team in the guidelines related to sexual activity is shown in a very timid way in the post-transplant guidelines, which may be contributing to a considerable prevalence of risky behaviors in these individuals. Supporting post-transplant providing guidance regarding sexual activity proves a coherent decision as a humanized practice. The deleterious clinical consequences of sexual risky behaviors, such as exposure to infections, can lead to cardiac impairment and reduced life expectancy in these patients. 109590 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING LUCAS LUCENA BEZERRA1, Mariana Diniz de Souza1, Guilherme Aragão Melo1, Cícero Samuel Tavares de Souza1, Maria Júlia Pimentel de Albuquerque1, Bruna Ferreira Apolinário Costa1, Lara Guedes Bezerra1, Michelle Alves de Farias1, Natália Lisboa de Carvalho Wanderley Cavalcanti1, Isabella Cristina Oliveira Pacheco1, Antonia Raiane Silva Claudino1, Diego Felipe Ferrão Pereira de Andrade Barros1 (1) FACULDADE DE MEDICINA DE OLINDA- FMO Introduction: Despite significant improvements in cardiopulmonary resuscitation (CPR), sudden cardiac arrest is one of the leading causes of mortality. Nowadays, Ultrasound is a widely available tool that can be used to evaluate the presence of cardiac wall motion during cardiac arrest, which can be used to determine prognostic of the patient during CPR. We performed a systematic literature review of the existing evidence of point-of-care ultrasound (POCUS) in Cardiopulmonary arrest (CPA). Objectives: The main objective of this study was to systematically review and synthesize the published literature on the benefits of POCUS in CPA. Methods: The reporting of this study follows PRISMA guidelines (Preferred Reporting Items for Systematic Reviews and Meta-analyses). The literature research that we performed covered 3 large databases (PUBMED, EBSCOHOST and ScienceDirect). The research was conducted gathering the articles from 2018–2022. We performed a systematic search in those databases up to 03/03/2022. Results: Initially, the literature search identified 120 articles, EBSCOHOST: 36; SCIENCEDIRECT: 15 and PubMed: 69. Furthermore, the updated withdrawal of duplicate articles was 74, leaving 22 articles selected. Finally, after the last selection, 6 articles were included in our review Among 15 papers, 9 were included and totalizes 356 patients. As for the articles included in our review, POCUS was used in order to identify the cause of CPA and the return of spontaneous circulation. When identified early, it increases patient survival. Thus, achieving greater diagnostic accuracy associated with less neurological and cardiac damage. Despite this, 2 of them suggest that POCUS should not be integrated into the protocols, so as to prolong pauses in CPR. They all also point out that POCUS depends on operator training and experience. Likewise, in one of the reports the instrumentator was trained in a four-hour training session, which included a didactic lecture and hands-on POCUS instruction. After this training, paramedics are prescribed to operate and enter the exam. Conclusions: POCUS has the potential to be an important tool in CPA and could potentially reduce health care costs. We conclude that POCUS is a powerful tool to assess reversible causes of cardiac arrest. However, it must be used in a protocolized and efficient way so as not to cause damage. 109592 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LUANA DE OLIVEIRA RIBAS1, Luana de Oliveira Ribas1, Silvia Nazaré Braga Pereira1, Rebeca Bittencourt Jaqueira Rios1, Aline Goneli de Lacerda1, Claudio Tinoco Mesquita1 (1) Universidade Federal Fluminense- UFF Introduction: In today’s globalized world, the dissemination of Fake News in the medical field represents a great risk for the population’s health. With the COVID-19 pandemic and the increasing of fake news circulation, including those of a general interest of medical nature, this phenomenon took on gigantic proportions, dominating social media. This scenario is propitious for the emergence of conspiratorial theories, pseudoscience and other misinformative speeches that represent a risk for public health. In this context, the purpose of this research is to survey and map the circulation of Fake News through videos in Portuguese that correlate COVID-19 vaccines and heart attack available on YouTube, popular and easily accessible data. Objective: Evaluate the veracity of Portuguese-language videos on the YouTube platform after a search relating the COVID-19 vaccine to heart attack. The initial objective was to evaluate the first 200 videos found. Methods: The data extraction was made through the YoutubeData Tools software through “Vídeo Network”, by the union of the key-words “vaccine” and “heart attack” utilizing the crawl depth 0 to identify the actors (videos) of most relevance about the subject. Then, the generated GDG file was imported to the software Gephi 0.9.2 responsible for the network analysis and visualization. Lastly, the 200 more relevant videos were analyzed for their content and labeled as truthful or containing Fake News. Results: 200 videos in Portuguese on the YouTube platform have been analyzed, obtained through the GDF file generated by the YoutubeData Tools. Of these, only 171 were classified as “True” or “Containing Fake News”. Of the classified videos, 7 of them had fake news in their content, representing approximately 4.09% of the total videos analyzed. The authors of these videos almost always used persuasion strategies, such as the use of technical terms, doctoral degrees or the use of lab coats, as a way to convey a sense of trust. At the time of the survey, the total views of videos classified as “Containing Fake News” was 2,944,884. Conclusion: In the midst of the advancement of technology and disinformation detection tools, Fake News are still a reality in our daily lives and a threat to Public Health. Although most of the videos analyzed have a scientific basis, the existence of Fake News in the health sector can represent a serious danger to the general population, being therefore a topic of scientific and social relevance. 109594 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH CAMILA SILVA DE OLIVEIRA1, Luiz Felipe Façanha Ramos1, Cecília Rodrigues Viana1, Aurea Nathalia Gomes de Souza1, Bianca Paula Miranda Martins1, Vinicius Maciel Vilhena1, Larissa Silva Ferreira,1, Marcos Roberto Marques da Silva Júnior1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá (UNIFAP) Introduction: Phlebitis presents as an inflammation of the intima of the veins, being caused by mechanical irritation, chemical or bacterial infection. Deep Vein Thrombosis (DVT), according to the Brazilian Society of Angiology and Vascular Surgery, is a disease caused by blood clotting inside the veins at an inappropriate place or time. And thrombophlebitis, according to the Brazilian Ministry of Health, consists of the inflammation of a clot formed when there is a thrombosis. In Brazil, data from the Ministry of Health put the three pathologies together and point out that women are the most affected due to risk factors such as pregnancy and use of contraceptives. Objective: To analyze the epidemiological profile for phlebitis, in all age groups, in Brazil, from 2017 to 2021. Method: Epidemiological study with a cross-sectional design of hospitalizations for phlebitis, venous thrombophlebitis and venous thrombosis in the regions of Brazil from 2017 and 2021. The investigated data were extracted from the SUS Hospital Morbidity System (SIM/SUS), from the database of the Department of Informatics of the Unified Health System (DATASUS). Results: In the investigated period, a total of 205,029 were documented. hospitalizations for phlebitis, venous thrombophlebitis and venous thrombosis. In 2017, the total was 40,238. The sum of hospitalization rates was higher in the Southeast region, which has about 55% of total hospitalizations in the period from 2017 to 2021, followed by the South region with 21.21%, the Northeast region with 15.93%, the Midwest and North region with7.74%. In the distribution of age groups, below 1 year to 19 years old are the least affected age with 1.61%, from 20 to 59 years old with 57.20% and 50 years or older with 40.85% of the total number of hospitalizations. It is worth mentioning that women are the most affected with about 154.34% more than men. When crossing the data with color/race, about 21.99% of the total hospitalizations were not identified, indigenous and yellow people with 1.97%, white people with 41.13% and black and brown people with 34% of cases registered. In total, 5292 deaths were recorded. Conclusion: Through the analyzed results, it was concluded that there was an increase in the rate of morbidity and mortality from phlebitis, venous thrombophlebitis and venous thrombosis in the period from 2017 to 2021. A higher prevalence was observed in white women aged 30 to 80 years. 109598 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY CLARA DE ASSIS KAROLINE OLIVEIRA1, Clara de Assis Karoline Oliveira1, Adilson Lima dos Santos Júnior1, Marcus Vinicius Guerra Canto1, Thácia Kiara Beserra de Oliveira2, Joelmir Lucena Veiga da Silva2 (1) PRODIIC- Faculdade de Medicina de Olinda; (2) DOCENTE – Faculdade de Medicina de Olinda Introduction: The cardiovascular diseases are evaluating due to risks and prevalence in world, as hypertension. The calcium-channel blockers (CCB) antihypertensives agents inhibit calcium-channel in the membrane of the vascular smooth muscle cells, it reduces calcium availability into cells impairing muscle contraction and decreasing vascular peripheral resistance. The flavonoid kumatakenin (5-hydroxy-2-(4-hydroxyphenyl)-3,7-dimethoxychromen-4-one), isolated from species vegetal Solanum paludosum, presented vasorelaxant effect in vitro by blocking the calcium channel (CaV). Thus, the researching by news drugs is a continuous process and natural products are a putative resource of medicines. Aim: To analyze the conformers and binding sites of kumatakenin on voltage-gated calcium channel by molecular docking. Methods: A quantitative and experimental research with an in silico approach that used the kumatakenin (CID 5318869) and nifedipine (CID 4485), standard drug, ligands from PubChem. To docking studies, atomics coordinates for the CaV, a structural basis of Ca2+ selectivity of a voltage-gated calcium channel, were retrieved from Protein Data Bank (ID 4MS2). The output conformers from Dockthor (GMMSB version 2.0), a receptor-ligand docking program, were ranked in order of increasing affinity with the protein. The affinity bonds (Ab) were obtained and compare using Test-t, where values p < 0.05 were significant. Results: There were three conformers that showed greater binding affinity with CaV and kumatakenin or nifedipine. The kumatakenin presented Ab of –8.353, –8.304 and –8.283, as the nifedipine of –8.021, –8.024 and –8.049. The comparing Ab into kumatakenin and nifedipine revealed significant difference (p < 0.05), showing that the flavonoid had more affinity than standard drug. The flavonoid conformer with the highest binding affinity occupies the most central region of the constriction, occupying a larger volume and justifying the events of greater amplitude of CaV blocker and greater reduction in the calcium conductance. The types of interaction involved in the connection between the three best docking solutions (flavonoid or nifedipine) and the CaV could be polar interactions, as salt bridge and carbon-hydrogen bond. Conclusion: The data confirm the blocking of voltage-gated calcium channel by kumatakenin in molecular insight, a putative CCB. It showed most affinity than nifedipine, a CCB antihypertensive. 109603 Modality: E-Poster Scientific Initiation – Non-case Report Category: PHYSIOTHERAPY ANNA JÚLIA LEAL RODRIGUES1, Heloisa Balotari Valente1, Lara Júlia Montezori Costa1, Felipe Ribeiro1, Carolina Takahashi1, Laís Manata Vanzella1, Luiz Carlos Marques Vanderlei1 (1) Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) Introduction: During cardiac rehabilitation programs (CRP) sessions patients may present signs and symptoms, which may be manifest prior to major adverse events, such as acute myocardial infarction and sudden cardiac death. Considering that high waist circumference is associated with a higher risk of major adverse events, it is important to investigate the relationship between waist circumference and the occurrence of signs and symptoms in CRP, since it may contribute to better conditions of cardiac risk stratification and to increased patient safety. Objectives: To investigate the relationship between waist circumference and the occurrence of signs and symptoms in CRP. Methods: 68 participants (65.81 ± 11.67 years) from an exercise-based CRP, diagnosed with cardiovascular disease and/or cardiovascular risk factors were included. Waist circumference was measured using a tape measure at the smallest point between the lower ribs and the iliac crest. The occurrence of signs and symptoms was assessed during 24 CRP sessions. The following signs and symptoms were assessed at the end of each step of the CRP session (initial rest, warm-up, resistance, and relaxation), and counted by session: arrhythmias, increased systolic (SBP) (>200 mmHg) and/or diastolic (DBP) (>120 mmHg) blood pressure during the exercise, tachypnea, pallor, angina, cramp, muscle pain, fatigue, dizziness, and nausea. Pearson’s correlation or Spearman’s correlation were used to evaluate the relationship between waist circumference and the occurrence of signs and symptoms, depending on the normality of the data (Shapiro-Wilk). The statistical significance level was set at <0.05. (CAAE: 79213417.0.0000.5402). Results: The average waist circumference found for the sample was 95.82 ± 9.72 centimeters (minimum: 70 centimeters; maximum: 119 centimeters). In total, 528 signs and symptoms were observed, which generated an average occurrence of 1 sign and symptom for each 3.09 hours of CRP session. No significant correlations were observed between waist circumference with total signs and symptoms (r = 0.111; p = 0.37), and with the main signs and symptoms observed in the 24 sessions: arrhythmias (r = 0.139; p = 0.26), muscle pain (r = 0.114; p = 0.35) and fatigue (r = –0.110; p = 0.37). Conclusion: Waist circumference is not related to the occurrence of signs and symptoms in CRP. 109610 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ISABELA ARAGÃO COLARES1, Mariana Salles Ballalai1, Helena Raquel Nogueira de Oliveira1, Gabriel Sousa Santos1, Renata Pinheiro Martins de Melo1, Gabriel Coelho Brito Dias1, José Levi Tavares Cavalcante1, Ane Karoline Medina Néri2, Weiber Silva Xavier3, João Luiz de Alencar Araripe Falcão1, Sandra Nívea dos Reis Saraiva Falcão1 (1) Universidade Federal do Ceará, Faculdade de Medicina, Curso de Medicina; (2) Universidade Federal do Ceará, Faculdade de Medicina, Hospital Universitário Walter Cantídio; (3) Programa de Educação em Reanimação Cardiorrespiratória Background: Abdominal Aortic Aneurysm (AAA) is a dilatation of that artery when its normal limit diameter of 2 cm is exceeded by 50%. It is considered a lethal event because of its risk of rupture, therefore Endovascular Aneurysm Repair (EVAR) is considered to be an important treatment for this condition, as it is indicated in the treatment of ruptured aneurysms and in elective endovascular procedure, if anatomically viable, according to the August 2016 recommendations of Brazilian Guideline for the treatment of AAA. Objectives: Observe the aspect of health care in the performance of EVAR, as well as in each Brazilian geographic region by verifying the data on hospitalizations, average hospital stay, mortality rate and death toll. Methods: The data available at DATASUS platform, provided by the Hospital Information System (SIH), from August 2016 to January 2022, were quantitatively and descriptively analyzed. Results: The EVAR with straight or conical endoprosthesis, of this period, performed electively, represents 135 hospitalizations, with a mean stay of 9.9 days, with a mortality rate of 5.10 and 7 deaths. As for the urgent care, there were 303 hospitalizations, with an average length of stay of 8.5 days, a mortality rate of 9.9, and 30 deaths. Furthermore, it was possible to verify the following information about each geographical region: the South had the highest number of hospitalizations, with 202 patients, and a mortality rate of 13.52, while the North had the lowest number of hospitalizations, 10 in total, and the second highest mortality rate, 14.82. Still, the Midwest Region showed the highest mortality rate, 15.12, among the 20 hospitalized patients. The Southeast presented 167 hospitalizations for the procedure in question, with a mortality rate of 13.02. The Northeast, with 42 hospitalizations, had a mortality rate of 13.03. Conclusion: EVAR is recommended for reducing early morbidity and mortality and is a technique that can be increasingly improved, as well as the care after the procedure. Moreover, it has a significant mortality rate in emergency treatment compared to elective due to the patient’s clinical condition, which is directly related to the anatomical evaluation and success of EVAR. Additionally, the differences in mortality rates in each Brazilian region may be related to the character of the care, the clinical status and recovery of the patient, as well as the existence of local reference services. 109635 Modality: E-Poster Scientific Initiation – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE VITHORIA VIDOTTI NEVES1, Julio Cesar Tolentino Junior1, Victor Pacheco Zanela Monte1 (1) Hospital Universitário Gaffrée e Guinle Introduction: Depression is associated with greater morbidity and mortality in hypertensive patients. Then, it is important to know potential protective factors for depression in these individuals. Studies have demonstrated that spirituality protects against depression in the general population. Therefore, we hypothesized that spirituality would be a predictor of depression. Additionally, higher spirituality could be related to less depression severity in this population. Objectives: 1) Investigate spirituality as a predictor of depression among hypertensive patients; 2) Analyze the association between spirituality and depression severity. Method: This cross-section study was carried out in hypertensive outpatients at a University Hospital. Depression was diagnosed by DSM-5 criteria. The Patient Health Questionnaire-9 evaluated depression severity. Spirituality was assessed through the Functional Assessment of Chronic Illness Therapy -Spiritual Well-Being (FACIT-Sp). In this widely validated questionnaire, total scores range from 0 to 48, with higher scores indicating higher spiritual well-being (SWB). With SPSS 25®, continuous variables were analyzed using the t-test and linear regression. The binary logistic regression model was performed to predict depression using sex, age, religious affiliation, and FACIT-Sp score as predictors. Data were presented with their respective 95% confidence intervals (95%CI) and odds ratios (OR), considering a significance level of 5%. Results: We included 150 hypertensive outpatients. The age ranged from 28 to 80 years (53.5 ± 14.9), and 69.3% were female. The prevalence of depression was 29.3%. The mean FACIT-Sp score was significantly lower in depressive patients than those without depression (39.5 and 30.2, respectively; p < 0.001). The logistic regression model was statistically significant [χ2(2) = 34.0, p < 0.001] and the Wald test indicated that female sex (OR = 3.884; 95%CI: 1.231–12.253; p = 0.02) and FACIT-Sp score (OR = 0.882; 95%CI: 0.836–0.930; p < 0.001) added significantly to the model. Then, SWB was the strongest depression predictor. The FACIT-Sp has a significant negative association with the PHQ-9 (F(1,148) = 52.205; p < 0.001; R = 0.51; R2 = 0.261). Conclusion: Spirituality was considered a protective factor for depression in hypertension outpatients. In addition, the higher SWB was associated with lower depression severity. These results highlight the importance of spirituality in the approach of hypertensive patients. 109638 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUIZ FELIPE FAÇANHA RAMOS1, Karen Tássia Façanha Ramos1, Hildeman Dias da Costa2, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia Introduction: Heart failure (HF) is characterized by complex syndromes that compromise tissue metabolic demands. There are about two million new diagnosed cases of HF per year in the world, constituting the leading cause of hospital admission in the elderly in Brazil. In this sense, it impacts on the increase in the costs of treating patients, thus being an important public health problem. Objective: To trace the epidemiology and treatment costs of HF from 2017 to 2021 in the northern region of Brazil. Methodology: This is an analytical ecological study of time series and geographical distribution about hospitalization cases and HF treatment costs in the northern region of the country, using secondary data by place of residence (from 2017–2021) from the Department of Informatics of the Unified Health System (DATASUS), through the Hospital Information System and the Hospital Morbidity System, for the evaluation of frequencies, case rates and association factors. Results: In the period 2017–2021, there were 52,963 hospitalizations for HF treatment in the North region of Brazil, which represented a cost of BRL 78,913,522.84 (with an average cost of BRL 1,489.97 per hospitalization) and with a growing trend in average spending on these services (1,658.32 in 2021). There was a drop in these hospitalizations in the period, and the State of Pará and Amapá, respectively, had the highest (42.14%) and lowest (2.2%) percentages of hospitalizations. In the region, there was an average length of stay of 8.2 days of hospitalization (ranged from 8.1 to 8.4 days) and a mortality rate of 11.97% (n = 6,319), with the year 2019 had the highest number of deaths (n = 1,415). The state of Roraima had the highest mortality rate from HF treatment (16.09%). It is noteworthy that associated factors that were more expressive are people of mixed race (n = 31,456), aged between 70 and 79 years (n = 12,608) and males (n = 29,921). Conclusions: During the study period, HF had a high number of hospitalizations in the northern region of the country, with an increase in average treatment costs over the years, with the highest average cost in 2021, although the number of hospitalizations has decreased. It is worth noting the State of Pará with the highest number of hospitalizations and the State of Roraima with the highest mortality rate due to HF, and 2019 was the most lethal year for HF. Finally, it was noticed that HF affects more brown men aged 70 to 79 years. 109642 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FELIPE SALIM HABIB BUHAMARA ALVES NASSER GURJÃO1, Felipe Salim Habib Buhamara Alves Nasser Gurjão1, Mateus de Sousa Cavalcante1, Bruna de Almeida Freixedelo1, Dara Medeiros Mendes1, Benedito Mesley Lima Portela1, João Marcos de Fontes Carneiro1, Daniel Salmito Chaves1, Paulo Roberto Matos Neto1, Luís Eduardo Rodrigues Reis1, Leandro Cordeiro Portela1 (1) Universidade Federal do Ceará – Campus Sobral Introduction: The third heart sound (S3) corresponds to a protodiastolic gallop, which results from the ventricular resistance to the rapid filling phase of diastole. In heart failure, S3 is a marker that indicates systolic dysfunction. Thus, the presence of S3 may be an important feature in the diagnosis and prognosis of patients with heart failure. Means: In this study, the objective is to analyze the relevance of the presence of S3 during hospital admission as a marker of mortality in patients at Hospital do Coração in Sobral with heart failure. Methodology: This is a prospective cohort study obtained with primary data, using as a sample patients admitted to the Hospital do Coração de Sobral with heart failure between the years 2015 to 2019. Thus, at moment of hospital admission the patients who participated in the study had auscultation performed, in order to determine the presence or absence of S3. Then the data was correlated with the in-hospital mortality of these patients. The informations were submitted to statistical analysis using the Chi-Square test, using the OpenEpi program, with a significance value of p = 0.0001. Results and discussion: This study obtained relevant data from 314 patients during this period. From those pacientes, only 21 (6.68%) had S3 at the time of hospital admission, from which 7 (2.23%) died. On the other hand, from the other 293 (93.31%) patients who were denied the presence of S3 at hospital admission, only 16 (5.09%) died. Thus, the risk of death in patients who were admitted with the presence of S3 in auscultation was 33.33%, while the risk of patients who were not admitted with S3 was 5.42%. Therefore, patients admitted with the presence of S3 on auscultation are 6.1 times more likely to die than patients without the presence of S3 during hospital admission. Conclusion: Based on the data analyzed, it can be seen that the presence of S3 during hospital admission of patients with heart failure may be an important marker of in-hospital mortality. Thus, larger studies on the subject are necessary. 109647 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ORLANDO SEIXAS ROCHA NETO2, Orlando Rocha Seixas neto2, Ellen Magnavita Seixas Santos2, Luiz Augusto Ferreira Alvarez2, Katarina Pereira do Lago2, Cinayra Daisy Fraga de Souza2, Ingrid Porto Gomes2, Beatriz Souza Bastos2, Ian Felipe Mariano Gonçalves2, Letícia Barbosa Machado de Lima2, Mauricio Santa Fé Lins Júnior2, Bruno Cunha Freitas2 (1) Universidade Salvador; (2) Liga Baiana de Cirurgia Cardiovascular Introduction: Rheumatic fever (RF) is an inflammatory, systemic disease triggered after an infection in pharyngotonsillitis and its etiology is group A beta-hemolytic streptococcus (ECGA). Early attention to pharyngitis coupled with antibiotic use can greatly reduce the risk of RF and its complications. In view of this scenario, one of the factors that may have implied a significant increase in this pathology in the national territory during the period 2010–2019 is the shortage of the main antimicrobial used in prophylaxis: benzathine penicillin. Objective: To assess the number of deaths from acute rheumatic fever in Brazil from 2010 to 2019 and to correlate the shortage of the main antibiotic used for the disease, benzathine penicillin, and poor adherence to treatment. Methods: A cross-sectional observational study of aggregated data, collected through the collection of information through the SUS Hospital Information System (SIH-SUS) by the SUS Department of Informatics (DATASUS), where the number of deaths in years from 2010 to 2019 related to acute rheumatic fever by macroregions of Brazil. The Kolmogorov-Smirnov test was performed for the subsequent adequacy of the tests of comparison of parametric or non-parametric groups. A linear regression in the 10 years surveyed was also performed to assess the increase in deaths in the Brazilian territory in this period. Results: In the linear regression from 2010 to 2019, throughout Brazil, there was evidence for an increase in deaths related to acute rheumatic fever, with the following results: β = –0.848; R² = 0.719; p = 0.002. Conclusion: Finally, it was possible to observe that, with regard to the mortality rate, the studies showed that in the period in which there was a shortage, the numbers of deaths increased, demonstrating a failure to adhere to treatment or a low continuity. of Streptococcus pyogenes prophylaxis in patients. However, even based on this assumption, the adoption of an adequate clinical approach for continuity in prophylaxis in order to avoid cardiac impairment and access to health resources for treatment is the most recommended, in order to modify these statistics. 110035 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LOUISE BUONALUMI TÁCITO YUGAR1, Tatiana Rubio Azevedo2, Larissa Costa Morete2, Beatriz Vaz Domingues Moreno1, Bruno Rodrigues1, José Fernando Vilela Martin2, Heitor Moreno Júnior1, Lúcia Helena Bonalume Tácito2, Juan Carlos Yugar Toledo2 (1) Faculdade de Ciências Médicas, UNICAMP; (2) Faculdade de Medicina de São José do Rio Preto, FAMERP Introduction: The golden ratio (φ, phi) is defined as a line divided into two parts of different size (a and b, in which a > b) so that the ratio of the whole length to the larger one is equal to the ratio of the larger number to the smaller. It is approximately 1,61. This number is found throughout the body as in fingers, uterus and the cardiovascular system. The blood pressure curve can be segmented in three parts: pulse pressure (PP), diastolic blood pressure (DBP) and systolic blood pressure (SBP), which is the sum of DBP and PP. In this paper, we aimed to analyze geometrically these values in the form of proportions (DBP/PP and SBP/DBP) to assess if the deviations from phi can be used as additional markers of cardiovascular risk. Material and methods: This retrospective study analyzed data of 171 normotensive patients (NT), 78 pre-hypertensive patients (PHTN), 46 hypertensive people (HTN) and 81 diabetic people (T2DM) from the study “Vascular hemodynamic alterations in HTN and T2DM type 2 patients”. The protocol was approved by the local ethical committee. We used age, sex, SBP, DBP, PP and pulse wave velocity (PWV) for the analysis. We made a descriptive statistical analysis along with t test and ANOVA in SPSS 24 (USA). Results: We observed lower SBP/DBP in PHTN and HTN when compared to NT (p = 0,0002 e p < 0,0001, respectively). Between PHTN and HTN, PHTN and T2DM, there was a significant difference. The DBP/PP ratio showed greater values in PHTN and HTN when compared to HTN (p < 0,0001 in both) and lower and we compared T2DM and NT (p = 0,0495). The comparison between PHTN and HTN, PHTN and T2DM, as well as HTN and T2DM showed significant difference. Multivariate regression in the different groups demonstrated that age and PWV are correlated to SBP/DBP in HTN and there’s a correlation with sex, age and PWV in T2DM. Additionally, we found significant correlation between SBP/DBP and DBP/PP with PWV and PP in the four groups. Conclusion: Deviations from phi were detected in PHTN, HTN and T2DM with different pattern among the groups and can be used as additional risk evaluators as well as markers of cardiovascular damage and are closely related to vascular accelerated ageing for its relation with PP and PWV. 109660 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIELA CUNHA FERNANDES2, Sylton Arruda de Melo1, Juliana Maria Gurgel Guimarães de Oliveira1, Ana Isabelly De Medeiros Tomaz1, Arthur Felipe Tertulino Cunha1, Pedro Nonato Silveira Costa1, Mateus Bessa Nogueira2, Sara Araújo de Oliveira Lima2, Maria Teresa da Fonseca Madruga2, Maria Laura Torres e Araújo2, Joice Raquel Urbano Nascimento2, Flávia Diógenes Forte Melo2 (1) Universidade Potiguar – UnP; (2) Liga Acadêmica de Cardiologia do Rio Grande do Norte (LiCordis-RN) Introduction: In view of the pandemic caused by SARS-Cov 2, a broad approach to the disease and its repercussions is extremely important. Unquestionably, COVID-19 has potentially serious outcomes: cardiovascular, pulmonary, systemic repercussions and death, but we must also consider its impact on the health system in general. Objective: To analyze the mortality rate from cardiovascular diseases (CVD) before and during the pandemic, noting the epidemiological situation in Brazil. Methods: Cross-sectional, retrospective and portal study, through the analysis of transparency data – civil registry. It was a survey on the number of deaths from CVD in Brazil, considering the pre-pandemic period of the year 2019 (from January), and the pandemic scenario of 2020, 2021 until March 15, 2022. Results: The period from January 2019 to March 15, 2022: in 2019 there were 275,032 deaths from cardiovascular causes, of which 37.2% (102,361) were due to stroke, while 36.4% (100,378) were due to acute myocardial infarction (AMI) and 26.2% (72,293) due to unspecific cardiovascular causes. In 2020, 293,963 deaths were observed, with 35.1% (103,238) from stroke, 32.6% (96,023) from AMI and 32.2% (94,702) from unspecific cardiovascular causes. In 2021, there were 318,602 deaths, 33.5% (106,817) from stroke, 32% (102,098) from infarction and 34.4% (109,687) from unspecific cardiovascular causes. In the year 2022, until March, 62,310 deaths were recorded, with 34.5% (21,522) deaths from stroke, 31.8% (19,831) from AMI and 33.6% (20,957) from unspecific cardiovascular causes. Conclusion: An increase in the number of deaths from cardiovascular causes was observed between 2019 and 2022, with a significant increase in the number of deaths from nonspecific cardiovascular diseases (26.2% to 33.6%). However, in 2022, until the analyzed period, a predominance of mortality from stroke was evidenced and that nonspecific cardiovascular causes remained close to the percentage numbers between 2020 and 2021, still above the observed in 2019. It’s concluded that there was an increase in deaths from CVD in a pandemic scenario in Brazil, which can be attributed to the fear of patients to seek medical care in the face of the risk of contamination, reaching more advanced stages or even dying outside the hospital environment. In addition, the increase in the number of deaths from unspecific cardiovascular causes can be largely attributed to mortality from the cardiovascular repercussions of COVID-19. 109663 Modality: E-Poster Scientific Initiation – Non-case Report Category: NURSING AMANDA CIRILO SILVA1, Andreia Correia de Menezes1, Ingrid Milani Nacaratto de Freitas1, Claudia Cristina Soares Muniz1, Everaldo Muniz Oliveira1, Fernanda de Mello Demai1 (1) Universidade Nove de Julho Introduction: The homeless population in Brazil is minority composed of women, which represent only 14.6% in the city of São Paulo¹, where they are commonly affected by the scarcity in which they live, without health information and lack of access to personal and oral hygiene. We used the nursing diagnoses and possible interventions aiming to produce quality of life and reduction of damage to their health. Objective: To evaluate cardiovascular risks to oral health in homeless women in São Paulo, associated with the Taxonomy of International Nursing Diagnoses Nanda I 2018/2019. Methods: This is a field research with quantitative method, exploratory and cross-sectional nature, approved by the Institutional Ethics Committee under protocol 036417, CAAE:21519413.40000.5511. Conducted in downtown São Paulo, the research counted among 173 evaluated, it was found 18 women volunteers in street situation between the months of November 2019 to March 2020, having between 18 and 59 years, previously selected by convenience and submitted to a questionnaire, being evaluated sociodemographic data pointing the risk factors for cardiovascular diseases (CVD), measurement of blood pressure (BP) and heart rate (HR), subsequently associated with the International Nursing Taxonomy Nanda I. Results: Of the women studied the mean blood pressure (BP) of 128 × 85 mmhg, and Heart Rate (HR) of 91bpm. Being that 18.1% reported having previous history for stroke, 9% for AMI and 76.6% could not inform previous history for AMI. Of these women, 18.1% reported having presented an abscess or edema in the last 6 months. And 27.2% reported toothache or pain in the mucosa region. It was observed that 72.70% of the women answered that yes, they use some illicit substance, 18.1% never used and 9% do not use. Thus, nursing diagnoses were listed during the analysis, using the NANDA I taxonomy, are: Related Ineffective Health Control, Impaired Dentition, and Risk of Impaired Oral Mucous Membrane Integrity. Conclusion: However, it is evidenced that oral hygiene has a direct connection with cardiovascular alterations in this population associated with AMI, stroke, and atherosclerosis. Thus, the importance of public politics for the improvement and eradication of this problem was observed, reducing the aggravations of CVD and aiming to improve the quality of life for this often underprivileged part of society. 109752 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUIZ FELIPE FAÇANHA RAMOS1, Karen Tássia Façanha Ramos1, Hildeman Dias da Costa2, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Leo Christyan Alves de Lima3, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia; (3) Centro Universitário São Lucas Introduction: Cardiovascular diseases are an important cause of mortality in the country, representing the main cause of mortality from all causes, especially cardiomyopathies. Objective: To analyze the evolution and profile of mortality from cardiomyopathies in Brazil in 3 consecutive years. Methods: This is an epidemiological, cross-sectional study on mortality from cardiomyopathies (ICD I42) from 2017 to 2019, in Brazil, using secondary data from the Department of Informatics of the Unified Health System (DATASUS). Results: In the analyzed period, there was a 2% increase in mortality from cardiomyopathies, from 2017 to 2019, totaling 23,061 deaths, with the year 2018 having the highest mortality in the period (n = 7,834). During this period, the southern region of the country had the highest number of deaths (n = 14,658) and the northern region had the lowest number (n = 651). Analyzing the age group, marital status, sex and color, there were more deaths, respectively, among people aged 60 to 69 years (n = 7,412), single (n = 8,683), male (n = 15,904) and white people. (n = 10,387). Conclusion: Thus, there was an increase in the number of deaths during the period studied, especially in 2018. In addition, there were more deaths in the south of the country, in the elderly, single and white. Therefore, there is an urgent need for public health policies for the southern region of the country to contain the advance of mortality from cardiomyopathies. 109687 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUIZ FELIPE FAÇANHA RAMOS1, Karen Tássia Façanha Ramos1, Hildeman Dias da Costa2, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia Introduction: The disease caused by the SARS-CoV-2 virus (COVID-19) has been responsible for high rates of hospitalization and high mortality. In addition to respiratory failure, coagulopathy has become a recurrent complication in infected patients, especially the most severe, thus increasing the number of arterial embolisms and thrombosis in the country. Objective: To analyze the effect of the COVID-19 pandemic on cases of arterial embolism and thrombosis in Brazil. Methodology: This is an ecological time series analytical study, using secondary data on arterial embolism and thrombosis (from 2017–2021) in the country based on the Department of Informatics of the Unified Health System (DATASUS), through the Information System of the Unified Health System (DATASUS). Hospital Morbidity. The data were tabulated in two spreadsheets – period 1 (P1): data from 2018–2019; period 2 (P2): 2020–2021 – of the Microsoft Excel software, which were analyzed by the GraphPad Prism software version 9, using the test of Wilcoxon and Pearson’s chi-square test to assess differences in rates, association factors, risk ratio (RiR) and their 95% CI, with a significance level of 5%. Results: Analyzing the two groups of data, there was an increase in mortality from arterial embolism and thrombosis in Brazil during the pandemic period (from n = 3736 to n = 4215; p = 0.05; RiR 1.00; 95% CI 0.99–1.00), also expressed by the difference in standardized mortality rates, since P2 had a higher rate than P1 (8.76 and 8.37, respectively; p = 0.18). It should be noted that the number of deaths was higher in the elderly in both periods (P1, n = 3125; P2, n = 3474), with P2 being the most lethal for people aged 60 years or older (p = 0.14; RiR 0, 95; 95% CI 0.89–1.01). It is noteworthy that the number of deaths occurred more in females in both temporal groups (p = 0.07; RiR: 0.95; 95% CI 0.91–1.00) and in white people (p < 0.0001; RiR: 0.89; 95% CI 0.85–0.93). Conclusions: During the pandemic, the indirect effect was the increase in the number of deaths and mortality rates from arterial embolism and thrombosis in the country, when compared to the same period 2 years before the beginning of the pandemic period. In addition, in the two analyzed periods, deaths occurred more among women, elderly people aged 60 years or older and white people. Thus, we can see the importance of investing in surveillance and effective treatment of this morbidity to possibly reduce the number of deaths. 110173 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARIA EDUARDA BERGAMO1, Maria Eduarda Bergamo1, Gustavo Henrique Ferreira Gonçalinho1, Nathalia Ferreira de Oliveira Faria1, Karen Lika Kuwabara1, José Rafael de Oliveira Nascimento1, Gean dos Santos de Sales1, Antonio de Padua Mansur1 (1) Insituto do Coração – HC FMUSP Introduction: Ischemic heart disease (IHD) is the leading cause of death in women. Despite advances in the treatment of IHD, little is known about how this knowledge is applied in current outpatient clinical practice. This study aimed to analyze the number of healthy metrics (HM) the clinical response to the recommended treatment in women with IHD. Methods: This cross-sectional study analyzed 462 women with IHD in outpatient care aged ≥30 years. IHD was diagnosed by cardiac catheterization for coronary lesions >70%. The response to clinical treatment was analyzed by quantifying the number of HM, namely: healthy eating (DASH-Dietary Approaches to Stop Hypertension diet), physical activity (PA), body mass index (BMI = 18.5–24.9 Kg/m2), blood pressure (SBP ≤140 and DBP ≤90 mmHg), non-smokers, LDL-cholesterol ≤50 mg/dL, glucose <126 mg/dl. Results: Mean age was 66.5 years; 340 (74%) women with chronic IHD had 3 and 4 HM outside the targets established by current guidelines; 47 (10%) had none or one HM; only 0.4% (n = 3) with all HM. Only 17 (3.7%) of the patients practiced physical activity (PA) regularly, and the entire study population did not have a healthy eating pattern assessed by the DASH diet. The table shows the prevalence of HM analyzed and the mean values of clinical and metabolic variables. Conclusion: The quality of outpatient care for women with chronic IHD did not meet the strategies recommended by current guidelines. 109693 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MARIA GABRIELA PIMENTA DOS SANTOS1, David Ferreira de Lima Duarte1, Gabriela Gama Zagni Jardim2, Julia Resende de Oliveira1, Antônio Carlos Eberienos Assad Filho1, Paola Pugian Jardim3, Andréa Vaospasse Cocco Faria3, Lilian Soares da Costa4 (1) Universidade Estácio de Sá/IDOMED, Campus Città; (2) Universidade Estácio de Sá/IDOMED, Campus Vista Carioca; (3) Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (4) Universidade Estácio de Sá/IDOMED, Campus Città e Vista Carioca; Instituto Estadual de Cardiologia Aloysio de Castro/IECAC Introduction: Arterial stiffness measured by pulse wave velocity (PWV) is considered the gold standard in different national and international guidelines, being a parameter of significant prognostic value of cardiovascular (CV) risk. Although the assertion that elevation of PWV increases the occurrence of CV events, CV mortality and death from all causes, its prognostic value in very high-risk individuals, such as heart failure with reduced ejection fraction (HFrEF), is not yet well established. Objective: Evaluate the oscillometric analysis of vascular pulsatile hemodynamics, using PWV, in patients with HFrEF. Materials and Methods: A cohort study evaluating patients with HFrEF, followed up at a tertiary cardiology hospital in the State of Rio de Janeiro. Blood pressure measurements, PWV, anthropometric measurements and chart analysis were performed. Results: PWV was analyzed in a series of sixteen consecutive cases of individuals with compensated HF and severe systolic dysfunction, comprising a cohort of 180 individuals at high CV risk, which 56% was male and mean age was 64.6 years (36–87 years). The subgroup is composed of sixteen patients, 75% male, mean age 63.4 years (44–78 years), HFrEF with mean EF 30.3% (18–40%). We emphasize that twelve individuals had three or more major risk factors or comorbidities, such as diabetes mellitus, arterial hypertension, dyslipidemia and coronary artery disease. A risk score for arterial stiffness (SAGE Score) was applied on the study population and demonstrated values within normal limits in 14/16 individuals. Blood pressure values within normal parameters were found in 5/16 participants and, in 2/16, arterial stiffness parameters were recorded above the median standards considered normal for the Brazilian population, adjusted for age, sex and presence of factors of risk. Conclusion: As assumed in the literature, a greater number of individuals is needed to confirm the role of this tool as a CV prognostic marker in this particular group of very high-risk individuals with HFrEF. The future perspective of our cohort is that the possibility of pharmacological interventions in the short and medium term can modify these parameters, in order to assess outcomes, for possible assessments of inferences about the role of PWV in CV prognosis. 109700 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY GUILHERME RODRIGUES VIANA1, Grasiele do Amaral Martins1, Marco Antônio Vinciprova Dall Agnese1, Adriano Louro Moreira1, Pedro Henrique Torres Tietz1, Bianca Brinques da Silva2, Caroline Engster da Silva1, Eric Seiji Kanai1, Estefany Karenine Rodriguez Casanova1, Letícia Vieira Senger1, Matheus Ribeiro Fretes1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre; (2) Universidade Luterana do Brasil Introduction: Extracardiac malformations (ECMs) associated with congenital heart disease (CHD) may increase the risk of morbidity and mortality of the child, often making surgical intervention risky. This association between ECMs and CHD may involve defects of different systems or tracts, such as the gastrointestinal tract (GIT). Purpose: To determine the frequency and types of GIT malformation in a sample of patients with CC. Methods: A retrospective study using Clinical Data from patients hospitalized for the first time in a cardiac intensive care unit of a reference pediatric hospital in southern Brazil. In the present study, ECMs and CHD data were obtained by filling out a standard protocol. Results: The sample was composed of 343 patients, 182 (53.1%) males, ages ranging from 1 day to 14 years and 6 months (60.1% <1 year). GIT changes were evident in 6 patients (1.7%) and consisted of esophageal atresia (n = 2), duodenal stenosis (n = 1), multiseptated gallbladder (n = 1), anteriorized anal canal (n = 1), and imperforate anus (n = 1). The most commonly observed CC among patients with GIT malformations were septal defects (n = 3), in particular ventricular septal defect (n = 2). Four patients were syndromic, and chromosomal alterations were observed in 5 patients. Conclusion: From the information of the presence of an associated GIT alteration, health professionals can perform a more detailed and directed evaluation of patients with CC, aiming at the best management of these patients and preventing future complications, mainly related to their prognosis. 109714 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUIZ FELIPE FAÇANHA RAMOS1, Karen Tássia Façanha Ramos1, Hildeman Dias da Costa2, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá; (2) Universidade Federal de Rondônia Introduction: The Family Health Strategy (FHS) stands out, among many factors, for its comprehensive care for people living with non-infectious chronic diseases, especially circulatory diseases, which represent the main cause of death and physical disability in the world. However, there are few studies that associate the advancement of the FHS to the parameters of these diseases, making systematic evaluations of its expansion necessary. Objectives: The objective of this study was to analyze mortality from circulatory diseases in parallel with the evolution of the FHS from 2017 to 2020 in Brazil. Methods: This is an ecological, retrospective study, based on the temporal evolution of the FHS, inhabitant coverage x FHS, and on standardized rates (per 10,000 inhabitants) of mortality from circulatory diseases in Brazil. Secondary data were taken from the SUS Hospital Morbidity System, from the Information Technology Department of the Unified Health System (DATASUS), and from the Primary Care Information and Management System, using the Spearman Correlation test, through the GraphPad Prism software version 9.3.1, for statistical association. Results: During the analyzed period, there was a 2% population increase in Brazil, a 4.7% increase in the number of FHS and a 20.5% increase in mortality from circulatory diseases. The population x FHS ratio increased from 4,965 (2017) to 4,834 (2020) people assisted (2.6% higher coverage). In 2020, the northeast region had the best population coverage by FHS, reaching 3,520 people per implemented unit. Even with the expansion of the FHS in the country, mortality from circulatory diseases increased in all regions, with the northern region having the highest increase (34%). When analyzing the data from the regions, it was noticed that in three there was a positive correlation between FHS and mortality from circulatory diseases (r > 0.7; p < 0.05) and the south and southeast did not present a significant correlation (r: 0, 2; p = 0.91). Overall, Brazil showed a positive correlation between FHS and mortality from circulatory diseases (r: 0.8; p = 0.33). Conclusion: Thus, this study demonstrated that, even with the increase in population coverage by the FHS in Brazil, there was no reduction in mortality from circulatory diseases and that they are a challenge for public health policies. In addition, there was a large increase in mortality from 2017 to 2021 that did not follow the growth of FHS coverage in the same proportion. 109745 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CAMILA THAYNÁ DE MENEZES CLEMENTE1, Mariane Leandro Ferro de Sousa1, Matheus Dantas Soeiro1, Carolina Jeronimo Magalhaes3, Jessica Myrian de Amorim Garcia1 (1) Faculdade Pernambucana de Saúde; (2) Real Hospital Português de Beneficência em Pernambuco; (3) Universidade de Pernambuco Introduction: Cardiovascular diseases (CV) is the most prevalent chronic conditions worldwide. The coronavirus disease 2019 (COVID-19) infection leads to heterogeneous clinical manifestations throughout the population. Therefore, it was necessary to establish the interplay between cardiovascular disease and COVID-19. Objective: Analyze the clinical profile and outcome of patients with COVID- 19 and previous CV disease compared with patients with COVID-19 without these comorbidities. Methods: A cross-sectional observational study of inpatients presenting with COVID-19. Conducted in a private Brazilian hospital from August 2020 and August 2021. The diagnostic criteria were a positive reverse transcriptase-polymerase chain reaction (RT-PCR) or ground-glass opacities on computerized chest tomography with compatible clinical manifestations of dyspnea, fever, and cough. The data were obtained from online medical records. Results: We studied 212 patients with COVID-19. The sample had a mean age of 58.4 years, and 55.7% were males. There was a 50.5% prevalence of previous CV disease, 98.1% of these group of patients had systemic arterial hypertension, 11.2% acute myocardial infarction, and 13.0% congestive heart failure. The patients with both COVID-19 and CV disease were associated with greater mean inflammatory markers D-Dimer (p = 0.03) and troponin (p < 0.01) (TABLE 1). Patients with COVID-19 and CV disease had a greater incidence of shock (p = 0.01), anemia (p < 0.01), bacteremia (p = 0.03), and worse outcomes marked by an extended hospital stay (p = 0.04) as well as 80% of the deaths (TABLE 2). Conclusion: Patients with previous cardiovascular disease infected by COVID-19 had a poorer prognosis and were subject to worse clinical course and complications during of hospitalization. 109755 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DANIEL DE OLIVEIRA MEIRELES 1, Sara Cristine Marques dos Santos1, Aline de Jesus Oliveira1, Thaís Lemos de Souza Macêdo1, Ivan Lucas Picone Borges dos Anjos1, Júlia Bardela de Oliveira1, Anderlúcia Côrrea Guedes1, Patrícia Rangel Sobral Dantas1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Acute myocardial infarction is when an obstruction of the coronary artery leads to oxygen deficiency in the myocardial supply, causing necrosis1. According to the Ministry of Health records, there was an increase in the occurrence of AMI in young people (aged 20 to 39 years) and a potential responsible for these cases, which despite being considered small when compared to the national context, are the unhealthy lifestyle habits associated with physical inactivity2. Infarctions in young people are more lethal due to the rapid evolution and their exuberant clinical condition. If there is a delay in starting treatment, complications such as heart failure and arrhythmias may occur2,3. Objectives: To analyze the panorama of the occurrence of AMI in people aged 20 to 39 years. Methodology: A literature review and an observational, descriptive, and cross-sectional collection of data on the occurrence of AMI, available at DATASUS – SUS Hospital Information System – December 2004 to December 2018, were carried out. Results: In the analyzed period, if 45,883 hospitalizations for acute myocardial infarction in people aged between 20 and 39 years, where the year responsible for the largest number was 2018, with 3,823, followed by 2017 with 3,778; on the other hand, the lowest number was 2004 with 2,351, followed by 2005 with 2,518. The total number of deaths was 2,704 in 15 years studied. As for sex, 33,462 affected males while 12,421 were females. The Southeast with 23,901 cases, the Northeast with 8,139, the South with 8,088, the Midwest with 3,295, and the North with 2,460. Among the federation units, São Paulo led with 13,377 hospitalizations. As for the character of the hospitalizations, 33,289 were considered urgent, where they obtained 1,946 deaths (mortality rate of 5.85), the electives totaled 2,373 with 73 deaths (mortality rate of 3.08) and for other causes, 3 with 50% mortality rate. Of the 1946 deaths in emergency care, 1,569 were in patients aged 30 to 39 years, corresponding to 80.6% of cases. Conclusion: It was possible to observe the percentage increase of 62% of the cases of AMI in individuals aged 20 to 39 years in 10 years, where 72.5% was attended to on an urgent basis, which obtained the highest mortality rate, mainly in the age range from 30 to 39 years. It is a disease prevalent in males, which accounts for 72.9% of cases. Where São Paulo has more than half of admissions across the Southeast. 109765 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY GIULIA VITORIA NASCIMENTO DA SILVA1, GABRIEL REZENDE NEVES1, LUCAS DIAS SILVA1, SAMARA GUILHERMINA DE SOUSA1, PAULO CESAR LOBATO MAGALHÃES2, LETICIA RIBEIRO DOS SANTOS1, MARIANA DOS SANTOS GUIMARÃES2, LARISSA DACIER LOBATO COMESANHA2, TEREZA MARIA MEIRELES FERNANDES DA SILVA1, MARCONDES TAVARES NEVES JUNIOR1 (1) Universidade do Estado do Pará (UEPA); (2) Universidade Federal do Pará (UFPA) Introduction: Due to the COVID-19 pandemic, logistical changes have occurred to meet the demand of patients affected by the SARS-CoV-2 virus. However, the expansion of beds for the hospitalization of infected people and the suspension of elective surgeries contributed to modifying the profile of cardiovascular interventions performed during the period. Objective: To analyze the impact of the COVID-19 pandemic on myocardial revascularization using extracorporeal circulation in the state of Rio de Janeiro (RJ). Methods: Observational, cross-sectional, analytical-descriptive and quantitative study. The numbers of myocardial revascularization surgeries using extracorporeal circulation, in RJ, from 2019 to 2021, were obtained via DATASUS – Hospital Information System of the Unified Health System (SIH/SUS). The amount of confirmed COVID-19 cases per month in RJ, from March 2020 to December 2021, were collected via Painel COVID-19 – the virtual platform of the Secretary of Health of the State of Rio de Janeiro. Results: COVID-19 cases in RJ emerged in March 2020 and the number of notifications grew abruptly in May, a period in which there was an increase of 44,687 cases when compared to the previous month. During this period, 77 myocardial revascularizations were performed with the use of extracorporeal circulation, 37.4% less than in the same month of the previous year. In May 2021, the second month with the highest number of new cases of infections in RJ throughout the pandemic, 88 procedures were performed, representing an increase of 14.3% when compared to the same month in 2020 and a decrease of 28.4% compared to the same period in 2019. In addition, in September 2021, the period with the highest peak of new cases of COVID-19 in RJ, 97 myocardial revascularizations were performed, 6.7% more than the same month in 2020 and 33.1% less than in 2019. September 2021 values were succeeced by only 64 procedures in the following month, October 2021, which configured a 34% decrease in surgeries performed between these months. Comparing October 2021 with the same month in 2020, there was a reduction of 27.3%, and in relation to the same period in 2019, a decrease of 56.7%. Conclusion: In view of the increase in cases of COVID-19, there is a significant decline in the volume of myocardial revascularization surgeries performed in the state of RJ, consequently hindering the medical therapy of atherosclerotic coronary disease. 109769 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CAIO PLUVIER DUARTE COSTA1, Mayara Gabriele Toledo1, Eduardo Thadeu de Oliveira Correia1 (1) Universidade Federal Fluminense Introduction: Monitoring volume status in patients hospitalized for heart failure (HF) is fundamental to provide therapeutic and prognostic guidance. Recently, the measurement of the internal jugular vein (IJV) by ultrasonography has been object of investigation by several studies. Therefore we aim to systematically review if IJV ultrasound is an useful parameter to predict central venous pressure (CVP) in patients hospitalized for HF. Methods: We conducted a systematic review following the PRISMA guidelines. Original studies indexed by Embase, Pubmed, Cochrane and LiLACS with the keywords: “ultrasound” or “ultrasonography” and “heart failure” and “jugular vein” published until March, 2022 and which matched the inclusion criteria were included. Two authors (C.P and M.T.) performed the screening and data extraction, in cases of discordance, a third author (E.T.) made the decision. Results: Thirteen studies were included, with a total of 898 patients analyzed. Due to the significant heterogeneity between studies, a meta-analysis was not conducted. In all included studies CVP was measured by right heart catheterization. For the ultrasound evaluation of CVP, 5 used the diameter of the IJV; another 4 used the cross-sectional area (CSA) of the IJV and 4 studies used the height of IJV collapse and added 5 centimeters to this measurement (uJVP). Studies that analyzed the diameter of the IJV showed an AUC of 0,74 to 0,84 to predict CVP. Regarding CSA, all 4 articles showed that the IJV ultrasound was useful to predict CVP. Zamboni et al. showed that the CSA was 50–60% better than clinical visualization of jugular vein, while Simon et al. demonstrated an AUC of 0,86 for a CSA variation higher than 66% during Valsalva, compared with rest. Moreover, for the uJVP method, all the included studies concluded that it was a significant predictor of CVP. Significantly, Wang et al. showed that uJVP was a significant predictor of CVP in obese patients, with an AUC of 0,81, although it had a better accuracy among non-obese patients. Conclusion: This systematic review shows that IJV ultrasound is a significant predictor of CVP measured by right heart catheterization. Also, this demonstrates the need for a standard ultrasonographic measurement method of the IJV, to diminish the heterogeneity of future studies. In conclusion, this study paves the way for future randomized trials that aim to investigate if IJV ultrasound can be used to guide diuretic therapy and discharge. 109776 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION OSVALDO CARLOS SILVA LEOPOLDINO1, Osvaldo Carlos Silva Leopoldino1, Camila Orge Rodrigues2, Jayne Milly Queiroz Santana1, Bernardo Oliveira Torres3, Juliana Almeida Frank3, Amanda Gabriela Rodrigues dos Santos de Souza1, Pedro Henrique Souza de Aragão3, Ana Luisa Soares Chiaretti3, Victória Valadares Andrade3, Cristiano Ricardo Bastos de Macedo4, Roque Aras Júnior4 (1) Universidade Salvador (UNIFACS); (2) Escola Bahiana de Medicina e Saúde Pública (EBMSP); (3) Universidade Federal da Bahia (UFBA); (4) Hospital Universitário Professor Edgard Santos (HUPES) Introduction: The relationship between hypertension and diastolic dysfunction with preserved ejection fraction (EF) is well established, yet this data is still nebulous in patients with Resistant Arterial Hypertension (RAH). It is uncertain whether other particulates in this group, such as high afterload, may interfere with EF or diastolic dysfunction. Objectives: Analyze the epidemiological and echocardiographic profile of patients with RAH. Methods. Open, Transversal, and Unicenter study from a University Hospital set in Brazil from June 2018 to March 2022 (last update). Eligible patients were those over 18 years and diagnosed with RAH (defined as medical office Blood Pressure (BP) ≥140/90 mmHg despite the use of ≥3 antihypertensive drugs, one of those being a diuretic, or controlled BP using ≥4 medication). Statistical tests (Chi-square test and Z-test) and dispersion measurements were performed using the graph pad and SPSS app. The BP was taken by standardized automated devices, and the echocardiographic exam was performed by the hospital’s staff. Results: There were evaluated 118 hypertensive patients, mostly black (n = 64) and brown (n = 48), with a mean age of 65.28 (±11.14) years and a predominance of women (n = 91). The mean systolic/diastolic BP values were 143.6 (±22.09 [P < 0.01])/82.98 (±16.42 [P < 0.01]) mmHg. At the echocardiographic, the mean EF was 66.00% (±13.05 [P < 0.01]), with only 6 (5.08%) having EF < 40% and 6 (5.08%) with EF 40–50%. The interquartile range of EF (Q1–Q3) was 9,19. Of this sample, 63.5% (n = 75) manifested diastolic dysfunction, with 93.33% (n = 70) being type 1 dysfunction and 6.66% (n = 5) type 2. Regarding the clinical profile, 55 patients declared themselves sedentary, 73 presented with metabolic syndrome, 23 with Chronic Kidney Disease, and 48 with Type 2 Diabetes Mellitus. Conclusion: This study presents some limitations, such as the low sample size and the echocardiogram limit (it is operator-dependent). Nevertheless, this work shows a high prevalence of preserved EF at the echocardiogram, revealing preservation of the left ventricle systolic function. A large proportion of the population had diastolic dysfunctions at an initial state, suggesting that, despite the severity of hypertension, the compromise to the cardiac functionality occurs at a rather late point in the natural history of the disease. At last, some individuals had low EF which suggests independent factors for systolic impairment. 109788 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LEANDRO LOURENÇO SILVA MONTEIRO1, Luiz Fernando Leite da Silva Neto1, Luig Matias Barreiros Pires2, Ana Beatriz Rezende dos Santos Corrêa2, Talles Levi Pereira Nogueira3, Ana Paula Correa de Lima2, Igor Lucas Farias Lima1, Fernando Tavares Brasil Teixeira1 (1) Universidade do Estado do Pará (UEPA); (2) Centro Universitário do Estado do Pará (CESUPA); (3) Universidade Federal do Pará (UFPA) Introduction: Cardiac Insufficiency, marked by heart failure in blood pumping, is still a common outcome for heart diseases of several etiologies and, despite optimized therapy, maintains high levels of hospitalization and in-hospital mortality of patients. In this sense, cardiac insufficiency has a high impact, since social determinants such as gender, age and socioeconomic conditions influence the clinical outcome of disease. Objective: To trace the epidemiological characteristics of deaths by Heart Failure in the Northern region of Brazil in 2020. Method: The study has ecological, descriptive and retrospective character. Data were collected from the Departamento de Informática do Sistema Único de Saúde (DATASUS) in the Sistema de Informação sobre Mortalidade (SIM) as to the number of deaths by Heart Failure in the Northern Region, and epidemiological characteristics of patients, in 2020. The following variables were analyzed: number of deaths, federation units, gender, age group, race/color, schooling and location. Results: In 2020, 1.510 deaths were recorded. The Federation Units with highest records are from Pará (46,42%), Amazonas (17,68%) and Rôndonia (15,56%), and Amapá with the lowest value (2,98%). Relating to age groups, the most prevalent were over 80 years (39,07%), 70 to 79 years (25,58%) and 60 to 69 years (18,27%). In addition, men received quantitative prominence with 58,14%. Regarding race/color, the most notified were brown (65,29%) and white (22,58%). A significant number of deaths occurred in individuals without schooling (32,05%), followed by 1 to 3 years of schooling (21,45%) and 4 to 7 years (16,88%), with the lower value in the classification corresponding to school formation over 12 years (3,5%). Finally, most occurred in a hospital environment (73,04%), followed by home location (19,4%). Conclusion: Cardiac Insufficiency is a disease with a five-year survival rate that is worrisome, because it generates functional loss and quality of life, gradually and eventually evolving to death. And yet, it is possible to notice that death from Heart Failure is prevalent in the Northern region, whose epidemiology is mainly related to age, gender, race/color and intra hospital environment according to data collected from DATASUS. Therefore, it is confirmed the importance of identifying the epidemiological characteristics of deaths for the implementation of effective public health policies in the prevention of such a pathology. 109795 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION DAVI DE SOUZA CATABRIGA1, Lucas Dalvi Armond Rezende2, Janaína Rodrigues Barbosa1, Beatriz Contarini Peluzzo Moraes1, Caio Lucas Franco Inocêncio1, Lorena Pádua de Moura Duarte3, Karolini Zuqui Nunes2 (1) Higher School of Sciences of the Santa Casa de Misericórdia de Vitória – EMESCAM; (2) Nursing Department, Health Sciences Center, Federal University of Espírito Santo – UFES; (3) Vila Velha University – UVV Introduction: Systemic arterial hypertension (SAH) is a risk factor for several diseases, including acute aortic dissection (AAD). AAD is defined by the entry of blood into the aortic wall, usually between the outer third and the inner two thirds of the tunica media, due to rupture of the intima. Objective: To elucidate the pathophysiology of aortic dissection and its relationship with systemic arterial hypertension, through the guiding question “What is the pathophysiological relationship of acute aortic dissection with hypertension?” Methods: A systematic review was performed, without meta-analysis, using the PRISMA protocol. Thus, the PubMed database and the Virtual Health Library (BVS) were used during the period of May 2021. The DeCS/MeSH descriptors “”Aneurysm, Dissecting”, “hypertension” and “Pathophysiology” were used. combined using the Boolean operator “AND”. Results: A total of 270 articles were found; of which, after applying the criteria for inclusion, exclusion of duplicates and exclusion by analysis of title, abstract and full text, 13 articles remained for analysis, all in English. AAD comprises the rupture of the tunica intima, which causes an infiltration of blood into the tunica media, so that this blood continues to flow and creates a false lumen. SAH decreases the blood flow to the vasa vasorum, responsible for nourishing the middle layer of the aorta. This situation results in an increasing stiffness of this vessel, which can cause a degeneration of the middle layer of the aorta and increase shear stresses, situations that favor the AAD. This degeneration involves mechanisms of loss of smooth muscle cells and cellular matrix and production of pro-inflammatory cytokines and metalloproteins. On the other hand, the rupture is related to sites exposed to greater shear stresses, which promote an endothelial alteration that delays the proliferative, apoptotic and remodeling processes of the aorta. Conclusion: It was found that the pathophysiological mechanism of AAD of hypertensive etiology is the degeneration of the aortic middle layer and the increased shear stress. These factors are caused by reduced flow of vasa vasorum, loss of smooth muscle cells, production of pro-inflammatory cytokinins and metalloproteins, and changes in proliferative, apoptotic and remodeling processes. We emphasize the need for further research on the pathophysiology of this process in order to define a better diagnostic and therapeutic approach to AAD. 109817 Modality: E-Poster Scientific Initiation – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE BEATRIZ MONTENEGRO OLIVEIRA1, Dra. Antoinette Oliveira Blackman1, Adda Cecília Batista de Carvalho Vieira1, André Luiz Vergamini Dias2, David Ricardo Bernal Lima Hernandez1, Felipe Romério Marques Durães Barbosa1, Maria Gabriela Alves da Silva1, Patrícia Brito de Almeida Borges1, Pedro Henrique Alves Miranda1, Suellen Keyze Almeida Lima1, Warllson Jesus dos Santos1, Wenderval Borges Carvalho Júnior3 (1) Centro Universitário do Planalto Central Apparecido dos Santos; (2) Universidade Nove de Julho; (3) Hospital Universitário de Brasília Introduction: Takotsubo Syndrome (TS), characterized by transient systolic dysfunction of apical contraction, which simulates Acute Myocardial Infarction (AMI) without obstruction in the corresponding territory, has in its management focus on pharmacological treatment. The pathogenesis is not completely known and the mechanisms suggested are: coronary spasm, microvascular dysfunction and excess of catecholamines, the latter being triggered by emotional stress. Thus, special attention must be given to the integral health of patients, corroborating the concept of health of the World Health Organization (WHO). Objective: To highlight the importance of Spirituality in the complementary management of TS. Methods: Narrative review with search in Pubmed and Scielo databases including case reports, cohort studies, randomized clinical trials and systematic reviews about TS in Portuguese and English languages. Results: Data on non-pharmacological treatment of TS are limited and do not show a relationship between spirituality and TS. Initially more prevalent (90%) in women with a mean age of 68.5 years, with the pandemic of COVID-19 there was an increase in prevalence in men (about 30% of cases), due to stress caused directly by myocardial injury resulting from SARS-CoV-2 or indirectly by the socioeconomic influences of the pandemic. The INTERHEART study (conducted in 52 countries) assessed the impact of cardiovascular risk factors for AMI and found that psychosocial factors (stress, depression, and life events) have a 32.5% risk. The PEACE-III study with post-AMI patients and positive psychology interventions showed substantial improvement in psychological and behavioral outcomes. For the spiritual anamnesis, we have the FICA, HOPE, SPIRIT and FAITH tools, besides the DUREL index; the FACIT-Sp-12 scale; the WHO quality of life instrument (WHOQOL-SRPB); the Spiritual Well-Being scale; among others, as instruments to assess the dimensions of spirituality. Final considerations: The importance of integrating Spirituality in a systematic way in clinical practice and the expansion of the look at the patient (integrality) to improve cardiovascular outcomes is evident, and the application of the WHOQOL – SRPB to measure the dimensions of spirituality should be enforced. More robust studies in the population in question are recommended in order to explore opportunities for future research on TS integrated with Spirituality. 109818 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION IGOR LUCAS FARIAS LIMA1, Luiz Fernando Leite da Silva Neto1, Wadilla Fiuza da Silva2, Larissa da Silva Cambraia1, Alexandre D‘Annibale Cartágenes1, Pietro Chaves Amaral Miralha3, Talles Levi Pereira Nogueira2, Vinícius Queiroz Silva1 (1) UNIVERSIDADE DO ESTADO DO PARÁ – UEPA; (2) UNIVERSIDADE FEDERAL DO PARÁ – UFPA; (3) CENTRO UNIVERSITÁRIO DO ESTADO DO PARÁ – CESUPA Introduction: Primary Hypertension is the term used to characterize Systemic Arterial Hypertension with no known cause, accounting for about 95% of cases. In this clinical entity, the patient has blood pressure levels that confer a significant increase in the risk of clinical events in the short and long term. As it is a highly prevalent disease, it is necessary to understand the hospital morbidity of this disease in the North region of Brazil. Objective: To characterize the epidemiological profile of patients with hospitalization outcome due to Primary Hypertension in the North region of Brazil between 2017 and 2021. Method: The following study is ecological, descriptive and retrospective. Data were sourced from the Departamento de Informática do Sistema Único de Saúde (DATASUS) regarding the epidemiological characteristics of hospital morbidity due to primary hypertension in Northern Brazil between 2017 and 2021. The following variables were analyzed: number of hospitalizations, year of hospitalization, federation units, sex, age group, hospitalization cost, hospitalization time and mortality. Results: A total of 28,331 hospitalizations were recorded in the verified period. The main years that received the most hospitalizations were: 2017 (22.24%), 2019 (22.07%) and 2018 (21.99%). As for the federation units, the most remarkable were: Pará (57.88%), Rondônia (17.38%) and Amazonas (14.57%). In concern to the profile of the hospitalized patient, the most prevalent were women (56.34%), with the most prevalent age groups from 60 to 69 years old (22.11%) and from 70 to 79 years old (19.66%). Furthermore, the medium cost of hospitalization was R$ 263.06, with an increase of 22.86% from 2017 to 2021. The average hospital internment was 3.3 days, with a higher value for males compared to females. Finally, the mortality rate is 2.02, with emphasis on the age groups over 80 years and 70 to 79 years. Conclusion: Besides noting that the number of hospitalizations showed an uneven distribution among the states in the region, there was an increase in the average cost of hospitalization for the health system during the period. Concerning the profile of patients, most are female, but males had higher mortality, in addition to a clear correlation between age and the outcome of death. This study presents data that can guide not only clinical decisions focused on populations at risk, but also provide the basis for public health actions and resource allocation. 109834 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ALICE ZANETTI DUSSIN1, Anna Paula Tscheika2, Luiz Claudio Danzmann3, Marcus Vinicius Simões4, Andrielle Dias Pinheiro2, Luiz Carlos Bodanese1 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS); (2) Hospital São Lucas da PUCRS (HSL); (3) Universidade Luterana do Brasil (ULBRA); (4) Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) Background: Congestion detected by lung ultrasound (LUS) through B-lines is related to prognosis in patients with heart failure (HF); performing it after physical stress tests can detect congestion even earlier. However, stress tests require equipment and specialized professionals. The 6-minute walk test (6MWT) is a submaximal exercise test; it is a feasible, inexpensive, and reproducible test to evaluate patients with HF. The dosage of NT-proBNP is also useful in the diagnosis and prognosis of patients with HF. There are no studies evaluating the use of lung US after 6MWT. Aims: To evaluate the correlation of the number of B-lines on LUS before and after the 6MWT with the measurement of the NT-pro-BNP in outpatients with heart failure with reduced ejection fraction (HFrEF). Methods: It is a cross-sectional analytical study with prospective inclusion of patients from three HF outpatient clinics. Inclusion criteria were: age ≥18 years, to have the diagnosis of HF for at least 6 months and to have echocardiogram with ejection fraction <40%. Blood samples were collected for NT-proBNP analysis before the 6MWT. Patients were submitted to the LUS before and after 6MWT. Results: From September 15, 2020 to November 30, 2021, 188 patients were included in the study. The mean age of the patients was 61.83 years (±12.13 years); most patients were male (63.8%); the median left ventricular ejection fraction was 31.73% (interquartile range 25–75% [IQR] 28–37%); the most prevalent etiology of HF was ischemic (52.7%). The NT-proBNP median value was 1,017.5 pg/mL (IQR 331.5–3,482.5); the median number of B-lines was 3 (IQR 1–9) at rest and 6 (IQR 2–13) after submaximal stress. The NT-proBNP value was correlated with the total number of B-lines at rest (r = 0.517; p < 0.001) and after submaximal stress (r = 0.585; p < 0.001), as shown in Figure 1. Conclusion: B-lines in LUS after submaximal stress and at rest were correlated to NT-proBNP in outpatients with HFrEF. Since congestion is one of the main factors that precedes hospitalization, the use of LUS seems to be a useful and easy-to-learn tool to evaluate outpatients with HF. 109839 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LUIZ FERNANDO LEITE DA SILVA NETO1, Leticia Lima Branco1, Kennedy Medeiros Cavalcante1, Adriano Leitão de Almeida1, Daniel Oliveira da Costa1, Maria Eduarda Silveira Bührnheim1 (1) Universidade do Estado do Pará Introduction: Acute Myocardial Infarction (AMI) is responsible for 12.1% of all cardiovascular diseases (CVD) deaths. This disease is marked by the impairment of the cardiac heart by the minimal blood supply due to total or partial obstruction of a coronary artery. In this context, an expressive severity of the pathology generates hospitalizations and deaths, denoting the need for epidemiological studies in the Brazilian territory to understand its prevalence. Objective: To evaluate the epidemiological profile of hospitalizations and deaths in Brazil due to myocardial infarction from January 2016 to December 2020. Methods: This study has a descriptive, transversal, quantitative and ecological character. Data was collected on Unified Health System Information Department, including information from january 2016 to december 2020. Thus, there were analyzed variables such as hospitalization year, region, sex, age group, color, type of care and deaths. Results: There were a total of 600.752 hospitalizations by AMI. The higher prevalence was found in the Southeast region (49,47%). Regarding the most affected group of people, white people (40,98%), from male sex (56,02%) and aged between 60 and 69 years old (30,75%) were in evidence. Furthermore, the hospitalization quantity increased from 2016 to 2019, although it decreased in 2020. Finally, most hospitalizations were categorized as emergencies (95.88%), and the number of deaths represented 10.31% of the total hospitalizations. Conclusion: Hospitalizations caused by AMI increased from 2016 to 2019 due to demographic transition, characterized by a life expectancy increase in the population. However, there was a decrease in cases in 2020 due to the COVID-19 pandemic, marked by a reluctance to seek medical care and structural limitations in hospital centers, hampering notification. Therefore, with these epidemiological data, it is possible to determine populations at risk and the location with the highest prevalence, guiding future research in this area and the creation of public health actions as well as new financial investments. 109850 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOGERIATRICS BRENO VINICIUS DIAS DE SOUZA1, Lauanda Ênia de Medeiros Rocha1, Emilie Queiroga Queiroga1, Gerson Barbosa do Nascimento1 (1) Universidade Federal do Rio Grande do Norte (UFRN) Introduction: Systemic Arterial Hypertension (SAH) is one of the main risk factors for cardiovascular diseases, which can lead to severe complications, target organ damage (TOD) and death, especially in the elderly. The disease control and consequent prevention of complications requires changes in lifestyle, with adherence to appropriate physical exercises for the age group. Objective: To review, based on the scientific literature, the impact of resistance exercise on blood pressure reduction in elderly hypertensive individuals. Method: Systematic review of articles published between 2011 and April 2021, in the BVS, LILACS, Cochrane and Pubmed databases with the descriptors “hypertension”; “resistance training” and “elderly”, with 579 articles found. There was the exclusion of duplicate articles, independent evaluation by two researchers of title and abstract, and analysis of the full text of original studies, according to the inclusion and exclusion criteria. Results: 10 articles were included and analyzed. All were unanimous in defending the safety of strength training in hypertension and 9 attested to its direct hypotensive effect in the elderly. The evaluation of resistance exercise in elderly hypertensive women stands out, with a relevant reduction in systolic blood pressure (BP). A 2016 study attested chronic reduction in systolic, diastolic and mean BP in women who acutely already had the hypotensive effect, establishing a relationship between them. It was also evidenced that the greater the duration or volume of exercises, the greater the hypotensive effect. These findings are in line with Brazilian Directive on Arterial Hypertension’s recommendations. Conclusion: Within the complexity of SAH treatment, the practice of resistance physical exercises was crucial for blood pressure control. Last decade’s studies with methodological rigor confirmed its role in the non-drug treatment of SAH, with safety, reduced BP and improvement in the hemodynamic profile in the elderly. 109852 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH RENAN GERONIMO SOUZA DA SILVA1, Renan Geronimo Souza da Silva1, Alexandre Akio Majima1, Ana Paula Cassetta dos Santos Nucera1, Fábio de Souza1, Wilson Braz Corrêa Filho1, Eduardo Marinho Tassi1, Davi da Silveira Barroso Alves1, Paulo Henrique Godoy1 (1) Federal University of the Rio de Janeiro State (UNIRIO) Introduction: Prevalence of heart valve diseases (HVD) is increasing and its etiological profile has changed over the last few decades. Objective: To analyze the trend of deaths due to HVD, according to gender and age group in Brazil, from 2000 to 2019. Method: Ecological study, in which information was collected from the Mortality Information System. The causes of death were categorized into: rheumatic valve disease (RVD); non-rheumatic valve disease (NRDV) and congenital valve disease (CVD), according to ICD-10. Age groups were: 0–29, 30–59, 60 or older. Crude and standardized rates were estimated according to sex and age. Results: A total of 113,570 deaths due HVD were found, 54.93% were female. The distribution by causes was: RVD – 36,018, RVD – 71,477 and CVD – 3,583. The female gender was predominant in the RVD, while in the CVD the male gender was prevalent. In the DVNR, the standardized rate was higher in males, although a greater magnitude of deaths occurred in females. The highest mortality rates due DVR and DVNR were observed in the group aged 60 years and over. In CVD, the mortality rate was higher in the younger age group. The trend for deaths due DVNR and CVD was increasing, while from DVR was decreasing. Conclusions: Trends in deaths from RVD and NRVD seem to reflect an epidemiological transition from these causes. The trend for DVC deserves attention. It seems to reflect higher mortality from congenital heart disease in the country. 109859 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH HILDEBRANDO ANTUNES DE CARVALHO NETO1, Bruno Oliveira Souza Prates1, Beatriz Souza Martins1, Beatriz Catarina dos Santos de Oliveira1, Gabriela Chateaubriand Campos1, Rafael Carlos Pereira1 (1) Universidade Federal da Bahia – UFBA Introduction: Systemic Arterial Hypertension (SAH) is a chronic multifactorial condition that constitutes the main modifiable risk factor for cardiovascular complications. It is estimated that this disease has high morbidity and mortality and affects about 30% of the world’s adult population, with higher prevalence in low- and middle-income countries, such as Brazil. In this sense, the epidemiological study of the disease presents itself as an important instrument for this control and prevention. Objective: To analyze the mortality rate and epidemiological profile of hospitalized patients with SAH in Brazil, between 2012 and 2021. Methods: This is an ecological time series study from secondary data collected by the Hospital Information System (SIH-SUS), between the years 2012 to 2021. The total and percentage distribution of elective and urgent care, mortality rate and demographic data were included. Results: In Brazil, in the years analyzed, 608,045 cases of patients hospitalized for SAH were reported, 93.9% of which were on an emergency basis. The year 2012 had the highest number of cases (85,646), and year 2021 had the lowest number (35,472). Females were the most prevalent (85.75%) and the age group most affected was the elderly between 60 and 69 years old (22.42%), followed by elderly people between 70 and 79 years old (20.34%). Most of the admissions were of brown patients (38.38%), followed by whites (24.24%). Within the years under review, the mortality rate ranged from 1.41 in 2012 and 2013 to 2.1 in 2021. Conclusions: Based on the data analyzed in a temporal sequence, there is a tendency to decrease the number of hospitalized patients each year, with the year 2021 having a total number of hospitalizations 58.58% lower than the beginning of the historical series. This decrease in hospital admissions happened gradually, and had its most significant drop in 2020 (24.99%). Elderly, female and brown patients had a higher prevalence in all analyzed years. Regarding the mortality rate attributed to SAH, among hospitalized patients, there was a trend towards stability, with an increase in the year (27.27%). 109863 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY ENZZO BARROZO MARRAZZO1, Luiz Fernando Moraes Pereira1 (1) Pontifícia Universidade Católica de Minas Gerais Background: Heart failure (HF) is occasioned by a functional or structural cardiac change. HF has been during years the major cause of mortality in the world. The treatment of chronic heart failure with reduced ejection fraction is mainly diuretics, neurohormonal antagonists and devices therapies. New pharmacological therapies have been developed as sodium-glucose cotransporter 2 inhibitors and vericiguat, which are promises a better prognosis. Aim: To clarify the impact of Vericiguat use on mortality and on hospitalization rates of patients with heart failure with reduced ejection fraction. Methods: A systematic review was carried out by using a sheared in the PubMed, Lilacs, and Embase databases to search for the descriptors “Vericiguat” e “Heart Failure, Reduced Ejection Fraction”. Only studies conducted on humans, clinical trials where participants were over 18 years old had been selected for inclusion. Studies, where was including patients with HF with preserved ejection fraction and studies focused on sacubitril/valsartan, were not included. To avoid bias was made a double-check in the entire filtering process by different authors. Results: A total of 116 articles were identified and analyzed, of which only 7 were eligible for this review. The use of vericiguat after 3 months of follow-up demonstrated a decrease in the incidence of cardiovascular deaths or hospitalization for HF, reducing these primary outcomes by 4.2 events per 100 patient-years. Despite the few studies, it is suggested that these results can be generalized to patients with worsening HF events in clinical practice. Furthermore, the use of vericiguat was not related to the occurrence of atrial fibrillation events after randomization, although these events are related to the number of deaths and hospitalizations for HF. The effect of vericiguat on the assessed outcomes appears to be better in younger patients, however there is no heterogeneity in the treatment effect of vericiguat compared to placebo on the composite primary endpoint of patients according to baseline systolic blood pressure. Conclusion: The use of vericiguat appears to be promising in reducing mortality and hospitalization rates in patients that developed HF with reduced ejection fraction. Despite recent and scarce studies, the drug demonstrates good safety in clinical management, not inducing the occurrence of events such as syncope, clinical manifestations resulting from hypotension and atrial fibrillation. 109868 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LARISSA ALMEIDA BUSNELLO1, Paola Gonçalves Moreira de Oliveira1, Bruna Karas1, Ana Carla Dlugosz1, Alice Magro Koscianski1, Francielle Nocera Viechineski1, Camilla Mattia Calixto1, Júlia Henneberg Hessman1, Mário Claudio Soares Sturzeneker1 (1) Universidade Estadual de Ponta Grossa (UEPG) Introduction: Heart failure (HF) has an estimated worldwide prevalence of 1–2%, reaching ≥10% in elderly people ≥70 years. In Brazil, hospitalizations for HF correspond to 4% of general hospitalizations and have high morbidity and mortality. During the COVID-19 pandemic, there was congestion of the health system, but during the 2nd half of 2021, about 70% of the population was vaccinated with the 1st dose and 40% with the 2nd dose. Objective: Evaluate the impact of the pandemic on HF mortality in the southern region of Brazil through the analysis of DATASUS data. Methods: Data regarding HF in the South region were collected in the DATASUS system: hospitalizations, deaths and average hospital stay. Lethality was calculated by dividing the number (N) of deaths by the N of hospitalizations. The periods of pre-pandemic (02/17–02/20), pandemic (03/20–06/21), peak of the pandemic (12/20–05/21), 1st and 2nd quarter of the peak (12/20–02/21 and 03–05/21) and mass vaccination (06–12/21) were evaluated. The means of each variable were calculated by sex and age, and each vaccination period was compared with the others, with the results manifested as a percentage. Results: There was a reduction in the number of hospitalizations in the period of mass vaccination in relation to the pre-pandemic period (25.88%) and an increase of 44,76% in comparison with 2nd trimester of the peak (2TP), and about hospitalizations and deaths, the largest percentage difference evaluated was seen in the 2TP. The evaluation by gender and age groups showed similar behaviour, including the largest increase in relation to the peak of the pandemic, reaching 55.06% in the population >80 years. Regarding the average hospital stay, there was an increase in the vaccination period compared to all other periods, except in the 20–29 age group. Regarding deaths, during the mass vaccination period, there was little variation when compared to the pre-pandemic period, but with significant increases compared to pandemic periods, especially when compared to the peak. There was a significant increase in lethality during the vaccination period when compared to the pre-pandemic period (39.42%), mainly in the age group of 40–49 years (62.43%) and 60–69 years (59.99%). Conclusion: Hospitalizations and inpatient deaths reduced during the COVID-19 pandemics, especially at the peak of the pandemy. However, currently, with mass vaccination, there is a tendency to return the parameters to pre-pandemic values. 109869 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA CARLA DLUGOSZ1, Larissa Almeida Busnello1, Camilla Mattia Calixto1, Bruna Karas1, Júlia Henneberg Hessman1, Alice Magro Koscianski1, Mário Claudio Soares Sturzeneker1, Paola Gonçalves Moreira de Oliveira1, Francielle Nocera Viechineski1 (1) Universidade Estadual de Ponta Grossa (UEPG) Introduction: The SARS-CoV-2 pandemic may have influenced heart failure (HF) morbidity and mortality due to a lack of vacancies or fear of contamination in the hospital. Objective: Assess the influence of the SARS-CoV-2 pandemic on HF morbidity and mortality in the Southeast region. Method: Data from 2017 to 2021 were collected on the DATASUS platform. Lethality was calculated by dividing the number (N) of deaths by the N of hospitalizations and expressed as a percentage. The periods denominated pre-pandemic (02/2017 to 02/2020), pandemic (03/2020 to 06/2021), peak of the pandemic (12/2020 to 05/2021), the peak’s 1st and 2nd quarter (12/2020 to 02/2021 and 03/2021 to 05/2021) and mass vaccination (06/2021 to 12/2021) were evaluated. The means of each variable were calculated in general by sex and age group. The pre-pandemic and mass vaccination periods were compared to the others, and the results were expressed as a percentage. Results: Compared to the pre-pandemic period, there was a reduction in the number of hospitalizations in the pandemic period (20.56%), mainly at the peak (34.31%). There was a reduction in the average hospitalization length in all periods of the pandemic, except for the 20–29 age group in the peak’s 1st quarter. There was also a reduction in deaths numbers, mostly in the peak’s 2nd quarter. There were increases in lethality in all periods of the pandemic. During the mass vaccination period, there were reductions in hospitalizations (15.77%), hospitalization length (3.05%) and mortality (3.06%) compared to the pre-pandemic period. However, compared to the pandemic period, there was an increase in hospitalizations (6.03%), hospitalization length (3.96%) and mortality (11.12%). As for lethality, there was an increase of around 15% in both genders and in all age groups compared to the pre-pandemic period and 6.32% compared to the pandemic period, except for women. Conclusions: Data suggests a significant impact of the pandemic on HF lethality in the Southeast region. The expressive reduction in the number of hospitalizations, associated with the reduction in the average hospitalization length, could justify a lower number of deaths and the divergent behaviour of lethality during the pandemic. Mass vaccination allowed these patients to have greater access to hospitals and, probably due to the increase in demand caused by the pandemic, there was an increase in hospitalizations, hospitalization length and mortality. 109874 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARIANA SALLES BALLALAI1, Helena Raquel Nogueira de Oliveira1, Isabela Aragão Colares1, Gabriel Sousa Santos1, Renata Pinheiro Martins de Melo1, Gabriel Coelho Brito Dias1, José Levi Tavares Cavalcante1, Ane Karoline Medina Néri2, Weiber Silva Xavier3, João Luiz de Alencar Araripe Falcão1, Sandra Nívea dos Reis Saraiva Falcão1 (1) Faculdade de Medicina, Federal University of Ceará (UFC); (2) Hospital Universitário Walter Cantídio (HUWC), Federal University of Ceará (UFC); (3) Programa de Educação em Reanimação Cardiorrespiratória (PERC) Introduction: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Acute chest pain is responsible for more than 5% of all emergency department visits. Although most of these patients are discharged with a diagnosis of non-cardiac cause, about 25% are affected with acute myocardial infarction (AMI). Objectives: To analyze the behavior of hospitalizations and deaths numbers due to acute myocardial infarction in the last decade in Brazil. Methods: Retrospective descriptive study with data from the Brazilian Unified Health System database(Tabnet-DATASUS) on hospital admissions and deaths from AMI, by region, in Brazil, from January 2012 to January 2022. Results: Annually, an average of 107,522.8 (±17,350.5) hospitalizations due to AMI occur in Brazil, and the southeast region has the highest average number of hospitalizations (55,005 ± 8003.8). There was an increase of 64% in the number of hospitalizations in 10 years, except in the year 2020 when there was a discrete reduction of 0.6%, which could be explained by the pandemic context. The most considerable increase occurred in the mid-western region, with 160.7% increase in the number of hospitalizations, followed by the northern region with 88.1%. On the other hand, the southern region had the shortest variation, with an increase of 51.7% in 10 years. About mortality, an average of 11,981.4 deaths (±936.26) occurred annually due to AMI. In the last decade, there was an increase of 28% in the number of deaths, except for the intervals 2016/2017 and 2019/2020, in which there was a subtle reduction respectively of 2.0% and 3.8% in the numbers of deaths. The period of greatest increase was 2020/2021 (8.6%). The region with the highest increase in mortality was the mid-west (37.8%). Although the Southeast region had the highest percentage of deaths, this region had the lowest variation in the number of deaths over the years (22.8%). Conclusion: We analyzed the numbers of hospitalizations and deaths due to AMI in the last decade and we found that AMI is still very prevalent in Brazil and that it is an important cause of death in this country. We also noted that there was a considerable increase in hospitalizations during the evaluated period, especially in the southeastern region. Thus, a greater strict prevention in populations at risk is necessary, and the importance of early recognition and adequate treatment of these patients is reaffirmed, to avoid worse prognosis. 109876 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY BRENDA GABRIELE SMANIOTTO RAULIK1, Jeronimo Antonio Fortunato Junior2, Juliana Fortunato2, Shema El-laden hammoud1, Letícia Gusso Scremin1, Nikolai Cernescu Neto1, Gabriel Fontana de Melo1, Thaísa Rodrigues Ferreira Basaglia1 (1) Universidade Positivo; (2) Hospital da Cruz Vermelha Brasileira – Filial Paraná Introduction: Coronary artery fistula (CAF) is defined as an abnormal communication of coronary arterial blood with other capillary structure, without passing through myocardial capillaries. The congenital origin is the most common, despite accounting for only 0.2 to 0.4% of congenital heart defects. Although rare, they can be fatal. Cardiac surgery allows the correction of most coronary anomalies, but thoracotomy and cardiopulmonary bypass support are associated with a higher number of per- and postoperative complications compared to less invasive videothoracoscopy techniques of surgical treatment. Objectives: Report 5 cases of patients diagnosed with CAF and describe the methodology used for diagnosis, the 5-year evolution of the patients, and the description of the first cases reported in the literature who underwent minimally invasive video-assisted thoracoscopic surgery (VATS). Methodology: We selected the medical records of five medical with CAF diagnosed by two-dimensional echocardiography, coronary angiotomography and cineangiocoronariography. Results: Patients who underwent VATS procedure had to sign an Informed Consent Form. In this study the male gender was more frequent, with 82% of cases, and the mean age was 56.8 years. All patients presented clinics of obstructive coronary insufficiency and only one patient evolved to heart failure NYHA III functional class, this same patient in angiographic studies presented, besides the CAF, an intramyocardial tract of the anterior descending artery. There were no intercurrences during the procedures and the primary success was confirmed with coronary angiographic studies to evaluate remaining fistulous branches or residual shunts. There was no mortality after 5 years of outpatient follow-up. Conclusion: The association of 3 diagnostic methods has fundamental importance to guide the treatment to be followed. With the minimally invasive technique and ligation of the CAF via thoracoscopy, there is a satisfactory surgical result, associated with better long-term clinical outcome and low morbidity. 109900 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES EDUARDA CHAVES SILVEIRA1, Mariana Goulart Almiron3, Jessica Luiza Pedroso da Silva1, Bruna Diehl1, Tiago da Rosa Rambo1, Helena Rocha Machado3, Alexander Romão Vieira Morinélli1, Patrícia Érika de Melo Marinho2, Dulciane Nunes Paiva1 (1) Universidade de Santa Cruz do Sul (UNISC); (2) Universidade Federal de Pernambuco (UFPE); (3) Hospital Santa Cruz (HSC) Introduction: Non-invasive ventilation (NIV) is widely used in the postoperative period (PO) of cardiac surgery (CS) to promote lung reexpansion. The orofacial mask, interface usually used in NIV, can cause air leakage and, in the Covid-19 pandemic, avoid aerosolization to the environment has become a priority focus. The adapted diving mask (Owner Mask) emerged as an alternative NIV interface and its effectiveness has been evaluated. Objective: To assess the satisfaction level and the perception of change in patients undergoing NIV with an Owner mask in the PO of CS. Methods: A cross-sectional study that evaluated patients undergoing coronary artery bypass graft or valve replacement surgery with clinical indication for NIV. The level of satisfaction and the perception of change was assessed using the Patients‘ Global Impression of Change questionnaire at discharge from the ICU. This instrument classifies the perception of improvement in 7 items: 1 = No changes, 2 = Almost the same, without any visible change, 3 = Slightly better, but without considerable changes, 4 = With some improvement, but the change did not represent any real difference, 5 = Moderately better, with slight but significant change, 6 = Better, and with improvements that made a real and useful difference, 7 = Much better, and with a considerable improvement that made all the difference. Data described in frequency of occurrence. Results: Sample (n = 29; 52% male) had comorbidities such as SAH (66.7%), diabetes mellitus (24.8%), ischemic heart disease (19.4%), CHF (16.7%) COPD (11.1%), dyslipidemia (11.1%) and obesity (11.1%). Of the sample evaluated, 17.2% reported “no changes”, 3.4% “Almost the same, no visible changes”, 20.7% “Slightly better, but without considerable changes”, 10.3% “With some improvements, but the change didn‘t make any real difference”, 10.3% Moderately better, with slight but significant change, 20.7% “Better, and with improvements that made a real and useful difference” and 17.2% reported as “Much better, and with a considerable improvement that made all the difference”. Conclusion: The level of satisfaction and the perception of change with the use of the Owner mask went from “Slightly better, but without considerable changes” to “Better, and with improvements that made a real and useful difference” in 41.4% of the sample evaluated. The comparison of these outcomes in relation to the conventional orofacial mask will be carried out with the expansion of the research. 109905 Modality: E-Poster Scientific Initiation – Non-case Report Category: NUTRITION RAQUEL SANTIAGO VITORINO1, Luana Azevedo de Aquino1, Letícia Martins Raposo1, Simone Augusta Ribas1, Michelle Teixeira Teixeira1 (1) Universidade Federal do Estado do Rio de Janeiro UNIRIO In an overwhelming way, the Sars-Cov2 virus changed the planetary routine starting in 2020. In order to contain the advance and mortality of the pandemic, the WHO and several entities and governments recommended restriction in social contact, resulting from the closing of schools, commerce and leisure areas. The domestic environment becomes the only space for social coexistence, limiting the activities of children and adolescents. The vulnerability of development in this age group maximizes the risks by interrupting the children’s routine. Objective: To describe the lifestyle of Brazilian children and adolescents, regarding food, physical activity, sleep quality, screen time, in the period of social restriction resulting from the COVID-19 pandemic. Methods: Cross-sectional study carried out between May and June 2020, with data collected through online forms, intended for parents and/or guardians of children aged 2 to 9 years and adolescents aged 10 to 18 years, in a random and non-probabilistic manner, in all Brazilian regions. Prevalence calculations were performed, with a 95% confidence interval, with the R software. Results: 1309 participated in the survey, 589 children and 720 adolescents. Of the total, half of the families were in social isolation (52%) and belong to the middle social class (56%). As for the practice of physical activity, 72% did not perform or performed less than one hour a day. In addition, the sedentary behavior of excessive use of electronic devices occurred among 75% of those evaluated, with adolescents being the users with the highest reported screen time. There was a change in sleep among 55%, with inadequacy for more (17%) or less (24%). As for eating habits, 30% used to replace meals with snacks, and there was a significant increase in the consumption of sugary drinks (25%), sweets (45%) and quick snacks (36%), especially among adolescents (p < 0.001). On the other hand, there was a reduction in the consumption of beans (10%), fruits (13%), vegetables (20%) and dairy products (10%). Conclusions: The social restriction imposed by the COVID-19 pandemic has imposed negative changes in diet and physical activity level that could possibly have a long-term impact on the health of children and adolescents. 109909 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM BIANCA MENDONÇA REIS1, Gabriela El Bazi1, Ravy Soares Álvares1, Millena Batistela Pereira1, Caroline Borges de Assis1, Bruno Carraro1, Humberto Graner Moreira1 (1) Universidade Evangélica de Goiás Introduction: The access to hospital care in Brazil has always been conditioned to each geographic region’s socioeconomic discrepancies. Following the isolation resulting of the pandemic of the new coronavirus, the access to health care has been compromised even further, especially regarding treatment for acute myocardial infarction (AMI). Until 2019, AMI represented the leading cause of deaths in Brazil, however, in 2020, with the unfolding of the pandemic, some countries observed a drop in the AMI notifications. Objectives: To describe the reduction in reported cases of AMI among the geographic regions of Brazil. Methods: Observational and descriptive study with a quantitative approach. Data collected using the databases of the Department of Informatics of the Unified Health System (DATASUS) from the Hospital Information System of the SUS (SIH/SUS) and the Mortality Information System (SIM) from January 2017 to December 2020, using the variables: number of hospital admissions; average length of hospitalization; hospital mortality rate and as the underlying cause myocardial infarction, codes I21 to I24 of the 10th International Statistical Classification of Diseases and Related Health Problems (ICD-10). Patients younger than 20 years old were excluded from the study. Results: Comparing data from 2020 to 2019, there was a decrease in cases of AMI in the North, Northeast and Southeast regions, while the South and Midwest presented an increase. In the North region, there was a progressive increase in cases between 2017 and 2018. In 2020, the reduction was of 1.61% cases compared to 2019. In the Northeast region, the reduction of 7.13% cases from 2019 to 2020, was more significant. However, in the Midwest region, there was an increase of 948 cases from 2019 to 2020 while in the Southeast region, there was a decrease of 427 cases. In the South, there was an increase of 3.01% from 2019 to 2020. Conclusion: Despite the expectations of a decrease in AMI cases in all geographic regions the Midwest and South regions presented increasing data. The reduction noticed in other regions is directly related to a sudden drop in the search for AMI treatment due to the imposed social isolation and the fear of contracting a respiratory infection in the hospital environment. Socioeconomic disparities between regions directly interfere with the result. 109915 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY JOÃO MARCOS DE FONTES CARNEIRO1, João Marcos de Fontes Carneiro1, Daniel Salmito Chaves1, Paulo Roberto Matos Neto1, Felipe Salim Habib Buhamara Alves Nasser Gurjão1, Mateus de Sousa Cavalcante1, Bruna de Almeida Freixedelo1, Benedito Mesley Lima Portela1, Leandro Cordeiro Portela1, David Carneiro Neto1 (1) Universidade Federal do Ceará – campus Sobral Introduction: Sustained alcohol consumption is closely linked to atrial fibrillation (AF), which is the most common heart rhythm disorder. This is due to electrical remodeling in the atrium, which facilitates the formation of arrhythmogenic portions. This condition is of great social relevance, as it is associated with the deterioration of the hemodynamic profile and cardiac output in affected patients, which is, therefore, a worsening factor in heart failure (HF). Objectives: To evaluate the relationship between alcohol intake and the incidence of AF in patients with decompensated HF. Methods: A cross-sectional study was carried out with 322 patients admitted to a cardiology hospital for acute HF decompensation. Information was obtained from a database collected by properly trained health professionals present at the time of hospital admission, with a signed consent form. Participants were divided into two groups: those who had the habit of drinking alcohol and those who did not. Electrocardiographic data were collected in both groups, and the appearance of Atrial Fibrillation was recorded. To verify the significance of the association between alcohol intake and the onset of AF, Fisher’s Exact Test was used. The calculation of statistical significance was performed on the OpenEpi platform, using a significance level of p < 0.05. Results: Of the 67 patients with atrial fibrillation at hospital admission, a total of 25 (37.31%) had the habit of drinking alcohol. On the other hand, of the 255 patients who were admitted without atrial fibrillation, only 65 (25.5%) reported drinking alcohol. Thus, there was a significant influence in both groups regarding the consumption of alcoholic beverages as a risk factor for the onset of atrial fibrillation at hospital admission, with a considerable significance level, with p = 0.04062. Conclusion: The habit of drinking alcoholic beverages is an important propensity factor for the development of AF in patients with acute decompensated HF, evidencing the importance of correct control of certain lifestyle habits for the prevention and control of the prevalence of factors that influence the aggravation of the symptoms and greater chance of complications that culminate in a worse prognosis of HF. 111117 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION AHYSLA GONÇALVES DURÃES1, Adson Renato Leite2, Antônio José Lagoeiro Jorge2, César Augusto da Silva Nascimento2, Maria Luiza Garcia Rosa2, Wolney de Andrade Martins2, Ana Paula Arriaga Carvalho2, Clara Mônica Figueredo de Lima1, Luan Andrade Carvalho3, Emilly Vendramini Mello1, Ana Leticia dos Anjos Sampaio1, Nathália Felícia Silva Frias1 (1) Universidade Federal do Sul da Bahia (UFSB); (2) Universidade Federal Fluminense (UFF); (3) Universidade Federal do Vale do São Francisco (UNIVASF) Obstructive sleep apnea (OSA) is a chronic, progressive disorder with high mortality and morbidity associated with cardiovascular diseases (CVD), including heart failure (HF). OSA-related pathophysiological alterations can directly impact the diastolic function of the left ventricle. Objective: To evaluate the relation between the risk of OSA, considered by the Berlin Questionnaire (BQ), and echocardiogram parameters, related to diastolic function, in individuals without HF assisted by the Family Medical Program of Niterói (PMF). Methods: Cross-sectional study that included 354 individuals (51% women) aged 45 years or older. The exclusion criteria were the presence of HF, non-completion of the BQ and obese patients with arterial hypertension and not classified as being at risk for OSA by another criterion. All the individuals selected for the study underwent an evaluation, performed in a single day, that consisted of the following procedures: consultation, filling out the BQ and clinical examination, performing laboratory tests and transthoracic doppler echocardiogram (TDE). Results: Of the 354 individuals analyzed, 63% were classified as being at risk for OSA. Patients at risk for OSA present significant alterations in the parameters that assess the diastolic function, which may indicate a worsening of this function in these patients. An adjusted multivariate gamma regression was performed. After adjustment (gender, age, BMI, fasting glucose, triglycerides, serum uric acid, urea albumin/creatinine ratio, systolic and diastolic blood pressure in their continuous forms), risk for OSA confirmed its positive and statistically significant association with indicators of diastolic dysfunction, LAV-i (p = 0.02); E‘/A‘(p < 0.01), A (p = 0.02), E/A (p < 0.01). Conclusion: Our data show that patients at risk for OSA present a worsening in the diastolic function parameters measured by the TDE. 109931 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GABRIEL COELHO BRITO DIAS1, Mariana Salles Ballalai1, José Levi Tavares Cavalcante1, Helena Raquel Nogueira de Oliveira1, Isabela Aragão Colares1, Gabriel Sousa Santos1, Renata Pinheiro Martins de Melo1, Ane Karoline Medina Néri2, Weiber Silva Xavier3, João Luiz de Alencar Araripe Falcão1, Sandra Nívea dos Reis Saraiva Falcão1 (1) Universidade Federal do Ceará (UFC); (2) Hospital Universitário Walter Cantídio (HUWC); (3) Programa de Educação em Reanimação Cardiorrespiratória (PERC) Introduction: Heart Failure (HF) is a syndrome in which the heart is unable to adequately pump blood to meet organic needs. Despite clinical advances, HF is the leading cause of hospitalization among circulatory system diseases. Objectives: To describe the morbidity of HF in Brazil and its impact on public health in the country. Methods: Observational descriptive study conducted using data obtained from DATASUS. The variables gender, age group, death and hospitalization costs of patients with HF between 2017 and 2021 were analyzed. Results: In the period, the total number of hospitalizations was 928,541, with the highest number occurring in 2017 recording 209,162 (22.53%) and decreasing annually until the lowest value in 2021 (16.27%). There was a predominance of male patients representing 480,691 (51.77%). The most affected age group was the elderly aged 70 to 79 years with 26.40% of hospitalizations. In addition, 108,692 deaths were registered in the period, totaling 11.71% mortality rate for HF within this time frame. Finally, the average amount spent per hospitalization was R$ 1,649.47. Conclusion: We analyzed the numbers of hospitalization and deaths due to HF in the last five years and we found that HF is still very prevalent in Brazil and, in despite of the decrease in the mean number of hospitalizations in 2021, it is an important cause of death in the country and a significant source of state expenses. Furthermore, we noticed that the hospitalized patients tended to be male and aged 70 to 79 years. Thus, a greater strict prevention in populations at risk is necessary and the importance of early recognition associated with an adequate treatment strategy is reaffirmed. 109933 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RAVY SOARES ÁLVARES1, Gabriela El Bazi1, Bianca Mendonça Reis1, Millena Batistela Pereira1, Caroline Borges de Assis1, Bruno Carraro1, Humberto Graner Moreira1 (1) Universidade Evangélica de Goiás Introduction: The search for medical assistance during the coronavirus pandemic in Brazil has declined significantly. Furthermore, the fear of contracting covid in hospitals and the prioritization of urgency and emergency care over elective consultations resulted in a decrease in the search for medical assistance during the pandemic. Faced with this new reality, some changes were noticed in the country’s morbidity and mortality rates, since the Acute Myocardial Infarction (AMI), the leading cause of death in the country until 2019, presented significant decrease during the pandemic. Objectives: To describe the incidence of AMI by sex and age group during the pandemic. Methods: Observational and descriptive study with a quantitative approach. Data was collected using the databases of the Department of Informatics of the Unified Health System (DATASUS) from the Hospital Information System of the SUS (SIH/SUS) and the Mortality Information System (SIM) from January 2017 to December 2020, using the variables: number of hospital admissions; average length of hospitalization; hospital mortality rate and as the underlying cause myocardial infarction and then stratified by age group, and sex, encompassing only individuals over 20 years old. Results: In 2020, the age group with the highest hospitalization rate for AMI was the one of 60 to 69 years old, while the lowest rate belongs to the age group of 80 years and older. In the age group of 80 years and older, females had the most significant reduction (3.3%) in hospitalizations while males decreased in 1.5% Previously to the pandemic, there was an annual progressive increase in mortality by AMI, however in 2020 there was an overall reduction of 6118 deaths when compared to 2019, being that the age group of 80 years and older presented a decrease of 56.15% of total deaths. Females presented a reduction of 2886 deaths in 2020 when compared to 2019 while males, in the age group of 80 years and older, presented a reduction of 30.9% of cases Conclusion: A reduction in hospitalizations and mortality by AMI was expected and confirmed in this study. However, it could be noticed that the main reason behind said reduction was not the improvement in people’s lifestyles and subsequent reduction in cardiovascular diseases, but the association between the lower search for hospital assistance and the fact that the AMI symptoms can be disguised by the coronavirus infection due to the latest’s greater expressiveness. 111850 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOGERIATRICS ALLEXA GABRIELE TEIXEIRA FEITOSA1, Gabriela de Oliveira Salazar1, José Icaro Nunes Cruz1, Cláudia Bispo Martins-Santos1, Lara Teles Alencar Duarte1, Cleovaldo Ribeiro Ferreira-Júnior1, Edvaldo Victor Gois Oliveira1, André Pinheiro Zylberman1, Eduardo José Pereira Ferreira1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe – UFS Introduction: The advance of age is an important risk factor for cardiovascular diseases, including ventricular dysfunction, which makes the elderly an important research and control group. Objective: To identify predictors of ventricular dysfunction in a population of elderly people and to evaluate the level of aerobic capacity of these patients. Methods: Cross-sectional study between January 2000 and January 2022 with individuals over the age of 60 years undergoing Physical Stress Echocardiography (SE) in a respected cardiological service. 1644 (68 ± 6 years) patients were categorized according to cardiorespiratory capacity (CRC) into low (MET < 7,9), intermediate (7,9 ≤ MET < 10,9) and high (MET ≥ 10,9) fitness. The chi-square test and logistic regression were used using SPSS Statistics software version 22.0. A significance level of 5% was admitted. Results: Among the patients analyzed, 40,8% had low CRC, 34,8% intermediate and 24,4% had high aerobic capacity. Of the total, 8,2% (135) had ventricular dysfunction. Low CRC was associated with the presence of: systemic arterial hypertension, sedentary lifestyle, obesity, alcohol consumption, female sex (p < 0,001) and family history (p = 0,01). Male sex (OR = 1,950; IC95% = 1,328–2,863; p = 0,001), diabetes mellitus (OR = 1,908; IC95% = 1,267–2,874; p = 0,002), smoking (OR = 2,540; IC95% = 1,409–4,577; p = 0,002), dyslipidemia (OR = 1,588; IC95% = 1,092–2,307; p = 0,015), low CCR (OR = 3,218; IC95% = 1,846–5,611; p < 0,001) and intermediate CCR (OR = 1,989; IC95%1,129–3,503; p = 0,017) were predictors of ventricular dysfunction in the elderly. Conclusions: Low aerobic capacity was associated with systemic arterial hypertension, sedentary lifestyle, obesity, alcohol consumption and the female sex, as well as being a predictor of ventricular dysfunction in the elderly. 110112 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BIANCA VASCONCELOS BRAGA CAVALCANTE1, Ana Lívia Gadelha Xavier da Nóbrega Pereira1, Beatriz Gadelha e Xavier1, Leticia Lacerda Burity1, Rayanne Alessandra da Silva Barreto1, Vinicius Vieira Leandro da Silva1 (1) FACULDADE DE MEDICINA NOVA ESPERANÇA – FAMENE Introduction: Chronic obstructive pulmonary disease is one of the main causes of mortality in the world and is closely related to cardiovascular diseases, with a risk of 4.5 times greater compared to the healthy population. There is a correlation between heart failure (HF) and COPD, which has been extensively investigated in recent years. Objective: To correlate arterial stiffness in patients diagnosed with COPD. Method and materials: This is a systematic review of the literature indexed between 2018 and 2022, published in the Scientific Electronic Library and PubMed databases. The following terms were applied to the Health Science Descriptors (DeCS): “Chronic Obstructive Pulmonary Disease” and “Aortic diseases”. Articles unrelated to the topic and inaccessible for free were excluded. Results: It is known that about 30% of patients with HF are also diagnosed with COPD, which corroborates the prognosis of increased hospitalizations and mortality in these people. There are few studies that report which biomarkers are related to the two diseases, but it has been seen that aortic pulse wave velocity (aPWV) is the gold standard measure to determine arterial stiffness and, therefore, is a predictor for elevated cardiovascular events. in COPD patients (defined as +0.7 m/s). In a meta-analysis, a +1 m/s increase in aPWV was reported to determine a 15% increase in the risk of heart disease. A plausible explanation for the relationship between aPWV and COPD is the pathological mechanism of degradation of elastin fibers in the lungs and large arteries, such as the aorta, since patients with COPD lose a large part of the cutaneous elastin, which generates severity in emphysema. and arterial stiffness. Conclusion: It is known that these data can influence the determination of the negative impacts of the two pathologies. Thus, there is a clear need for further studies on biomarkers related to COPD and heart disease. 111395 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH AMANDA GABRIELA FREITAS RODRIGUES1, Matheus Ricardo Malveira Camacho2, Lucas dos Santos Fontes1 (1) Universidade Federal do Pará; (2) Universidade Estadual do Pará Introduction and/or Fundamentals: Acute myocardial infarction (AMI) is responsible for significant mortality rates in Brazil and worldwide. Despite the implementation of national policies to prevent precursor diseases of cardiovascular importance such as hypertension and diabetes, mortality rates from AMI remain high compared to the rates of developed countries. Therefore, the analysis of the epidemiological profile of deaths caused by AMI in Brazil is crucial, so that the strategy towards reducing mortality from this disease is more accurate. 2. Objectives: Trace an epidemiological profile of deaths caused by acute myocardial infarction in the state of Pará, Brazil, from 2016 to 2020. 3. Methods: Analysis of data provided by the digital platform of the Department of Informatics of the Unified Health System (DATASUS), in the category “deaths from preventable causes from 5 to 74 years old”, ICD-10 category “I21 Acute myocardial infarction”, selecting the sections according to the search parameters, such as “sex” and “education”. 4. Results: In the period evaluated, there was a trend of growth in the number of cases of deaths caused by AMI; 1577 were registered in 2016; 1709, in 2017; 1721, in 2018; 1840, in 2019; and 1863, in 2020; totaling 8710 deaths. There was a greater involvement of men (n = 5953/68.4%) and less of women (n = 2757/31.6%). Regarding self-declaration of color, most deaths were registered in brown people (n = 6702/76.9%), followed by white (n = 1181/13.5%) and black (n = 605/6.9%). People aged between 60 and 69 years were more affected (n = 3343/38.4%), followed by people aged between 50 and 59 years (n = 2159/24.8%) and people aged between 70 and 74 years old (n = 1697/19.5%). In addition, in relation to schooling, there was a higher occurrence among people with 4 to 7 years of schooling (n = 2193/25.2%), followed by people with 1 to 3 years of schooling (n = 2138/24.5%) and people with 8 to 11 years of schooling (n = 1614/18.5%). 5. Conclusion: This study has found an important social stratum in the data in question; people with up to 7 years of schooling symbolize 49.7% of the total number of cases registered and analyzed, indicating an influence of social status in this scenario, as well a stronger the prevalence of AIM between males. Therefore, public policy should be more aware of epidemiological data to define strategies to prevent AIM. 109957 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RAFAEL AUGUSTO SILVA CABEÇA1, Ingrid Jade Muniz Wanderley1, João Lucas Silva Sales1, Israel Figueira Lemos1, Ivan Cuoco Sampaio1, Maria Eduarda Dantas da Veiga1, Mariana Lassance Maya Palheta1, Juliana de Sousa Tavares1, Luiz Carlos Figueiredo Filho1, Luma Maria Favacho Bordalo1, Paula Larissa Baía Lima1 (1) Universidade do Estado do Pará Background: HELLP Syndrome (hemolysis, elevated liver enzymes and low platelets) is a multisystemic disorder that usually arises from a serious condition of pre-eclampsia, which is present in 7,1% of pregnant women with hypertensive disorder. The presence of this condition is responsible for the development of endovascular endothelial damage by the intravascular platelet activation, which leads to many cases of maternal, perinatal and neonatal morbidity and morbimortality. Objective: Describe, through a systematic review, the available evidence about the maternal disorders arising from HELLP syndrome. Methods: A systematic review with articles collected at MEDLINE, Scielo and LILACS databases published from 2017 to 2022, using the descriptors “HELLP” and “HELLP Syndrome”. Were included cohort retrospective studies, full text available, in english, spanish and portuguese languages, while studies with tematic deviation, duplicated and articles that haven‘t published results. This review was based on preferred reporting items for systematic reviews and meta-analysis (PRISMA) standards and procedures. Results: 310 articles were collected, and after the selection, analysis and the application of exclusion and inclusion criteria, 11 articles were selected. In the period of study, the analysis of publications demonstrated the prevalence of three poor outcomes on pregnant women’s clinical conditions: liver damage, kidney damage and hypertension, which were classified as primary outcomes. Furthermore, the studies described the following secondary outcomes: premature birth, placental abruption and pulmonary infection, uterine infection and gastrointestinal infection. Were observed the presence of gestational diabetes and congenital abnormalities caused by placental disorders. In addition, cerebrovascular morbidities were detected in patients with HELLP syndrome, and epigastric pain was the main isolated symptom on pregnant women, despite some patients related to having both symptoms in considerable part of the cases. The outcomes associated with cardiac disorders were concentric hypertrophy of the left ventricle and diastolic dysfunction. Conclusion: In this aspect, although HELLP syndrome is a rare obstetric condition, it was possible to observe that the outcomes resulted by, particularly related to hepatic, kidney and vascular damages, may leave after-effects at short and long term in the life of the pregnant. 109965 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH BRUNO OLIVEIRA SOUZA PRATES1, Hildebrando Antunes de Carvalho Neto1, Beatriz Souza Martins1, Beatriz Catarina dos Santos de Oliveira1, Gabriela Chateaubriand Campos1 (1) Universidade Federal da Bahia – UFBA Introduction: Atherosclerosis is a chronic inflammatory disease of multifactorial origin and is one of the main causes of morbidity and mortality worldwide. Its prevalence has been increasing rapidly in developing countries, such as Brazil, and this growth is closely linked to the lifestyle adopted by the population. In this way, the epidemiological study of deaths associated with atherosclerosis is presented as a tool to assess the efficiency of the treatment instituted. Objective: To analyze the epidemiological profile of patients who died from atherosclerotic disease in Brazil, between 2012 and 2021. Methods: This is an ecological time series study from secondary data collected by the Hospital Information System (SIH-SUS), between the years 2012 to 2021. The total and percentage distribution of deaths, the mortality rate and sociodemographic data were included. Results: A total of 7.927 deaths were recorded during the analyzed period, with 2012 presenting 572 cases and 2021 with 1.006. The mortality rate presented an oscillatory character, varying in a range between 3.63 (in 2017) and 4.11 (in 2021). There was no significant difference between genders, but the age group most affected was the elderly over 60 years (89.29%). Furthermore, the Southeast region has the highest number of cases with 3,967 (50.04%) followed by the Northeast region with 2,169 (27.36%). Conclusions: As can be seen from the data obtained, despite the fact that the main risk factors for the development of atherosclerosis, as well as their treatment, are currently very well established, the numbers of deaths continue to increase in Brazil. And this increase is not just due to population growth, since the highest mortality rate in the historical series was recorded in 2021. As for sociodemographic data, the need for greater attention to elderly patients was once again reinforced, since they account for almost 90% of deaths. Finally, the data obtained prove that atherosclerosis remains an important and preventable cause of death in Brazil. 109968 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CAMILLA MATTIA CALIXTO1, Alice Magro Koscianski1, Ana Carla Dlugosz1, Bruna Karas1, Francielle Nocera Viechineski1, Julia Henneberg Hessman1, Larissa Almeida Busnello1, Paola Gonçalves Moreira de Oliveira1, Mário Claudio Soares Sturzeneker1 (1) Universidade Estadual de Ponta Grossa (UEPG) Introduction: Acute myocardial infarction (MI) is the primary global cause of death, and accounted for 7.06% of all deaths in Brazil in 2017. Chest pain is the cause of up to 40% of hospital admissions; and 10% of non-traumatic visits to the emergency department. The pandemic could reduce access to healthcare due to hospital overcrowding and the reduced seeking of the healthcare system, although the evidence is limited. Objective: Estimating the possible influence of the SARS-CoV-2 pandemic on morbimortality related to acute MI in the Southeast region through analysis of DATASUS data from 2017 to 2021. Methods: Data on the number of admissions, mortality, lethality, and duration of inpatient stay was collected. The lethality was calculated as the ratio of deaths over admissions and expressed as a percentage. We studied the periods pre-pandemic (02/2017 to 02/2020), pandemic (03/2020 to 06/2021), pandemic’s peak (12/2020 to 05/2021), peak’s 1st trimester (12/2020 a 01/2021), peak’s 2nd trimester (03/2021 a 06/2021), and mass vaccination (06/2021 a 12/2021). We calculated the mean of each variable for sex and age group. We compared the mass vaccination period to the others and recorded the difference between means as a percentage. Results: During mass vaccination, there was an increase of 6.69% in admissions compared to the pre-pandemic period, 10.35% compared to the peak, and 20.4% for the 2nd trimester, particularly among women (5.87%, 12.03%, and 23.06% respectively). The duration of stay dropped by 15.34% compared to the pre-pandemic period in a similar way between the sexes. Regarding the pandemic’s peak, the 2nd trimester showed a 4.66% increase. The mortality increased, especially comparing the peak’s 2nd trimester with the vaccination period (18.66%). The lethality showed mixed results with a slight upward tendency. There was a 9.44% increase compared to the 1st trimester and a 1.46% drop for the 2nd trimester. The sex and age analysis had similar results, except for an increase in all comparisons in the age group 40–49 and a reduction in the 30–39 age group. Conclusion: After the mass vaccination period, there was an increase in mortality and hospital admissions. The duration of inpatient stay decreased compared to the rates before the pandemic and increased compared to the pandemic’s peak. The lethality had a slight upward tendency. 110011 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY SHEMA EL-LADEN HAMMOUD1, Juliana Fortunato2, Jeronimo Antonio Fortunato Junior2, Leticia Gusso Scremin1, Nikolai Cernesco Neto1, Gabriel Fontana de Melo1, Brenda Gabriele Smaniotto Raulik1 (1) Universidade Positivo; (2) Hospital Cruz Vermelha Brasileira – Filial do Paraná Introduction: In Brazil, valvulopathies represents a significant portion of hospitalizations by cardiovascular disease. Rheumatic Fever is the main etiology, representing 70% of all cases, unlike developed nations, where degenerative diseases (DD) are the main cause. For patients that have DD, mitral valvuloplasty with a ballon catheter (MVBC) is still the most recommended treatment. However, in the last decades the minimally invasive cardiologic surgery (MICS) assisted by video, recieved a major boost and has become a possible choice of treatment to the diseases of the mitral valve. In pacients with a rheumatic disease (RD), the inicial treatment is farmacologic, reserving the surgical intervention on refratary cases. Objective: To compare the results of mitral valvuloplasty performed using the MICS techinique in patients with DD and RD; and also assess the initial surgical results with a five year evolution from the same patients. Methods: Between 2005 and 2019, patientes with Degenerative Desease (43) and Rheumatic Desease (36) undergoing MICS were selected totalizing 79 patients. Results: Among the patients selected, 56% were female and the average age was 49 years. Of all valve alterations, the rheumatic etiology prevailed in mitral valve stenosis while the degenerative disease prevailed in mitral valve insufficiency. There were 97,7% of plasties in DD, against 72,2% in RD. Sequential echocardiography showed minimal mitral regurgitation in 88,4% in patients with DD, versus 61,5% in RD. Left ventricular diastolic dimension was 57,8 mm in DD pacients, versus 51 mm in RD. Valve replacement after plasty failure occurred in the initial postoperative period from one pacient with RD and another pacient with DD, one year later the procedure. Five years later, one pacient with DD was submitted to a new surgical procedure due to mitral endocarditis. Late survival after 10 years of follow-up was 91,67% in RD versus 88,4% in DD. Conclusion: As observed in the world literature, the surgical results in pacients with DD was satisfactory. In pacients with RD the surgical results were also promising, with increased survival. 110013 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY GABRIEL FELIPE GONZAGA SILVEIRA1, Antonio Luiz Pinho Ribeiro1, Marcelo Martins Pinto-Filho1 (1) Universidade Federal de Minas Gerais UFMG; (2) Universidade Federal de Minas Gerais UFMG; (3) Universidade Federal de Minas Gerais UFMG The electrocardiogram (ECG) is a simple and useful clinical tool, used in population studies as a tool for disease prognosis estimation. The Minnesota code (CM) is used for better standardization of the ecg reading. The degree of ECG variability in the same patient (as well as the variation of codes) over time is not well established, with some reporting significant variations including pathological Q waves. The aim of the present study is to evaluate the discordance and causes of these discordances in major Q-wave CM in patients from The Longitudinal Study of Adult Health (ELSA-Brasil) over a 4-year follow-up period. The ELSA-Brasil is a multicenter cohort that included more than 15,000 participants aged between 35 and 74 years at baseline. The ECG is one of the tests performed in the study. For the present study, the presence of pathological Q wave was evaluated in a total of 26,516 ECGs performed in the years 2013 (13,877) and 2018 (12,639), using the major criteria of group 1 of the Minnesota Code (CM 1.1 a 1.2). The ECGs were performed in a sequence called waves 1, 2 and 3. The ECGs of wave 3 were fully lauded and coded with manual review and therefore were considered the gold standard for comparison. Waves 2 and 3 ECGs were compared in order to observe the discordance and causes of these discordances in the larger Q wave CM. We called Major Disagreement when there were changes in the pathological Q wave category, that is, the major Q wave pathological changes changed to minor or non-existent pathological changes between the groups in the CM. Minor disagreement: when there are changes in the CM, but no change in the Q wave category. Total agreement when the CM is identical and New pathological Q wave: when new pathological Q wave events have occurred. Patients without ECG were excluded. From the 26,516 electrocardiograms performed, we found 1,562 major changes in the Q wave (CM 1.1 and 2.1). When comparing the electrocardiograms performed in 2013 (13,877) and 2018 (12,639) we found 121 (1.0%) total agreement. Minor disagreement: 94 (0,7%). Major disagreement: 5 (0.04%) and New pathological Q wave:713 (5.6%). In conclusion, there was little Total agreement between the 2013 and 2018 ECGs. Most of the disagreements are due to Minor disagreement, which is possibly due to millimeter variations in measurement and the New pathological Q wave, which possibly occurred due to the aging of the population of the study. 110032 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT SOFIA HELENA VITTE1, Idrys Henrique Leite Guedes2, Joanna Sousa da Fonsêca Santana3, Marclébia Quesado Borges4, Marcelle Rodrigues Carneiro de Souza Reis5 (1) Pontifícia Universidade Católica de Campinas; (2) Universidade Federal de Campina Grande; (3) Escola Bahiana de Medicina e Saúde Pública; (4) Universidade Federal do Recôncavo da Bahia; (5) Centro Universitário do Planalto Central Apparecido dos Santos Introduction: Congestive heart failure is a complex syndrome that is a result of either functional or structural disorder, which can be divided into caused by heart failure such as ischemic heart disease and systemic hypertension. During the pandemic, it was possible to see a scenario that got worse in a country that faced serious consequences due to the coronavirus onset, with higher mortality data. Thus, our main goal was to show this impact on the statics of heart failure in an emergent country. Methodology: A cross-sectional quantitative study was carried out using the open database of the Brazilian Ministry of Health (DATASUS). Information on hospitalizations and mortality rates for heart failure, as well as hospitalizations for ischemic diseases and primary hypertension from January 2018 to December 2021, were analyzed. Results: There were 201,040 and 199,858 hospitalizations for heart failure in Brazil in 2018 and 2019, respectively, while in 2020 and 2021 these values were 167,375 and 151,106, which represents a reduction of 20.5% between the averages of the two biennia. In these same periods, heart failure mortality rates ranged from 11.12 and 11.41 in 2018–2019 to 12.11 and 13.54 in 2020–2021, resulting in a 13.8% increase between the two periods. Finally, average hospitalizations for other ischemic diseases and primary hypertension in Brazil decreased by 23.7% and 30.7% in 2020–2021 compared to 2018–2019. Conclusion: From the available data, a significant reduction in the number of hospitalizations for heart failure after the beginning of the pandemic, as well as for hypertension and ischemia, can be observed. Conversely, the mortality rate for the pathology grew, which demonstrates a possible relationship between the limitations imposed in the period on the adequate treatment of the two main etiologies of heart failure in the country. 110945 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION CATARINA RAQUEL GASPAR DOS SANTOS1, Patrícia Coelho1, Francisco Rodrigues1 (1) Instituto Politécnico de Castelo Branco Introduction: It is characterized as Orthostatic Hypotension when there is a drop in systolic blood pressure greater than 20 mmHg and/or 10 mmHg in diastolic blood pressure. Its symptoms are related to cerebral hypoperfusion, such as blurred vision, nausea, palpitations, headache, neurological changes, and loss of balance. Its prevalence is associated with increasing age. Objective: To determine the prevalence of orthostatic hypotension in the population of Sertã. Methodology: Prospective study with sample collection based on the cluster method, through the random selection of streets in the municipality of Sertã. Initially, anthropometric data were collected and, as a base, an assessment of blood pressure was carried out with the individual sitting and at rest, then, with an interval of 3 minutes, after this medication, blood pressure was reassessed in the sitting position. orthostatic. Statistical treatment and data analysis were performed using the SPSS Statistics program. Results: The sample is composed of 1000 individuals, of legal age and residing in the municipality, 515 (52%) male and 485 (48%) female. The mean age was 56.04 years with a standard deviation of ±16.04 years. It was found that 7.4% of the sample has orthostatic hypotension, with 54.1% being male and 45.9% female. Regarding the age groups, it was between 61 and 70 years old that stood out the most with this diagnosis, with a represented value of 41.9%. Discussion: Like the results found in the present study, there is an article on the study of Orthostatic Hypotension in the municipality of Proença-a-Nova, in which a prevalence of 5.3% of the population with this diagnosis was found, having been more prevalent in females, contrary to what was observed in the municipality of Sertã. Conclusions: Despite the low prevalence, it is still important to carry out its diagnosis in health units since this risk factor is also associated with cardiovascular disease. 110072 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MAYARA GABRIELE TOLEDO1, Caio Pluvier Duarte Costa1, Eduardo Thadeu de Oliveira Correia1 (1) Universidade Federal Fluminense Introduction: Tools that aid the differentiation of dyspnea from cardiovascular and noncardiovascular etiology are fundamental in the emergency department (ED). In this study we will systematically review if internal jugular vein (IJV) ultrasound can serve as a screening method for the diagnosis of heart failure in the ED. Methods: We conducted a systematic review following the PRISMA guidelines. Original studies indexed by Embase, Pubmed, Cochrane and LiLACS with the keywords: “ultrasound” or “ultrasonography” and “heart failure” and “jugular vein” published until March, 2022 and which matched the inclusion criteria were included. Two authors (C.P and M.T.) performed the screening and data extraction, in cases of discordance, a third author (E.T.) made the decision. Results: Eight studies were included, with a total of 931 patients analyzed. The ultrasound examination of the IJV was used to evaluate patients with dyspnea in ED, using different methods. Due to significant heterogeneity between studies, a meta-analysis was not performed. All of the included studies showed that IJV ultrasound had a high sensitivity for differentiation of dyspnea on the ED. Jang et al defined 10 cmH2O of central venous pressure (CVP) as a good cutoff to define HF by IJV ultrasound for patients without jugular vein distension on physical examination, showing a sensitivity of 98.2% and a specificity of 42.9% for IJV ultrasound in dyspneic patients comparing with pulmonary edema on initial Chest x-ray, a sensitivity of 99%, specificity of 59% compared with echocardiography, and finally a sensitivity of 100%, specificity of 43% compared with a BNP >= 500 pg/ml. Tzadok’s study evaluated the use of the change of the IJV area with respiration in patients with dyspnea to detect acute HF, showing that the respiratory area change of the IJV had a specificity and sensitivity of nearly 70% rate to identify HF. Conclusion: Our systematic review demonstrates that IJV ultrasound is a complementary tool that provides important information for the diagnosis of HF in patients with dyspnea in the ED. Further studies are needed to define a standard measurement method to apply IJV ultrasound in clinical practice. 110064 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY SOFIA HELENA VITTE1, Idrys Henrique Leite Guedes2, Joanna Sousa da Fonsêca Santana3, Marclébia Quesado Borges4, Marcelle Rodrigues Carneiro de Souza Reis5, Allan Christian Cardozo Cembrane6 (1) Pontifícia Universidade Católica de Campinas; (2) Universidade Federal de Campina Grande; (3) Escola Bahiana de Medicina e Saúde Pública; (4) Universidade Federal do Recôncavo da Bahia; (5) Centro Universitário do Planalto Central Apparecido dos Santos; (6) Hospital Alvorada Brasília Cardio-oncology is a field focused on detection, monitoring and treatment of cardiovascular disease occuring due to chemotherapy cardiotoxicity. Not only that, but cardiovascular diseases and cancer share multiple risk factors, such as increased age, smoking and obesity, and the presence of one condition worsens the other. Even though cardio-oncology is a new field, the most researched topics are focused on chemotherapy’ toxicity and cardiac assessment during treatment, leaving very little room to discuss heart malignancies. 25% of malignant cardiac tumors are cardiac sarcoma, and secondary tumors are 40 times more likely to appear. Therefore, the aim of this study is to analyze the profile of heart, mediastinum and pleural cancer in Brazil. This is a cross-sectional descriptive population based study with values obtained from the database of the Ministry of Health of Brazil (DATASUS) during the period from 2018 to 2021. The analyzed variables were: treatment time, sex, year and age group. The tables were obtained and exported to the IBM SPSS 26 statistical software for the purpose of comparing the variables. A total of 4438 diagnoses were accounted, with 2143 corresponding to males and 2295 to females. In the annual distribution, we can observe an increase of approximately 93.6% from 2018 to 2019, and a decrease of 12% from 2019 to 2020 and of 16.5% from 2020 to 2021. The most affected age group was from 60 to 69 years old. Finally, regarding the average treatment time, it can be observed that in 2,020 cases, treatments lasted up to 30 days, while 71 cases were between 31 and 60 days and 123 cases were for more than 60 days. Data shows a decrease in the number of diagnosed neoplasms in Brazil in the post-pandemic period, with a small increase between 2020 and 2021. Among those affected, elderly people, under 70 years old, are the age group with greater expression of the disease. However, it is not possible to establish a relationship between sexes, due to the small difference in cases. Regarding the treatment, most of it was done within a period of up to 30 days, showing a small time rate of tratament. 110065 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARCIO CÉSAR RIBEIRO MARVAO1, Giovanna Coutinho Jardim2 (1) Instituto Evandro Chagas (IEC); (2) Universidade Federal do Pará (UFPA) Introduction: Chagas disease is an anthropozoonosis caused by the parasite Trypanosoma cruzi that is considered a public health issue in several countries of The Americas, being endemic in 21 countries of the continent, reaching about 7 million people. There are several pathophysiological mechanisms that surround Chagas disease, so that there is permanent tissue repercussion, autosomal abnormality and impaired coronary microcirculation, the latter can generate decreased myocardial performance and impair the functional capacity of the affected person. The relationship between Chagas and deforestation can be addressed by the theme of destruction of the vector habitat, causing its migration to areas with human presence. The state of Pará, northern Brazil, leads deforestation rates in the Amazon region, in addition to suffering from chagas disease endemics. Objective: To perform a comparative analysis between deforestation data and incidence of acute chagas disease in the state of Pará from 2015 to 2020. Methodology: Cross-sectional, observational and descriptive study, with data from the Notifiable Diseases Information System (SINAN/DATASUS) on the frequency of acute chagas disease in the state of Pará. The linear regression test was used using the PASR4.03 software to verify the statistical relationship. The variables of annual forest loss, forest cover rate and forest deforestation rate were analyzed. Results: During the period described, the highest incidence occurred in 2016, marking 3.3 new cases per 100,000 inhabitants. In relation to the forest situation, the greatest loss of forest cover (hectare) marked the year 2020, representing a loss of 675591.3, with a forest deforestation rate of 24.93%, followed by 2016, with 597199 of area loss and with a rate of 24.04%. The statistical correlation showed R2 = 37% of events, demonstrating a variability of 63% of the relationship occurring by other factors. Non-significant P-value (0.19997). Conclusion: Although deforestation is a major problem for the onset of diseases and a decrease in the quality of life of the population. The data collected did not bring a significant relationship between deforestation and the incidence of chagas in the described region, not ruling out this relationship in other locations. 111668 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH VALENTINA BRATTI DE NADAL1, Pedro Van Der Sand Germani2, Juliana Nichterwitz Scherer1 (1) Universidade do Vale do Rio dos Sinos (UNISINOS); (2) Pontifícia Universidade Católica do Rio Grande do Sul Introduction: AVS is, in children and adults, the most common cause of left ventricular outflow obstruction. When the severity of Aortic Valve Stenosis is low to moderate, the disease is well tolerated and is usually not accompanied by symptoms. When it progresses to severe, AVS is associated with significant mortality. The classic manifestations when the disease is in terminal stage are dyspnea on exertion, syncope and angina. It is important to identify the profile of patients who died from such condition in order to provide more adequate screening and to identify risky groups. Objective: To describe the profile of patients who died from AVS in Brazil in the last 10 years. Methodology: An ecological study was conducted through the collection and analysis of data from the Departamento de Análise de Saúde e Vigilância de Doenças Não Transmissíveis (DASNT) system, covering the period from January 2011 to December 2021. The variables evaluated were: number of deaths for AVS, geographic region and place of occurrence, age group, race/color and sex. All data were extracted into an excel spreadsheet, and the descriptions of the variables were performed by analyzing absolute and relative frequencies. Results: A total of 32,094 deaths due to AVS were recorded in Brazil between 2011 and 2021. Of these, 53.6% were females and 46.3% were males. Considering age group, 96.1% of deaths occurred in individuals over 50 years old, with 47.3% of total deaths being aged 80 years old or over. The lowest rate occurred in patients aged 05 to 09 years, totalizing 0.06%. About race/color, 73.8% of deaths occurred in white patients, 17.8% in brown and 3.5% in black, while the lowest rate occurred in indigenous people, 0.09%. Regarding region, the Southeast had the highest rate of deaths (50.9%), followed by the South (26.3%) and the Northeast (14.7%). The North and Midwest accounted for 8.1% of deaths. Conclusions: As 96.1% of deaths caused by AVS occurred in patients over 50 years old, it should be noted that there is a need for medical management to identify these patients before they become symptomatic, which improves survival. Public policies that propose regular monitoring of these patients are extremely important to monitor hemodynamic progression and the development, mainly in the Southeast, where there were 50.9%% of deaths. 110074 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LOUISE BUONALUMI TÁCITO YUGAR1, Larissa Costa Morete2, Tatiana Rubio Azevedo2, Elizabeth do Espírito Santo Cestario2, Bruno Rodrigues1, Sílvia Elaine Ferreira Souza Melo1, Carolina Manzano2, Lúcia Helena Bonalume Tácito2, José Fernando Vilela Martin2, Heitor Moreno Júnior1, Juan Carlos Yugar Toledo2 (1) Faculdade de Ciências Médicas, UNICAMP; (2) Faculdade de Medicina de São José do Rio Preto, FAMERP Introduction: The wave pressure profile can be separated into three segments: pulse pressure (PP), diastolic blood pressure (DBP) and systolic blood pressure (SBP), which corresponds to the sum of the previous ones. Although the measurement of these values has advanced, there are still few studies on the relationship between them. The golden ratio (φ, phi, or Fibonacci number) is present in several organisms and is defined by dividing a straight line into two segments a and b (a being greater than b) so that the ratio between a and b is equal to the ratio of the sum of the quantities by the larger segment. The value of this equality is approximately 1.61. In this sense, we try to relate SBP, DBP and PP to phi. In this study, we evaluated and compared the deviations from the golden ratio during the periods of ABPM: 24h, daytime and nighttime in resistant hypertensives (RHTN) and type 2 diabetics (T2DM) using the SBP/DBP and DBP/PP ratios for comparison. Material and Methods: We retrospectively analyzed data from RHTN participants (n = 54) from the ResHypOT ClinicalTrials.gov study, ID: NCT 02832973 and T2DM patients (n = 81) from the study “Changes in vascular hemodynamics in patients with RHTN and T2DM. The data used were: age, sex, SBP, DBP and PP. Descriptive statistical analysis, t-test and ANOVA were performed with SPSS 24 softwares (IBM-USA). Results: We observed elevated blood pressure values in RHTN and normal values in T2DM in the 24-hour periods, daytime and nighttime with attenuation of the systolic and diastolic nocturnal dipping, being more pronounced in the RHTN group. The SBP/DPB ratio in the 24-hour period and nighttime was significantly higher in the T2DM group when compared to the RHTN group (p = 0.0063 and p = 0.044, respectively), above phi in both. The SBP/DBP ratio in daytime was higher in the RHTN group (p < 0.0001) (figure 1). The DBP/PP ratio in RHTN was lower in the 24-hour period and during nighttime in T2DM (p = 0.0012; p = 0.0189, respectively) (figure 2). There was no significant difference between T2DM and RHTN groups for DBP/PP ratio in daytime. Conclusion: The phi deviations found in T2DM are greater than those observed in RHTN. These differences may be attributed to the difference in blood pressure values between the groups and reduced nocturnal SBP dipping in patients with RHTN. Future studies using the deviations of the golden ratio are needed to validate the information obtained from this mathematical proposal. 110081 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LARISSA SILVA FERREIRA1, Aurea Nathallia Gomes de Souza (Souza, ANG)1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira (Oliveira, CS)1, Cecília Rodrigues Viana1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Vinícius Maciel Vilhena1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá (UNIFAP) Introduction: Cardiovascular diseases are the major causes of death in Brazil. Among the most likely risk factors for the development of these diseases are: arterial hypertension (SAH), dyslipidemia, diabetes mellitus (DM), obesity and physical inactivity. With the changes in life habits in Brazil and in the world, high-calorie dietary changes and the decrease in physical activity among children and young people stand out, which raises an alert for the increased probability of developing diseases in the cardiovascular system in the first two decades of life. Objective: To analyze the mortality rate of children and adolescents due to heart failure in children and adolescents in Brazil, with data from 2021 and 2022. Method: Cross-sectional epidemiological study on the mortality rate of individuals aged 0 to 19 years, due to heart failure cardiac arrest, based on data extracted from the Information Technology Department of the Unified Health System (DATASUS). Results: Between 2021 and 2022, the mortality rate from heart failure before the age of 19 was 7.51% and increased numbers were observed in children under 1 year (9.18%) and between 15 and 19 years (9.83%). With regard to Brazilian regions, the Midwest region has the highest mortality rate (10%), while the Northeast region has the lowest percentage (6.97%). In addition, the rate is higher in children under 1 year old (10.67%) and decreases with age, increasing again from 10 to 14 years old (6.29%) and until reaching 15 to 19 years old (8.75%). This rate is higher in males (8.57%). Finally, the indigenous are the most affected (18.75%) and the yellow ones the least (5.26%). Conclusions: In the child and adolescent population, there was a predominance of males and individuals aged less than 1 year and 15 to 19 years in all data evaluated. Thus, it is understood that changes in habits are increasingly affecting a younger population. This shows the need to invest in health promotion policies for the prevention of cardiovascular diseases, a need reinforced by the high cost of assisting modifiable factors such as diet and physical activities. 110360 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BEATRIZ JUNQUEIRA MATTAR1, Bruno Henrique dos Reis Souza Oliveira1, Káthia Maria Duarte Ono1, Thiago Lima Pereira2, Renata Margarida Etchebehere2, Rodrigo César Rosa2, Sanívia Aparecida de Lima Pereira1 (1) Universidade de Uberaba UNIUBE; (2) Universidade Federal Do Triângulo Mineiro UFTM Introduction: Atherosclerosis is an inflammatory disease of the arteries responsible for a high rate of morbidity and mortality worldwide. Objective: To associate the histopathological and immunohistochemical aspects of tongue inflammation with aortic and coronary atherosclerosis in autopsied humans. Methodology: A total of 4,378 autopsy reports were analyzed and all cases where there were stored fragments of the tongue, aorta and coronary artery of the same individual were selected (n = 16). Morphological and immunohistochemical evaluation was performed for IL-1 beta, IL-6, TNF alpha and IFN gamma. Results: IL-1 in the aorta was associated with the following parameters evaluated in the tongue: IL-6 (p = 0.031); inflammation (p = 0.047); spongiosis (p = 0.018); lymphocytic exocytosis (0.003). IL-6 in the tongue was associated with IL-1 (p = 0.031), IL-6 (p = 0.016) and TNF-alpha (p = 0.016) in the aorta. Tongue exocytosis was associated with IFN gamma in the aorta (p = 0.003). Conclusion: Inflammation and higher immunostaining by IL-6, TNF-alpha and IFN gamma on the tongue are associated with higher immunostaining by IL-1 in atherosclerotic plaques of the aorta arteries. Therefore, knowing that IL-1 is a pro-inflammatory mediator associated with the development of atherosclerosis and that infectious agents are the major causes of inflammation in the tongue, we suggest that prevention and treatment of lingual infections could reduce the risk of atherosclerosis. Thus, these simple and low-cost measures could help prevent the atherosclerotic process, reducing the morbidity and mortality rates associated with this disease. However, further studies are needed in order to corroborate our findings. 110356 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION CAMILA PIENTZNAUER SOARES MONTE VIANNA1, Claudio Tinoco Mesquita1, Juliana Cadilho da Silva Abrantes1 (1) Universidade Federal Fluminense (UFF) Introduction and/or Fundamentals: 3D printing has proven to be a resource in healthcare through the creation of models of organs and structures with various applications. In cardiology it is possible to use this technology both in the educational area and in surgical planning. In education, an important application of this tool is the printing of three-dimensional embryological models of the formation of the heart, in order to provide a better understanding of human embryology and congenital heart defects. Thus, 3D printed embryological models can provide active learning by visualizing the stages of heart formation in a three-dimensional way, being an innovative didactic resource in embryology and cardiology classes. Objective: We aimed to describe and analyze the improvements of the application of 3D printing in teaching cardiac embryology to health care students. Materials and Methods: At the university we have a digital fabrication laboratory (Fab Lab) that has a GT MAX 3D FDM printer, which builds models by heating and extruding PLA and ABS filaments. These models (Figure 1) were digitally modeled by the lab’s multidisciplinary team using the Sculptris software and saved in STL format by analyzing images from books and articles in the area. Three embryological cardiac structures have already been printed: cardiac tubes and cardiac tube folding. The printed models are being used for teaching embryology to undergraduate students through face-to-face seminars in the lab. Results: The classes resulted in in-depth teaching of the structures of the heart at different stages of its formation, focusing on the visualization of structures, as well as stimulating the sciences and technologies applied to medicine. Through the three-dimensional visualization of the structures, the students obtained a better understanding of the embryological development of the heart, as well as of its malformations due to failures in this process. In addition, the understanding of the initial formation of the heart helped to better understand the anatomy of the adult heart. Conclusions: The Fab Lab has been of great value and interest to the academic-scientific community. The experience with the 3D models of the heart in teaching embryology and anatomy shows the great potential of this tool allowing a multidisciplinary integration that serves as a stimulus to attract young students. 110371 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JADE ZARICHTA COSTA1, Pietro Preis Casagrande2, Vitor Henrique Macarini2, Josué Barbosa2, Franciely Vanessa Costa3, Maíra Cola4, Roberta de Paula Martins1 (1) Laboratório de Bioquímica Patológica, LABIP, Departamento de Ciências da Saúde, Centro de Ciências, Tecnologias e Saúde, Universidade Federal de Santa Catarina, UFSC, Campus Araranguá.; (2) Acadêmico do Curso de Graduação em Medicina, Departamento de Ciências da Saúde, Centro de Ciências, Tecnologias e Saúde, Universidade Federal de Santa Catarina, UFSC, Campus Araranguá.; (3) Departamento de Ciências da Saúde, Centro de Ciências, Tecnologias e Saúde, Universidade Federal de Santa Catarina, UFSC, Campus Araranguá.; (4) Grupo de Gestão em Riscos e Desastres, Departamento de Ciências da Saúde, Centro de Ciências, Tecnologias e Saúde, Universidade Federal de Santa Catarina, UFSC, Campus Araranguá. Introduction: Pediatric patients may develop Multisystem Inflammatory Syndrome (MIS-C) in severe cases of COVID-19, a clinical condition that is poorly described yet and may cause cardiovascular changes, persistent fever, skin manifestations, and coagulopathies. Objective: To determine whether elevated D-dimer levels are a risk marker for the development of thromboembolic events in the pediatric population with COVID-19. If so, D-dimer levels could be used to determine prophylactic anticoagulation measures if needed. Methods: This systematic review was conducted in accordance with the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyzes (PRISMA) and registered in the PROSPERO Database of Systematic Reviews under CRD42022298330. The last update of the database search was on December 14, 2021, resulting in 79 documents for analysis. Inclusion criteria were: academic articles (case series, cohort studies, and case controls) on patients aged up to 21 years, in English, Portuguese, and Spanish; the databases used were PubMed, Scopus, MEDLINE/Bireme (Virtual Library of Health – VLH), Web of Science, and EMBASE, queried by topic, keywords, or abstract. Bibliographic reviews, books, nonacademic research, case reports, and content in languages other than those indicated were excluded. All articles were independently reviewed by all authors using the Joanna Briggs Institute (JBI) checklist and critical appraisal for risk of bias. Disagreements were resolved through discussion within the group. Six of the articles used were rated as low risk of bias and one was rated as moderate risk. Results: From the 79 articles found, only 7 were selected for analysis. Among these seven articles, only one had patients with thromboembolic events. In the other articles, D-dimer levels were elevated but were considered controversial in predicting events, with no clear association between the magnitude of D-dimer change and the magnitude of thrombosis risk, as the level tended to increase in all inflammatory diseases. Conclusions: Although D-dimer is used for adults, it was not a good parameter for assessing the risk of thromboembolic events in individuals younger than 21 years. The main shortcomings are that D-dimer is elevated in many patients even without the occurrence of thromboembolic events and increases with any type of inflammation, therefore is not a specific marker. 110836 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION KELLY ANNY FERNANDES MASCARENHAS 1 (1) Instituto Politécnico de Castelo Branco Introduction: Diabetes are defined as a metabolic disorder characterized by insufficient insulin production or by the incapacity of the body in using them. Consequently, it results in an increase in glucose levels in the blood, being type II diabetes the most common one. It’s considered an important risk factor for cerebrovascular pathologies development such as acute myocardial infarction and cerebral vascular accident. In Cabo Verde, according to the National Survey on the Risk Factors of the Non-Transmitted Diseases (SNTD II) 2020, 3.9% of the adult population of Cabo Verde was diagnosed with diabetes, being the females the most predominant one, and 16.6% of them with the range between 60–69 years of age. Objectives: To compare the results of measured diabetes through questionnaire application as well as evaluated diabetes through capillary glycemia of the adults from the island of Santiago – Cabo Verde. Methodology: The transversal study of population background of 599 participants over 18 years old, from both genders. The data collection took place in October and November 2021, in the nine municipalities of Santiago Island- Cabo Verde. It was excluded from the study the individuals of Cape Verdean nationality who have been living for over a year abroad and that have just arrived on Santiago Island in less than 30 days, as well as the individuals with any kind of disabilities that may affect their participation in the study. The data were collected through questionnaires; As for diabetes, it was evaluated through a glucometer. Results: The sample was made of 599 individuals with the age range between 18 and 99 years old with an age average of 42 years of age, 54.8% were female and 45.2% were male. Through a questionnaire, it was possible to verify that 7.5% of the participant population answered they have a diabetes diagnosis 71.1% of females. In the diabetes evaluation carried out through capillary glycemia, we found a prevalence of 4.5% of diabetes, of which 63% are females. In both cases, it was verified that there’s a meaningful statistical relationship between diabetes and the age group. Furthermore, only measured diabetes by questionnaire showed there’s a meaningful statistical relationship with gender. Thus, we could infer that 3% of diabetics have controlled glycemia. Conclusion: There is a greater rate of prevalence in evaluated diabetes by questionnaire, with the women presenting greater predominance in the adminis. 110459 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ISABELLA VICTÓRIA DA CUNHA PEIXOTO RIBEIRO1, Eduardo Augusto Victor Rocha1, Maria Paula Parreira1, Arthur de Vasconcellos Rocha2, Vitor Lanza Avelar Almeida2 (1) Faculdade Ciências Médicas de Minas Gerais – CMMG; (2) Faculdade da Saúde e Ecologia Humana – FASEH Introduction: Cardiovascular surgeries with cardiopulmonary bypass (CPB) have great associated risk, therefore prognostic markers are very important to help in medical decision. In this study we tried to search for markers that help in the decision making process. Objective: To evaluate variables and relate them to morbidity/mortality in order to find prognostic markers for cardiovascular surgery with CPB. Method: Observational retrospective cohort study through consecutive analysis of 113 medical records of patients over 18 years old undergoing cardiovascular surgery with cardiopulmonary bypass from January 1 to December 31, 2015, at the Hospital Universitário Ciências Médicas de Minas Gerais, in the city of Belo Horizonte, MG – Brazil. We evaluated 32 variables relating them to intensive care stay, hospital stay and mortality. Results: EuroScore II, preoperative creatinine, on intensive care unit (ICU) admission and at hospital discharge, reintubation, surgical infection and return to CPB were statistically significant variables. The number of lymphocytes at ICU admission, above 1263.9 was a statistically significant variable in relation to mortality. These results, except the lymphocyte values, were expected to be related to death. However, the values of lymphocytes collected at the ICU entrance, is an interesting finding and is not in the algorithms for prognostic evaluation in cardiovascular surgery. The study has some limitations due to the small number of participants in the study, because it was done in a single center, and because it has only one year of data collection. However, this study brings a new data, which may be an important prognostic marker in cardiac surgery. Conclusion: The value of lymphocytes above 1263.9/mm3, in addition to Euroscore II, Creatinine in the pre, per and postoperative, infection, return to CPB, showed a significant relation with mortality in cardiovascular surgery with CPB. 110468 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MATHEUS CANDIDO BARBOSA1, Willian Cirillo1, Fernando Piza1, Marcio J.O. Figueiredo1, Odilson M. Silvestre2, Miguel M. Fernandes-Silva3, Roberto Schreiber1, Matheus F.R.A. Oliveira1, Otavio R. Coelho-Filho1, José R. Matos-Souza1, Andrei C. Sposito1, Wilson Nadruz Jr.1 (1) Department of Internal Medicine, School of Medical Sciences, State University of Campinas, São Paulo, Brazil.; (2) Federal University of Acre, Rio Branco, Brazil.; (3) Federal University of Parana, Curitiba, Brazil. Background: In-hospital delays in permanent cardiac pacemaker (PPM) implantation are common and may result in in-hospital infection among patients waiting for PPM implantation (pre-PPM-HI). This study investigated the predictors and prognostic impact of these events. Methods: We retrospectively evaluated 905 consecutive patients (68.2 ± 16.0 years; 54% males) who underwent PPM implantation. Clinical characteristics, pre-PPM-HI and 30-day mortality were recorded and a risk score for pre-PPM-HI was generated using multivariable logistic regression coefficients. Results: Eigthy-nine patients (10% of the sample) developed pre-PPM-HI. Multivariable logistic regression analysis identified implantation of temporary pacemaker, diabetes, hospitalization >3 days, heart failure and complete atrioventricular block as independent predictors of pre-PPM-HI. The generated score (range 0–9.9) played a better role in predicting pre-PPM-HI than individual factors, yielding an area under the curve [95%CI] of 0.702 [0.653–0.751]. Patients with score ≥7.5 had 12-fold greater risk of developing pre-PPM-HI than those with score <2.5. Furthermore, multivariable Cox-regression analysis showed that patients who developed pre-PPM-HI had greater 30-day mortality after PPM implantation (hazard ratio [95%CI] = 2.86 [1.16–7.06], p = 0.022) as compared with their counterparts. Conclusions: This study reveals that pre-PPM-HI is an independent predictor of early mortality after PPM implantation. In addition, a clinical score developed from simple clinical variables accurately identified patients at high risk of pre-PPM-HI. In scenarios where delays in PPM implantation are unavoidable, such as reference hospitals with high demand, the use of this tool can potentially help in the hierarchy of patients and in the reduction of this adverse event. 110476 Modality: E-Poster Scientific Initiation – Non-case Report Category: PHYSIOTHERAPY LUCAS CONSOLAIO DE ASSIS1, Lucas Consolaio de Assis1, Liz Maria Xavier de Oliveira2, Cristiane Rodrigues Pedroni3 (1) Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Marília, Faculdade de Filosofia e Ciências (FFC – Marília, SP); (2) Centro de Estudos e Reabilitação (CER – UNESP, Marília – SP); (3) Laboratório de Pesquisa em Ortopedia e Recursos Terapêuticos (LAPORT – Unesp, Marília) Dysfunction or fatigue of the triceps surae muscles can lead to a defect posture, muscle contracture, fiber ruptures, contusion and injuries related to excessive and repetitive dorsiflexion. Photobiomodulation is a well-studied resource for reduce fatigue and the risk of muscle injuries, being a treatment option and a effective method also for improving muscle performance. Purpose: To investigate the effect of photobiomodulation on muscle performance and recovery after the installation of triceps surae fatigue in physically active people. Methods: Randomized double-blind study, with 14 healthy and physically active subjects, of both sexes, aged between 18 and 30 years, randomly divided into two groups: irradiation and placebo. were carried out the Y Balance Test, manual dynamometry and the electromyographic exam to verify the lower limb performance, strength and electrical activity respectively. In Then, the adapted Bruce protocol and fatigue protocol were applied to the triceps surae, and then active photobiomodulation or placebo according to the group. used the equipment Cluster LASER infrared with 4 diodes, wavelength 808 nm, 120 mW output power, continuous mode emission and total energy (cluster – 4 points) equal to 60J. The effect of therapy between the groups was verified through the values of performance on YBT, strength values by dynamometry and by RMS and index values of fatigue calculated by the median frequency of the electromyographic signal. Results: No significant differences were observed in the intergroup comparison and when compared pre and post in the evaluations of YBT (p = 0.46), muscular strength (p = 0.73) and in the electromyographic behavior of mean RMS values (p > 0.05) and fatigue index (p > 0.05). Conclusion: The photobiomodulation protocol applied after muscle fatigue proposed for the gastrocnemius muscle was not enough to change the parameters of lower limb performance, muscle strength and electromyography. 112438 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIA HELOÍSA BEZERRA VILHENA1, Bárbara Vilhena Montenegro1, Lorena Souza dos Santos Lima1, Hillary Ferreira Parnaiba1, Natália Fernandes Ribeiro1, Michelle Sales Barros de Aguiar1 (1) Instituto Michelle Sales Introduction: Use of antihypertensive medication in Marfan Syndrome (MFS) aims to prevent one of the worst outcomes, aortic dissection. Beta-blockers are well-established drugs in this context, however, a possible response to the use of losartan has attracted the attention of researchers. Objective: To describe the effect of beta-blockers and losartan for preventing aortic dissection in MFS. Methods: This systematic review is based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) protocol. PUBMED and Cochrane databases were searched, using the descriptors “Aortic dissection”, “Beta-blocker” and “Marfan Syndrome” combined with the Boolean operator “AND”. Inclusion criteria were: published in the last 5 years, full text available, clinical trials, randomized trials, books and documents. Exclusion criteria were articles on Loeys-Dietz Syndrome. 116 articles were identified, of which 7 met the inclusion criteria. After checking the studies, a careful reading was carried out, verifying the PRISMA protocol checklist, in order to obtain the qualitative results of the systematic review. Results: According to a randomized study carried out in 2018, the use of atenolol for 3 years by patients with MFS caused a decrease in aortic root stiffness by lowering heart rate (P = 0.01). In the losartan group, no significant differences were observed (p = 0.31). However, a clinical trial was carried out in 2018, in which no significant differences were observed in the increase in the diameter of the aortic root between losartan group (95% CI: 0.2 to 0.5 mm/year) and atenolol group (95% CI: 0.3 to 0.6 mm/year). A study carried out in 2020 in the Netherlands reports that the group of patients using beta-blockers exclusively (n = 124) had 11 cases of aortic dissection, while the group using combined therapy with beta-blockers and losartan had 3 evolutions for this event during the eight years of study. In another 2020 finding, patients with MFS who had the CC genotype and were in use of atenolol had a better rate of improvement than patients who were using losartan (interaction P = 0.002; –0.20 ± 0.02 vs –0.07 ± 0.02; P < .001). Conclusion: Combined therapy of atenolol and losartan has beneficial effects when compared to losartan alone, although there are no significant clinical differences on the exclusive use of losartan. There are insufficient randomized studies to prove the effectiveness of this therapy in acute aortic dissection in MFS. 110549 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION NATÁLIA MERHEB HADDAD1, Suhen Aquino de Liz2, Marcello Ricardo Paulista Markus3, Maria Eduarda Venera4, Ernani Tiaraju de Santa Helena5, Clóvis Arlindo de Sousa6, Alan de Jesus Pires de Moraes1 (1) Universidade do Vale do Itajaí; (2) Programa de Pós-Graduação Stricto Sensu em Saúde Coletiva. Universidade Regional de Blumenau; (3) Department of Internal Medicine B, University Medicine Greifswald, Greifswald, Germany. German Centre for Cardiovascular Research (DZHK), partner site Greifswald, Greifswald, Germany. German Center for Diabetes Research (DZD), partner site Greifswald, Gre; (4) Departamento de Educação Física. Universidade Regional de Blumenau, Blumenau, Santa Catarina, Brasil; (5) Departamento de Medicina. Programa de Pós-Graduação Stricto Sensu em Saúde Coletiva, Universidade Regional de Blumenau; (6) Departamento de Educação Física, Programa de Pós-Graduação Stricto Sensu em Saúde Coletiva, Universidade Regional de Blumenau Background: Carotid intima-media thickness (CIMT) is a marker of atherosclerosis and a predictor of cardiovascular disease, however, there are few studies on factors associated with CIMT in specific populations. Objective: The goal was to analyze the prevalence and factors associated with CIMT among adults and the elderly people in a German city in southern Brazil. Method: Cross-sectional population-based study, with 2488 people between 20 and 79 years old, from the Study of Health in Pomerode – SHIP-Brazil. The CIMT was evaluated by ultrasonography and the altered thickening was values ≥0.90 mm. The independent variables involved sociodemographic aspects, lifestyle, health conditions and chronic diseases. Crude and adjusted Poisson regression with estimates of prevalence ratios (PR) and confidence intervals were used. Results: The prevalence of high CIMT was 10.9%, higher in the elderly (42.5%) when compared to adults (4.7%). Among adults, male (PR = 1.75), ex-smoker (PR = 2.06), smoker (PR = 2.15), high waist-to-hip ratio (PR = 2.09), hypertension (PR = 2.19), and carotid plaque (PR = 2.34) were associated with elevated EMIC. In the elderly, male (PR = 1.33), insufficiently active (PR = 1.39), high waist-to-hip ratio (PR = 1.34) and plaque (PR = 1.54). Conclusion: Actions to prevent and control high CIMT involve increase the level of physical activity, smoking cessation, preventing of hypertension and excess abdominal fat. 111061 Modality: E-Poster Scientific Initiation – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE JOSÉ ICARO NUNES CRUZ1, Adelle Cristine Lima Cardozo1, Giulia Vieira Santos1, Jade Soares Dória1, Mariano César de Souza Reis1, Gabriela de Oliveira Salazar1, Bruna Souza Matos de Oliveira1, Juliana Maria Chianca Lira1, Diego Maldini Borba de Lima1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital; (3) Primavera Hospital Introduction: Resilience refers to the ability to develop adequately, even when facing difficulties, and can be an important factor in health promotion, especially in chronic diseases. Objectives: To evaluate how resilience behaves among patients with Chronic Coronary Syndrome (CCS) seen in the public and private healthcare networks. Methods: This is a cross-sectional, analytical study, whose sample included patients with CCS assisted at cardiology outpatient clinics of two public hospitals and one private hospital. The patients were divided into two groups, according to the healthcare system: I) Public; II) Private. Participants completed the Connor-Davidson Resilience Scale (CD-RISC-10), composed of ten items, each one presented as an ordinal scale from 0 to 4. The total resilience level comprises the sum of the responses from each item, and thus ranges from 0 to 40. The results were described in terms of means and standard deviations and the statistical analysis was performed using Fisher’s Exact test and Student’s T-test, with significance level set at 0.05. Results: 53 patients with CCS were included, 69.8% from the Public group and 30.2% from the Private group. The gender distributions were not different between groups (p > 0.05), however the Public group had a lower mean age than the Private group (60.2 vs. 66.9 years; p < 0.05). Patients with CCS assisted at the public system showed a lower level of total resilience than patients assisted at the private healthcare system (26.4 vs. 31.9; p < 0.05). The CD-RISC-10 showed that patients in the Public group believed less that they could achieve their goals compared to the Private group (3.0 vs. 3.8; p < 0.05). When presented with the statement “I am not easily discouraged by failures”, Public group patients had lower mean scores on the scale (2.9 vs. 3.7; p < 0.05), which denotes that this statement is less true for this group compared to the Private group. Conclusions: The results show that patients with CCS assisted in the public healthcare system have a lower level of resilience than patients assisted in private settings. Differences in resilience levels between the groups are concentrated in the belief in the possibility of achieving personal goals (lower for the Public group), and in discouragement in the face of failures (higher for patients in the Public group). 110589 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIA JÚLIA DA SILVEIRA MARQUES1, Maria Júlia da Silveira Marques1, Audrei Pavanello1 (1) Unicesumar Congenital heart diseases (CHDs) describe a set of abnormalities of the heart and great vessels which are present at birth and are associated with great physiological impact on the neonate. The etiology of those malformations is multifactorial, including genetic and environmental factors, maternal obesity, pre-gestational diabetes mellitus and the use of potentially teratogenic drugs like retinoic acid. These causes seem to influence the development of the fetal cardiovascular system, causing cardiac and great vessel anomalies and consequently, a functional disorder of this system. Considering these factors, the objective of this study is to correlate the risk factors associated with poor fetal cardiovascular development, through an analysis of the Unified Health System database (DATASUS). The data used were collected from the DATASUS Information System for Live Births (SINASC) platform with the help of Software R (version 4.1.3) and RStudio (version 2021.09.0 + 351). For data collection, the R microdatasus package was used, with the referring period of 2007 to 2020 in the state of Paraná. Data were filtered using the R tidyverse package, with 1135 births with congenital heart anomalies registered during this period (ICD Q200 to Q250). For comparison, 5676 observations of non-anomaly neonates chosen from random data were used. The analysis was performed using the rpart package, where a classification tree was constructed crossing the variables: weeks of pregnancy, maternal age, birth weight, sex and race against the presence of congenital anomalies. Data were plotted using the rpart.plot package. With the analysis of the data, through the softwares R and RStudio, the following results were obtained: in order of greater to lesser relevance, the factors that influence the development of CHD are the birth weight (184.497256), the type of delivery (40.688717), gestational age (29.954032), mother’s age (21.551755), race (2.277806) and sex of the neonate (1.405119). Greater attention is necessary to these factors, aiming possible prevention and early management of this condition. 110627 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MARIA EDUARDA DE ALMEIDA OLIVEIRA1, Daniele Araujo de Azevedo Coutinho2, Sergio Augusto Antonio3, Davi Cassiano Costa4, Vanessa da Costa Rodrigues5, Raphael Carreiro Moura6, Maria Auxiliadora Saad7, Carlos Roberto Moraes de Andrade Junior8, Priscila Pollo Flores9, Débora Vieira Soares10 (1) Universidade Federal Fluminense; (2) Universidade Federal Fluminense; (3) Universidade Federal Fluminense; (4) Universidade Federal Fluminense; (5) Universidade Federal Fluminense; (6) Universidade Federal Fluminense; (7) Universidade Federal Fluminense; (8) Universidade Federal Fluminense; (9) Universidade Federal Fluminense; (10) Universidade Federal Fluminense Background and Aims: Non- alcoholic fatty liver desease (NAFLD) is the most frequent cause of hepatic disease, with a world prevalence of 25%. There seems to be a connection between the gravity of NAFLD, endothelial dysfunction, atherosclerosis and the increase in cardiovascular mortality events. This study aimed to asses cardiovascular risk profile of individuals with NAFLD. Methods: Prospective observational analytical study. Adults with established risk for the development of NAFLD were selected, such as: type 2 Diabetes Mellitus (DM), metabolic syndrome and obesity are selected. Non-invasive assessment of liver steatosis and fibrosis was performed by hepatic ultrasound (FLI-score) and transient elastography. We assessed the frequency of the cardiovascular disease, according to the clinical history, common carotid artery intima-media thickness (IMT) using an ultrasound examination of the carotids and a stratification of the cardiovascular risk by a specific algorithm. Results: All data are presented in median (IQR) and n(%): 31 individuals, 25(80,65) female, performed the ultrasound examination of the carotids. Chronological age 67,5(53,75–66,5) years, vascular age 85(46–85)years. Right IMT 0,805(0,54–0,79)cm and left IMT 0,85(0,53–0,75)cm, 8(25,81) had atherosclerotic plaques. We observed: Hepatic Steatosis in 28(90,32), being 6 (19,35) mild, 12(38,71) moderate and 10(32,25) severe. Liver Fibrosis in 8(25,81), being 2(25,00) moderate and 6(75,00) advanced or cirrhosis. Systemic Arterial Hypertension 24(77,42), DM 18(58,06) and Dyslipidemia 22(70,97). Conclusion: Our data shows a high frequency of hepatic fibrosis and atherosclerotic disease in the collected sample. 110660 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY AMANDA BERGAMO MAZETTO1, Eduarda Druck Magadan2, Rafael Aprecido Garcia2, Hellen Cristina Cinesio1, Abdalla Hoelz1, Julien Ramos Stein1, Berta Paula Napchan Boer1, Paula Lavitola1, Ana Flávia Diez de Andrade1, Flávio Tarasoutchi1 (1) Instituto do Coração da Faculdade de Medicina da USP; (2) Escola de Medicina da PUCRS Introduction: Valvular heart disease is a public health problem in Brazil, mainly due to the high prevalence of rheumatic fever, resulting in the need for surgical procedures to repair the valvular disease. Choosing the type of prosthesis in some situations remains a challenge, since it is necessary to consider the patient’s age, preferences and socioeconomic conditions, besides thromboembolic, hemorrhagic and new valve replacement risks. With the development of the Valve-in-Valve procedure, the risk of reoperation has decreased considerably and, therefore, the ideal type of prosthesis for the patient remains a controversial topic. Objectives: The objective of the present study is to analyze the type of prosthesis that is most chosen in the surgical treatment of valve replacement in aortic or mitral position in patients operated in a tertiary health center. Methods: Medical records were reviewed of patients undergoing valve replacement surgery in aortic and mitral position between 2010 and 2021. Results: There were 5333 patients with a mean age at the time of surgery of 57.8 years. Among the analyzed patients, 49.9% of the individuals were female. Regarding the indication of the procedure, elective surgery was the indication in 82.2% of the cases, and urgent and emergency cases, in 17.8% of the cases. In total, 91.4% of biological prostheses and 8.6% of mechanical prostheses were placed. A total of 3042 (57%) aortic valve replacements were performed alone, of which 2876 (94.5%) were biological and 166 were mechanical (5.4%). In the mitral position, there were 1889 (35.42%) valve replacements alone, of which 1654 (87.5%) were biological and 235 (12.4%) were mechanical. In the study, 402 (7.53%) patients with double lesions (aortic and mitral) underwent surgery, 354 (88.1%) being biological and 48 (11.9%) mechanical. Conclusion: Biological prosthesis is the model of choice in our service. The main factors that justify this choice are the age of our patients, the socioeconomic and educational conditions that often limit the possibility of anticoagulation, the better quality of life without the need for anticoagulation, and the lower risk of current reoperation with the Valve-in-Valve procedure in biological prostheses. 110676 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES PABLO RIBEIRO MIRANDA BARBOSA1, Brunna Machado Medeiros1, Gabriel Dias de Oliveira1, Gustavo Fornachari1, Matheus Giacomelli da Trindade1, Marcelle Telasca Patzlaff1, Lucas Álvares de Souza1, Ricardo Augusto Oliveira Mendes1, Mikaelle Tainá Bertoli1, Bruno Noschang Blaas2 (1) Federal University of Pelotas (UFPEL); (2) Federal University of São Paulo (UNIFESP) Introduction: An aneurysm is the dilation of a blood vessel that is greater than 50% of its normal diameter. It induces a fragility in the arterial wall and can trigger severe outcomes, such as the compression of adjacent structures or rupture of the blood vessel itself[1]. In Brazil, the most prevalent cases of aneurysms come in the form of an abdominal aortic aneurysm (AAA). This is more frequently found in elderly patients, with other risk factors in addition to age being: smoking, systemic arterial hypertension (SAH), dyslipidemia and diabetes mellitus. It is 4 to 6 times more frequent in males. Furthermore, ultrasonography (USG) AAA screening is recommended for men between the ages of 65 and 75, who are smokers or who have first-degree relatives with a history of AAA. As for women, screenings are recommended for those with a family history of AAA where a 1st degree relative has had a diagnosis.[2] Objective: Describe the annual prevalence in the number of hospitalizations for the treatment of aortic aneurysms (AA) in Brazil between January of 2011 and December of 2021. Methodology: Descriptive cross-sectional study based on a quantitative approach to the frequency of hospital admissions for treatment of aortic aneurysm in Brazil between January of 2011 and December of 2021. Data regarding hospitalizations was collected from the Hospital Information System of the Unified Health System in Brazil, colloquially known as SIH/SUS. Results: In 2011 there were 2,892 registered hospitalizations for the treatment of AA, followed by 2,906 in 2012, 3,184 in 2013, 3,440 in 2014, 3,815 in 2015, 4,061 in 2016, 3,907 in 2017, 4,184 in 2018, 4,456 in 2019, 3,733 in 2020 and 4,092 cases in 2021. Thus, we conclude an increase of 41.4% in the number of cases between 2011 and 2021. Conclusion: We observed an increase of more than 40% in hospitalizations for the treatment of AA during the study period. However, during the same period, it is important to note that there was a population increase of 10.9%.[5] As a result, we can speculate that there was an increase in diagnostic screening, in addition to a higher incidence of comorbidity such as obesity, diabetes mellitus, dyslipidemia and hypertension. In conclusion, this article is not able to infer specific causality for the number of hospitalizations, thus suggesting that further studies in this topic are needed. 110768 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA FLÁVIA BOTELHO1, Caroline Link1, Larissa Almeida Busnello1, Paola Gonçalves Moreira de Oliveira1, Francielle Nocera Viechineski1, Bruna Kara1, Ana Carla Dlugosz1, Alice Magro Koscianski1, Camilla Mattia Calixto1, Julia Henneberg Hessman1, Mário Claudio Soares Sturzeneker1 (1) Universidade Estadual de Ponta Grossa Introduction: Worldwide, ischemic heart disease is the most common cause of death, and, in this context, acute myocardial infarction (AMI) is a significant public health problem. The SARS-CoV-2 pandemic caused changes in the dynamics of care in emergency units and consequently influenced morbidity and mortality from AMI. Purpose: To estimate the impact of the SARS-CoV-2 pandemic on morbidity and mortality from AMI in the Northeast region through the analysis of DATASUS data. Method: In the period from 2018 to 2021, subdivided into pre-pandemic (11/2018 to 02/2020), pandemic (03/2020 to 06/2021), the peak of the pandemic (12/2020 to 05/2021), first and second trimester of the peak (12/2020 to 02/2021 and 03/2021 to 05/2021) and mass vaccination period (06/2021 to 12/2021), were evaluated in general and by gender, hospitalizations, deaths, mortality rate (percentage obtained by dividing the number of deaths by the number of hospitalizations), calculating the average of each variable and comparing the respective periods, with differences expressed as percentages. Results: In the pandemic period, the number of hospitalizations reduced by approximately 4% compared to the pre-pandemic period. There is an increase in the number of hospitalizations in the second trimester of the peak and the period of mass vaccination, with an increase of 2% and 15%, respectively. Compared to the pre-pandemic period, the average hospital stay was lower in the pandemic and first trimesters of the peak periods (12.3%). There was a reduction in the number of deaths in the pandemic (1%) and the first trimester of the peak (5%) compared to the pre-pandemic period. There is an increase in the number of deaths in the second trimesters of the peak, especially in the mass vaccination period (13%) compared to the pre-pandemic period. Regarding gender, the number of hospitalizations was higher in men, and the mortality rate was higher in women throughout the period evaluated. Conclusions: Social isolation and functional changes in the health system caused by the SARS-CoV-2 pandemic influenced morbidity and mortality from AMI in the Northeast region of Brazil, and mass vaccination culminated in the return to characteristics similar to the pre-pandemic period. 110789 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION JULIA FARENZENA ZAPELINI1, Manuela Matheus1, Samantha Cristiane Lopes1, Franciani Rodrigues da Rocha1, Ottávia de Vasconcelos Zainho Helbok1, Caroline de Oliveira Fischer Bacca1 (1) UNIDAVI – Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí Introduction: Considering that some cardiac death occurs in asymptomatic patients, it is very important to identify subgroups of people with a high risk of cardiovascular arterial diseases (CAD). The coronary artery calcium (CAC) is a marker that identifies the atherosclerotic burden, which is related to the incidence of CAD. Objectives: Evaluate the prevalence of subclinical atherosclerosis defined as altered CAC, recognizing the most prevalent risk factors. Methods: Cross-sectional study with 547 patients who underwent CAC between Jan/16 and Dec/21. Statistical analyses were performed using SPSS employing Fisher’s exact test or Pearson’s chi-square test. The level of statistical significance was set at p < 0.05. Results: The mean age was 52.3 ± 8.9 years. Including CAC, the mean age was 70.5 ± 13.4 years and the CAD risk was 9.5 ± 8.8%. There was correlation between hypertension (SAH) and CAC > 400 (p = 0,04; p < 0,05); and diabetes (DM) and CAC > 400 (p = 0,01; p < 0,01). When the CAC was adjusted in percentiles, the presence of SAH was relevant with the percentile >75 (ra = 4,8; p < 0,01), and dyslipidemia with the percentile 50–75 (ra = 2,7; p < 0,01) and >75 (ra = 2,5; p < 0,01). The absence of SAH (ra = 4,3; p < 0,01) and dyslipidemia (ra = 4,1; p < 0,01) were protective factors, since they showed significance with the 0 percentile. Conclusions: Elevated CAC showed relevance with DM, SAH and dyslipidemia, while the absence of SAH and dyslipidemia were cardiovascular protection factors. 110797 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOGERIATRICS ANA GABRIELA PONTE FARIAS1, Arnóbio Dias da Ponte Filho4, Francisca Tatiana Moreira Pereira4, Vera Marques4, Luís Gustavo Bastos Pinho1, Maria Jacqueline Batista1, Eduardo Augusto Quidute Arrais Rocha3, Ana Rosa Pinto Quidute1, Marcela Albuquerque de Holanda3, Aston Alves de Freitas3, Arthur Holanda Dantas3, Eduardo Arrais Rocha4 (1) Universidade Federal do Ceará; (2) Universidade de Fortaleza; (3) Centro Universitário Christus; (4) Centro de Arritmia do Ceará Introduction: In the elderly, there are several causes of syncope or presyncope, making etiological diagnosis challenging. The relevance of the Head-Up Tilt Test (HUT) in this population has been questioned because of a high incidence of cardiac causes with risk of sudden death. However, dysautonomic causes are also common in this age group, affecting morbidity and mortality. Objective: The aim of this study was to compare the results of the HUT between the elderly (≥60 years old) and non-elderly age groups. Methods: This was a retrospective cohort study, carried out from 2016 to 2021. For comparisons, non-parametric Mann-Whitney/Wilcoxon tests were used, with a significant p-value <5%. The protocols were Westminster or Italian, with the sensibilization phase (1.25 mg sublingual isosorbide) used according to the medical decision during the examination. Results: A total of 2347 tilt tests were analyzed, 61.7% were female, with a median age of 51.1 (31–71). The overall positivity rate was 33.3%, with 43.3% with pharmacological sensibilization (p < 0.01). There were 972 (41.82%) in the elderly group (EG) and 1352 (58.18%) in the non-elderly group (NEG). The positivity rate of EG was lower (p < 0.01); 266 (11.45%) in the EG had positive HUT × 496 (21.24%) in the NEG. The positivity rate with sensibilization in the EG was 27.37% × 20.52% in the NEG (p = 0.008). In the EG, 48 patients (5.04%) had a dysautonomic response × 10 (0.73%) in the NEG (p < 0.001). In the EG, the rates of vasovagal response were 209 (21.97%), distributed as follows: vasodepressor (15.23%); cardioinhibitory (0.92%); mixed (5.86%), while in the NEG, the rates were 466 (33.94%), being vasodepressor (16.94%), mixed (15.90%), and cardioinhibitory (1.26%) (p < 0.001). The rate of complications in the EG was 1.54% × 1.63% in the NEG (p = 0.02), but without serious events. Prodromes were more common 39.86% (539) in the NEG × 31.10% (302) in the EG (p < 0.001). Conclusion: The HUT in the elderly population showed a lower incidence of positivity in the passive phase, however with a higher rate in the sensibilization phase, compared to the non-elderly. The elderly had a higher incidence of dysautonomic responses and a lower rate of prodromes and complications. This method should remain, therefore, as a complementary test in the syncope or presyncope investigation also in the elderly. 110799 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LUCAS BONACOSSA SANT’ANNA1, Eduardo Amar Ferreira1, Sérgio Lívio Menezes Couceiro3, Daniel Chamié4, Fernando Mendes Sant‘Anna2 (1) Fundação Técnico-Educacional Souza Marques (RJ); (2) Universidade Federal do Rio de Janeiro (Macaé); (3) Hospital Santa Izabel (Cabo Frio); (4) Instituto Dante Pazzanese de Cardiologia (SP) Introduction: Fractional flow reserve (FFR) is the gold standard to evaluate severity of coronary stenosis. Quantitative flow ratio (QFR) is a new angiography-based method used to infer FFR. Studies have shown >90% agreement between QFR and FFR. Objective: To conduct a systematic review and diagnostic accuracy analysis of QFR using individual vessel data. Methods: This review follows PRISMA guidelines. MEDLINE, EMBASE and Cochrane Library of Clinical Trials were searched for QFR accuracy studies published until Oct 2020. Inclusion criteria: (a) QFR vs FFR; (b) QFR diagnostic capacity; (c) agreement data between QFR/FFR expressed as dot plots or individual data tables. Graphic data were digitized using a semiautomatic software. QFR/FFR values were dichotomized using cutoff values of ≤0.80 for ischemia. QFR diagnostic accuracy was assessed by two logistic regressions superimposed on the same graph to ensure the probability of agreement between QFR and FFR for any QFR value. FFR was the reference standard. Results: 20 studies comprising 5,318 vessels from 4,429 patients were included. Most patients were male (64%) at an age of 66.8 ± 5.2 years. Figure 1A shows FFR distribution and QFR diagnostic accuracy for different QFR ranges. QFR overall accuracy, sensitivity, specificity, PPV and NPV are displayed in Figure 1B. A diagnostic accuracy of 87% was reached for QFR cutoff values <0.71 or >0.86, and 95% or 98% with cutoffs <0.66 or >0.91 and <0.62 or >0.94, respectively (Figure 1B). Conclusions: A very good diagnostic accuracy of QFR measures was observed using individual vessel data. QFR can be used to evaluate the severity of coronary stenosis. At less accurate values addition of FFR can improve precision. 110835 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION KELLY ANNY FERNANDES MASCARENHAS MASCARENHAS1 (1) Instituto Politécnico de Castelo Branco Introduction: It is estimated that about 1.13 thousand million people around the world are affected by Hypertension (HTN), which causes about 45% of heart disease deaths and 51% of the CVA deaths, more than 8 million deaths per year, and 92 million years of life disability. In Cabo Verde, according to the National Survey about the Risk Factors of Non-Transmitted Diseases (NTDS II) 2020, 20,1% of the population suffer from hypertension. Objectives: To study the prevalence of hypertension in adults residing in Santiago Island – Cabo Verde. Methodology: The transversal Study of the population background in which the data collection took place in October and November 2021, in the municipality of Santiago IslandCabo Verde. It was excluded from the study the individuals of Cape Verdean nationality who have been living for over a year abroad and that have just arrived on Santiago Island in less than 30 days, as well as the individuals with any kind of disabilities that may affect their participation in the study. The data were collected through questionnaires; As for hypertension, it was evaluated using an automated sphygmomanometer. Results: It was studied 599 individuals with the age range between 18 and 99 years old with the age average of 42 years of age, while 54.8% female and 45.2% male. The findings showed that 32.6% of the participants in the study had high blood pressure; 53.8% are female and 46.2% are male. Out of 195 hypertensive participants, it’s realized that 34.6% had stage I HTN, 26.2% had stage II HT, 14.1% had stage III HTN and 25.1% had systolic Isolated hypertension. Thus, it was also found that there is a meaningful statistical relationship between HTN, diabetes, and the age group. Conclusion: There is a high prevalence of HTN in the population of Santiago Island – Cabo Verde. 110867 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES LUANN GABRIEL PORPINO SALES1, Marcelo Augusto Araújo Castro1, Pedro Felipe Alves de Souza1, Valéria Duarte de Almeida1, Eduardo Bulhões Leopoldo da Câmara1, Suyane Maria Paiva Pimenta1, Micássio Fernandes de Andrade1, Cléber Mesquita Andrade1, Thales Allyrio Araujo de Medeiros Fernandes1, Amanda Estevam Carvalho1, Felipe Alves de Lima1 (1) Universidade do Estado do Rio Grande do Norte (UERN) Introduction: The neutrophils are capable of interacting with Trypanosoma cruzi, the etiological agent of Chagas Disease (CD), and release NETs (extracellular Neutrophils traps) causing damage to the myocardium. Objective: To evaluate the correlations between the amounts of NETs released by patients with CD, their clinical forms, with the echocardiographic parameters of these patients. Methods: Retrospective cross-sectional study with conventional sampling. A total of 35 volunteers participated in the research, being 11 healthy individuals and 24 patients with Chagas disease (15 of the indeterminate clinical form and 9 of the cardiac form). The quantification of the formation and release of NETs was performed by means of fluorescence. The identification of clinical forms and the analysis of the echocardiogram were performed by analyzing the filed medical records. The data obtained were processed and statistically analyzed using the GraPad Prism 7.00. Results: Neutrophils from healthy individuals, when stimulated with soluble T. cruzi antigen, release NETs greater than the cells of chagasic patients, both with the clinical cardiac form (* p = 0.0014), as for the indeterminate clinical form (* p = 0.0142). When correlating the concentration of NETs released with the parameters of the Echocardiogram, a negative correlation was observed with the systolic diameter of the left ventricle (r = –0.07998; * p = 0.0097) and with the left ventricular mass index (r = –0.7743; * p = 0.0086), in addition to a positive correlation with the left ventricular ejection fraction (r = 0.6539; * p = 0.0403). Conclusions: Neutrophils from patients with CD release less NETs than neutrophils from healthy individuals and may be associated and contribute to the clinical course of Chagas cardiomyopathy. 110877 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOGERIATRICS CAROLINA DE CARVALHO MOURY FERNANDES1, Maria Eduarda Borges Matias1, Carolina Jeronimo Magalhães3, Matheus Dantas Soeiro1, Sabrina Barreto Braga Pires1, Ellen Beatriz Sobral1, Jessica Myrian de Amorim Garcia2, Francisco Bandeira3 (1) Faculdade Pernambucana de Saúde; (2) Hospital Agamenon Magalhães; (3) Universidade de Pernambuco Introduction: The incidence of heart failure (HF) has steadily increased especially due to the aging of the population. One of its main etiologies is the coronary artery disease (CAD). In the elderly there is a high prevalence of cognitive dysfunction, which can be analyzed by mini cog, a short cognitive impairment screening exam. This cognitive dysfunction may be associated with both coronary and cerebrovascular disfunction. Objectives: Analyze the prevalence of cognitive dysfunction in hospitalized patients with ischemic heart failure. Methods: Cross-sectional observational study of inpatients aged ≥65 years, hospitalized with HF. The study was conducted in a public Brazilian cardiology teaching center from August 2020 to December 2021. The diagnostic criteria for ischemic heart failure was based on coronary angiography findings, while cognitive dysfunction was evaluated using the Mini-Cog. Results: Our total sample consisted of 198 elderly patients diagnosed with HF, we studied 98 of them that also had CAD, showing the prevalence of ischemic heart failure. The mean age was 72.7 years ranging from 65 to 90, and 56.1% were male. Among the comorbidities, 42.9% had diabetes and 89.8% hypertension. The prevalence of cognitive dysfunction was 59.2% based on the Mini-Cog. Conclusion: There was a high prevalence of cognitive dysfunction among the elderly patients with CAD and HF, that was evident when analyzing using the Mini-Cog. 111798 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MAYURI AKEMI RODRIGUES HIGASHI1, Gabriel Ribeiro de Souza1, Rafaela Andrade Penalva Freitas2 (1) UNIVERSIDADE DE SANTO AMARO; (2) INSTITUTO DANTE PAZZANESE DE CARDIOLOGIA Introduction: Despite significant scientific advances and public prevention policies, acute myocardial infarction corresponds to a high rate of hospitalizations and morbidity and mortality. Objective: To analyze and describe the profile of patients who are hospitalized in the city of São Paulo for Acute Myocardial Infarction in the year 2021. Methodology: It consists of a descriptive, retrospective, longitudinal epidemiological study, based on the analysis of a secondary source of data, with the aim of the studies being hospital admissions in the Unified Health System of the city of São Paulo, in the year 2021, for causes related to diseases. hypertensive As inclusion criteria, all hospital admissions were selected through the Hospital Information System (SIH), through DATASUS. The period studied was the year 2021. The morbidities were separated and selected according to the main hospitalization diagnosis and classified according to the International Disease Code 10 (ICD10), the following being selected: I10 -Essential hypertension, I11 – Hypertensive heart disease, I12 – Hypertensive kidney disease, I13 – Hypertensive heart and kidney disease, I15 – Secondary hypertension. Gender, race/color and age group were also taken into account. Results: During the study period, 2219 hospitalizations were performed for hypertensive diseases, of which 49.2% were male and 50.8% female. Among the target audience of the study, when considering race/color, 35.8% of patients consider themselves white, 8% black, 29.5% brown and 0.2% yellow. 26.5% of the registered admissions had no description of race/color. Regarding the age groups studied, there is a gradual increase in the number of hospitalizations as the observed age group also increases, with the group of patients over 80 years of age having the highest number of hospitalizations, corresponding to 14% of all the hospitalizations. Conclusion: Hospitalizations in the city of São Paulo due to hypertensive syndromes were slightly higher in males, most of whom were white and aged over 80 years. 110903 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES NÍCOLAS DE ALBUQUERQUE PEREIRA FEIJÓO1, Mariana Giorgi Barroso de Carvalho1, Thatyane Veloso de Paula Amaral de Almeida1, Ingrid Paiva Duarte1, Léo Rodrigo Abrahão dos Santos1, Rafael Quaresma Garrido2, Giovanna Ferraiuoli Barbosa2, Clara Weskler2, Wilma Golebiovski2, Bruno Zappa2, Marcelo Goulart Correia2, Cristiane Lamas2 (1) Unigranrio/Afya, Rio de Janeiro, Brasil; (2) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (3) Instituto Nacional de Infectologia Evandro Chagas, Fiocruz, Rio de Janeiro, Brasil Introduction/Objectives: Enterococci have increasing frequency as agents in infective endocarditis (IE) due to the population’s increasing life expectancy. They are also strongly related to infections acquired in healthcare scenarios. The present study aims to describe episodes of Enterococcal Endocarditis (EE) in a cardiac quaternary referral center, while also comparing them to other cases IE within the cohort. Materials and Methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Patients were identified prospectively. Categorical variables of the EE group were compared to the other patients IE in the cohort by test of proportions. Statistical significance was set in p < 0,05 threshold. Results: Enterococcal Endocarditis was observed in 48 out of 435 (11%) cases, in which 2 out of 48 (4.2%) cases were identified as E.faecium, while E.faecalis accounted for the vast majority (46/48, 95.8%). Patients with EE were significantly older (median age 59 years, IQR 46–65.3) than the remaining of the cohort (median age 46.0, IQR 32.5 –61.0). A higher incidence of EE was observed in patients with coronary artery disease (13/48, 27.1% vs 47/380, 12.4%, p = 0.006), previous cardiovascular surgery (26/48, 54.2% vs 144/385, 37.4%, p = 0.025), coronary artery bypass graft surgery (6/47, 12.8% vs 19/383, 5%, p = 0.031), diabetes mellitus (12/48, 25% vs 43/387, 11.1%, p = 0.006), chronic renal failure, CRF (16/48, 33,3% vs 75/385, 19.5%, p = 0.026), and cerebrovascular disease (6/48, 12.5% vs 23/386, 6%, p = 0.087). EE patients often acquired the infection in hospital (23/48, 47.9% vs 89/386, 23.1%, p < 0.001). Furthermore, data demonstrated that early Prosthetic Valve Endocarditis (PVE) was proportionally more common in patients with EE (9/48, 18.8% vs 38/387, 9.8%, p = 0.06). New conduction abnormalities (11/43, 25.6% vs 43/349, 8.3%, p = 0.017) were more frequent in patients with EE. Discussion: The frequency of EE in our cohort was similar to the literature (5 to 20%). The results define a profile of older patients with multiple comorbidities, especially CRF and diabetes, and a higher proportion of early-PVE. The observed complication of new conduction disturbances may result from PVE. We hypothesize vascular accesses were the portal of entry for enterococci, and special care must be taken with with them, especially in the perioperative period. 110905 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES NÍCOLAS DE ALBUQUERQUE PEREIRA FEIJÓO1, Thatyane Veloso de Paula Amaral de Almeida1, Mariana Giorgi Barroso de Carvalho1, Ingrid Paiva Duarte1, Léo Rodrigo Abrahão dos Santos1, Rafael Quaresma Garrido1, Giovanna Ferraiuoli Barbosa2, Clara Weksler2, Wilma Golebiovski2, Bruno Zappa2, Marcelo Goulart Correia2, Cristiane Lamas2 (1) Unigranrio/Afya, Rio de Janeiro, Brasil; (2) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (3) Instituto Nacional de Infectologia Evandro Chagas, Fiocruz Introduction/Objectives: Fungi, especially candida spp., have become a frequent cause of bloodstream infections, and Fungal Endocarditis (FE) has a bad prognosis with high in-hospital mortality rates. This study aims to describe cases of FE in a cardiac quaternary referral center and compare them to other infective endocarditis (IE) cases within the cohort. Materials and methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Patients were identified prospectively. Categorical variables of the EF group were compared to the other patients with IE within cohort by test of proportions. Statistical significance was set in p < 0,05 threshold. Results: FE occurred in 14/434 (3.2%) of IE cases; Candida spp accounted for 12/434 (2.7%), of which 8/12 (66.6%) were C. parapsilosis, 2/12 (16.6%) C. albicans, 1 (8.3%) C. tropicalis and 1 (8.3%) C.famata, and Trichosporum spp for 2/434 (0.4%). A higher frequency of FE was observed in patients who had previously been submitted to cardiovascular surgery (8/12, 66.7% vs 162/421, 38.5%, p = 0.07). Patients with FE often acquired the infection in hospital (7/12, 58.3% vs 105/422, 24.9%, p = 0.016). Vascular embolic events were more frequent in FE (9/12, 75% vs 196/412, 47.6%, p = 0.079). Specifically, peripheral embolization (3/12, 25% vs 35/421, 8.3%, p = 0.079) and mycotic aneurisms (4/12, 33,3% vs 43/420, 10.2%, p = 0.032). A higher rate of mortality was observed in patients with Fungal Endocarditis (6/12, 50% vs 103/413, 24.9%, p = 0.05). There was no difference between the rates of surgical indication (91.7% vs 86%) and performed surgery (63.6% vs 80.3%). Discussion: The incidence of FE within the cohort of IE in adults was similar to studies published by other centres (2 to 4%). Non-albicans candida predominated (91.7%), especially C.parapsilosis, which differs from international studies but is similar to other centres in South America. This is probably related to hospital acquisition and the affinity of parapsilosis to catheters and prosthetic material. In fact, previous heart surgery and nosocomial IE were more frequent in FE. Emboli, particularly manifested as mycotic aneurisms, and peripheral emboli were two common complications of FE, which highlights the strong emboligenic dissemination of these agents. Mortality was higher in FE, at 50%, which is similar to the published literature. 110908 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES NÍCOLAS DE ALBUQUERQUE PEREIRA FEIJÓO1, Thatyane Veloso de Paula Amaral de Almeida1, Mariana Giorgi Barroso de Carvalho1, Ingrid Paiva Duarte1, Léo Rodrigo Abrahão dos Santos1, Rafael Quaresma Garrido2, Giovanna Ferraiuoli Barbosa2, Clara Weksler2, Wilma Félix Golebiovski2, Bruno Zappa2, Marcelo Goulart Correia2, Cristiane C. Lamas2 (1) Unigranrio/Afya, Rio de Janeiro, Brasil; (2) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (3) Instituto Nacional de Infectologia Evandro Chagas, Fiocruz, Rio de Janeiro, Brasil Introduction/Objectives: S.aureus has become the mostly encountered pathogen in series of patients with infective endocarditis (IE) from developed countries in recent years, but its role and behaviour is not so well defined in developing countries. S.aureus IE (SAIE) is described as more severe and lethal. Our aim was to describe cases of SAIE in a developing country and compare it to other cases of IE in a cohort presenting to a cardiac surgical referral center. Materials and methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Patients were identified prospectively. Categorical variables were presented as frequencies and percentages, and the SAIE group was compared to the other patients with IE in the cohort by test of proportions. A p-value less than 0.05 was considered statistically significant. Results: S.aureus accounted for 49/434(11.3%) episodes of IE. Resistance to methicillin was seen in 8/49(16.3%) strains (5 MRSA, 3 community-acquired MRSA or ca-MRSA). Patients with SAIE more often had chronic renal failure (15/49, 30.6% vs 76/384, 19.8%, p = 0.08). Rheumatic valve disease (RVD) was less often seen in SAIE (16.3% vs 33.5%, p = 0.015). Patients with SAIE more often had hospital-acquired IE (46.9% vs 23.1%, p < .001), and devices were more often affected (24.5% vs 5.5%, p < 0.001). No differences were seen regarding the frequency of paravalvular abscess (18.4% vs 15.1%), valve perforation (14.3% vs 18.8%) or fistulae (4.1% vs 4.2%). Complications more often found in SAIE were persistent bacteremia (23.9% vs 5.6%, p < .001), peripheral emboli (18.8% vs 7.5%, p = 0.01) and recurrent emboli (19.1% vs 5.1%, p < .001). Acute renal failure 44.7% vs 32.5%, p = 0.097) and need for hemodialysis (52.2% vs 25.0%, p = 0.008) were more often seen in SAIE. Rates of surgical indication (83.3% vs 86.5%), surgery (72.7% vs 80.7%) and mortality (28.6% vs 25.3%) were similar between groups. Discussion: S.aureus was the third mostly encountered causative agent in IE in our center, and was mostly associated with healthcare acquisition and intracardiac devices. Surgical rates and mortality were similar to other IE, possibly because of referral bias, but recurrent emboli, persistent bacteremia and acute renal failure were more frequent in SAIE, highlighting its acute presentation with sepsis. 110914 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION CATARINA RAQUEL GASPAR DOS SANTOS1, Catarina Santos1, Patrícia Coelho1, Francisco Rodrigues1 (1) Instituto Politécnico de Castelo Branco Arterial hypertension (HTA) is one of the main risk factors for cerebrovascular diseases, representing a high incidence rate in the world population. In this way, it becomes increasingly important to make people aware of the importance of hypertension and the risk factors that it entails. As such, the objective of this investigation was to assess the prevalence of hypertension in the municipality of Sertã. The present research study is of the prospective type. The sample collection was based on the cluster method, through the random selection of streets in the municipality of Sertã. Anthropometric data were collected, 3 blood pressure assessments were performed, with an interval of 3 minutes between each, and a survey on risk factors was applied. Statistical treatment and data analysis were performed using the SPSS Statistics program. The sample is composed of 1000 individuals, of legal age and residing in the municipality under study, with 515 (52%) of the participants being male and 485 (48%) females. The mean age was between 56.04 years. Regarding the prevalence of arterial hypertension, a percentage of 43.6% was found, and this result includes all individuals who presented mean SBP and/or DBP values above normal values (140/90 mmHg, respectively) and all individuals who take antihypertensive medication. About individuals who reported performing associated therapy, 165 individuals (60.2%) had controlled hypertension, however, 109 (39.8%) had uncontrolled hypertension. Regarding risk factors, the most affluent were obesity (61.7%), physical inactivity (48.2%) and family history of hypertension (42.3%). It is concluded that the present study found that there is a significant prevalence of hypertensive patients in this region, which may be related to the lack of control of risk factors. 110920 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY GABRIELA DANTAS MACHADO LEAL1, Fernanda Dantas Machado Leal1, Marília Menezes Gusmão1 (1) Escola Bahiana de Medicina e Saúde Pública Introduction: The prevalence of depression and the use of antidepressive agents have been increasing worldwide over the last few years. Hypertension is another clincal condition of high prevelence in which it is common to be associated with drepression. Objective: Avaluate by a systematic review, the likelihood of antidepressive agents effecting blood pressure, the study will also evaluate what are the classes of antidepressive agents that are related to the deacrease or increase of blood pressure value. Methods and materials: searches were conducted in databases such as PubMed and Scielo, and selected papers in humans, which included the use of antidepressive agents for depression treatment and blood pressure mesurement. Researches that have used other medications that were not antihipertensive agents, but could have potentially altered blood pressure measurement, were excluded. The review protocol was registeres in PROSPERO. The protocol registration number is CRD42020194118. Results: The final sample consisted of seven articles. In three of them, the use of antidepressive agents did not cause significant changes on the value of blood pressure. One study detected blood pressure drop after the use of antidepressive agents. While three other studies detected blood pressure spike after the use of antidepressive agents. Conclusion: Selective serotonin reuptake inhibitors (SSRIs) e serotonina and norepinephrine reuptake inhibitors (SNRIs) are not capable of influencing and altering blood pressure value, however, GSK372475 is capable of increase blood pressure value. 111006 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING CAROLINA MENDES PEREIRA1, Camila Pereira de Carvalho2, Clara Alice Lima Leal1, Luis Augusto Oliveira Santos1, Breno Carvalho Cirne de Simas1, Ferdinand Gilbert Saraiva da Silva Maia1, Josivan Gomes de Lima1, Lucia Helena Coelho Nóbrega2, Ricardo Luiz de Medeiros Lima2 (1) Federal University of Rio Grande do Norte; (2) University Hospital Onofre Lopes Introduction: Acromegaly is a rare disease characterized by high levels of growth hormone (GH) and insulin-like growth factor (IGF-1). Cardiovascular disease, especially heart failure, is a common complication in acromegaly. Objectives: Perform a comprehensive echocardiographic assessment of acromegalic patients and relate systolic function indexes with disease activity. Methods: Bidimensional echocardiographic assessment of left ventricular geometry, Ejection Fraction (EF, determined by Simpson’s method), Global Longitudinal Strain (GLS, using speckle-tracking technique) and diastolic function were performed in on-treatment acromegalic patients without established cardiovascular disease. Patients with poor acoustic window, atrial fibrillation and oncologic treatment (systemic chemotherapy or thoracic radiotherapy) were excluded. Ejection fraction and GLS were compared in patients with active and remitting disease. Results: 28 acromegalic patients were included (17 women and 11 men; mean age 42,8 ± 11,5 years; mean time from diagnosis 7 ± 4.9 years). Thirteen patients were in biochemical remission, 11 had active disease and 4 had elevated GH level with normal IGF-1 (indeterminate pattern). Structural and/or functional cardiac alterations were present in 16 patients (57.8%). Eleven patients (39.28%) exhibited abnormal geometry (7 with eccentric hypertrophy, 3 with concentric hypertrophy and 1 with concentric remodeling). Mean EF was 58.8% (±6.3). Only one patient had mildly reduced EF. Mean GLS was 20.5 ± 2.8. Seven patients (25%) had below normal GLS (6 patients had borderline and 1 had reduced GLS). Four patients (14.28%) presented alteration in diastolic function indexes (2 with type 1 and 2 with indeterminate diastolic dysfunction). EF (57.4 ± 7.39 vs 59.5 ± 6.2, p = 0.44) and GLS (19.8 ± 3.1 vs 20.4 ± 2.7, p –0.60) were not different in patients with active or remitting disease. Conclusions: In this cohort of on-treatment acromegalic patients without established cardiovascular disease, structural and/or functional echocardiographic alterations were frequent. Left ventricular hypertrophy is the most frequent alteration. Ejection fraction and global longitudinal strain were not different in patients with active or remitting disease. 110923 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES LUANN GABRIEL PORPINO SALES1, Pedro Felipe Alves de Souza1, Felipe Alves de Lima1, Marcelo Augusto Araújo De Castro1, Henrique Rangelly Gabriel de Melo1, Leonardo Lacerda do Amaral1, Rafaela Germana Cavalcanti da Nóbrega2, Micássio Fernandes de Andrade1, Amanda Estevam Carvalho1, Valéria Duarte de Almeida1, Cléber Mesquita Andrade1 (1) Universidade do Estado do Rio Grande do Norte (UERN); (2) Centro Universitário de João Pessoa (UNIPÊ) Introduction: Neutrophils are cells with a typical polylobulated nucleus, capable of modulating the immune response in Chagas disease (CD). In some occasions, these cells may have a ring shaped nucleus, typical of immature leukocytes with different performing mechanisms, and your greater incidence be associated with solid tumor cancers and CD. Objective: Evaluate the morphology of the peripheral blood neutrophils nucleus of patients with Chagas disease, in the different clinical forms of the disease. Methods: Transversal study with non-probabilistic sampling. A total of 32 volunteers participated in the research, being 11 individuals with indeterminate clinical form of the disease and 21 individuals with cardiac clinical form. The analysis of the morphology of the nucleus of the polymorphonuclear cells were performed by light microscopy. The identification of the clinical forms and the analysis of the echocardiogram were performed by analyzing the filed medical records. Spearman and Pearson tests were used to determine the correlations between the absolute number of ring neutrophils and echocardiographic parameters. Results: A negative correlation was observed between the absolute number of ring neutrophils in the patients‘ peripheral blood and the left ventricular (LV) ejection fraction (*p = 0.0031 and r = –0.7087). However, regarding the other echocardiographic parameters of LV Systolic Diameter (*p = 0.0009 and r = 0.8040), LV Diastolic Diameter (*p = 0.0250 and r = 0.5942) and Mass Index of the LV (*p = 0.0351 and r = 0.5655), a positive correlation was noticed concerning the absolute number of annular neutrophils. Conclusion: Ring shaped neutrophils from patients with CD may contribute to the physiopathology of Chronic Chagas Heart Disease. 110948 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION KELLY ANNY FERNANDES MASCARENHAS1, Patrícia Coelho1, Francisco Rodrigues1, Júlio Rodrigues2 (1) Instituto Politécnico de Castelo Branco; (2) Ministério de Saúde Cabo Verde Introduction: Cerebrocardiovascular diseases represent a meaningful death rate worldwide, making a big challenge to public health and the economy. Many African countries like Cabo Verde are facing an epidemiologic transition from transmitted diseases to non-transmitted diseases, demographic changes as well as socio-economic changes, and are facing rapid growth in terms of cardiovascular diseases and are linked to rising in cardiovascular risk. Objectives: This present study had as its objective to identify the prevalence of risk factors of cardiovascular diseases in adults residing on the island of Santiago-Cabo Verde. Methodology: The transversal study of a population background of individuals over 18 years of age, from both genders. The data collection took place in October and November 2021, in the nine municipalities of Santiago Island- Cabo Verde. It was excluded from the study the individuals of Cape Verdean nationality who have been living for over a year abroad and that have just arrived on Santiago Island in less than 30 days, as well as the individuals with any kind of disabilities that may affect their participation in the study. The sample size was calculated based on the population projection of the year of Santiago Island, for those over 18 years of age according to the appropriate epidemiologic sample. The field data (domiciliary) were obtained through a questionnaire application where ages, sex, race, weight, height, risk factors, (diabetes presence, dyslipidaemias, hereditariness, physical exercise practices, smoking habits, cerebrovascular antecedent events, weight control habits, and blood pressure) are considered. Results: The sample of the study is mostly made of females representing 54.8%. Furthermore, the age group with the highest sample number was between 18–27 years old, corresponding to (21%). 42.2% with BMI ≥ 25 kg/m2; 65.1% of physical inactivity; 7.3% with Tabaco, 14.4% with alcohol, 6.5% with cardiac diagnosed diseases, 19.9% with close family with cardiac pathology, 2.7% have had cerebral vascular, 0.1 has already suffered an ischemic transitory accident, 0.5% myocardial infarction, and 23.5% had a periodic habit of checking the blood pressure, 32.6% hypertensives and 4.5% of diabetics. Conclusion: The result of the study allowed us to affirm that there’s a high prevalence of risk factors for cerebrovascular accidents in the island of Santiago-Cabo Verde, especially among women. 110988 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES THATYANE VELOSO DE PAULA AMARAL DE ALMEIDA1, Mariana Giorgi Barroso de Carvalho1, Nícolas de Albuquerque Pereira Feijóo1, Ingrid Paiva Duarte1, Léo Rodrigo Abrahão dos Santos1, Rafael Quaresma Garrido2, Giovanna Ferraiuoli Barbosa2, Clara Weksler2, Wilma Golebiovski2, BRUNO ZAPPA2, Marcelo Goulart Correia2, Cristiane C. Lamas3 (1) Unigranrio/Afya, Rio de Janeiro, Brasil; (2) Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil; (3) Instituto Nacional de Infectologia Evandro Chagas, Fiocruz, Rio de Janeiro, Brasil Introduction: Prosthetic valve infective endocarditis (IE) has high morbidity and mortality. Late prosthetic valve IE (LPVE), defined as that acquired 12 or more months after prosthesis implantation, often has as causative agents’ community pathogens. Our objective was to describe cases of LPVE and compare them to other cases of IE. Methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Statistical analysis was performed using the Jamovi and R software. Results: Of the 435 patients with IE, 77 (17.7%) had LPVE. They were older, with a median age of 52 years (IQR 38–68), compared to 47 years (IQR 32–61). Enterococci were more frequent in LPVE (29.2% vs 16.3%, p = 0.027), but negative blood cultures were less frequent (11.9% vs 20.3%, p = 0.036). In LPVE, infection was community-acquired in 75.3%, and hospital-acquired (HA) in 16.9%. Patients with LPVE more often had a history of coronary artery disease (21.1% vs 12.5%, p = 0.051), coronary artery bypass surgery (12.2% vs 4.5%, p = 0.01), congestive heart failure (59.2% vs 35.9%, p < 0.001), and cerebrovascular disease (17.1% vs 4.5%, p < 0.001); chronic renal failure was less frequent in LPVE (11.7% vs 23.0%, p = 0.027). Rheumatic valvular heart disease (RVD) was observed more frequently in LPVE (57.9% vs 25.7%, p < 0.001). Aortic and mitral involvement was frequent in both groups. The frequency of abscess (23.7% vs 13.7%, p = 0.029) and conduction disturbances (25.7% vs 11.2% p = 0.001) was higher in LPVE. Heart failure secondary to acute valve regurgitation was equally frequent (56.6% vs. 61.6%), as were neurological events (40.5% vs. 50.0%), including mycotic aneurysms (6.5% vs 11.8%). Splenic emboli were frequent in both groups (32.5% vs. 35.8%). Surgical indication occurred less frequently (78.9% vs 87.7%, p = 0.046), as well as surgery (67.2% vs 82.4%, p = 0.005). In-hospital death was higher in LPVE (39.5% vs 22.6%, p = 0.002). Conclusion: LPVE had a frequency of 17.7%, like other series in the literature (10–30%). However, it showed a high prevalence of VHD, as in other developing countries where it is the main reason for valve replacement. Clinical features were like other IE, except for the presence of abscesses, and mortality was very high, possibly due to patients being older, having more underlying illnesses and less often having valve replacement surgery for IE. 110991 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES GUILHERME DANTAS CAMPOS PINTO1, Silvia Marinho Martins2, Cristina de Fátima Velloso Carrazzone2, Maria das Neves Dantas da Silveira Barros1, Carolina de Araújo Medeiros2, Gabriela Arcoverde Wanderley1, Gustavo Sales Santa Cruz1, Gabrielly Nascimento de Lima1, Virginia Maria Barros de Lorena3, Luciane de Freitas Firmino3, Maria da Gloria Aureliano de Melo Cavalcanti2, Wilson Alves de Oliveira Júnior2 (1) Universidade de Pernambuco (UPE); (2) Ambulatório de Doença de Chagas e Insuficiência Cardíaca – PROCAPE/UPE; (3) Fundação Oswaldo Cruz (Fiocruz) Introduction: Changes in the electrocardiogram (ECG) are markers of severity and prognosis in chronic Chagas disease (CD). However, in the acute form, the prognostic value of the ECG before and after treatment with benznidazole, as a tool to analyze serological evolution, has not been well established yet. Objective: To verify the association between electrocardiographic changes and serology evolution in patients with acute CD. Methodology: This is a cohort study with 25 patients infected by T. cruzi in an outbreak in a city in the Brazilian Northeast. The population was followed up at a referral outpatient clinic in Pernambuco State, from acute phase treatment until 24 months (+/- 6) after benznidazole administration. The diagnosis was confirmed by serological methods and/or molecular tests. ECGs and serological tests were performed before and after treatment. Then, the remission, maintenance, and appearance of electrocardiographic changes were determined, as well as the serological evolution of each patient. The ECGs were evaluated by two professionals – one of whom is an expert in the area. P-value < 0.05 indicates a statistically significant association. Results: The population was mostly female (56%), with a mean age of 30 years (SD = 11.5) and without comorbidities (92%). The prevalence of ECG changes after 30 and 60 days of the infection was 56%. They were: altered cardiac repolarization (ACR) (20%), electrical axis deviation (20%), low QRS voltage (20%), incomplete right bundle branch block (IRBBB) (8%), left anterior fascicular block (LAFB) (8%) and first-degree atrioventricular block (8%). On reassessment of the ECGs after 2 years, 48% had some alteration: low QRS voltage (32%), IRBBB (12%), LAFB (8%), left anterior fascicular block (8%), and chaotic atrial rhythm (8%). When follow-up ECG was compared to pretreatment exam, 7 remained normal; 5 remained altered; 6 which were previously altered were normalized, and 7 presented some new alteration. Fifteen patients (60%) showed positive IgG for T. cruzi. In the acute phase, 100% of patients who had ACR before treatment had positive serology tests after 2 years (p = 0,061). There was no statistical association between serology results and the follow-up ECG. Conclusions: ECG changes in the acute phase showed no power of association with post-treatment CD serology. It is considered relevant to elucidate the predictive value of ACR in the acute phase for seroconversion, through novel studies. 111000 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES ADMILSON LEMOS DA COSTA FILHO1, Marcelo Alves Maia2, Ester Cerdeira Sabino3, Ana Isabel Nobre Maia4, Artur Lima Sendin1, Kássia Burini1, Fellipe Colares P G Versiani4, Antonio Luiz P Ribeiro2, Maria Carmo P Nunes2 (1) School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.; (2) Post Graduation Program in Infectious Diseases and Tropical Medicine, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.; (3) Instituto de Medicina Tropical e Departamento de Moléstias Infecciosas e Parasitarias da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.; (4) Health Science Program, Universidade Estadual de Montes Claros, Montes Claros, Brazil. Background: Chagas cardiomyopathy is an important cause of heart failure in endemic areas. Natriuretic peptides are well established in the diagnostic of heart failure, but their prognostic value is not well defined, especially in community-based Chagas patients. Objective: This study aims to assess the prognostic value of N-terminal pro-brain natriuretic peptide (NT-proBNP) in predicting mortality in patients with Chagas cardiomyopathy from remote areas. Methods: Patients with Chagas disease from endemic areas who had heart failure defined as left ventricular ejection fraction (LVEF) <50% and/or NT-ProBNP >300 pg/ml were eligible for the study. Clinical data were obtained using a standardized questionnaire. A resting 12-lead electrocardiogram was recorded at baseline. A range of readily obtained echocardiographic measures were collected using portable equipment at public health primary centers. The end point was all-cause mortality. Results: The study cohort consisted of 370 patients with a mean age of 66.2 ± 12.7 years, and 210 patients (57%) were women. The majority of the patients had left ventricular (LV) systolic dysfunction with mean ejection fraction of 41 ± 12%. During a mean follow-up of 31 months, 133 patients died (36%) with overall mortality incidence rate of 39.6 deaths per 100 patient-years. Four key echocardiographic parameters were predictors of mortality, including LVEF (HR 0.97, 95% CI 0.95 to 0.98), right ventricular (RV) end-diastolic area (HR 1.06, 95% CI 1.03 to 1.09), E/e’ ratio (HR 1.03, 95% CI: 1.01 to 1.05), and left atrial (LA) volume (HR 1.01, 95% CI 1.00 to 1.01). The inclusion of NT-ProBNP on top of echocardiographic parameters resulted in significant improvement in model performance. Conclusions: In a contemporary cohort of patients with Chagas cardiomyopathy, NT-proBNP was a strong predictor of death, independently of LV dysfunction severity and RV involvement. NT-proBNP assessment may be used in a clinical setting to improve the risk prediction model for mortality in patients with Chagas cardiomyopathy. 111010 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES THIAGO FONSECA DE AZEVEDO1, Ádria Rayane Lima Cascaes1, Leonardo Rodrigues Ferreira Diogo1, Pablo Rodrigues Nunes de Souza1 (1) Universidade do Estado do Pará (UEPA) Introduction: In the research about cardiac pathologies, cardiogenic shock (CS) stands out, given its complexity and high incidence (between 5 and 15% of patients with acute myocardial infarction). In general, CS is the inability of the heart to perform a tolerable cardiac output to oxygenate and nourish tissues. In this sense, understanding the processes of this cardiological condition is important, as there is still a high mortality associated with it (about 50%). Despite this, it is observed that the treatment of cardiogenic shock is not standardized and doubts persist regarding its therapeutic protocol. Therefore, it is important to understand the therapeutic methods associated with cardiogenic shock, in order to manifest a logical and temporal framework in the progression of the disease and in the improvement or worsening of the patient. Objective: To assess the most used clinical treatment protocols for cardiogenic shock. Methods: Systematic review without meta-analysis, using the descriptors “Clinical protocol” and “Cardiogenic shock”. The search was carried out in the PubMed, LILACS and Cochrane databases. As inclusion criteria, articles in English limited to the last five years (2018–2022) were accepted. Studies such as randomized clinical trials, cohort, case-control and observational studies were selected. Articles without abstract or full text available and duplicates were excluded. Results: Critical analysis of the collected data revealed the absence of a standardized protocol for CS across centers, together with a high post-discharge mortality despite advances in the field; however, some factors, such as early invasive hemodynamics and percutaneous coronary intervention, as well as shock team-based protocols, were correlated with higher rates of survival and better prognosis, while the use of intra-aortic balloon pump didn’t show advantages in several studies, and is accordingly not recommended anymore for management of CS. Conclusion: The present study concludes that ECMO, Inotropic Agents, Impella 5.0 and percutaneous hemodynamic support were prevalent in the studies analyzed. In Addition, intra-aortic balloon pump and the creation of a Shock Team were used as a way to improve prognosis in patients with cardiogenic shock. The disparity of results showed the necessity of clinical trials and a clearer Cardiogenic Shock clinical protocol in order to establish a better CS clinical management. 111002 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY EDUARDO AUGUSTO QUIDUTE ARRAIS ROCHA1, Marcela Sobreira Kubrusly1, Luís Gustavo Bastos Pinho3, Vera Marques4, Francisca Tatiana Moreira Pereira4, Bruna Sobreira Kubrusly3, Davi Sales Pereira Gondim2, Fernanda Pimentel Arraes Maia3, Rodrigo Carvalho Paiva1, Maria Eduarda Quidute Arrais Rocha2, Arnóbio Dias da Ponte Filho4, Roberto Lima Farias3 (1) Centro Universitário Christus (Unichristus); (2) Universidade de Fortaleza (Unifor); (3) Universidade Federal do Ceará (UFC); (4) Centro de Arritmia do Ceará (CACE) Introduction: Postural Orthostatic Tachycardia Syndrome (POTS) represents one form of clinical presentation among neurally mediated syndromes, with several peculiar characteristics. Many aspects of this syndrome still remain unknown. This study aimed to analyze the characteristics of patients with clinical suspicion of POTS. Methods: Retrospective cohort study, conducted in the period 2016–22. Statistical analysis was performed in the stats models module. To obtain odds ratios, we used logistic regression models (for positivity as a response) and multinomial logistic regression models (for the type of response). Other comparisons were made using the Mann-Whitney test and Fisher’s exact test, with p-value of 5% considered significant. Results: The mean age for the 36 patients with a POTS response was 35.3 ± 0.45 years; 72.2% (26) were male. Among the symptoms that motivated the TT, in the POTS group, we had 69.4% (25) with syncope, 8.3% (3) presyncope, 8.3% (3) with a previous suspicion of POTS, 8.3% (3) with dizziness and 5.5% (2) unknown. None of the patients had associated pathology or reported previous medication use. The complication rate was 2.7% (1). The total number of tilt tests performed were 2462, with 115 exclusions, 37.5% of which were male, with a median age of 51.1 (31–71) years, with 66.31% TT with normal response and 33.69% positive TT. The types of response were 16.22% (377) hypotensive, 11.7% (272) mixed, 2.49% (58) dysautonomic, 1.55% (36) POTS, 1.11% (26) cardioinhibitory, 0.47% (11) psychogenic. A total of 14 patients had a previous suspicion of POTS, with 42.8% (6) presenting a positive TT, while 57.1% (8) a negative one. In the group that did not have a suspicion of POTS, the values were, respectively, 32.7% (756) and 67.2% (1554) (p = 0.47). The symptom reproduction rate in patients with a POTS response was 52.7% (19) versus 47.2% (17). Conclusion: The Postural Orthostatic Tachycardia Syndrome represented a small percentage of the TT exams performed, predominating in young patients, with a positive response on the TT, even in the absence of clinical suspicion, with reproduction of symptoms in most cases and a low rate of complications in the exam. POTS is usually a challenging diagnosis. 111057 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MANOEL PEREIRA GUIMARÃES1, Carlos Dornels Freire de Souza1, Rodrigo Feliciano do Carmo1, Ricardo Khouri2, Sávio Luiz Pereira Nunes3, Jandir Mendonça Nicacio1, Ana Marice Teixeira Ladeia4, João Augusto Costa Lima5, Anderson da Costa Armstrong1, Manoel Barral-Netto2 (1) Universidade Federal do Vale do São Francisco (UNIVASF); (2) Instituto Gonçalo Moniz/FIOCRUZ; (3) Universidade de Pernambuco (UPE); (4) Escola Bahiana de Medicina e Saúde Pública; (5) Johns Hopkins University Introduction: Atherosclerotic disease and its risk factors show relationships with inflammatory changes. The inflammatory mediation of cardiovascular risk in indigenous groups undergoing a rapid process of urbanization is still unknown. Objective: To describe the inflammatory dynamics of two indigenous groups in Northeast Brazil with and without arterial hypertension. Methods: Cross-sectional ancillary study (2016–2017) involving 302 indigenous people from the Fulniô (n = 210) and Truká (n = 92) ethnic groups (Pernambuco, Brazil). Sociodemographic data and samples of blood to measure the inflammatory markers IL-8, MPO, Tenascin-C2, Trail, FGF-2 and Lox-1. For the analysis, multivariate network analysis was used by the Leats absolute shrinkage and selector operator (LASSO)- with non-parametric transformation. This project is part of the PAI-Study (Integral Indigenous Care Project). Results: The prevalence of arterial hypertension was 23.5%. (n = 71) [20.0% in Fulniô and 31.5% in Truká] in hypertensive patients, a positive correlation was observed between Tenascin-C2 and FGF-2, which was not present in non-hypertensive indigenous people. There is a stronger positive correlation between TRAIL and IL-8 than the obs herbed in non-hypertensive patients. On the other hand, the correlation between FGF-2 and PAI-1 was stronger in non-hypertensive individuals. Conclusions: Differences were found in the inflammatory dynamics of hypertensive and non-hypertensive indigenous people, with emphasis on the positive correlation between Tenascin-C2 and FGF-2. 111062 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MANOEL PEREIRA GUIMARÃES1, Antônio Marconi Leandro da Silva1, Elder Gil Alves da Cruz2, Matheus Pereira Barreira1, Everardo Joaquim Gonçalves dos Santos1, Frank Land Carvalho1, Fernando Marcos França3, Audes Diógenes de Magalhães Feitosa4, Rodrigo Feliciano do Carmo1, Jeová Cordeiro de Morais Júnior1, Carlos Dornels Freire de Souza1, Anderson da Costa Armstrong1 (1) Universidade Federal do Vale do São Francisco (UNIVASF); (2) Clínica do Coração Dr. Elder Gil; (3) CARDIOVASF – Instituto do Coração do Vale do São Francisco; (4) Universidade Federal de Pernambuco (UPE) Introduction: Validated home blood pressure monitoring (HBPM) is not widely available in Brazil. In the places HBPM is available, it is unknown how Brazilian inequalities in access to health may impact on hypertension diagnosis and control. Objectives: To compare hypertension diagnosis and control in public and private health sectors in an unfavored outland area in Northeast Brazil (Sertão; Pernambuco). Methods: Longitudinal, multicenter study. Information on sex, age, body mass index (BMI), antihypertensive medications, office BP and HBPM was collected using the TeleMRPA platform, between September 2017 and February 2022. All equipment was validated and calibrated. All participants were classified according to the Brazilian Guidelines on Hypertension and grouped following the health sector (public vs. private). Data was also collected in recurrent exams up to six months. For data analysis, the Shapiro-Wilk, Mann-Whitney and chi-square tests were used. Results: 2,261 individuals were included. People from the public sector were younger (58.3 ± 15.7 vs 55.5 ± 15.2 years; p = 0.001), had higher BMI (29.6 ± 5.8 vs 28.1 ± 5.2 g/m2; p < 0.001), and used higher number of BP pills (1.6 ± 1.3 vs 0.9 ± 1.0 pill; p < 0.001). Male participants were more prevalent in the private sector (37.2%vs31.4%; p = 0.019). In the first HBPM performed, 54.9% of the public sector group had BP out of control, compared to 44.0% (p < 0.001) in the private sector. During follow-up, 50 participants enrolled in the new HBPM within six months of the first exam. For the second HMBP, 47.1% of the public sector group had BP out of control compared to 54.5% of the private sector (p = 0.616). Conclusion: Hypertensive participants enrolled from the public health sector had a more severe disease profile in the initial assessment but were similar to those from private sector in the 6-month follow-up period, when HBPM was used. The wide use of HBPM may aid reducing inequalities in hypertension treatment in Brazil. 111103 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIELA ARCOVERDE WANDERLEY1, Guilherme Dantas Campos Pinto1, Gustavo Sales Santa Cruz1, Felipe José Gomes Pereira de Lucena1, Larissa Cassiano de Araújo1, Maria da Glória Aureliano Melo2, Maria Elisa Lucena Alves2, Maria das Neves Dantas Silveira Barros2, Wilson Alves de Oliveira Júnior2, Silvia Marinho Martins Alves2 (1) Universidade de Pernambuco; (2) Casa do Paciente Portador de Doença de Chagas e Insuficiência Cardíaca (PROCAPE) Introduction: Heart failure is a high mortality clinical syndrome. The electrocardiogram (EKG) is an assessable exam with low cost, very useful in the evaluation of heart failure patients. The QRS axis, determined by EKG, is a possible prognostic tool. Objective: To verify the prognostic value of the QRS axis in patients with heart failure with reduced ejection fraction (HFrEF). Methodology: Cohort study with 110 patients with HFrEF (EF ≤40% in a last year echocardiogram -ECHO). QRS axis was determined by the Hexaxial Reference System and was classified in: normal (–30° to +90°), left axis deviation (–90° to –30°), right axis deviation (+90° to +180°) and northwest axis (–90° to –180°). To prognostic analysis was considered: funcional class (New York Heart Association), left ventricular end-diastolic diameter (LVEDD) on ECHO and right ventricle systolic function (RVSF), evaluated by tricuspid annular plane systolic excursion on ECHO and classified in reduced RVSF and normal RVSF. P-value <0,05 indicates a statistically significant association and a 95% confidence interval was considered. Results: The population was predominantly male (58%) and brown (“pardos”, 68%) with a mean age of 57 years (29–82, SD = 11,5). Low education levels were present (70% of the patients dropped out in Primary or Secondary School). The most prevalent comorbidities were overweight/obesity (67%), arterial hypertension (61%), dyslipidemia (46%) and diabetes (31%). HFrEF most frequent etiologies were Chagas Cardiomyopathy (31%), Hypertensive Heart Disease (19%) and Isquemic Heart Disease (17%). Mean ejection fraction was 29% (12–40%, SD = 6,8). Mean LVEDD was 65 mm (30–90, SD = 8,5) and 38% of the patients had reduced RVSF. About QRS axis: 48% had normal axis, 36% had left axis deviation, 10% had right axis deviation and 5,5% had the northwest axis. Neither the functional class (p = 0,174) nor LVEDD (p = 0,623) were statistically associated with QRS deviation. QRS axis deviation was associated with RVSF (p = 0,013) and patients with right axis deviation (OR = 9,5) or left axis deviation (OR = 1,3) had more chances of presenting reduced RVSF when compared with patients with normal axis. However, the northwest axis was less likely to have reduced RVSF when compared with normal axis (OR = 0,03). Conclusion: QRS axis deviation was only associated with RVSF. 111278 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY GEORGIA MARQUES JARDIM1, Bianca Brinques da Silva2, Guilherme Rodrigues Viana1, Carolina Guimarães Herzog1, Eric Seiji Kanai1, Grasiele do Amaral Martins1, Carolina Andreatta Gottschall2, Egídio Junior Lorenzetti Ruggini1, Helena Guedes da Rocha1, Letícia Vieira Senger1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA; (2) Universidade Luterana do Brasil – ULBRA Introduction: Congenital heart diseases (CHDs) are abnormalities in the structures or functions of the heart that can compromise the individual in all age groups. In this sense, they are a public health problem, since early diagnosis is crucial for patient survival. Objective: To evaluate the use of antihypertensive drugs according to their risk classification in pregnancies of fetal CHD patients. Method: The study sample consisted of 198 patients, who were consecutively evaluated during their first hospitalization in a cardiac intensive care unit. The participants were submitted to a protocol that evaluated the use of antihypertensives during their pregnancy. These were divided according to their fetal risk, following the classification proposed by the Food and Drug Administration (FDA). The data analysis considers high risk the exposure of children to drugs that have pharmacological classes D and X. Results: Of the total sample, 103 patients (52%) were male, with ages ranging from 1 to 4934 days. The interview for data collection was carried out with the patient’s mother in 48.5% of cases and with both parents in 42.9%. Most of the patients are users of the brazilian national health service. The most frequently observed CHDs were ventricular septal defect (16.1%) and atrial septal defect (16.1%). Furthermore, 124 patients (62.6%) were exposed to at least one drug during pregnancy. 18 mothers (9%) reported using antihypertensive drugs during pregnancy, which included methyldopa (n = 7–3.5%), enalapril (n = 4–2%), hydrochlorothiazide (n = 3–1.5%), verapamil (n = 2–1%) and propranolol (n = 2–1%). Regarding the use of enalapril (n = 4), two pregnant women used it in the first trimester of pregnancy and two in the second. As for propranolol, one did it in the first trimester and the other in the third. According to the FDA classification, 3 cases (16.7%) were class B medications, 12 (66.7%) class C and 3 (16.6%) class D (2 cases of enalapril, in which the use was performed in the second trimester and one of propranolol, in the third trimester). Conclusion: The use of antihypertensives belonging to class D among pregnant women in our study, suggest that these exposures may have relevance in the origin of CHD detected in patients later. The incidence and severity of clinical cases of CHD highlight the importance of prevention and education of health professionals as well as patients regarding the use of these medications in pregnancy. 111131 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CAROLINE LINK1, Ana Flavia Botelho1, Francielle Nocera Viechineski1, Bruna Kara1, Ana Carla Dlugosz1, Alice Magro Koscianski1, Camilla Mattia Calixto1, Larissa Almeida Busnello1, Paola Gonçalves Moreira de Oliveira1, Julia Henneberg Hessman1, Mário Claudio Soares Sturzeneker1 (1) Universidade Estadual de Ponta Grossa – UEPG Introduction: Heart failure (HF) remains poorly prognostic and has a high morbidity and mortality rate. The SARS-CoV-2 pandemic may have influenced these parameters mainly due to the overload of health services, although regional characteristics may have been influential. Objective: To assess the impact of the SARS-CoV-2 pandemic on HF morbidity and mortality in Northeast Brazil through the analysis of DATASUS data. Method: The average hospital stay, the number (No) of hospitalizations and deaths, and the mortality rate (calculated by dividing the number of deaths by the number of hospitalizations) were evaluated from 02/2017 to 12/2021. The respective period was subdivided into pre-pandemic (02/2017 to 02/2020), pandemic (03/2020 to 06/2021), peak (12/2020 to 05/2021), first and second trimester of the peak (12/2020 to 02/2021 and 03/2021 to 05/2021) and mass vaccination (06/2021 to 12/2021). The subdivisions of the total period evaluated, named pre-pandemic and mass vaccination, were compared with the others, and the results were expressed as a percentage. Results: Compared to the pre-pandemic period, there was a reduction of approximately 33% in the pandemic period and 15% in the mass vaccination period. Compared to the peak period, there was a 50% increase in hospitalizations during the mass vaccination period. There was a reduction in the monthly average of deaths (21–28%) in the pandemic period when compared to the pre-pandemic period, with this average being similar in the mass vaccination and pre-pandemic periods. However, there was a significant increase in the mortality rate in the pandemic period (12.45%) when compared to the pre-pandemic period (10.9%), with the highest percentage observed in the mass vaccination period (12.79%). Mean hospital stay in days was highest in the first trimester (8.5) and lowest in the second trimester of the peak (7.7) and 8.1 in the pre-pandemic period. The mortality rate was higher, and the average length of stay was lower in all periods for the female gender. Conclusions: There were relevant consequences of the SARS-CoV-2 pandemic on HF morbidity and mortality in the Northeast region of Brazil, with a lower number of hospitalizations and an increase in mortality compared to the pre-pandemic period. During the vaccination period, the increase in hospitalizations and the higher mortality rate may reflect both the difficulty in monitoring the disease in previous periods and the greater access to health services. 111146 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ARTHUR MAROT DE PAIVA1, Ana Luísa Guedes de França-e-Silva1, Beatriz Caldas Gonçalves1, Felipe Thomé Arradi1, Gustavo Elias Gomes de Oliveira1, Daniela do Carmo Rassi1, Salvador Rassi1, Aguinaldo F. Freitas Jr1 (1) Universidade Federal de Goiás – UFG Introduction: Chagas disease (CD) affects about 6 to 7 million people in Latin America with a dramatic increase in areas such as United States and Europe. Cardiomyopathy is the most frequent clinical form of chronic CD, occurring in 20% to 30% of chronically infected individuals, with heart failure (HF) being one of its main manifestations. Objective: Compare echocardiographic and clinical profile of patients diagnosed with chagasic and non-chagasic HF. Methods: Analytical cross-sectional study with outpatients treated at an University Hospital between June 2020 and February 2022. Were included patients with a previous diagnosis of HF whom underwent echocardiography in the last 5 years. For the analysis of independent samples, the student’s t test was performed (5% significance). Results: A total of 91 patients were included, of which 51 have HF because of CD (CHAGAS group) and 40 have non-chagasic HF (NCE group). Of the NCE patients, 10 were ischemic etiology, 9 hypertensive, 1 post-chemotherapy, 13 idiopathic, 1 trachycardiomyopathy, 2 peripartum, 1 myocarditis, 10 valvular and 6 alcoholic. Comparing the age of the two groups, the mean of CHAGAS was 64.90 (±10.67) and NCE (±15.05) (p = 0.04). When analyzing the heart rate, the CHAGAS group had a mean of 64.72 (±10.81) and the NCE 72.37 (±12.92) (p = 0.0028). Regarding systolic blood pressure (SBP), the mean of CHAGAS group was 104.78 mmHg (±19.99) and of NCE group was 117.60 mmHg (±30.82) (p = 0.01). About the diastolic blood pressure (DBP), the mean of CHAGAS group was 67.74 mmHg (±11.37) and of NCE group was 75.27 mmHg (±21.77) (p = 0.03). Regarding the echocardiographic measurements, the mean of ejection fraction in CHAGAS group was 31.07% (±74.16) and in NCE group was 33.64% (±101.80) (p = 0.22), the left atrium in the CHAGAS group had an average of 43.77 mm (±7.79) and the NCE group 42.00 mm (±7.28) (p = 0.27). The right ventricle had an average of 29.06 mm (±8.04) in the CHAGAS group and 25.75 mm (±6.58) in the NCE group (p = 0.04). The mean of left ventricular mass index in CHAGAS group was 134.97(±39.40) and in the NCE group was 142.91 (±39.69) (p = 0.36). Conclusion: About clinical measures, there was a significant difference in the values of heart rate, SBP and DBP between the two groups, with CHAGAS showing lower values than NCE group. Among the echocardiographic measurements, the measurement of the right ventricle was the only one that showed a significant difference, with CHAGAS group showing higher values. 111139 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MARIA EDUARDA QUIDUTE ARRAIS ROCHA1, Luís Gustavo Bastos Pinho2, Bruna Sobreira Kubrusly2, Roberto Lima Farias2, Vera Marques4, Arnóbio Dias da Ponte Filho4, Francisca Tatiana Moreira Pereira2, Maria Jaqueline Batista2, Juvêncio Santos Nobre2, Maria Camila Timbó Rocha3, Camila Pinto Cavalcante2, Eduardo Arrais Rocha2 (1) Universidade de Fortaleza (Unifor); (2) Universidade Federal do Ceará (UFC); (3) Centro Universitário Christus (UNICHRISTUS); (4) Centro de Arritmia do Ceará (CACE) Introduction: The tilt table test (TT) provides relevant information on individual susceptibility to neuro-mediated hypotension and bradycardia. Its importance has been questioned with the advent of implantable cardiac monitors. In this work, we analyzed the results and safety of the Tilt Test in the investigation of syncope, presyncope or postural dizziness in the general population. Methods: Retrospective cohort study, with analyzes of TT exams performed by 5 specialists in cardiac arrhythmias, from 2016 to 2021, in a syncope unit. Comparative analyses were performed using the Mann-Whitney tests, multiple logistic regression and ROC curves, with a p-value of 5% considered significant. The protocols used were the Westminster or Italian protocol, with a sensitization phase with 1.25 mg of sublingual isosorbide, used as assessed by the physician during the examination. Results: A total of 2462 tilt tests performed were analyzed, with 115 exclusions, 61.7% of which were female, with a median age of 51.1(31–71) years. The overall positivity rate was 33.3%, with 43.3% with pharmacological sensitization (p < 0.01). In patients with tests requested to investigate syncope, positivity was 34.2% (477) × 30.65% (285) for other symptoms, with a significant difference in relation to the overall rate (p < 0.001), while evaluating syncope and pre-syncope together, the difference was 37.55%(623) × 20.9%(139) for other symptoms (p < 0.001). Positivity rates were higher in: males (p < 0.01; OR = 1.40 (1.16–1.69)), older patients (p < 0.01; 1.01(1.009–1.02)), in the sensitized tests (p < 0.01; 2.01(1.64–2.38)), in patients with early orthostatic hypotension with symptoms (p < 0.01; 9.68 (4.13–27.44)) or without symptoms (p < 0.01; 2.93(2.07–4.18). Previous use of hypotensive drugs or the pathologies presented were not significant as predictors of response. The complication rate was 4.85%, without hospitalization or death. Conclusions: The Tilt Table Test continues to be an important and safe methodology in the investigation of patients with suspected neurally mediated syndromes in clinical practice in Brazil. 111142 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ARTHUR MAROT DE PAIVA1, Ana Luísa Guedes de França-e-Silva1, Beatriz Caldas Gonçalves1, Felipe Thomé Arradi1, Gustavo Elias Gomes de Oliveira1, Daniela do Carmo Rassi1, Salvador Rassi1, Aguinaldo F. Freitas Jr1 (1) Universidade Federal de Goiás – UFG Introduction: Chagas disease (CD) is a zoonosis whose main manifestation is heart failure (HF). One of the drugs most related to a better prognosis in HF are beta-blockers. However, the use of this class in patients with heart failure because of CD may be limited due to the patient’s heart rate. Objective: Compare the tolerability of beta-blockers in patients with chagasic and non-chagasic HF Methods: Analytical cross-sectional study with patients with HF who used beta-blockers in outpatient clinic of an University Hospital between June 2020 and February 2022. For the analysis of independent samples, the student’s t test was performed (5% significance). Results: 78 patients were included in the study, being 27 male and 51 female. The non-chagasic etiology group (NCE) had 35 patients and the chagasic etiology group (CHAGAS) 43. Of the NCE group, 8 of them were ischemic etiology, 1 valvular, 6 alcoholic, 8 hypertensive, 1 post-chemotherapy, 6 idiopathic, 2 peripartum and 1 myocarditis. In CHAGAS group, 33 were in use of metoprolol with a mean dose of 67 mg and 10 carvedilol with a mean dose of 6 mg. In the NCE group, 27 were in use of metoprolol with an average dose of 90 mg and 8 carvedilol with an average dose of 6 mg. In the CHAGAS group, 48% were in use of amiodarone, 4% calcium channel blockers, 25% angiotensin receptor blockers (ARB), 20% digoxin, 83% loop diuretic, 86% spironolactone, 32% ACE inhibitor, 11% SGLT-2 inhibitor and 23% sarcobitril-valsartan. In the NCE group, 17% were in use of amiodarone, 14% calcium channel blockers, 34% ARB, 11% digoxin, 71% loop diuretic, 82% spironolactone, 45% ACE inhibitors, 20% ISGLT-2 and 17% used sarcobitril -valsartan. When comparing heart rate, the NCE group had a mean of 72.94 (±13.46) and the CHAGAS group 64.67 (±10.59) p = 0.0033. The NCE group had a mean systolic blood pressure (SBP) of 117.8 (±32.13) and the CHAGAS group had a mean SBP of 107.18 (±19.82) p = 0.07. The mean NCE ejection fraction was 33.64% (±10.09) and the CHAGAS mean was 31.07% (±8.61) p = 0.22. Of the patients in the CHAGAS group, 6 were on a maximum dose of beta-blocker and 37 were not. In the NCE group, 9 were on the maximum dose and 26 were not, with a prevalence ratio of 0.86 (0.6–1.0). Conclusion: The CHAGAS group had a lower heart rate than NCE group. SBP of CHAGAS group showed a tendence to have lower values than NCE group. The number of patients using maximum dose of beta-blockers was lower in CHAGAS group than NCE group. 111144 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH BEATRIZ XIMENES BANDEIRA DE MORAIS1, Larissa de Oliveira Beltrão1, Luiza Dias Aguiar1, Bianca Guirra Matos de Oliveira1, Arycia Laís Nascimento Cunha1, Paloma Gomes Tavares Sette1, Michelle Lima de Carvalho Silva1, Lucas Sandes de Lima1, Renato Dias Aguiar1, Augusto França Cruz Ximenes2, Andreza Leite Marques de Sá Souza1, Alexandre Duram de Lima Júnior3 (1) Faculdade Pernambucana de Saúde (FPS); (2) Universidade Católica de Pernambuco (UNICAP); (3) Instituto de Medicina Integral Professor Fernando Figueira (IMIP) Background: Rheumatic Heart Disease (RHD) is the major cause of morbidity and mortality from severe or multiple episodes of acute rheumatic fever (ARF), a serious public health problem, especially in low and middle-income countries. These patients need continuous cardiac care and, in severe cases, heart valve surgery. During the COVID-19 pandemic, access to these services was limited, leading to adverse clinical outcomes to many patients living with RHD. Objectives: This study aims to assess the impact of the COVID-19 pandemic on Hospital Admissions (HA), In-Hospital Morbidity and Mortality Rate from RHD in Brazil. Methods: A time-series, observational study, using the public database from the Brazilian Unified Public Health System (DATASUS), to conduct a comparative analysis of HA, Morbidity and Mortality Rate from RHD, in Brazil, in 2020 and 2021, using as reference values from 2016 to 2019, and linear regression projection for 2020 and 2021. Results: Compared to 2019, HA for RHD decreased in 31.5% in 2020 and 30.65% in 2021, In-Hospital Morbidity decreased in 19.15% in 2020 and 28.24% in 2021, In-Hospital Mortality Rate was 9.21 in 2020, a 16.43% increase, and 8.05 in 2021, a 1.77% increment. The number of valvuloplasties and valve replacement surgery fell, there was a reduction of 31.66% in 2020 and 34.64% in 2021, compared to 2019, whilst their mortality rate grew from 12.02 in 2019 to 13.59 in 2020, 13.05% increase, and was at 12.26 in 2021. All the 2020–2021 rates differed from their projected trend, specially 2020 values. Conclusion: Resource prioritization and COVID-19 contention strategies, paired with patient’s reluctance to seek medical help, corroborated with precautionary postponement of elective procedures and reduction in HA and In-hospital morbidity for RHD, leading to delays in care and assistance, and a consequent increase of in-hospital mortality rates. Meanwhile, postponement of procedures implicated in the delay and decrease of valvuloplasties and heart valve replacement surgeries, leading to higher procedural mortality rates and severe consequences to the clinical outcome of RHD patients. 111161 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GEORGIA FIGUEIREDO RAMOS1, Francis Ribeiro de Souza2, Patricia O. Guimaraes3, Renato D. Lopes4, Cicero P. Albuquerque2, Alexandre Soeiro2, Renata M. do Val2, Ludhmila A. Hajjar2, Maria Janieire de Nazaré Nunes Alves2, Roberto Kalil Filho2 (1) University Santo Amaro (UNISA), São Paulo, SP, Brazil; (2) Heart Institute (InCor), Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil; (3) Hospital Israelita Albert Einstein, São Paulo, SP, Brazil; (4) Duke University Medical Center – Duke Clinical Research Institute, Duke Health, Durham, United States of America; (5) Hospital Beneficência Portuguesa Mirante, São Paulo, SP, Brazil; (6) Hospital Sírio Libanês, São Paulo, SP, Brazil Introduction: Previous studies showed that patients with prior history of cardiovascular disease present worst clinical outcomes when infected by Coronavirus Disease 2019 (COVID-19). Moreover, elevated levels of troponin and N-terminal pro B-type natriuretic peptide (NT-proBNP), are important prognostic markers associated with poor outcomes. However, whether patients with COVID-19 without prior structural heart disease may present cardiovascular alterations are poorly understood. Objectives: We aimed to evaluate if individuals with COVID-19 without prior history of structural heart disease may present cardiovascular manifestations and/or cardiac dysfunction. In addition, we also evaluated the factors that could be associated with a worst clinical outcome. Methods: This is an unicentric, retrospective, secondary analysis of the previous study (CoronaHeart study). For this analysis, 1198 patients were assessed. However, only those patients without prior history of structural heart disease who presented alterations in echocardiogram (ECHO) at admission were included. Additionally, logistic regression models to evaluate the association between clinical variables and in-hospital mortality were performed. Results: A total of 40 participants had cardiovascular manifestations and were included (mean age 60 ± 14 years; 62% male). Overall, at hospital admission, 60% were admitted to intensive care units (ICU), and needed mechanical ventilation, 75% had elevated troponin levels, and 60% had elevated D-dimer levels. In-hospital mortality was 48%. The strongest factors associated with in-hospital mortality were elevated NT-proBNP levels (hazard ratio [HR] 1.0, 95% confidence interval [CI] 1.0–1.0 p = 0.002) and length of ICU stay (HR 0.87, 95% CI 0.77–0.98 p = 0.024). Additionally, in the hospital admission ECHO, 30% had acute pulmonary hypertension in the absence of thromboembolic events. Further analyses showed a higher rate of mortality in those individuals who presented an increased Systolic Pulmonary Artery Pressure (PAPs) compared to those individuals who presented normal values of PAPs [median, 95% CI, 44 (34–51) vs 30 (24–40) mmHg, respectively, p = 0.027]. Conclusion: Patients with COVID-19 even in the absence of prior history of structural heart disease may present cardiovascular manifestations and cardiac dysfunction associated with higher rate of mortality. Additionally, the increasing PAPs was more evident in the subjects with worst outcome. 111173 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION GIULIA MICALI1, Tuany Mageste Limongi Zamperlim2, Luciana Gonzales Auad Viscardi1, Camila Martins1, Giovanna Dolder1, Pricila Helena de Souza1, Rodrigo Moreno de Oliveira1, Thamyres Oliva Bueno1, Higor Quintas Gonçalves1, Leila Dal Poggetto Moreira1, Adriana Sarmento de Oliveira1 (1) UNIVERSIDADE ANHEMBI MORUMBI -UAM; (2) UNIVERSIDADE FEDERAL DE JUIZ DE FORA-UFJF Introduction: Frailty syndrome is the progressive decline of physiological systems. One of its dysfunctions is reduction of heart rate variability (HRV), which is related to adverse cardiovascular effects, a leading cause of death in the elderly. Objective: To evaluate the impact of advanced fragility syndrome on cardiac autonomic control in the elderly. Methods: In this cross-sectional prospective study, 18 Pre-Fragile (PF) and 9 Fragile (F) patients, of both genders aged over 65 years, were evaluated at Centro Integrado de Saúde. Using Fried’s criteria, participants were grouped as F, PF or NF if presenting with 3, ≤2, or nil criteria features, respectively. Staging focused on unintentional weight loss, low level of physical activity, exhaustion, reduced walking speed, and decreased grip strength. Isometric exercise protocol was performed at 30% maximum handgrip strength with participants in supine position during a 3 minutes isometric exercise. Analyses in the time domain used heart rate indices (MNN, SDNN, RMSSD, and pNN50). Low frequency (LF: 0.04–0.15 Hz) and high frequency (HF: 0.15–0.49 Hz) indices in absolute (m2) and normalized (un) units and the LF/HF ratio representing the sympathovagal balance were used in frequency domain analyses. Student t-test and Mann Whitney U test were used to compare differences between two groups. Statistical significance was set at p < 0.05. Results: For time domain, there were no significant difference between groups for interval between heart beats (Mean RR; PF: 829.2 ± 61.4 vs F: 828.5 ± 85.7;p = 0.98), heart rate (Mean HR; PF: 72.8 ± 5.3 vs F: 73.4 ± 8.4;p = 0.83) and in parasympathetic modulation indices like SDNN [PF: 17.4 (13.1–19.5) vs F: 17.0 (10.0–20.1);p = 0.41], RMSSD [(PF: 17.9 (12.6–19.0) vs F: 15.6 (12.9–17.9);p = 0.41] and pNN50 [(PF: 1.6 (0.0–5.1) vs F: 0.4 (0.0–1.2);p = 0.28]. Conversely, for frequency domain parasympathetic modulation was lower in the F group compared to PF group [HFms; PF: 106.1 (56.8–165.7) vs F: 67.6 (19.8–85.6); p = 0.03]. There were no significant differences between groups for sympathetic modulation [LFms; PF: 144.8 (84.7–163.0) vs F: 86.9 (36.9–129.7); p = 0.12], LFun (PF: 56.2 ± 11.4 vs F: 63.5 ± 10.9; p = 0.11), HFun related to parasympathetic modulation (PF: 43.7 ± 11.5 vs. F: 36.4 ± 10.9; p = 0.11) and the sympathetic-vagal balance LF/HF ratio (PF: 1.5 ± 0.8 vs. F: 2.0 ± 0.9; p = 0.12). Conclusion: During exercise, fragile individuals show poorer parasympathetic modulation compared to pre-fragile elderly. 111182 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT VANDO DELGADO DE SOUZA SANTOS1, Elisama Quintino Sales1, Saul Rassy Carneiro1 (1) Universidade Federal do Pará – UFPA Introduction: Chronic Rheumatic Heart Disease (RHD), associated with reduced socioeconomic development in a given country, is a sequela of rheumatic fever, which is a complication of pharyngotonsillitis caused by Streptococcus pyogenes and results from a late immune response to this infection in populations genetically predisposed. Objectives: To analyze the epidemiological profile of hospital admissions and the RHD mortality rate in the northern region of Brazil from 2011 to 2021. Methods: This is a descriptive longitudinal epidemiological study based on secondary data from the Department of Informatics of the Unified Health System regarding the number of hospitalizations, gender, age group and RHD mortality rate in northern Brazil, between January from 2011 to December 2021. Results: In the analyzed period, 3,841 hospitalizations for RHD were recorded in the northern region, with the highest number of hospitalizations in 2017 (n = 468) and the lowest in 2021 (n = 246). The state of Pará had the highest rate of hospitalization for CRC (39.47%), followed by the state of Amazonas (30.82%), the state of Roraima had only 14 cases (0.36%). Females were the most affected (52.17%), the state of Amazonas was the only one with the highest rate of hospitalizations of the male population compared to females (59.97%) and the state of Tocantins was the one with the highest rate of hospitalizations females compared to males (59%), the age group most affected was 50 to 59 years (17.65%). Regarding the number of deaths, a total of 361 deaths from RHD were recorded, the year 2014 had the highest number (n = 55) and 2013 the lowest (n = 21), the state of Roraima had the highest mortality rate (21, 43%) and the state of Tocantins the lowest (9.06%). The female population had the highest mortality rates (54.02%) compared to the male population (45.98%), the most affected age group was 50 to 59 years (21.33%). Conclusion: The study showed that the states Pará and Amazonas had the highest numbers of hospitalizations and deaths in the analyzed period. The low rate of hospitalizations and mortality in the most geographically distant states was noticeable, indicating an inefficiency in access to health. The data showed a higher number of hospitalizations and deaths in women and in the population over 30 years of age. With this, the impact of RHD on the health of population and the need for prevention, diagnosis and early treatment policies can be seen. 111183 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS MARIA LUIZA DE CASTRO AMARAL1, Maria Luiza de Castro Amaral1, Manuella Bernardo Ferreira1, Maria Eduarda Valente da Luz Fontes1, Cecília Meirelles Gaspar Coelho Tomazzoni1, Larissa Rovaris de Quevedo1, Roberto Léo da Silva2, Tammuz Fattah2, Daniel Medeiros Moreira2 (1) Universidade do Sul de Santa Catarina (UNISUL); (2) Instituto de Cardiologia de Santa Catarina (ICSC) Objective: This study evaluated whether there is an association between previous smoking load and the incidence of cardiovascular events 30 days after the first myocardial infarction (AMI). Methods: This is a preliminary analysis of data from the Catarina Heart Study, a prospective cohort that assesses patients diagnosed with first-time AMI. Results: There was a significant association between smokers and non-smokers age (56 ± 9.2 vs 62.0 ± 11.5) and Body Mass Index (BMI) [26.6 (23.6–29.3) vs. 27.9 (25.0–31.1)]. There was an increase in thrombosis and reinfarction (AMI) at 30 days in smokers vs. non-smokers, with an incidence (2.5% vs. 0.2%) and (3.7% vs. 0.7%), respectively. There was an association between previous smoking load to the first AMI with reinfarction [OR 1.028 (CI 1.013–1.043)] and in-stent thrombosis [OR 1.023 (CI 1.004–1.043] at 30 days among smokers. In the smoking population, there was a significant association between previous smoking load and post-infarction ventricular function evaluated with left ventricular ejection fraction (LVEF) (r = 0.093, P = 0.047). Conclusion: Patients with previous smoking load had more re-infarction and in-stent thrombosis. Smokers had better post-infarction LVEF, fitting into the smoking paradox. 111191 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIANA GIORGI BARROSO DE CARVALHO1, Thaissa dos Santos Monteiro2, Thatyane Veloso de Paula Amaral de Almeida1, Nícolas de Albuquerque Pereira Feijóo1, Leo Rodrigo Abrahão dos Santos1, Wilma Golebiovki2, Clara Weksler2, Giovanna Ferraiuoli Barbosa2, Bruno Zappa2, Rafael Quaresma Garrido2, Marcelo Goulart Correia2, Cristiane C. Lamas3 (1) Universidade do Grande Rio – Unigranrio; (2) Instituto Nacional de Cardiologia – INC; (3) Instituto Nacional de Doenças Infecciosas Evandro Chagas – Fiocruz Introduction: Congenital heart disease is among the several risk factors for infective endocarditis (IE) and it has distinct clinical features. Objectives: Our objective was to describe cases of IE in patients with congenital heart disease (CHDIE) in a developing country and compare it with other cases of IE in a cohort presented to a referral center for cardiac surgery. Methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Statistical analysis was performed using the Jamovi and R software. Results: There were 435 episodes of IE, of which 61 (14%) were CHDIE. Of these, 21/61(34.4%) were bicuspid aortic valves. There was no difference in the proportion of gender between CHDIE and other IE. Coronary artery disease, diabetes mellitus, systemic arterial hypertension and chronic renal failure were observed less frequently in CHDIE (3.3% vs 15.8%, p. = 0.008; 4.9% vs 13.9%, p = 0.06; 27.6% vs 51.6%, p < 0.001; 6.6% vs 23.4%, p = 0.002, respectively), but previous heart failure was frequent for both groups (39.3% vs 40.1%). Patients with CHDIE less frequently had hospital-acquired IE (14.8% vs 27.6%, p = 0.033). Aortic regurgitation was more frequent in CHDIE (51.7% vs 36.7%, p = 0.027) as were aortic vegetations (62.3% vs 38.2%, p < 0.001), while mitral regurgitation was less frequent (14.8% vs 50.7% p < 0.001). No differences were observed regarding the frequency of valve perforation (18% vs 18.3%) or fistulae (4.9% vs 4%), however paravalvular abscess was more frequent (32.8% vs 20.9% p = 0.004). The frequency of fever, embolic and immunological phenomena, splenomegaly, CRP and ESR levels were not different between groups. There was no difference in the proportion of microorganisms (S.aureus, viridans group streptococci, enterococci or fungi) identified in blood cultures. The rates of surgical indication (91.7% vs 85.3%) and surgery performed (81% vs 79.6%) were similar. Mortality was lower for CHDIE (19.3% vs 26.6%) but this was not statistically significant (p = 0.238). Conclusion: In our cohort, bicuspid aortic valves were the most frequent CHD found. There were fewer comorbidities in patients with CHDIE, and a lower mortality, possibly because it presents usually in young adults. The aortic valve was more affected, corroborating the most common malformation in congenital heart diseases, which is the bicuspid aortic valve. 111203 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH DAVI FIALHO SILVA1, Davi Fialho Silva1, Fabio Leandro dos Santos Correia1, Anselmo Araújo Oliveira1, Lucas Perales Borbon1, João Gabriel Neves Lopes1 (1) Universidade do Estado da Bahia Introduction: Cardiovascular diseases (CVD) and Diabetes Mellitus (DM) are currently among the main challenges for Public Health in Brazil. Systemic Arterial Hypertension (SAH) is a multifactorial disease that affects the blood circulation, causing serious damage to target organs. DM is an endocrine-metabolic disease that mainly affects carbohydrate metabolism, causing systemic and target organ impairment. The proper diagnosis and treatment of these diseases, as well as an effective therapeutic adherence is essential for the patient’s quality of life. Failures resulting from incorrect management demand high public expenditures. To reduce the impact of these comorbidities, the HiperDia program was created through Ordinance No. 371/2002 of the Ministry of Health. Objective: To verify the effect of the HiperDia Program on mortality from SAH, DM and CVD in Bahia. Methods: Data were obtained from the Mortality Information System database (SIM-DATASUS), and deaths related to codes I10, E10, E11, E13, E14 and chapter IX of the ICD-10 were collected. Mortality coefficients and annual percentage change rates (APC) of SAH, DM and CVD were calculated in Bahia in the period, before and after the HiperDia Program. Result: There was an increase in the median mortality rate in all the variables studied after the implementation of the program (Table 1). There was a statistically significant reduction in the growth of deaths from DM and CVD (Table 2). Conclusion: The increase in the median mortality coefficients indicates how challenging it is for the Health System to properly manage these diseases. However, there was a deceleration in the growth of mortality from CVD and DM in Bahia after Hiperdia, suggesting a possible effect of the program on mortality from these diseases. 111211 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION CLEOVALDO RIBEIRO FERREIRA-JUNIOR1, Cláudia Bispo Martins-Santos1, Lara Teles Alencar Duarte1, Allexa Gabriele Teixeira Feitosa1, Edvaldo Victor Gois Oliveira1, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe (UFS) Introduction: Cardiovascular disease (CVD) in women differs from CVD in men because it has sex-specific risk factors related to reproductive health and hormonal changes. In the menopausal transition phase, estrogen levels decrease, and this is a moment of acceleration of the risk of CVD, with CVD being the main cause of morbidity and mortality in postmenopausal women. According to a meta-analysis involving studies from 24 countries on 6 continents, the global mean age of menopause is 48,78 years. Objective: To evaluate menopause as an additional factor to the classic risk factors for myocardial ischemia (MI) in women undergoing Physical Stress Echocardiography (ESE). Methods: Cross-sectional study performed between January 2000 and January 2022 with women who underwent ESE at a cardiology referral service in Sergipe. A total of 7276 women (age: 58.6 ± 10.9 years) were included, divided into two categories according to the ESE result: positive or negative for MI. Patients over 48 years of age were considered menopausal women. For analysis, binary logistic regression was used to identify whether menopause and other risk factors were independently associated with MI. To enter the model, the significance level was p < 0.10 and, to remain, p < 0.05. Finally, the chi-square test was performed to assess factors associated with menopause. Results: The frequency of MI was 19.5% with a 95% confidence interval of 18.6–20.3. The factors independently associated with MI were: menopause (OR = 1.72; 95%CI 1.42–2.08), dyslipidemia (OR: 1.90; 95%CI 1.66–2.16), diabetes (OR = 1.54; 95%CI 1.30–1.82), hypertension (OR = 1.22; 95%CI 1.07–1.39) and family history (OR = 1.58; 95%CI 1.39–1.79). The group of postmenopausal women had a higher presence of comorbidities, such as hypertension (p < 0.0001), diabetes (p < 0.0001) and dyslipidemia (p < 0.0001); however, it presented better habits regarding the practice of physical activity (p = 0.002) and a lower frequency of smoking (p = 0.007). There was no difference between women before and after menopause in the distribution of obesity and family history (p > 0.05). Conclusions: Menopause is an additional factor to consider when evaluating for MI in women. The classic risk factors dyslipidemia, diabetes, hypertension and family history were also associated with MI. 111218 Modality: E-Poster Scientific Initiation – Non-case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES MARIANA GIORGI BARROSO DE CARVALHO1, Thatyane Veloso de Paula Amaral de Almeida1, Nícolas de Albuquerque Pereira Feijóo1, Ingrid Paiva Duarte1, Leo Rodrigo Abrahão dos Santos1, Wilma Golebiovki2, Clara Weksler2, Giovanna Ferraiuoli Barbosa2, Bruno Zappa2, Rafael Quaresma Garrido2, Marcelo Goulart Correia2, Cristiane C. Lamas3 (1) Universidade do Grande Rio – Unigranrio; (2) Instituto Nacional de Cardiologia – INC; (3) Instituto Nacional de Doenças Infecciosas Evandro Chagas – Fiocruz Introduction: Early Prosthetic Valve Infective Endocarditis (EPVIE), defined as infective endocarditis (IE) occurring up to 1 year after valve replacement surgery, is a feared complication and usually involves hospital pathogens. Our aim was to describe cases of EPVIE in a developing country and compare it with other cases of IE in our cohort. Methods: Adult patients with definite IE according to the modified Duke criteria were included from 2006–2021 using the International Collaboration in Endocarditis case report form. Statistical analysis was performed using the Jamovi and R software. Results: EPVIE was responsible for 10.8% of episodes of IE (47/435). Median age of patients with EPVIE was 54 years [34.5–64.5], significantly different to other IE, where it was 47 years [33–61.3]. No differences were found regarding gender, diabetes mellitus, chronic renal failure, congenital heart disease and rheumatic valve disease (RVD). However, patients with EPVIE more frequently had coronary artery disease (25.5% vs 12.6%, p = 0.016), coronary artery bypass graft surgery (15.2% vs 4.7% p = 0.004), heart failure (58.7% vs 37.7% p = 0.006), cerebrovascular disease (12.8% vs 5.9% p = 0.077) and arterial hypertension (69.8% vs 45.5% p = 0.003). Fever, embolic and immunological phenomena, splenomegaly, CRP and ESR levels were not different between EPVIE and other cases of IE, although the presence of new murmurs was less frequent (28.3% vs 57.8%, p < 0. 01). Most found complications in EPVIE were myocardial or paravalvular abscess (32.6% vs 21.4%, p < 0.01), acute renal failure (ARF) (48.9% vs 32%, p = 0.024) and need for hemodialysis (HD) (45.8% vs 82.4%, p < 0.01). The microorganisms most frequently associated with EPVIE were enterococci (18.8% vs 9.8%, p = 0.06) and coagulase-negative staphylococci, but not S.aureus (4.1 vs 11.7%). Surgical indication rate was 58.7% (vs 89.4%, p < .001) and surgery (56.4% vs 80.7%, p < .001) in patients with EPVIE were lower when compared to other cases of IE, while the mortality rate (31.1% vs 25%) was higher, but not statistically different between groups. Conclusions: Myocardial abscess, which is an absolute surgical indication, occurred in almost 1/3 of EPVIE. Possibly because of poor clinical status and frequent comorbidities, patients with EPVIE had less surgical indication and less surgery than the others in the cohort, evolving with high mortality. 111227 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION CLEOVALDO RIBEIRO FERREIRA-JUNIOR1, Lara Teles Alencar Duarte1, Allexa Gabriele Teixeira Feitosa1, Edvaldo Victor Gois Oliveira1, Cláudia Bispo Martins-Santos1, Enaldo Vieira de Melo1, Antônio Carlos Sobral Sousa1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe (UFS) Introduction: Cardiovascular disease (CVD) has become increasingly prevalent in women, although it is underestimated when compared to other diseases specific to women. It differs from CVD in men in terms of pathophysiology, with traditional risk factors predicting higher or lower risk of CVD in women than in men. Objective: To evaluate the clinical factors associated with Chronotropic Incompetence (CI) in women undergoing Exercise Stress Echocardiography (ESE). Methods: Cross-sectional study between January 2000 and January 2022 with women who underwent ESE at a cardiology referral service in Sergipe. A total of 7049 women (age: 58.6 ± 10.9 years) were included, divided into two categories according to the chronotropic index: chronotropic competent and incompetent. Patients with a chronotropic index less than 0.8 were considered chronotropic incompetent. For analysis, binary logistic regression was used to identify factors that were independently associated with CI. Results: We compared 6430 (91.2%) competent women and 619 (8.8%) incompetent women. Dyslipidemia, diabetes, family history of CVD, obesity and beta-blocker use (withdrawn 3 days before the exam) were not independently associated with CI (p > 0.05). Factors independently associated with CI were: sedentary lifestyle (OR = 2.49; 95%CI 1.93–3.21), smoking (OR: 2.84; 95%CI 1.60–5.05), former smoking (OR = 1.90; 95%CI 1.29–2.79), age 60 years or older (OR = 1.55; 95%CI 1.24–1.95), hypertension (OR = 1,39; 95%CI 1.09–1.78) and previous presence of symptoms such as chest pain – typical or atypical – or dyspnea in the clinical history (OR = 1.45; 95%CI 1.14–1.85). Conclusions: Bad habits such as smoking and physical inactivity had the highest odds ratios for CI. Patients who did not quit smoking were more likely to have CI than former smokers. Beta-blocker discontinued 3 days before the exam was not independently associated with CI. 111658 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH BEATRIZ CUNHA LISBOA DE MEDEIROS NUNES1, Luísa Silva de Azevedo2, Júlia Freitas Carneiro2, Kimberlly Aparecida de Sousa Ferreira2 (1) Universidade Potiguar – UNP; (2) Universidade Do Estado Do Rio Grande Do Norte – UERN Introduction: Chronic non-communicable diseases (CNCD) have gained space in the epidemiological and social context, constituting a global problem responsible for 74% of annual deaths. In Brazil, which follows this pattern, it is still possible to single out ischemic heart diseases (IHD) as the main cause of death in all states. To endure this reality, the Strategic Action Plan for Combating CNCD was established which was not able to achieve its goals. Objective: To analyze the number of deaths due to IHD in Brazil, from 2010 to 2020, and correlate it with the goals of the Strategic Action Plan to Combat CNCDs in force. Methods: Based on a time-series study on the number of deaths from IHD that occurred in Brazilian macro-regions, from 2010 to 2020. Data were taken from the Mortality Information System of the Informatics Department of the Unified Health System. To obtain the approach, the International Classification of Diseases was used, specifically the codes I20 to I25. Results: From 2010 to 2020, 1,208,022 total deaths from IHD were registered in Brazil, with a percentage increase of approximately 10% of the cases recorded in the studied period. Furthermore, there is an increase in the number of deaths recorded by four out of the five regions in the country, with emphasis on the North, which shows an increase of 56.13% in its percentage of deaths. In 2011, the Health Ministry of Brazil launched the Strategic Action Plan to Combat CNCD as a way to contain the rising trend predicted in 2010 for the following decades. In the opposite direction of the efforts, it can be observed, by the exposed data, that the purpose is not close to being achieved. On the contrary, the numbers in most Brazilian regions are in full advance, as can be seen in the figures in Table 1. Conclusions: IHDs still represent an important obstacle to public health in Brazil. The disparities in rates among the states reveal the need for actions consistent with the reality and local singularities. Not reaching the goals established in the plan has undeniable impacts on the health of the Brazilian population. 111253 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY PAOLA BITAR DE MESQUITA ABINADER1, Ana Flávia Oliveira de Souza2, Ana Josefina Gonçalves Salomão3, Camila Rodrigues Maciel4, Cecília Gomes da Silva5, Daniel Oliveira da Costa6, Davi Gabriel Barbosa7, Gabriel de Siqueira Mendes Lauria8, Juliana Ayumi Azevedo Kurosawa9, Lucas Guimarães Junqueira10, Rafaela Oliveira Cardoso11, Luis Eduardo Werneck de Carvalho12 (1) Centro Universitário do Estado do Pará (CESUPA); (2) Centro Universitário do Estado do Pará (CESUPA); (3) Centro Universitário do Estado do Pará (CESUPA); (4) Centro Universitário do Estado do Pará (CESUPA); (5) Universidade Federal do Pará (UFPA); (6) Universidade Estadual do Pará (UEPA); (7) Universidade Estadual do Pará (UEPA); (8) Centro Universitário Metropolitano da Amazônia (UNIFAMAZ); (9) Centro Universitário do Estado do Pará (CESUPA); (10) Universidade Estadual do Pará (UEPA); (11) Centro Universitário do Estado do Pará (CESUPA); (12) Clínica Oncológica do Brasil Introduction: The cardiac tumors can begging in the cardiac tissues of the internal coating, muscular layer and pericardium. Such neoplasms can show secondary symptoms and pathologies, such as: obstructions to the blood flow, dyspnea, chest pains or syncope, embolizations and arrhythmias. These clinical pictures usually show up late, and are non- specific to characterize these tumors, therefore hampering the clinical diagnosis. However, the image examinations crucial in this diagnosis are: transesophageal echocardiography, thorax tomography, magnetic resonance of the heart and even the fetal echocardiography, which allows for a diagnosis in the intrauterine phase. Therefore, the diagnosis methods in conjunction with the clinical interfere directly on the early choice of treatment and prognosis of the patients, showing the importance of the epidemiology on this pathology. Objectives: Characterize the epidemiology of the diagnosis of heart neoplasia, mediastinum and pleura in Brazil between the years of 2013 and 2020. Method: It’s and epidemiological study about malignant neoplasias of the heart, mediastinum and pleura in Brazil between the years of 2013 and 2020. The data was collected on the database of the Departamento de Informática do Sistema Único de Saúde (DATASUS). The variables analyzed here were the year and number of notifications, gender and age group. Results: In total, 3954 cases of malignant neoplasias of the heart, mediastinum and pleura were registered between the years of 2013 and 2020, with a greater incidence on males (50,9%) and a lesser in females (49,01%). Regarding the age groups, in males there was a greater prevalence of cases between 60–64 years (48,3%), while in females, the cases concentrated on the age group of 55–59 years (56%). Besides that, it was observed that 2019 was the year with the biggest number of cases, counting 1420 cases (35,9%), distributed between males and females. Conclusion: Based on the results obtained, malignant neoplasias of the heart, mediastinum and pleura affect, most of all, elderly men, with the biggest number of cases being observed in 2019. Taking on account the noted epidemiology and the difficult of identifying the risk factors and symptomatology of this type of cancer, this work puts emphasis on the need of more studies on the topic aiming to diagnose and treat the afflicted individuals early. Besides that, is primordial that the population achieves a greater access to the means of diagnosis. 111564 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LUCAS MÜLLER PRADO1, Jessica Tamires Reichert1, Gustavo Sarot Pereira1, Jorge Tadashi Daikubara1, Michelle Bozko Collini1, Rafael Moretti1, Matheus Bissa Duarte Ferreira1, Eduardo Leal Adam2, Miguel Morita Fernandes da Silva2 (1) Universidade Federal do Paraná; (2) Complexo Hospital de Clínicas, Universidade Federal do Paraná. Background: In patients with acute ST elevation myocardial infarction (STEMI), primary percutaneous coronary intervention (PCI) is the first-choice reperfusion therapy, but fibrinolysis is recommended in non-PCI centers, when the expected time-to-PCI is expected to be more than 120 minutes (min). Objectives: To assess the time from chest pain (CP) to PCI (CP-to-PCI time) and the reasons for delay in patients with STEMI undergoing PCI in a tertiary hospital from the public health system in Brazil. Methods: We prospectively included patients admitted for STEMI in a Tertiary Hospital in Curitiba, Brazil, from Oct/2019 to Dec/2021. CP onset was the beginning of the last symptom that motivated the patient seeking care. For patients first admitted in non-PCI centers, we collected the moment the patient arrived in the emergency care unit (ECU) before being referred to the tertiary hospital. Results: We included 71 patients (61 ± 13 years old, 31% women), with 46 (65%) referred from non-PCI centers. Overall, the CP-to-PCI time was 378 [272.0, 628.0] min, and there was no significant difference between patients referred from non-PCI centers and those directly admitted in the tertiary hospital (p = 0.54). CP-to-ECU time was shorter in patients in non-PCI centers than those directly admitted in the tertiary hospital (104.5 [47.0, 206.5] vs 170.0 [123.0, 339.0] min, p = 0.012), but this was offset by a delay between the non-PCI center ECU and the tertiary hospital (172 [116.0, 335.0] min), with 74% of patients taking longer than 120 min (Figure 1). Conclusion: We found important delays in primary PCI in patients with STEMI treated in the public health system in a city of Brazil, with time of referral from non-PCI centers to the tertiary hospital being largely responsible for these delays. 111957 Modality: E-Poster Scientific Initiation – Non-case Report Category: DYSLIPIDEMIA GABRIEL IRISMAR RODRIGUES SCHWAMBACK1, RAIMUNDO BENÍCIO DE VASCONCELOS NETO1, REBECCA SHAIANE SOARES NUNES RIVOREDO1, BRENDA DOS SANTOS RODRIGUES1, SOFIA DOS SANTOS SOUZA1, JADE GOMES DA COSTA MEDEIROS1, LIANA MIRANDA PEREIRA1, ANA JÚLIA OMODEI RODRIGUES MARTIM1, ANTONIETA RELVAS PEREIRA2, JULIANA DE SOUZA ALMEIDA ARANHA CAMARGO1, SERGIO DE ALMEIDA BASANO1, LUIS MARCELO ARANHA CAMARGO1 (1) Centro Universitário São Lucas – UNISL; (2) Centro Universitário Aparício Carvalho – FIMCA Introduction: Measuring the prevalence of dyslipidemia and its risk factors is necessary to enable the design of interventions and management of population health. However, it is observed that these data are scarce in the Western Amazon, especially regarding the monitoring of children living in riverside communities. Objectives: To estimate the prevalence of dyslipidemia and associated risk factors in children and adolescents residing in riverside communities in the Western Amazon, Brazil. Methods: The present cross-sectional study was carried out with individuals aged between 6 and 16 years, living in the riverside region of the Madeira River, in Humaitá (S 6o 58’62” and W 62o 50” 08W), Amazonas State, Brazil. The project has been approved by the research committee of the Brazilian Research Center for Tropical Medicine in Rondônia (CEPEM). For the research, a clinical-epidemiological questionnaire was applied, as well as 10 mL of blood samples were collected and analyzed, as established in the parameters of the V Brazilian Directive on Dyslipidemia and Prevention of Atherosclerosis. The analysis was performed by applying the prevalence ratio (PR), Mantel-Haenszel chi-square test, with statistical significance p < 5%. Results: It was possible to analyze the data of 131 (82%), among 160 children and adolescents, being 63 females and 68 males. Eleven blood samples were found with serum values consistent with dyslipidemia, representing an absolute prevalence of 8.4%. The mean age of the sample was 11 years, ranging from 6 to 16 years. The average family income reported was calculated at R$ 809.16. For risk factors, the PR between dyslipidemia and female sex was equal to 2.0 (p = 14%); for a family history of dyslipidemia, the PR was 1.66 (p = 21%); for obesity the PR was 1.9 (p = 28%); and for sedentary lifestyle and systemic arterial hypertension, PR values were lower than 1, with p > 5%. Conclusion: Dyslipidemia is poorly studied disease in remote communities, especially when the target audience is children and adolescents. The data point to a prevalence of dyslipidemia of almost 10%, considered high for children and requiring intervention by the health system to mitigate the occurrence of chronic non-communicable diseases in adulthood. In this study, no proven risk factors associated with dyslipidemia were found. 111633 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY IDRYS HENRIQUE LEITE GUEDES1, Kleber Oliveira de Souza1 (1) Universidade Federal de Campina Grande Introduction: In the global pandemic scenario and crisis in the Brazilian health system, the restriction of medical care for diseases other than COVID-19 infection triggered vast effects in the treatment of various pathologies. Therefore, it is important to analyze the consequences of this situation specifically on the treatment and management of patients with conduction disorders and cardiac arrhythmias, given the complexity of these conditions. Objectives: Collect and analyze hospitalization, hospital stay, and death data for conduction disorders and cardiac arrhythmias in Brazil. Methodology: Cross-sectional quantitative study on the treatment of conduction disorders and cardiac arrhythmias in Brazil from January 2018 to December 2021, based on data obtained from the SUS Hospital Information System (SIH/SUS), through the Ministry of Health database (DATASUS), and analyzed using IBM SPSS statistical software. The following variables were included: year of care, hospitalizations, the average length of stay, and the number of deaths. Results: There were 64790 and 69673 hospitalizations due to conduction disorders and cardiac arrhythmias in Brazil in 2018 and 2019, respectively, while in 2020 and 2021 there were 60362 and 59834 hospitalizations, resulting in a 14% difference between the highest and lowest values and a P-value <0,001. The average hospital stay in the pre-pandemic biennium increased from 4.75 days to 4.3 days (P-value of 0.978), while the number of deaths increased from 7153 and 8500 to 8576 and 8674, with a respective P-value < 0,001. Conclusion: According to the analysis, there was an indication of a significant reduction in the number of hospitalizations, a likely result of the low demand for medical care in the pandemic period. It is still possible to observe a lower number of days of hospital stay in the post-pandemic period, probably contributing to the growth in the number of deaths observed, which is also favored by the greater severity in the evolution of patients who delayed medical follow-up for too long. 111513 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION MARIA RAFAELA ALVES NASCIMENTO1, Yure Batista Sousa1, Letícia Rego Borborema1, Victoria Liery Ribeiro Alves1, Lara Carneiro Magalhães1, Livia Caroline Bemquerer Veloso1, Karolina Campos Sampaio Lopes1, Fernando Guimarães Fonseca1, Lanuza Borges Oliveira1, Maria das Mercês Borem Correa Machado1 (1) Centro Universitário FIPMOC – Afya Introduction: Systemic arterial hypertension (SAH) is a considerable risk factor for cardiovascular diseases. Resistant hypertension (RH) diagnosis can be reached through the management of elevated levels of blood pressure (BP), despite the use of three or more antihypertensive medications of different classes, including diuretic ones. The sympathetic nervous system (SNS) plays an essential role in elevating BP through peripheral and central mechanisms which have an impact on the kidneys, the heart and blood vessels. The use of invasive procedures for RH treatment has recently been proven to be effective. Renal denervation modifies the sympathetic interactions between the kidneys and the central nervous system through a percutaneous procedure by connecting a catheter to a radiofrequency device. Objective: To observe the impact of renal sympathetic denervation to the treatment of resistant hypertension. Method: Systematic review has been used, with articles found in the SciELO database as well as in the Biblioteca virtual em saúde, LILACS, and Medline databases. The keywords “renal denervation”, “resistant hypertension”, and “sympathetic nervous system” have been used in research, with the use of PRISMA methodology. Nineteen papers published in the last ten years have been selected; ten of which published in Brazil and nine others published internationally. Results: Based on the positive results of the pilot study, a new study has been encouraged: the Symplicity HTN-2. In this trial, 106 RH patients were randomly selected for renal sympathetic denervation (RSD) (n = 52, initial average BP of 178/96 mmHg) or underwent previous treatment (n = 54, initial average BP of 178/97 mmHg). After six months of observation, it was possible to determine a decrease of systolic blood pressure higher than 10 in 84% of patients subjected to RSD. In the control group, only 35% of patients showed positive results. Finally, 20% of patients from the intervention group showed decrease in the amount of medication used, as compared to the control group, which showed only 6% of patients. Conclusion: With the data collected from the study, it is possible to conclude that RSD is an efficient procedure in light of RH diagnosis, which showed no relevant complications or adverse effects and has proven to provide significant increase in the quality of life of patients who have been subjected to such treatment. 111479 Modality: E-Poster Scientific Initiation – Non-case Report Category: SPIRITUALITY AND CARDIOVASCULAR MEDICINE BRUNA SOUZA MATOS DE OLIVEIRA1, Adelle Cristine Lima Cardozo1, José Icaro Nunes Cruz1, Camille Marques Aquino1, Jade Soares Dória1, Giulia Vieira Santos1, Juliana Maria Chianca Lira1, Gabriela de Oliveira Salazar1, Philipi Santos Soares1, Antônio Carlos Sobral Sousa2, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira2 (1) Federal University of Sergipe; (2) Rede D’Or São Luiz – São Lucas Hospital; (3) Primavera Hospital Introduction: Several studies analyze the influence of spirituality and religiosity (S/R) on the lifestyle of individuals. It is known that sedentarism, smoking and high alcohol consumption are risk factors for cardiovascular disease, the leading cause of global morbidity and mortality. Objectives: To analyze the relationship between S/R levels with the variables sedentarism, smoking and alcohol consumption. Methods: Observational, cross-sectional, analytical study. The sample included patients from cardiology outpatient clinics of three hospitals in Sergipe. Three natures of religiosity were assessed according to Duke Religiosity Index (DUREL): organizational (OR), non-organizational (NOR) and intrinsic (IR). The cut-off point for defining low or high level of OR and NOR was 4 (<4: low; ≥4: high), while the cut-off point for low or high level of IR was 13 (<13: low; ≥13: high). The variables alcohol consumption, smoking, and sedentary lifestyle were assessed by applying a self-questionnaire and their prevalence was compared between the OR, NOR and IR groups using Fisher’s exact test, with the significance level set at 0.05. Results: 130 patients were included in the study, of which 62.3% were female. The mean age was 60.6 ± 11.2 years. In this sample, 73.8% were hypertensive, 59.2% were dyslipidemic, 40.8% had chronic coronary syndrome, and 35.4% had diabetes mellitus. Patients with low level of NOR had higher prevalence of alcohol consumption when compared to high level patients (71.4% vs. 28.9%; p < 0.05). Individuals with low level of IR showed higher prevalence of alcohol consumption compared to individuals with high level (61.9% vs. 25.0%; p < 0.05). There was no difference between patients with low or high level of OR for the prevalence of alcohol consumption, smoking, and sedentary lifestyle (p > 0.05). There was no difference between patients with low or high level of religiosity for the prevalence of smoking and sedentary lifestyle (p > 0.05). Conclusions: Patients with high levels of NOR and IR had lower prevalence of alcohol consumption compared to patients with low levels. The prevalences of the risk factors evaluated were not different between patients with high and low OR. There was no difference between patients with low or high levels of religiousness for the prevalence of smoking and sedentary lifestyle. Future studies with larger numbers of participants are needed to better understand this association. 111580 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH RENATTA KAROLINE BEKMAN VOGAS1, Jonatas Benarroz da Silva1, Davi da Silveira Barroso Alves1, Paulo Henrique Godoy1 (1) Federal University of the State of Rio de Janeiro – UNIRIO Introduction: Hypertensive diseases (HD) have a high prevalence and are a risk factor for cerebrovascular and coronary diseases. They are one of the main causes of death in Brazil, however, its participation as the underlying cause of death (UCD) may be underestimated due to the selection rules of the International Classification of Diseases (ICD-10). Objective: To analyze the trend in mortality by underlying cause and by mentioned cause due to hypertensive disease, according to age and sex, in Brazil, from 2000 to 2019. Methods: Ecological study with a description of a historical series on deaths due to HD, reported as UCD or mentioned cause (MC) in lines A to D, recorded in Mortality Information System databases, in Brazil, from 2000 to 2019, based on ICD-10 codes for HD (I10–I15). Crude and standardized mortality rates were estimated for UCD and MC, according to sex and age groups. Results: There were 814,722 deaths whose UCD was HD and 3,714,982 deaths recorded as MC. The number of deaths was 4.5 times higher in the case of MC than when considered the UCD. Mortality rates as MC were significantly higher than when considered as UCD and there was a proportional increase according to age groups, especially in males, aged 30–59 years. Trends observed in the 0–29 age group were the ones that most differed, for the sexes, according to UCD and MC. Conclusions: It seems reasonable to use mentioned cause of death as a method to analyze mortality due to HD and minimize possible underestimations due to UCD selection rules, especially when considering younger groups. 111358 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MAYARA GABRIELE TOLEDO1, Mayara Gabriele Toledo1, Caio Pluvier Duarte Costa1, Eduardo Thadeu de Oliveira Correia1 (1) Universidade Federal Fluminense Introduction: One of the key therapeutic aims of the outpatient management of heart failure (HF) is to treat peripheral and pulmonary edema. However, although clinical signs can identify patients with congestion, it may still be present subclinically, which is an indicator of a worse prognosis. Therefore, this study aims to systematically evaluate the use of internal jugular vein (IJV) ultrasound to detect congested HF patients seen in outpatient clinics. Methods: We conducted a systematic review following the PRISMA guidelines. Original studies indexed by Embase, Pubmed, Cochrane and LiLACS with the keywords: “ultrasound” or “ultrasonography” and “heart failure” and “jugular vein” published until March, 2022 that matched the inclusion criteria were included. Two authors (C.P and M.T.) performed the screening and data extraction, in cases of discordance, a third author (E.T.) made the decision. Results: Six studies were included with a total of 1236 patients analyzed, excluding control patients. Due to the significant heterogeneity between studies, a meta-analysis was not conducted. For the ultrasonic assessment of volume status, 4 studies used the variation of the IJV diameter at rest and during the Valsalva maneuver, 1 used the variation of cross-sectional area (CSA) at rest and after the Valsalva maneuver before and after the use of diuretics, and 1 study compared the measurement of right atrial pressure (RAP) estimated by IJV ultrasound with the value obtained during right heart catheterization. One of the studies that analyzed the variation of IJV diameter at rest and during the Valsalva maneuver showed that jugular vein distention (JVD) is a valid option to assess fluid status of patients and provides diagnostic and prognostic information similar to natriuretic peptides. Pellicori et al, in their 2 studies showed that resting JVD was lower in controls (0.16 and 0.17 cm) than in HF patients (both 0.23 cm) and the JVD ratio was higher in controls (both 6.3) than in HF patients (4.5 and 4.4). In one of his studies, he showed that JVD was similar during Valsalva (1.03 cm vs 1.08 cm) in controls and HF. Conclusion: This systematic review shows that IJV ultrasound is useful for examining intravascular volume status and identifying congestion. Future clinical trials evaluating whether IJV ultrasound could be used to guide diuretic therapy are needed. Finally, further studies are needed to define a standard IJV ultrasound technique to examine volume statu. 111754 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY REBECCA SHAIANE SOARES NUNES RIVOREDO 1, Rebecca Shaiane Soares Nunes Rivoredo1, Raimundo Benício de Vasconcelos Neto1, Gabriel Irismar Rodrigues Schwamback1, Paulo Roberto de Oliveira Molina4, Isabel Bussinguer Gomes1, Ingrid da Silva Morais Freitas4, Francisco Siosney Almeida Pinto2 (1) Centro Universitário São Lucas/Afya UNISL; (2) Instituto Cardiovascular- INCARDIO; (3) Centro Universitário Aparício Carvalho Introduction: Transcatheter aortic valve implantation (TAVI) is a minimally invasive procedure that, since its debut in 2002, evolved to become the standard care for patients with aortic stenosis (AS) at intermediate and severe risk to surgery once conventional surgery as the femoral approach can be safely implemented. Over the years, there has been a significant improvement in the development of transcatheter heart valves (THV), in addition to the refinement of the skills involved in the implant process, which made it possible to increase the spectrum of patients who benefit from TAVI. Currently, studies are investigating the role of TAVI in patients at low surgical risk, new indications and new alternatives for perioperative management. Objectives: This is a descriptive study of TAVI cases performed in a high complexity hospital located in the Western Amazon. Methods: The data presented were obtained through research via medical records, complementary exams and databases such as PubMed and Google Academy. Results: In the period between September 2019 and November 2021, 11 TAVIs were performed by the cardiology team of a high complexity hospital in the Western Amazon. Patients undergoing TAVI are 5 women and 6 men, with an average age of 65.8 years, of which only one underwent the procedure on an emergency basis. The main symptom reported by patients before implantation was dyspnea on minor exertion and at rest, this symptomatology is justified by the diagnosis of severe aortic stenosis associated with heart failure NYHA > 3. The implants were performed using the Inovare® expandable balloon prostheses, introduced by catheter via the femoral artery with a unanimous duration of approximately 2 hours per procedure. A total of 7 patients evolved with a good prognosis without aortic regurgitation and after 24 hours of hospitalization in the intensive care unit, they were discharged from the hospital six days after the procedure and consequent progressive decrease in associated symptoms. From the sample space, 4 patients died, due to non-surgical causes. Conclusion: The data presented show the effectiveness of the procedure in patients of high and medium complexity. Therefore, the preparation and efficiency of the cardiology team responsible for managing cases and modifying the prognosis of patients who benefit from TAVI in this region of the country is of remarkable importance. 111405 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FRANCIELE ROSA DA SILVA1, Lucio Padilla2, Antonio Carlos Botelho3, João Eduardo Tinoco de Paula4, Carlos Augusto Homem de Magalhães Campos,5, Marco Alcantara6, Ricardo Santiago7, Franklin Leonardo Hanna Quesada8, Félix Damas de Los Santos9, Marcia Moura1, Pedro Piccaro de Oliveira1, Alexandre Schaan de Quadros1 (1) Instituto de Cardiologia do Rio Grande do Sul (ICFUC); (2) Instituto Cardiovascular de Buenos Aires; (3) Hospital São Jose do Avai; (4) Instituto Cardiovascular de Linhares (UNICOR); (5) InCor; (6) Centro Médico 20 de Noviembre ISSSTE; (7) Hospital Pavia Santurce; (8) Clinica Comfamiliar; (9) Instituto Nacional de Cardiologia Ignacio Chavez Background: Chronic total occlusion (CTO) percutaneous coronary interventions (PCI) has been increasingly performed worldwide, but there is few information in Latin America. Objectives: To assess contemporary CTO PCI in the daily practice of Latin America. Methods: Centers volunteered to participate, and no minimal number of procedures was required. The databank was managed in a REDCap platform, and clinical, procedural and outcome data were evaluated. Predictors of unsuccessful procedures were assessed by multivariable analysis. Results: Data from 2.968 CTO PCIs performed in 63 hospitals from Argentina, Brazil, Bolivia, Chile, Colombia, Costa Rica, Ecuador, Mexico and Puerto Rico was analyzed. Most patients were male (78%) with a mean age of 64.0 ± 10.7 years. CTO PCI was performed mainly for angina control (83%) and/or to treat significant ischemia (30%). Radial access, dual injections and a microcatheter were used in most cases. The overall success rate of CTO PCI was 85%. The successful strategy was antegrade wire escalation in 80%, retrograde approach in 10% and antegrade dissection reentry in 10%. Proximal cap ambiguity, blunt stump, calcification, and previous attempt were predictors of unsuccessful procedures. In-hospital major adverse cardiovascular events occurred in 1.6% of the cases, and at the one-year follow-up 82% of the patients remained free of angina. Conclusions: CTO PCI in Latin America has achieved a high rate of angiographic and clinical success with low complication rates in centers dedicated to these procedures. 111406 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MATHEUS RICARDO MALVEIRA CAMACHO1, Amanda Gabriela Freitas Rodrigues2, Wellen Sampaio Ferreira1, Izaura Maria Vieira Cayres Vallinoto2 (1) Universidade do Estado do Pará; (2) Universidade Federal do Pará Introduction: The association between diabetes and hypertension is a public health problem with great socioeconomic impact, being responsible for great morbidity and mortality – generating high rates of hospitalization and burden on the Brazilian Unified Health System. Among the most common late complications of hypertensive and diabetic patients is amputation, a condition that requires a considerable amount of resources in its treatment and is associated with a significant reduction in the quality of life of patients. Added to this, recent studies demonstrate that the association between smoking, diabetes and hypertension may be responsible for early death due to macrovascular complications. Objectives: To analyze the influence of smoking on the occurrence of amputations among the population that has both hypertension and diabetes. Methods: A quantitative research was carried out in a secondary database using the digital platform of the Department of Informatics of the Unified Health System (DATASUS). Data referring to hypertension associated with diabetes between the years 2003 and 2013 in the geographic scope of the state of Pará were selected, analyzing the following variables: smoking and amputation. The data were worked with the help of tabulators for the Internet and for Windows (TABNET and TABWIN). The data found were analyzed by Pearson’s chi-square statistical test using the Biostat software. Results: During the study period, 56,974 cases of hypertension concurrent with diabetes were reported in the state of Pará. Among the registered cases, 1632 (2.86%) were smokers, while 55,342 (97.14%) were non-smokers. Among the total of smokers, 578 progressed to amputation, making 35.42% of the participants in this group. On the other hand, among non-smokers, amputation occurred in 12,049 patients, a figure that represents 27.83% of this group. When calculating the p-value of the data, a value <0.0001 was obtained; therefore, there is a statistically significant difference in the occurrence of amputations between smokers and non-smokers. Conclusions: There is an important relationship between the occurrence of amputations in patients with hypertension concomitant to diabetes and smoking in Pará. This data is in agreement with the current literature and signals the need for public policies related to the prevention of hypertension and diabetes, as well as encouraging smoking cessation in the state. 111407 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY KARLA SANTOS PINTO1, Luiz Carlos Passos1, Rodrigo Morel1, Diogo Azevedo1, Jackson Brandão1, Clara Salles1, Mariana Baraúna da Silva1, João Pedro Martins Moreira Granja1, Beatriz Barbosa Viana1, Elias Soares Roseira1, Livia Maria Goes Lemos1, Raissa Barreto Lima1 (1) Hospital Ana Nery Introduction: The surgical technique with Extracorporeal Circulation (ECC) is used as the technique of choice in most cardiac surgeries, whether myocardial revascularization or valvular revascularization. However, CEC acts as an aggressor agent due to the diversion of blood to an artificial circuit. This tool puts the patient at a greater risk of developing coagulopathies, infection and vasoplegia, due to the inflammatory response. Therefore, new technologies are being developed to minimize these risks, such as ECC with endothelial biological material coating. Goal: To assess the impact on mortality and in-hospital outcomes of the use of a covered circuit for cardiopulmonary bypass Methods: This is a prospective cohort with all patients undergoing cardiac surgery using ECC from March 2021 to February 2022, in a referral hospital in Salvador, Bahia. For analysis, they were divided into group 1 (use of the coated circuit) and group 2 (use of the standard circuit); there was no prerequisite for the use of the coated circuit, it was defined in a semi-random way in the distribution of operating rooms. Surgical mortality, length of stay in the intensive care unit (ICU), length of mechanical ventilation (MV) and time of vasoactive drug use (VAD) were evaluated. Statistical significance was considered for p < 0.05. Results: A total of 292 patients were included, 104 (40.5%) using an ECC coated circuit. Of the patients, 128 (43.8%) were female and 152 (52.0%) of the surgeries involved valve approach and 140 (48.0%) underwent myocardial revascularization. The Euroscore II mean was 1.77% (±1.5). There was no difference in mortality, in the duration of mechanical ventilation: 7 (4–12) versus 5 (3.8–9); p = 0.058, DVA use: 23 (0–46.5) versus 24 (2.8–48); p = 0.950 or length of ICU stay: 3 (2–4) versus 3 (2–3); p = 0.057, respectively, for groups 1 and 2. Conclusion: The use of a coated cardiopulmonary bypass circuit did not reduce mortality, duration of vasoactive drug use, length of stay in the ICU or time of use of mechanical ventilation. 111821 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH SARA CRISTINE MARQUES DOS SANTOS1, Maria Luiza Silva Barbosa1, Alice Machado de Sales Silva1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras UV Introduction: Cor triatriatum, or triatriated heart, is a rare congenital anomaly, representing 0.1% to 0.4% of congenital heart diseases¹. Embryologically, it occurs when the pulmonary vein leaves a remnant in the left atrium, dividing it into three chambers. Physiologically, there is a similarity to mitral stenosis and other obstructive pathologies of the right ventricle2. This study aims to analyze the current panorama of Cor Triatriatum correction procedures performed in Brazil for 10 years and to correlate the current epidemiology with the results obtained. Methods: A systematic literature review and an observational, descriptive, and cross-sectional collection of triatriatum color correction data, available at DATASUS – SUS Hospital Information System (SIH/SUS) for a period of ten years – December 2008 to December 2018. Results: In the analyzed period, 61 hospitalizations were observed for the performance of color correction triatriatum procedures. The total expense was R $ 937,420.58, with the year 2017 being responsible for the highest cost: R $ 179,379.02. The 61 procedures were considered highly complex, of which 31 were performed on an elective basis and 30 were urgent. The total mortality rate in the 10 years studied was 12.90, corresponding to 8 deaths, a mortality rate of 50 was identified in the years 2010 and 2018, representing the highest, while the years 2009 and 2017 had the lowest rate, 11,11. The average total hospital stay was 14.6 days. The Brazilian region with the highest number of hospitalizations was the Southeast with 17 hospitalizations, followed by the Northeast region with 15, South, and Midwest with 12 and, finally, the North region with 5 hospitalizations. Among the units of the federation, the states of São Paulo and Minas Gerais concentrated the majority of hospitalizations, accounting for 8 each. The North region had the highest mortality rate (20.0), followed by the South region (16.67). The Midwest region had the lowest rate, with a value of 7.69. Conclusions: From the present study, it can be observed that the North region, despite having the lowest number of hospitalizations, has the highest mortality rate when compared to other regions. It is worth noting that it is a rare congenital malformation and therefore, little discussed. Also, highlight the need for the correct notification of procedures, due to the absence of certain information, to improve the current epidemiological analysis. 111418 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY KARLA SANTOS PINTO1, Elias Soares Roseira1, João Pedro Martins Moreira Granja1, Mariana Baraúna da Silva1, Beatriz Barbosa Viana1, Taina Viana1, Rodrigo Morel1, Ana Karenina1, Luiz Carlos Passos1, Clara Salles1, Livia Maria Goes Lemos1, Raissa Barreto Lima1 (1) Hospital Ana Nery Introduction: Pulmonary hypertension (PH) is one of the factors associated with high morbidity and mortality, especially in valve surgeries. It is generally associated with more severe valve disease or with an evolved natural history. Objective: To describe the association between PH and mortality in heart valve surgery, with main emphasis on patients with suprasystemic pulmonary artery pressure (PASP) with values above 100 mmHg. Methods: This is a prospective cohort including all individuals over 18 years of age who underwent heart valve surgery using ECC from January 2020 to December 2021 in a referral hospital in the city of Salvador-Ba. For univariate analysis, chi-square test was used for categorical variables and t test for continuous variables. Multivariate analysis with logistic regression was performed, in which predictors with p < 0.05 in univariate analysis or those with biological plausibility were included. A value of p < 0.05 was considered statistically significant. Results: A total of 474 individuals were included, with a mean age of 48.1 ± 14.4 years, 180 (38%) were male and the main etiology of valvular heart disease was rheumatic heart disease: 244 (51.5%), mortality estimated by Euroscore II was on average 2.34 ± 2.53. Mean PASP was 49.8 ± 19.6 and mean ejection fraction was 60.3 ± 12.1. PASP as a continuous variable was associated with higher mortality: RR 1.06 (CI 1.05–1.07); p < 0.001 and severe PH (considered as PASP > 70 mmHg) was associated with higher mortality: RR 3.7 (1.6–8.5); p = 0.005. Seven patients presented PASP above 100 mmHg, none of which evolved to surgical death. Conclusion: Severe pulmonary hypertension was independently associated with higher mortality in heart valve surgery, which is an important factor to be considered in decision making regarding the surgical risk of patients who are candidates for surgery, with perspectives of specific conducts directed to the care of pulmonary circulation and function. of right chambers in the perioperative period. 111427 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY KARLA SANTOS PINTO1, Aurea Maria Lago Novais1, Elias Soares Roseira1, Larissa Xavier Gomes da Silva1, Raissa Barreto Lima1, Ana Luísa de Aguiar Almeida Silva1, Tainá Viana1, Rodrigo Morel1, Luiz Carlos Passos1, Clara Salles1, Diogo Azevedo1, Ana Karenina1 (1) Hospital Ana Nery Introduction: The need for blood transfusion in the postoperative period of cardiac surgery is associated with increased surgical morbidity and mortality. For this reason, it is important to identify predictors so that we can try to optimize them or try to solve them before the procedure. Objective: To identify predictors of need for blood transfusion in the postoperative period of cardiovascular surgery using cardiopulmonary bypass (CPB). Methods: This is a prospective cohort including all individuals over 18 years of age who underwent cardiac surgery using ECC from January 2020 to December 2021 in a referral hospital in the city of Salvador-BA. For univariate analysis, the chi-square test was used for categorical variables and the t test for continuous variables. Multivariate analysis with logistic regression was performed, in which predictors with p < 0.05 in univariate analysis or those with biological plausibility were included. A value of p < 0.05 was considered statistically significant. Results: A total of 1668 individuals were included, with a mean age of 54.5 ± 14.4 years, 889 (53%) were male, 412 (25%). The majority underwent isolated valve surgery 738 (44.2%), followed by coronary artery bypass graft surgery in 672 (40.3%). The mean EuroSCORE II was 1.96 ± 2.26 and the mean baseline blood count (Hb) was 12.9 ± 2.0. There was a need for blood transfusion in 506 (30.3%). Blood transfusion was associated with higher mortality: RR 2.7 (CI 1.8–4.0); p < 0.001. The predictors of blood transfusion in the multivariate analysis were male gender: RR 1.66 (1.31–2.11) and preoperative Hb: RR 1.19 (1.10–1.28). Conclusion: Blood transfusion in the postoperative period of cardiovascular surgery with the use of ECC was associated with higher mortality. Transfusion predictors were sex and preoperative hemoglobin. Therefore, it is important to identify patients with lower levels of basal hemoglobin to attempt optimization, with possible impact on surgical outcomes 111443 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY LETÍCIA RODRIGUES GATTI PEREZ1, Carlos Alberto Pastore2, Nelson Samesima2, Leonardo Paschoal Camacho Varoni2, Mirella E. Facin2 (1) Universidade Municipal de São Caetano do Sul – USCS; (2) Instituto do Coração (InCor) – Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo Background: The clinical presentations during the chronic phase of Chagas disease may be chest pain, thromboembolism, cardiac arrhythmias, sudden death and chronic heart failure. Although the electrocardiogram (ECG) has a crucial role in the diagnosis/prognosis of chronic Chagas cardiomyopathy (CCC) complications, there is no report of any ECG aspect associated with left ventricular (LV) apical aneurysms (present in 21%–46% of cases). Objective: To characterize the ECG pattern of LV apical aneurysms in the chronic chagasic cardiomyopathy. Methods: Cross-sectional study of patients with CCC, with and without LV aneurysm. From 2012–2019, 3377 patients were consecutively enrolled. Those with a time interval of ≤6 months between cardiac magnetic resonance imaging and ECG were included. Exclusion criteria were pacemaker, left bundle-branch blocks (LBBB), tachyarrhythmias, premature ventricular contractions, aneurysm in inferior wall, previous cardiac surgery and manifest coronary artery disease. Seventy-six patients were studied, 34% with aneurysm. The ECG analysis evaluated the presence of T-wave with plus-minus pattern in V2 and/or V3 and/or V4, in addition to DI and aVL leads, to identify those with LV aneurysm. In patients with LBBB+LSFB, the aneurysm was identified by the finding of ST-segment elevation in V2 ≥ 1 mm and a positive T-wave. Results: A T-wave plus-minus pattern in V2 and/or V3 and/or V4 and/or DI and aVL identified LV apical aneurysm with sensitivity, specificity, positive (PPV), and negative predictive value (NPV) of 58%, 100%, 100% and 82%, respectively. ST-segment elevation in V2 ≥ 1 mm (in the presence of LBBB+LSFB) showed respectively 45%, 98%, 83% and 89%. The concomitant utilization of both criteria resulted in 77%, 98%, 95%, 89%, 91%, and 38 for sensitivity, specificity, PPV, NPV, accuracy and likelihood ratio, respectively. Conclusions: The presence of a T-wave with plus-minus pattern in V2 and/or V3 and/or V4, and/or DI and aVL leads, in addition to ST-segment elevation in V2 ≥ 1 mm with positive T-wave (in the presence of LBBB+LSFB) showed high accuracy to identify LV apical aneurysm in patients with chronic Chagas cardiomyopathy. Further prospective studies should evaluate the relationship between these new ECG findings and morbi-mortality clinical outcomes. 111446 Modality: E-Poster Scientific Initiation – Non-case Report Category: NEGLECTED CARDIOVASCULAR DISEASES RAFAEL MATOSO DE OLIVEIRA FIGUEIREDO1, Paula Gouvêa Abrantes1, Samuel Barud Massensine1, Rodrigo Máximo Silveira1, Moisés de Toledo Vilela1, Gabriela Godinho Rezende1, Amanda Gonçalves Vieira Martins1, Eliane Ferreira Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Cardiology Department, Medical School, Federal University of Juiz de Fora, Minas Gerais – UFJF Introduction: Obstructive sleep apnea (OSA) and smoking have been established as prevalent and relevant risk factors for cardiovascular outcomes, such as stroke, acute myocardial infarction and death. Although their reported synergic effect, OSA can be an underdiagnosed and undervalued as a co-risk factor. Objective: Assess the prevalence of OSA in a smoking cessation group and the correlations with comorbidities and other known risk factors. Methods: A transversal study was conducted from 2021 to 2022, with six consecutive smoking cessation groups. The participants received comprehensive evidence-based support, including a multidisciplinary team care with pharmacological treatment. A STOP-Bang score of at least 5 was defined as high-risk screening for OSA, due to its high sensibility (>90%) and accuracy. Other clinical data were collected and analyzed for knowledge between the OSA positive and negative screening groups. Results: A sample of 36 smokers, 66.7% female, 56.63 ± 10.8 years old and 42.9% with low schooling (<8 years). Comparing users with measured positive OSA with the negative OSA, it was possible to verify that the prevelance of arterial hypertension (p < 0.0001), cardiac arrhythmias (p < 0.012), abdominal obesity (p < 0.049), cognitive decline (p < 0.001), anxiety (p < 0.035) and a high tendency for depression (p < 0.077) were significantly higher for the OSA group. Regarding smoking history, nicotine dependence (p < 0.002), prevalence of the smoking triggers: stress, habits and coffee use (p < 0.001, p < 0.021 and p < 0.013, respectively) and use of marijuana and crack (p < 0.002) were significantly higher in the positive screening for OSA group. Conclusion: We observed that tobacco use might be a potential factor for aggravating or developing obstructive sleep apnea. Therefore, the synergic effect goes beyond both being relevant for cardiovascular outcomes and death. These findings may suggest how both can impact the global patient health, as isolated or, more importantly, combined risk factors. 111448 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MARIA GABRIELA MEDEIROS CUNHA DE ARAUJO1, Anna Luiza Fragoso Guimarães Costa2, Beatriz Cunha Lisboa de Medeiros Nunes3, Eduarda Caroline Lopes de Freitas4, Felipe Andrade de Lima Trindade2, Felipe José Cavalcanti de Albuquerque Holanda2, João Victor Bezerra Ramos1, Lívia Farias de Holanda Furtado1, Mateus Ribeiro Fernandes Teixeira5, Quezia Valerio Brito6 (1) Universidade Federal da Paraíba; (2) Faculdade de Ciências Médicas da Paraíba; (3) Universidade Potiguar – UnP; (4) Centro Universitário Fametro; (5) Centro Universitário de João Pessoa – UNIPÊ; (6) Universidade Nilton Lins Introduction: Electronic cigarettes or e-cigarettes (EC), used as an alternative to smoking cessation, have become popular despite the uncertain cardiovascular risk. Although toxic-carcinogenic substances are present at lower levels in ECs compared to tobacco cigarette (TC) smoking, the products in e-liquid can be harmful to health and worryingly are often consumed by a nonsmoking public, especially young people. Objective: To provide current insight into the relation of e-cigarettes and their effects and or risks of developing cardiovascular disease. Methods: This is a systematic review, using the PubMed and Scopus databases. The Mesh descriptors used were: Electronic Nicotine Delivery Systems, Heart Disease Risk Factors, Cardiovascular Diseases and their synonyms. The initial search generated a total of 1301 articles, which after using filters resulted in 113 and after inclusion and exclusion criteria, 35 articles, used for the present study, for which the PRISMA methodology was followed. Results: It is notorious the divergence on the effects and cardiovascular risks of ECs. Some studies show damage in cellular repolarization, increase in heart rate and rhythm, and mean arterial pressure, while others do not. Furthermore, most users make dual use of electronic cigarettes and combustible cigarettes, making them more vulnerable to cardiovascular risk factors than smokers of tobacco alone or never smokers, with a higher prevalence of Metabolic Syndrome, for example. Switching from conventional smoking to the use of electronic devices is associated with a potential lower cardiovascular risk compared to continuing conventional tobacco, but some studies show no useful change in biomarkers of oxidative stress. On the other hand, users who started electronic devices after conventional ones, have a substantial higher cardiovascular risk than those who have ceased smoking. However, according to imaging methods, both conventional cigarette smokers and EC users have more carotid plaque disposition and similar alteration of vascular stiffness, with no difference in inflammatory degree between them, when compared to nonsmokers. Conclusion: The use of tobacco and the use of ECs cause damage to the cardiovascular system and alter inflammatory biomarkers. However, there is still a lack of evidence-based studies on this subject, since many of the studies are based only on acute and not chronic alterations, and the irreversibility of the disease is unknown. 111449 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION FERNANDA ABRAHAM LEÃO1, Maria Eduarda S. Barbosa1, Raquel P. Cabús1, Marco Antonio Mota Gomes1, Waléria Dantas Pereira1, Márcia Sâmia Fidelix1, Annelise Machado Gomes de Paiva1 (1) Centro Universitário Cesmac Background: The onset of CVD is most often associated with genetic predisposition, sex and age, in addition to a lifestyle that includes alcoholism, smoking, physical inactivity and diet; factors that can also influence the development of Type 2 Diabetes, Dyslipidemia and Arterial Hypertension. Given this situation, it is pertinent to analyze the relation between blood pressure parameters and cardiovascular risk variables. Objectives: To analyze the relation of different risk factors with PWV as an outcome. Methodology: Identification and behavioral data were formed, risk factors were verified, and the study variables were: age, sex, smoking, sedentary lifestyle, alcoholic beverages, presence of hypertension, diabetes, dyslipidemia, in addition to peripheral parameters and blood pressure (blood pressure and pulse wave velocity) which was created by the Mobil-O-Graph® equipment. An anthropometric assessment (weight, height) was performed and then the calculation of the body mass index (BMI) was verified. Results: When all risk factors were observed, 58.3% of the sample had hypertension, one of the most relevant pathologies for increasing PWV and corrected for age, it was noted that SBP with a p-value of <0.01, alcoholism with a p-value of 0.04 and smoking with a p-value of 0.02 were significant in relation to PWV. Conclusion: It appears that age and sex are the main causes of increased PWV, but they are not unique components, analyzed as risk factors, and it was noted that it is not an innate factor, but a set of mortifiable conditions. 112103 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION ENRICO DE FRANCISCO MAGNANI1, Jhessica Macieira Pereira1, Danielle Aparecida Gomes Pereira1, Rafael Dias de Brito Oliveira1, Thayrine Rosa Damasceno1, Suelen Cristina Silva de Oliveira1, Denise Mayumi Tanaka2, Eduardo Elias Vieira de Carvalho2, Júlio César Crescêncio2, Eduardo Rubio Azevedo2, Marcus Vinícius Simões2, Luciano Fonseca Lemos de Oliveira1 (1) Universidade Federal de Minas Gerais – UFMG; (2) Faculdade de Medicina de Ribeirão Preto – FMRP/USP Introduction: Heart failure (HF) is one of the main disorders that characterize chronic Chagas cardiomyopathy (CCC), and it is directly related to diffuse myocarditis, ventricular dilatation and reparative fibrosis. With its development, there is a consequent reduction in functional capacity and the appearance of symptoms such as fatigue and dyspnea on exertion. However, little is known about the factors that determine such reduction in functional capacity in CCC. Objective: To evaluate the functional capacity and identify the determining factors of the reduction of the maximum oxygen consumption (VO2peak) in patients with CCC. Methods: Observational study using 101 patients with CCC, who underwent clinical examination, cardiopulmonary exercise test (CPET) and echocardiography as part of the standard clinical evaluation. Data normality was analyzed using the Kolmogorov-Smirnov test. The association between VO2peak and clinical or echocardiographic variables were analyzed by Pearson or Spearman correlation coefficients. Multivariable linear regression analysis was used in order to identify independent clinical and echocardiographic predictors associated with peak VO2max. Results: Mean age of study patients was 56.2 ± 13.4 years. The mean left ventricular ejection fraction (LVEF) was 42.6 ± 18%, and mean VO2peak was 16.8 ± 6.1 ml/kg/min–1. VO2peak was associated with age (r = 0.44; p < 0.001), male gender (p < 0.001), functional class (FC-NYHA, r = 0.53; p < 0.001), LVEF (r = 0.51; p < 0.001), LV diastolic diameter (r = –0.38; p < 0.001), left atrium diameter (r = –0.34; p < 0.05), E wave (r = –0.32; p = 0.01) and LV mass index (r = –0.28; p = 0.006). In the multivariate analysis, only age, male gender, LVEF and E wave remained independently associated with VO2peak with adjusted R2 = 0.66. In patients who were able to assess pulmonary artery systolic pressure (PASP, n = 45) and tricuspid annular plane systolic excursion (TAPSE, n = 55), the VO2peak correlated significantly with PASP (r = –0.60, p < 0.001) and with TAPSE (r = 0.28, p = 0.04). Conclusion: In patients with CCC, disease severity (FC-NYHA), male gender, LV systolic and diastolic function, as well as pulmonary hypertension influence functional capacity. 111520 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH VANESSA FARIA DE ALMEIDA SCHNEIDER1, Larissa Lima Nunes1, Inaiê Maiala De Almeida Miranda1, Ana Gabriela da Silva Farias1, Leandro Rozin1 (1) Faculdades Pequeno Príncipe Introduction: Congenital Heart Diseases (CHD) are abnormalities in cardiocirculatory structure or function that occur in the intrauterine period, being diagnosed during pregnancy or after birth. They range from communications between cavities that regress spontaneously, to large malformations that require several invasive procedures, which can result in intrauterine death, in childhood or even in adulthood. Objective: To estimate the prevalence of CHDs and their most frequent subtypes in Brazil, from the years 2017 to 2021. Methods: An observational epidemiological survey was carried out using data from the Information System on Live Births (SINASC) and DataSUS, available by the Ministry of Health. We analyzed, through descriptive statistics, the incidences at birth and the prevalence in that period, bringing information according to the country’s regions for every 10,000 live births. Results: In the total of 5 years researched, 14,748 cases of Congenital Malformations Of The Circulatory System (Q20–Q28) occurred in Brazil. Its highest incidence (18.59) was in 2017 in the Southeast region. Among the subtypes of the disease, the Southeast region continues in highlight due to Congenital Malformations Of The Cardiac Septum (Q21), with a prevalence of 7.79, reaching its highest incidence (8.87) in 2017. The greatest rates of this subtype correspond to Atrial Septal Defect (Q21.1), with a prevalence of 5.09 and its highest incidence of 6.04 occurred in 2020. Another highlighted subtype concerns Other Congenital Heart Malformations (Q24), which reached a prevalence of 4.74 in the South of the country and its highest incidence of 5.17 in 2020 – its highest rates account for Unspecified Heart Malformations (Q24.9), with a prevalence of 3.77 and its highest incidence of 4.02 occurred in 2018. A fact that draws attention is that, in the sum of the 5 years, more than 65% of cases of CHD occurred in the Southeast region, information that opens valuable precedents for new research. Conclusions: It is a fact that the difficulty in diagnosing Congenital Heart Diseases represents a significant Global Health Problem, mobilizing public policies for early diagnosis, which in Brazil has the “Teste do coraçãozinho” (Little Heart Test), Morphological Ultrasound Exams, and Fetal Echocardiography. Despite this, it is worth mentioning that there is a disparity in access to these resources, which represents another challenge to the issue and may indicate underreporting of cases. 111550 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY CAMILA ALENCAR DE ANDRADE1, Gabriel Irismar Rodrigues Schwamback1, Bruna Katharine Cavalcante Nascimento1, João Pedro de Oliveira Vicente1, Leo Christyan Alves de Lima1, Fabiana Cristina Schabatoski Passos2, Alexandre Venturelli da Silva2, Luiz Henrique Gasparelo2 (1) Centro Universitário São Lucas – UNISL; (2) Angiocenter Introduction: First introduced more than 40 years ago by Japanese surgeon Kanji Inoue, percutaneous balloon mitral valvuloplasty (PBMV) is performed using a balloon inflated with high pressure in a stenotic heart valve. Mitral stenosis (MS) is a serious disease that can progress to elevated left atrial pressure and pulmonary hypertension, formation of emboli, among other complications. Objectives: This is a descriptive study of registration of cases of percutaneous mitral valvuloplasty with Inoue balloon, performed in a highly complex service in the state of Rondônia, in the Western Amazon. Methods: Data obtained through research via medical records and complementary exams were used. Results: In this case series, four patients were followed, three female aged 24, 42 and 54 years and one male aged 28 years. All patients were symptomatic (complaints of fatigue and/or dyspnea). On echocardiography, they showed significant MS with a mean gradient of 13 to 20 mmHg. The procedures were performed through the right femoral vein with an Inoue balloon, through transseptal puncture performed with the aid of transesophageal echocardiography. Patients had a hospital stay of less than two days. In the one-year follow-up with echocardiographic control, they had a favorable evolution of less than moderate stenosis and insufficiency. Discussion: Correction of MS is indicated for patients with severe symptomatic mitral stenosis (dyspnea with New York Heart Association II to IV) or asymptomatic patients with complicating factors, including pulmonary hypertension or new-onset atrial fibrillation. The patient should preferably have an echocardiographic score less than or equal to 8 (with subvalvular apparatus and calcification less than or equal to 2). Patients who present pre-procedure echocardiographic score greater than 8, post-procedure mitral valve area with a value of less than 1.5 or post-procedure mitral regurgitation with a value greater than or equal to 2, will have the need for of mitral valve replacement surgery at 2 years after of the procedure. Conclusion: Although it is a recent procedure for the region when compared to large centers, performing PBMV for the treatment of severe MS in the state of Rondônia promotes a safe procedure, capable of improving the patient’s quality of life and increasing life expectancy, with a relatively low risk. 111936 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY BRUNA VIEIRA SILVA OLIVEIRA2, Ana Paula Fernandes Pereira2, Marla Thais Fernandes Teodoro3, Davi Marques de Souza1, João Victor Silva Souza1, Lara Oliveira Santana Rocha1 (1) Universidade estadual do sudoeste da Bahia; (2) Universidade federal da Bahia; (3) Faculdade Santo Agostinho Introduction: Dextrocardia is a congenital malformation that consists of a shift of the heart axis to the right side of the chest, contrary to physiological and derives from a change in the embryological process. In the literature, the overall incidence is about 10:100,000, and may be underdiagnosed in most cases. Objectives: To determine the annual incidence of the malformation in each Brazilian region during the last 10 years. Methods: Data collection in the platform of the SUS Department of Informatics – Information System on Live Births (DATASUS – SINASC), separated by region – north, northeast, south, southeast, and center-west – and year – 2010 to 2019 – with statistical calculations of the Brazilian incidence rate per 100,000 inhabitants, in order to make the data more tangible. Results: The Brazilian region with the highest incidence was the southeast, with 92 cases, and the lowest was the midwest, with 8. The year 2016 had the highest number of cases, totaling 16. Table 1 shows the relative data by region in each year. The incidence rate was about 0.5:100,000 population. Conclusions: Dextrocardia is still an underdiagnosed condition in Brazil, since the incidence rate was lower than the global rate. A result that corroborates this statement is that the southeastern region had the highest incidence in all years, perhaps due to greater access to diagnostic methods or because of a lower prevalence of complications related to the condition. 111573 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANTONIO MAYCON DA SILVA SOUSA1, Carlos Eduardo Batista de Lima2, Victor Eulálio Campelo2, Ginivaldo Victor Ribeiro do Nascimento2, Newton Nunes de Lima Filho2, Thiago Nunes Pereira Leite2, Jônatas Melo Neto2, Maurício Giraldi2, Lucas Teixeira Dias2, Paulo Márcio Sousa Nunes2 (1) Universidade Federal do Piauí (UFPI); (2) Hospital Universitário da Universidade Federal do Piauí (HU-UFPI) Introduction: Telemedicine experienced strong growth after the Covid-19 pandemic. Objective: Evaluate the clinical demographic profile, rate and probability of occurrence of electrocardiographic abnormalities in the population studied in relation to sex and age. Methods: The telehealth center of the University Hospital of the Federal University of Piauí (THC UH-FUPI) uses an analysis system that allows receiving exams from peripheral units. From November 2020 to March 2022, data from patients who underwent electrocardiogram (ECG) in basic health units sent to THC UH-FUPI were evaluated. The variables analyzed were: age, gender and electrocardiographic diagnosis: unspecific changes in ventricular repolarization (UCVR), old infarction, bundle branch block, ischemia, intraventricular conduction disorder (IVCD), arrhythmias and chamber overloads. For statistical analysis, Anderson-Darling, Mann-Whitney and logistic regression tests were used with a significant p-value <0.05. Results: ECG of 27955 patients were evaluated, aged between 51.9 ± 17.3 years and predominantly female (63.0%). Of the ECGs analyzed, 6075 (21.7%) had an abnormality, with a higher occurrence with advancing age (p < 0.0001). UCVRs were more prevalent (2337; 7.96%), followed by conduction disorders (1949; 6.63%), arrhythmias (1401; 4.77%), bundle branch block (594; 2.02%), chamber overload (481; 1.64%), old infarction (359; 1.22%) and ischemia (30; 0.1%). Atrial fibrillation (AF) was observed in 138 cases (0.5%); 13% in <60 years; 87% in >60 years. Male patients were more likely to have an ECG change (OR 2.25, 95%CI 1.77–2.78), however, adjusted for advancing age, this probability cancels out. As a factor regardless of age, males had a lower chance of UCVR (OR 0.85; 95%CI 0.78–0.93), and a higher chance of arrhythmias (OR 1.29; 95%CI 1.15–1, 44), old infarction (OR 1.69; 95%CI 1.37–2.09), and ischemia (OR 1.93; 95%CI 0.93–3.99). Conclusion: In the population studied, the rate of abnormal ECGs was high (21.7%), increasing with age, with females being more prevalent. The rates of occurrence of AF and ischemia were low (0.5% and 0.1%, respectively). The main alteration was UCVR with a higher chance of occurrence in females and males were more likely to have arrhythmias, old infarction and ischemia. In this initial experience, it was observed that the tele-ECG is feasible and can be implemented, with a wide reach, even in regions where there is greater difficulty in accessing health care. 111571 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH RENATTA KAROLINE BEKMAN VOGAS1, Jonatas Benarroz da Silva1, Davi da Silveira Barroso Alves1, Paulo Henrique Godoy1 (1) Federal University of the State of Rio de Janeiro – UNIRIO Introduction: Secondary hypertension (SH) is a condition in the group of hypertensive diseases, which has a lower prevalence. Knowing mortality from SH is important, however, the lower prevalence can influence its analysis in death records. Objective: To analyze mortality due to SH, when registered as the underlying cause of death (UCD) and mentioned cause (MC), according to age group and sex, in Brazil and regions, from 2000 to 2019. Methods: Ecological study on deaths due to SH, registered as UCD and MC in lines A to D, of death certificates, from the Brazilian Mortality Information System. Code I15 of the International Code of Diseases 10 (ICD-10) was used. Crude and standardized mortality rates were estimated for UCD and MC, for Brazil and regions, from 2000 to 2019, according to sex and age groups. Results: For UCD 14 deaths were recorded, the standardized rates were similar for both sexes. The highest frequency of deaths occurred in the age group of 60 years or older. In the Midwest region, no deaths were recorded, while the Northeast accounted for 57% of deaths. As for MC, 16,892 were registered due to HS. This result is 1,200 times greater than that found in the records by UCD. There was greater disparity between the sexes than in UCD analysis, with 43% of the standardized rate for males higher than females. Standardized rates were lower in women and crude rates increased with age. The North region presented the highest standardized rate and the Southeast the lowest, in both sexes. Conclusions: It is possible that the selection rules, defined by the ICD-10, for UCD underestimate deaths from HS. Thus, it may be useful to consider MC as a method for analyzing mortality from less prevalent diseases. 111701 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY MARCELLA OLIVEIRA MONTE SANTO1, Afonso Moraes Melo Junior3, Ana Flávia Oliveira de Souza3, Camila Rodrigues Maciel3, Cecília Gomes da Silva1, Lucas Guimarães Junqueira2, Mainã Cristina Santos dos Santos2, Rafael Silva Lemos2, Rafaela Oliveira Cardoso3, Paola Bitar de Mesquita Abinader3, Luis Eduardo Werneck de Carvalho4, Davi Gabriel Barbosa2 (1) UNIVERSIDADE FEDERAL DO PARÁ (UFPA); (2) UNIVERSIDADE ESTADUAL DO PARÁ (UEPA); (3) CENTRO UNIVERSITÁRIO DO PARÁ (CESUPA); (4) ONCOLÓGICA DO BRASIL Introduction: Within the thoracic region there are 3 main sites of development and metastasis of malignant neoplasms: the heart, the mediastinum and the pleura. Although neoplasms in the cardiac muscle tissue are rare, there are cases of primary and secondary tumours, representing 0.05% and 1–2% of neoplasms. In contrast, pleural tumours are more common, the main cancer of which is mesothelioma in its diffuse form. In general, most tumours present in these organs are metastases from other neoplasms in advanced stages. Objectives: To establish an overview of mortality from malignant neoplasms of the heart, mediastinum and pleura in Brazil, through a time-series study, between 2011 and 2020. Methodology: This is a cross-sectional study, evaluating the information on mortality from malignant neoplasms of the heart, mediastinum and pleura in the years 2011 to 2020. A search was performed in the tabnet DATASUS system. The ICD C38 (heart, pleura and mediastinum) was selected to perform the search and comparison between the regions of Brazil. It established the stratification by age range affected, the division by gender and the total number of cases per year in the selected period. Results: In the period 2011–2020 about 7585 deaths from cancer of the heart, mediastinum and pleura, and 2019 was the year with the highest number of deaths; 867 were recorded. In 2019, the southeastern region had the highest number of deaths (45.7%). Of the total absolute deaths in the regions, 5859 (77.3%) were in ages 50 years and above, with the most prominent age group being 60–69 years with 1809. Comparing the sexes, there were more male deaths, with 4,249 (56.01%) registered, compared to the female, with 3,332 deaths (43.92%). Both genders presented a higher number of deaths in 2019, with men representing 54.09% and women 45.91%. Moreover, the highest number of deaths of both sexes was registered in the Southeastern region and the most affected age group was 60–69 years. Women presented a greater constant of deaths along the years than men. Conclusion: It appears that neoplasms of the heart, mediastinum and pleura in Brazil have a higher incidence at ages between 60–69 years. There was a predominance of male deaths, besides the southeastern region accounting for most of the deaths. Thus, it is necessary to invest in prevention actions directed to this public, in order to reduce the number of deaths from these neoplasms. 111604 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY FABIO ANTONIO SERRA DE LIMA JUNIOR1, Fábio Antônio Serra de Lima Júnior1, Ana Beatriz Venâncio de Paula Bezerra1, Renan Furtado de Almeida Mendes1, David Cesarino de Sousa1, Raphael Patrik Borges da Costa1, Isaac Newton Guimarães de Andrade1, André Telis de Vilela Araújo1 (1) Universidade Federal da Paraíba Introduction: Cardiac surgery, through sternotomy, can cause intense postoperative pain, most commonly in the immediate postoperative period, affecting 50% of patients and may lead to significant short- and long-term consequences, as well as chronification. Despite this, between 50% and 75% of patients do not receive adequate analgesic management. Few randomized controlled trials have addressed the efficacy of pregabalin administration in the perioperative period for pain control, delirium, and postoperative recovery, obtaining variable results. Objectives: This study aimed to evaluate the preemptive use of pregabalin to decrease pain perception in patients undergoing cardiac surgery in the first three postoperative days. Methodology: Patients, undergoing cardiac surgery, were randomized into two groups: one group will preemptively use pregabalin 75 mg on the day of surgery until the second postoperative day, and the other group will use placebo for the same period. After postoperative extubation, they were evaluated at 24, 48, and 72 hours for visual analog scale (VAS) and analgesic use. The study was registered in ClinicalTrials.gov through NCT04173390. Results: Twenty-three patients were included, with 11 receiving pregabalin and 12 placebo, presenting, respectively, visual pain scale means of 1.75 ± 2.188 vs. 2.27 ± 2.412 (p = 0.634) at 1st PO day; 0.88 ± 1.246 vs. 2 ± 2.049 (p = 0.188) at 2nd PO day; and 1.75 ± 2.915 vs. 1.64 ± 1.804 (p = 0.918) at 3rd PO day. There was no statistically significant difference in the use of simple analgesics, weak or strong opioids on any of the postoperative days. Conclusion: In this partial evaluation of the study, pregabalin did not affect patients‘ postoperative pain scores, nor did it affect their analgesic consumption. The study will continue to randomize and collect patients until it reaches the anticipated sample size for more adequate statistical power. 111609 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY MATHEUS COELHO TORRES1, Juliana Góes Martins Fagundes2, Luis Fabio Barbosa Botelho1, Thiago Lins Fagundes de Sousa5, Alinne Fernanda Amaral Verçosa3, Rodrigo de Carvalho Flamini3, Eudanusia Guilherme de Figueiredo2, Emilio Carlos De Arruda Lacerda2, Igor Lemos Duarte2, Jean Fabrício De Lima Pereira2, Marcelo Dantas Tavares De Melo1, Silvia Moreira Ayub Ferreira4 (1) Hospital Universitário Lauro Wanderley – UFPB, João Pessoa, Brazil; (2) Oncoclinicas Paraiba (Centro Paraibano de Oncologia), Joao Pessoa, Brazil; (3) Nova Diagnostico por Imagem, Joao Pessoa, Brazil; (4) Instituto do Coração, Sao Paulo, Brazil; (5) Hospital Universitário Alcides Carneiro, UFCG, Campina Grande, Brazil Background: The rise of Immune Checkpoint Inhibitors (ICI) as a cancer treatment has been followed by the increase of cardiotoxicity events reports. Retrospective studies with anthracycline revealed a relationship between increased myocardial 18F-FDG uptake (MGU) and a slight drop in Left Ventricular Ejection Fraction values, suggesting that this may be a useful tool for early detection of cardiotoxicity. Our study is intended to evaluate the prognostic impact of the variation in MGU in advanced lung cancer undergoing treatment with ICI. Methods: This is a unicentric, retrospective study, that evaluated lung cancer patients treated with ICI from 2016 to 2021 and submitted to at least two positron emission tomography (PET-CT). The primary objective was to evaluate the MGU variation during ICI treatment (ΔSUV) and its impact on survival outcomes. Results: 59 patients fulfilled all inclusion criteria. 51% received Immunotherapy in combination with chemotherapy and 49% received only immunotherapy. Global median ΔSUV was +0.05; Among patients in the disease control group, the median ΔSUV was +0.48, while the median ΔSUV of the disease progression group was –0.66. Considering patients with positive myocardial ΔSUV versus negative ΔSUV, there was a median increase of progression-free survival (PFS) of 161 days in favour of the positive myocardial ΔSUV group (p = 0.066) and increase of 281 days on Overal Survival (p = 0.256). There was an increase of 759 days in the OS of diabetic patients (p = 0.023), and better results in metformin users (p = 0.015), in addition to an increase of 285 days in favor of beta-blocker (BB) users (p = 0,886). The trend data found indicate that BB and/or metformin could perhaps have a supporting role in the treatment of ICI. These findings correlate with recent evidence showing an increase in MGU after BB use and that BB may function as immunomodulatory agents improving immunotherapy results. Conclusions: The increase in MGU was associated with better outcomes, suggesting that perhaps it could be studied as a predictor of treatment response, in addition to the trend of benefit also shown with the use of metformin and BB, which strengthens their possible synergistic effect in treatment. However, more robust studies are needed to better understand the MGU role on ICI treatment. 111642 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION PAOLA DE OLIVEIRA DAS CHAGAS1, Camila Alvarez Alonso1, Luz Alcira Avila Rincon Alves1, Fabiane Rosa Rezende Honda Marui1 (1) Universidade Municipal de São Caetano do Sul – USCS Introduction: Hypertension in adolescents has become more and more frequent due to the increase in the childhood obesity rate. In addition to obesity and overweight, stress and excessive salt intake are also factors related to increased blood pressure in this age group. The increase in cases of hypertension in adolescence has been a recent concern, since until the mid-1970s, the measurement of Blood pressure in asymptomatic children was not frequently performed, after updates to the guidelines, it is recommended that in the absence of comorbidities, the Blood pressure is measured annually. ABPM, which is the gold standard for monitoring, has been the most recommended for confirming the diagnosis of AH, but despite the recommendation, ABPM has high costs and risks of failure. Home blood pressure monitoring has shown great efficacy and sensitivity when compared to ABPM, although there are not enough studies. Method: The bibliographic survey was carried out from articles collected on the LILACS and MEDLINE platforms. Articles published between 2012 and 2021, in Portuguese, English and Spanish were eligible. The articles were initially selected by reading the titles and abstracts for categorization according to the study objectives. Subsequently, the selected ones were read in full. Results: Considering the analysis of the studies, it was identified that the ABPM obtained values similar to the ABPM when performed in adolescents and presented a better cost-benefit ratio, in addition to being more effective in identifying White apron effect and masked hypertension. Studies have shown that this accuracy prevents teenagers from being misdiagnosed and that they consequently involve unnecessary treatment with antihypertensive drugs. Final considerations: Although evidence on HBPM is still limited, the literature review points out that the values obtained through home monitoring proved to be accurate when compared to other methods and also provided information on the daily variability of the assessed public, showing be a precise alternative in confirming the diagnosis of hypertension and young people. 111648 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GABRIEL NERY LIMA1, Arthur Afonso Ferreira Rebelo1, Thassio Marcyal Bento Ferreira1, Gisely Seguchi Spinassé1, Matheus Ricardo Malveira Camacho1, Amanda Gabriela Freitas Rodrigues2, Israel Figueira Lemos1 (1) Faculdade de Medicina da Universidade do Estado do Pará; (2) Faculdade de Medicina e Cirurgia do Pará Introduction: Systemic Arterial Hypertension (SAH) is a clinical condition that consists in high and sustained levels of blood pressure, generating effects in the heart, brain, kidney and blood vessels. The SAH is related with several metabolic disorders, which leads to elevated risk of cardiovascular fatal and non-fatal events. Nowadays, in Brazil, the SAH affects approximately 30% of the population and corresponds to 40% of deaths from stroke and 25% of deaths from Chronic Arterial Disease. Therefore, there is great relevance in studying the epidemiology of this disease in the country, seeking to understand which social groups are most affected by it and, thus, need greater attention from Public Health. Objective: To describe the epidemiological profile of hospitalizations and deaths due to Systemic Arterial Hypertension in the state of Pará. Methods: A cross-sectional, descriptive and quantitative study was carried out using the database of the Department of Informatics of the Unified Health System (DATASUS). Data referring to patients hospitalized or who died from SAH in the period from 2008 to 2020 in the state of Pará were selected, considering the following variables: age, sex and race. Data were processed using Excel and Biostat software. Results: During the study period, 55,503 hospitalizations and 508 deaths from SAH were reported in the state of Pará. Hospitalizations were more frequent among women (58.5%), black and brown people (57.6%), and in the 50–69 age group (42.6%). On the other hand, deaths occurred mainly among men (51.9%), black and brown people (43%), and aged over 70 years (54.6%). It is worth mentioning that the analysis of the “race” criterion was hampered by a large number of cases without information in the system (23,097 records). Conclusion: There is greater morbidity and mortality among the non-white population, which is in agreement with the current literature, which states that there is a greater prevalence and severity of SAH among black and brown people, mainly due to genetic factors. However, it is known that in Brazil there is a historical marginalization of this population; this factor may be related to the difficulty in accessing the right to health, aggravating the risk factors for the development of SAH in this social group. In this sense, greater efforts must be made in order to reduce social inequality and promote universal access to heahealth thus minimizing unfavorable outcomes due to lack of assistance. 111649 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH MARCOS OLIVEIRA1, Solange Lima Gomes1, Franciane de Paula Fernandes1 (1) Universidade do Estado do Pará (UEPA) Introduction: Acute Myocardial Infarction (AMI) is an important cause of mortality in society. In indigenous peoples, this illness gains prominence due to barriers that hinder access to health. Despite advances, AMI remains among the main causes of death in these peoples. In view of this, it is necessary to investigate how this situation affects the occurrence of this disorder in traditional populations. Objective: To analyze the mortality due to AMI in indigenous and non-indigenous peoples from Brazil over the past decade. Methods: This is a descriptive study, with a quantitative, cross-sectional, and retrospective approach. Data from public domains of the Mortality Information System of the Informatic Department of the Unified Health System (SIM/Datasus) were analyzed. Results: 974,862 deaths were reported in non-indigenous peoples and 1,775 in indigenous peoples. Among non-indigenous peoples, the highest number occurred in 2019. In the same group, 59% were male and 40.98% female. Furthermore, the age group of 80 years or older was predominant (26.04%) and the majority of deaths occurred in hospitals (51.24%). Among non-indigenous peoples, the highest number of deaths occurred in 2020. There was a predominance of males with 59.15%, while 40.78% were female. The most affected age group was 80 years or older (29.35%). It was also noted, a higher frequency of notifications at home (47.49%). Conclusion: This context may be a reflection of greater difficulty in accessing health facilities for the traditional population. It is evident the need for health access strategies that consider the cultural particularities of each group, in addition to awareness campaigns with a preventive approach, and research that explores prevention strategies and control of risk factors in health units. 111680 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JOSÉ RENAN DE MATOS PAIN1, José Renan de Matos Pain1, Jorge Tadashi Daikubara Neto1, Matheus Bissa Duarte Ferreira1, Rafael Moretti1, Jessica Tamires Reichert1, Lucas Muller Prado1, Gustavo Sarot Pereira da Cunha1, Leornardo Henrique dos S. Melo1, Michelle Bozko Collini1, Raphael Henrique Déa Cirino1, Miguel Morita Fernandes da Silva1 (1) Universidade Federal do Paraná (UFPR) Background: Comorbidities are associated with worse quality of life and prognosis in chronic heart failure. However, there is lack of data on the impact of comorbidities in acute heart failure (AHF). Objective: To assess the association between comorbidities and the and prognosis of patients with AHF. Methods: Patients diagnosed with AHF, admitted to a tertiary hospital in southern Brazil, between 2019 and 2021 participated in the study. We selected the following comorbidities: hypertension, diabetes mellitus, dyslipidemia, obesity, smoking, alcohol consumption, anemia, stroke, atrial fibrillation, coronary artery disease (CAD), peripheral vascular disease (PVD), chronic obstructive pulmonary disease (COPD) and chronic kidney disease (CKD). The outcome was a composed of all-cause death or re-hospitalization within 6 months. We performed a stepwise forward selection with Cox regression analysis to identify the comorbidities independente associated with the outcome. Results: We analyzed 175 patients with AHF were included, of which 66 died or were re-hospitalized during the study period. The mean age of the patients was 67 years, 88 (50%) patients were female, the mean ejection fraction was 43.8, and the ejection fraction extremes were 61.6–26.1. The five most prevalent comorbidities were: hypertension (79%), diabetes (38%), atrial fibrillation (36%), dyslipidemia (31%), CAD (29%) and COPD (18%). In the stepwise multivariate regression, the only comorbidity that was independently associated with the composite outcome was COPD (Hazard ratio 1.83, 95% confidence interval, 1.04–3.23, p = 0.035). Ejection fraction (EF) was not significantly associated with the outcome (p = 0.48). Conclusion: In patients with AHF, COPD was the only comorbidity independently associated with the risk of events in a six month follow up. 111691 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM JÚLIA SCHOUCAIR NEVES1, Marília Menezes Gusmão1, Laís Araújo Carneiro de Campos1 (1) Escola Bahiana de Medicina e Saúde Pública – EBMSP Introduction: The COVID-19 pandemic brought impacts that may have had a great influence on the incidence and lethality of Acute Coronary Syndromes (ACS). Since the actions to contain the pandemic have regional differences, it is necessary to know if the increase in the number of cases of COVID-19 was linked to an increase in the number of cases and mortality from ACS in Salvador. Objective: To investigate whether there was an association between cases of ACS and cases of SARS-CoV-2 infection, during the first wave of the COVID-19 pandemic, in Salvador. Methodology: Observational study of comparative incidence and temporal trend carried out using secondary data from a local public ambulance service (SAMU) registries and the COVID-19 database provided by Health Secretary of Bahia State. The monthly incidence, lethality and assessment of the association between its incidences and Pearson’s correlation coefficient were performed in Excel software. Results: From April to December 2020, there were 490 new cases of ACS, attended by SAMU, in Salvador. Compared with the number of cases in the same period of pre-pandemic years (2017, 2018 and 2019), respectively 341; 484 and 496 cases, it was observed that the incidence remained constant. There was no significant correlation between the monthly incidence of COVID-19 and ACS (r = –0.1) or lethality among these two variables (r = –0.13), from April 2020 to April 2021. Conclusion: There was no increase in ACS cases registered by SAMU between April 2020 and April 2021, during the COVID-19 pandemic, in Salvador, when compared to the same period of the last 3 years and there was no significant correlation between the incidence of ACS cases and COVID-19 cases. 111705 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION RAIMUNDO BENÍCIO DE VASCONCELOS NETO1, Raimundo Benício De Vasconcelos Neto1, Rebecca Shaiane Soares Nunes Rivoredo1, Gabriel Irismar Rodrigues Schwamback1, Brenda dos Santos Rodrigues1, Sofia dos Santos Souza1, Maria Eduarda Brotto de Souza1, Bárbara Barbosa Pires1, Antonieta Relvas Pereira2, Sergio de Almeida Basano3, Juliana de Souza Almeida Aranha Camargo4, LUÍS MARCELO ARANHA CAMARGO4 (1) Centro Universitário São Lucas- UNISL; (2) Centro Universitário Aparício Carvalho- UniFIMCA; (3) CEMETRON; (4) ICB-5 USP Introduction: Systemic arterial hypertension (SAH) is a chronic non-communicable disease (CNCD) with high worldwide prevalence. It is a disease that has its onset, often in childhood and adolescence, and can lead to serious future consequences. However, it appears that there is a data scarcity about the occurrence of SAH in Western Amazon communities, especially riverside communities. Objectives: To estimate the prevalence of SAH and associated risk factors in public school students residing in riverside communities in the Western Amazon, Brazil. Methods: This work is a cross-sectional study, carried out with individuals aged between 4 and 21 years old, without gender distinction, living in a riverside region in Humaitá (S 6o 58’62’’ re W 62o 50’’ 08W), Amazonas State, Brazil. The project was approved by the research committee of the Brazillian Center for Research in Tropical Medicine (CEPEM). For the blood pressure measurement, the patients were prepared according to the protocol of the Brazilian Ministry of Health, 3 measurements were performed, excluding the first and applying the average of the last two in each arm. The analysis was performed using the prevalence ratio (PR), Mantel-Haenszel chi-square test, with statistical significance p < 5%. Results: 160 public school students were randomly selected for the study, of which 152 (95%) had their data analyzed, 82 male and 70 female. Twenty-five individuals were found with blood pressure levels consistent with SAH, representing an absolute prevalence of 16.5%. The mean age of hypertensive students was 12 years, ranging from 5 to 21 years. The average income reported was calculated at R$ 862.00. Regarding risk factors, the PR between SAH and males was 1.5 (p = 13%); for family history of SAH, the PR was 1.2 (p = 35%); for SAH and sedentary lifestyle, the PR was 1.3 (p = 30%); for the association between SAH and obesity in this population and SAH and dyslipidemia, the PR was less than 1, but with p > 5%. Conclusions: A few decades ago, CNCD, such as SAH had significance only among the adult population, now it also affect children and adolescents in a similar way. The data from this analysis point to a prevalence of more than 16% of SAH among students aged 5 to 21 years, which is considered high for this age group, requiring an intervention by the health system in order to attenuate the occurrence of NCDs in adulthood. There was no association between risk factors and SAH in this study. 111733 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION REBECCA SHAIANE SOARES NUNES RIVOREDO1, Rebecca Shaiane Soares Nunes Rivoredo1, Raimundo Benício de Vasconcelos Neto1, Gabriel Irismar Rodrigues Schwamback1, Jade Gomes da Costa Medeiros1, Liana Miranda Pereira1, Bárbara Barbosa Pires1, Ana Júlia Omodei Rodrigues Martim1, Antonieta Relvas Pereira2, Sergio de Almeida Basano3, Juliana de Souza Almeida Aranha Camargo4, LUÍS MARCELO ARANHA CAMARGO4 (1) Centro Universitário São Lucas/Afya UNISL; (2) Centro Universitário Aparício Carvalho FIMCA; (3) CEMETRON; (4) ICB-5 Schools in the Western Amazon Introduction: The current Brazilian epidemiological profile is undergoing a process of transition in the most gentrified regions of the country, where infectious diseases predominated, has been showing an increase in non-communicable chronic diseases. Obesity is a chronic disease considered a serious public health problem, defined by excess body fat. Its prevalence may contribute to the emergence of cardiovascular diseases. This study aimed to identify the prevalence of obesity and its risk factors in riverine schoolchildren in the Western Amazon, Brazil. Methodology: This is a cross-sectional study, carried out with individuals aged between 6 and 16 years old who attend public schools. The sample space consisted of 152 individuals belonging to 22 riverside communities along the Madeira River in Humaitá, Amazonas State, Brazil. For the research, a clinical-epidemiological questionnaire was applied, in addition to obtaining anthropometric data (weight, height, waist circumference), body mass indices (BMI) were calculated, considering the guidelines of the Brazilian Ministry of Health as a standard of normality. The statistical analyzes performed were the prevalence ratio (PR), Mantel-Haenszel chi-square test, with statistical significance p < 5%. Result: The 152 individuals evaluated corresponded to 95% of the sample population aged between 6 and 16 years. The study showed 7 individuals with BMI values consistent with obesity, demonstrating an absolute prevalence of 4.6%. The age of obese individuals ranges from 6 to 16 years old, and the average family income reported was R$ 809.16, most of which came from financial Federal Government Programs. Among the risk factors, the association between obesity and family history for obesity, the PR is 0.4 (p = 40%), the PR for males is 4.84 (p = 5.7%) and finally, sedentary lifestyle and obesity have a PR that is below 1 (p = 13%). Conclusion: Little is known about the culture and epidemiological profiles of riverside communities, but with the data collected, it is observed that even in remote areas, cases of obesity are already identified. It is important to develop public policies aimed at these populations, with the objective of improving knowledge about nutrition and health, physical activity and diet, ensuring good nutrition throughout life, promoting health in addition to reducing public spending on health problems. triggered by excess weight. 111718 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH NESLAYNE LOUISE CAMPIOL1, Andreisa Prieb1, Thiago Santos Souza1, Monique Veloso Lima1, Nathalia Freire Gilo1, Maurício Antônio Batista Cavalcante1 (1) Universidade de Gurupi – UNIRG Introduction: Non-transmissible cardiovascular diseases represent a major public health problem worldwide. In Brazil, Acute Myocardial Infarction (AMI) is the leading cause of death in the country, according to DATASUS, and constitutes a process of ischemic necrosis caused by acute obstruction of a coronary artery. Therefore, a greater awareness of the measures for prevention and control of AMI is necessary, in addition to the stratification of cardiovascular risk in the population. Objective: Analyze the epidemiological profile of hospitalizations for AMI among Brazilian regions from 2011 to 2021. Methods: A retrospective, observational, descriptive, cross-sectional study was conducted on data from hospitalizations for AMI, available at DATASUS – SUS Hospital Information System (SIH/SUS), in the period from January 2011 to December 2021, evaluating several variables such as the number of hospitalizations, mean value, mean length of stay, number of deaths and mortality rate. Results: In the period studied, 1,187,982 hospitalizations for procedures to treat AMI were observed, representing a total expenditure of R$ 4,378,770,679.45. The Southeast region recorded 593,441 hospitalizations, corresponding to half of the entire country, followed by 233,365 in the Northeast (19.6%), 232,330 in the South (19.6%), 81,086 in the Midwest (6.8%), and 47,760 in the North (4.02%). The total number of deaths in Brazil was 130,209, being higher in the Southeast with 63,517 (48.8%) and in the Northeast with 28,506 (21.9%). The mortality rate in Brazil was 10.96, with the Northern and Northeastern regions above the national average, 12.22 and 12.04, respectively; and the other regions below the national average, Southeast (10.70), South (10.36), and Center-West (10.32). The national average stay was 7.3 days, while in the North it was 8.2, followed by 7.7 in the Northeast and Southeast, 7.3 in the Midwest, and 5.6 in the South. Concerning the average value of hospitalization, the highest value was in the South, R$4,417.68, followed by the Southeast R$3,695.58, Center-West R$3,503.92, Northeast R$3,194.34, and North R$2,716.46. Conclusions: It is important to emphasize the importance of promotion and prevention in primary health care and early diagnosis to avoid sequelae and reduce the risk of death in patients. In addition, it reinforces the need for correct notification so that the information can help in the development of public health policies. 111726 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH LÉO RODRIGO ABRAHÃO DOS SANTOS1, INGRID PAIVA DUARTE1, ANDRÉ MELO DE FARIA1, MATHEUS SOUZA DE MOURA RIBEIRO1, NATALIA MELO DUARTE DE ALMEIDA1, LUANA TEIXEIRA DA SILVA1, LUIZA FURTADO AREAS1, THATIANA DA SILVA PIRES1, DANIEL ALBUQUERQUE MARTINS ZANIBONE1, THIAGO CAVALHEIRO TAVARES1, LUIS ORLANDO POZES PEREIRA FILHO1, OGI JANDERSON ANTUNES DE CASTRO BRITO1 (1) Unigranrio-Afya, Rio de Janeiro, Brazil Introduction/Objectives: Artificial Cardiac Pacemakers are devices capable of replacing electrical impulses through multiprogrammable stimulation to achieve physiological cardiac electrical activity. They are classified by clinical need as Temporary or definitive, by transcutaneous route, transvenous or thoracotomy, by the types of endocavitary and epicardial cable, single chamber stimulated chambers, double, triple or the four chambers. The study aims to know epidemiologically the population that underwent pacemaker implantation in thecity of Rio de Janeiro. Materials/methods: The collection of data was made in the hospital information system of the municipality of Rio de Janeiro from January 2010 to December 2020, researching race, age, type of pacemaker performed, hospitalizations, mortality and expenses. The present study is observational, descriptive and cross-sectional. Results: The study found records of 5031 patients who underwent the procedure, 4063 (81%) were Transvenous Double Chamber Pacemakers, 564 (11%) of Transvenous Single Chamber, 219 (4%) of Transvenous Multi-site, 73 (2%), of Epimyocardial Double Chamber, 56 (1%) Temporary Transvenous, 30 (1%) of Single Epimyocardial Chamber, 21 (<1%) of Multi-site Epimyocardial by Thoracotomy for Thoracotomy for electrode implant and 5 (<1%) multi-site Endocavitary with reversal to epimyocardial. The majority were attended at the Instituto de Cardiologia Aloysio de Castro (IECAC) which totaled 1524 (30.3%) procedures. 3238 (64%) performed the implant electively. 73% were over 65 years old. 44% were White. 2547 (51%) were Men. The average length of hospital stay was 5.44 days and an average expenditure of US$ 2,552. In addition, 2499 (49.7%) had a daily ICU with an average expenditure of US$ 3,125. The most expensive procedure was the Transvenous Multi-site pacemaker with an average cost of US$ 5875.6. There were 84 deaths in general, 61% in Transvenous Double Chamber Pacemakers, the highest mortality rate was found in patients who underwent temporary transvenous pacemaker implantation with 25% of deaths. Discussion/Conclusion: Patients undergoing pacemaker implantation have a profile mainly of elderly males, whites who perform Transvenous Double Chamber Pacemakers mostly in high complexity cardiac hospitals. These data, in addition to characterizing users, show how costly, complex and common the use of these devices is, and the need for investment in cardiology services in the city. 111746 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH IGOR LUCAS FARIAS LIMA1, Luciano Moura de Assunção3, Giulia Vitoria Nascimento da Silva1, Ariane Lobato Moraes2, Marcos Aurélio Vieira da Costa Filho2, Wanda Maria de França Pires2, Larissa Dacier Lobato Comesanha2, Júlia de Moura Carvalho Faria1 (1) UNIVERSIDADE DO ESTADO DO PARÁ – UEPA; (2) UNIVERSIDADE FEDERAL DO PARÁ – UFPA; (3) FUNDAÇÃO SANTA CASA DE MISERICÓRDIA DO PARÁ Introduction: Chronic rheumatic heart disease (RHD), a sequel of rheumatic fever (RF), is caused by lesions that mainly affect mitral and aortic valves, and late diagnosis results in major complications such as heart failure, pulmonary hypertension and atrial fibrillation¹. The primary prevention of RHD consists of the diagnosis and treatment of streptococcal pharyngitis and secondary prevention in long-term antimicrobial prophylaxis in individuals with a previous diagnosis of RF². Objective: To assess the prevalence and analyze the epidemiological profile of RHD hospitalizations in the Northern Region of Brazil from 2017 to 2021. Methods: Observational, cross-sectional, descriptive and quantitative study based on data from the SUS Hospital Information System (SIH/SUS) – DATASUS. The variables analysed were: number of hospitalizations, age, sex, average hospital stay, average cost per hospitalization and mortality rate, referring to hospitalizations due to RHD in the Northern region of Brazil between 2017 and 2021. Results: In the analyzed period, 1744 hospitalizations for RHD were reported in the Northern Region of Brazil, with the highest number of hospitalizations in the state of Pará (697). In the North Region, the largest amount occurred in 2017 with 468 cases, with a gradual decrease until 2021 with 246 hospitalizations. Regarding gender, 51.15% of the hospitalized patients were female and 48.85% were male. The most affected age groups were 50 to 59 years old, with 308 (17.6%) notifications, and 40 to 49 years old, with 297 (17%) cases. The average length of stay in 2017 to 2021 was 13.1 days, being highest in Rondônia (15.1 days) and lowest in Roraima (7.3 days). In the North region, the lowest average time (11.1 days) occurred in 2020 and the highest (14.7 days) in 2017. The mortality rate was 7.91%, being the lowest in 2018 (6.14%) with gradual growth until 2021 (10.16%). The state with the highest mortality rate was Roraima (28.57%) and the lowest was Amazonas (6.53%). A total of R$16,878,115.65 was spent in the considered period, with an average cost of R$9,677.82 per hospitalization. Conclusion: The study reports that the highest number of hospitalizations occurred in 2017, mostly in the state of Pará. Mortality rate due to RHD is rising and the highest rate comes from Roraima. In addition, the most affected age group was 50 to 59 years, and there was no significant difference in prevalence between genders. 111751 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM LUANA LIVELLI BECKER1, Kletey Mendes Silva1, Odilson Marcos Silvestre1, Miguel Fernandes da Silva Morita2, Dhayn Cassi Freitas1, Roberta Gabriela da Silva1, Laís Vitória de Andrade Miranda1, Sabrina da Silva Medeiros1, Emanuel Silva de Abreu1, Jéssica Borsoi Maia do Carmo1, Leonardo Buranello1, Wilson Nadruz3 (1) Federal University of Acre; (2) Federal University of Parana; (3) University of Campinas Introduction: The impact of COVID-19 infection in patients with heart disease remains little explored. Object: To investigate the incidence of physical inactivity in individuals with heart disease after 12 months of SARS-CoV-2 infection. Methods: We prospectively evaluated 837 individuals who practiced regular physical exercise infected with SARS-CoV-2 in Rio Branco, Acre. Sociodemographic information, diseases of interest (cardiopathies, hypertension and diabetes mellitus) and details about the acute phase of the disease were collected after COVID-19 infection. In a second 12-month follow-up questionnaire, participants were asked about their physical exercise. To identify independent predictors of physical inactivity, multivariate logistic regression with stepwise strategy was used. Results: The mean age of the sample was 39 ± 11 years, 54% were male. A total of 14 participants were identified with heart disease (cardiac arrhythmia, n = 7; heart failure, n = 5; congenital heart disease, n = 1; third-degree atrioventricular block and pacemaker, (n = 1) and 42 participants with diabetes mellitus. A total of 243 (29%) participants were inactive after 12 months of SARS-CoV-2 infection. Compared to individuals without heart disease, those with heart disease had a higher occurrence of physical inactivity after 12 months of COVID-19 (28% vs 71%, respectively). After adjusting for multiple confounders, heart disease, diabetes mellitus, and female gender were predictors of long-term physical inactivity (figure 01). Conclusion: After 12 months of infection by SARS-CoV-2, 71% of patients with heart disease did not practice physical exercises. Female sex, diabetes mellitus and heart disease were independent predictors of long term physical inactivity. 111752 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION RAIMUNDO BENÍCIO DE VASCONCELOS NETO1, Gabriel Irismar Rodrigues Schwamback1, Rebecca Shaiane Soares Nunes Rivoredo1, Maria Eduarda Brotto de Souza1, Archimedes Fernandes Alves de Santana1, Julia Maria de Lourdes Balsan1, Sofia dos Santos Souza1, Renata Gonçalves Silva Santos1, Izabella Gurgel do Amaral Pini1, Gesanaje da Paz Carvalho1, Fernanda Gabry Scazuza Gomes de Souza1, Byron Maia Feitosa1 (1) Centro Universitário São Lucas- UNISL Introduction: Characterized as Chronic Non-Communicable Diseases (CNCD), Systemic Arterial Hypertension (SAH) and Diabetes Mellitus (DM) affect a large part of the Brazilian population, even if undiagnosed. Despite having different mechanisms of action, when associated, these CNCD considerably increase the risk of developing cardiovascular and renal pathologies. Therefore, assessing the prevalence of these comorbidities is necessary to establish intervention and management strategies for the population, especially in remote communities. Objectives: To estimate the prevalence of the association of Systemic Arterial Hypertension and Diabetes Mellitus in individuals living in a riverside community in the Western Amazon, Brazil. Methods: This is a cross-sectional, quantitative, retrospective study carried out with individuals aged between 1 and 87 years, regardless of gender, living in a riverside region in Porto Velho, Rondônia, Brazil. Home visits were carried out in which the residents answered an individual epidemiological questionnaire, each form had the information tabulated. Results: 268 individuals were randomly selected for the study, of which 55 (20.52%) had informed SAH, 26 (9.7%) had informed DM and 23 (8.58%) claimed to have both associated chronic non-communicable diseases. Females had the highest prevalence of the association between Systemic Arterial Hypertension and Diabetes Mellitus, with 14 cases (60.86%). The average age of individuals with both chronic non-communicable diseases is 58 years old. Conclusion: Systemic Arterial Hypertension and Diabetes Mellitus are the most frequent non-communicable chronic comorbidities in the world population. Nevertheless, the significant prevalence of these health conditions in the Amazon riverside population observed in this study reinforce the importance of monitoring cardiovascular health and associated risk factors. 111763 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION CLARISSA MARIA TITO BELTRÃO1, Waléria Dantas Pereira Gusmão2, Alana Costa Machado Gomes1, Fernanda Abraham Leão1, Silvia Dandara Coutinho de Souza Lins Machado1, Giulia Abraham Leão1, Isabele Rejane de Oliveira Maranhão Pureza1, Annelise Machado Gomes de Paiva1, Marco Antonio Mota Gomes1, Claudia Roberta de Castro Moreno2 (1) Centro Universitário CESMAC; (2) Universidade de São Paulo Introduction: Blood pressure (BP) oscillations are regulated by neural, endocrine, endothelial and hemodynamic pathways in a circadian pattern. Although environmental and behavioral phenomena can mask the circadian rhythm of BP, circadian variability has higher levels in the morning, a plateau throughout the day and lower values at night during sleep. Poor quality or sleep deprivation can potentiate greater insulin resistance, chronic inflammatory processes, increased cortisol and catecholamines and exacerbation of stress, can disorganize the structure of the vascular wall, which can increase pressure. Objective: To determine if there is an association between subjective sleep quality and BP values. Methods: This is a cross-sectional study, carried out in a cardiology outpatient clinic, from Sep. to Dec. 2021, in which sociodemographic and clinical data were collected and a visual analogue sleep quality scale was applied through a structured questionnaire. Pressure values were estimated using an Arteris-AOP® oscillometric device. Results: 133 adults (19–89 years, mean 53.49 ± 14.01 years) were included in the study, 77.44% were female and 22.56% were male, the mean body mass index (BMI) was 29.30 ± 4.60 Kg/m2 (18.7–42.02 Kg/m²), the mean peripheral BP values were 128.51 ± 15.91 (99–181) mmHg of systolic BP, 83.05 ± 9.74 (62–115) mmHg of diastolic BP and 45.46 ± 13.55 (25–94) mmHg of pulse pressure. The BP central mean values were 118.63 ± 14.25 (116.19–121.08) mmHg of systolic BP, 84.62 ± 9.79 (64–116) mmHg of diastolic BP and 31.04 ± 11.25 (32.08–35.94) mmHg pulse pressure. To analyze the association between subjective sleep quality and peripheral and BP central values, linear regressions were performed with adjustment for age, sex and BMI. Subjective sleep quality was inversely proportional to systolic BP (β: –0.16; 95%CI: –2.76; –0.05; R2 = 0.19) and diastolic BP (β: –0. 28; 95%CI: –2.36; –0.59; R2 = 0.08), as well as systolic centrals BP (β: –081; 95%CI: –2.58; –0.20; R2 = 0.22) and diastolic centrals BP (β: –0.278; 95%CI: –2.35; –0.57; R2 = 0.08) with no effect on peripheral or central pulse pressure. The findings indicated an association between subjective sleep quality and peripheral BP levels in the studied population, that is, the worse the sleep quality, the higher the BP, constituting a promising area for study. Conclusion: Hypertensive patients should investigate sleep quality, since there is an association between poor sleep quality and high BP. 111932 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH VANDO DELGADO DE SOUZA SANTOS1, Saul Rassy Carneiro1 (1) Universidade Federal do Pará – UFPA Introduction: Chronic Rheumatic Heart Disease (RHD) is a sequela of rheumatic fever, which is a complication of pharyngotonsillitis caused by Streptococcus pyogenes and results from a late immune response to this infection in genetically predisposed populations, having a direct relationship with the development of services. of health in the country. All the financial resources made available to the Unified Health System are made directly by the National Health Fund (NHF) for each Federation Unit. Objectives: To relate the total financial transfers from the NHF and the amount of the Family Health Strategy (FHS) with the number of hospital admissions for RHD in northern Brazil from 2011 to 2020. Methods: This is a descriptive longitudinal epidemiological study based on secondary data from the Department of Informatics of the Unified Health System, the Portal of the National Health Fund of the Federal Government and the Portal of the Secretariat of Primary Health Care. RHD hospital morbidity and mortality data were correlated with the financial resources allocated to each FU and with the total number of FHS in each FU. Results: In the analyzed period, there were a total of 3.595 hospitalizations for RHD and a total of 336 deaths in the northern region, that is, a mortality rate of 9.35%. The state of Pará had the highest rates of hospitalizations (38.58%), followed by the state of Amazonas (32.54%), and the state of Roraima had the lowest rates (0.39%). In the same period, Pará and Amazonas were the ones that received the most financial transfers and had the largest number of FHS, and Roraima was the state that received the least financial resources and had the lowest number of FHS. Conclusion: The study showed that the greater the availability of NHF resources for the FU and the greater the number of FHS, the greater the hospitalization rates and, consequently, the mortality rate, this is due to the greater screening of heart diseases and greater access to health. 111777 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY SARA CRISTINE MARQUES DOS SANTOS1, Maria Luiza Silva Barbosa1, Alice Machado de Sales Silva1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: Myocardial revascularization surgery (CABG) is one of the treatments for obstructive atherosclerotic coronary artery disease accompanied by myocardial ischemia. The procedure can be performed with or without cardiopulmonary bypass (CPB). The present study aimed to analyze the current panorama of CRM procedures, with and without CPB and use of 1 or more grafts performed in the city of Vassouras for 10 years and correlate the current epidemiology with the results obtained. Methods: A systematic literature review and observational, descriptive and cross-sectional data collection of myocardial revascularization procedures, available at DATASUS – SUS Hospital Information System (SIH/SUS) for a period of ten years – December 2008 to December 2018 – evaluating the value of public spending, complexity, mortality rate, deaths, permanence, and character of care and articles available in Scielo, Lilacs, and PubMed. Results: In the analyzed period, 306 hospitalizations were observed for the performance of myocardial revascularization procedures, representing a total expenditure of R $ 3,228,341.33, with 2010 being the year with the highest number of hospitalizations (80) and 2011 being the year responsible for the largest amount spent during the period (R $ 687,969.79). Of the total procedures, 162 were performed on an elective basis and 144 on an urgent basis, with 306 being considered highly complex. The total mortality rate in the 10 years studied was 10.46, corresponding to 32 deaths, 2016 being the year with the highest mortality rate, 18.18, while 2012 had the lowest rate, 4.00. The mortality rate for elective procedures was 9.26 compared to 11.81 for urgent procedures. The average total hospital stay was 14.2 days. Conclusions: It can be seen, from the present study, that CABG is a procedure considered to be highly complex, regardless of whether it is performed urgently or not. A progressively considerably high mortality rate has been identified. Also, the need for correct notification of procedures is highlighted, aiming to improve the current epidemiological analysis. 111780 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION BIANCA DE FÁTIMA PEREIRA1, Ramon José Moreira Silva1, Beatriz Stephan Farhat Jorge1, Pedro Drumond Maia1, Fernanda Silva Mota1, Gabriela Godinho Rezende1, Moisés de Toledo Vilela1, Paula Gouvea Abrantes1, Marcela Rabelo1, Hélio Brito de Lima Júnior1, Eliane Ferreira Carvalho Banhato1, Arise Garcia de Siqueira Galil1 (1) Cardiology Department, Medical School, Federal University of Juiz de Fora, Juiz de Fora – Minas Gerais. Introduction: Smoking is a preventable and controllable chronic disease, in addition to high morbidity and mortality. Encouraging smoking cessation is a priority in Public Health and represents a cost-effective intervention. Objective: To describe the profile of smokers assisted by a treatment group for smoking cessation. Methodology: Longitudinal study, “Livres do Tabaco” group. Users with multimorbidities were assisted between September/2021 and March/2022, in consecutive treatment groups, mixed intervention (face-to-face and telemedicine), multidisciplinary team, behavioral cognitive approach sessions, drug treatment and follow-up. Definitions: Low level of schooling, <8 years. Normal systolic blood pressure (SBP) <130 mmHg. Depression, score Patient Health Questionnaire (PHQ-9) ≥9 points. High risk for Obstructive Sleep Apnea Syndrome (OSAS), Stop Bang questionnaire ≥5 points. Abusive use of alcohol, Audit-C ≥5 points. Cognitive deficit, Montreal Cognitive Assessment <26 points. High nicotine dependence, Fagerstrom test ≥5 points. Results: Sample with 36 patients, 6 consecutive treatment groups. Age, 56.6 ± 10.8 years; elderly, 44%; female, 66.70%; low level of schooling, 42.9%. Dyslipidemia, 52.90%; sedentary lifestyle, 61.5%; family history of coronary artery disease, 88.20%. Arterial hypertension, 68.6%; coronary insufficiency, 25.7%; diabetes mellitus, 17.1%; chronic obstructive pulmonary disease; 31.4%; previous history of cancer, 8.6%. High risk for OSAS, 31.4%, depression, 55.90%, cognitive impairment, 79.4%, alcohol abuse, 20%. Daily consumption of 17.9 ± 12.2 cigarettes; pack-years, 40.7 ± 29.3; high nicotine dependence, 68.6% and 55.6%, heavy smokers. Smoking triggers, stress, 85.7%; coffee, 60% and alcohol intake, 48.6%, were the most frequent. Smoking cessation until the 12th week of accumulated treatment, 33.3%. Conclusion: The initial data showed a high prevalence of both risk factors and comorbidities that most increase morbidity and mortality worldwide. It was also found a high prevalence of women, depression, cognitive impairment, in addition to high nicotine dependence and heavy smokers, data considered as weaknesses within the smoking cessation process. Even so, smoking cessation was observed to be within the average for smokers without associated comorbidities. 111783 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION GUSTAVO PEDROZO1, Sérgio Eduardo Soares Fernandes1, Pedro Henrique Santos de Medeiros1 (1) Escola Superior de Ciências da Saúde ESCS Introduction: Cardiac auscultation as a semiological tool has been widespread for more than four centuries, but only recently, with the popularization of computer processors, some of them with GPIO (General Purpose Input/Output) interfaces, it was possible to bring technological evolution to low-cost research centers. Objectives: To develop a low-cost cardiac and pulmonary audio signal capture prototype for future tests with continuous monitoring. Method: A long literature review was carried out about concepts of acoustics, stethoscope chest-piece modelling, sound signal capture and articles with similar prototypes of digital stethoscopes. Then, a chest-piece was built with Autodesk Inventor® software and printed with antimicrobial PLA (Polylactic Acid) filament. The prototype was assembled with a digital microphone INMP441® connected to a raspberry® pi 3b board (figure 1). The signal capture was performed using Python 3.10.4 code with pyaudio and mathplot libraries. The tests were carried out exclusively among the research team and validation tests about the method have not yet been carried out until this submission date. Results: The prototype was able to capture satisfactorily cardiac and pulmonary signals which were converted into phonograms (figure 1). The cost of materials used with the initial prototype was $67.32 including taxes and shipping. The cost proved that the reproduction of this prototype is viable for future larger tests. Likewise, it was possible to create new conceptual prototypes with potential for significant cost reduction, which will be produced in later phases of the study. Conclusion: The prototype allowed the analysis of production feasibility, however, to proceed the project with validation of methods for continuous monitoring, safety studies will still be necessary and, as well, the development of a continuous analysis code with intelligence to alert critical findings. This study allowed not only the production of a viable prototype, but also perspectives of cost reduction and the means for the continuity of the project with the validation of a monitoring system by auscultation. 111869 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KARLA SANTOS PINTO1, Tainá Viana1, Raissa Barreto Lima1, Ana Luísa de Aguiar Almeida Silva1, Eduarda Luciana Mendes Borges1, Larissa Xavier Gomes da Silva1, Aurea Maria Lago Novais1, Luiz Carlos Passos1, Mariana Baraúna da Silva1, Elias Soares Roseira1, Karla Santos Pinto1, João Pedro Martins Moreira Granja1 (1) Hospital Ana Nery Introduction: Heart failure (HF) is a complex and highly prevalent clinical syndrome that limits the quality of life of many of its patients. Patients with heart failure reduced ejection fraction (HFrEF) are subject to higher mortality, given the potential reduction in cardiac function. The objective of this study is to describe the clinical and epidemiological profile of patients with HFrEF in a reference hospital in Salvador-BA. Methods: This is a prospective cohort that included patients with HFrEF treated at a heart failure outpatient clinic of a cardiology referral hospital in Salvador from September 2020 to July 2021. The outcome variables evaluated were mortality and hospitalization for any cause in 1 year. Variables with nonparametric distribution were described by median and interquartile range and categorical variables were described as frequency and percentage. Results: We included 196 patients with HFrEF with a median age of 60.5 (49–68) years, most of them men (54.1%), and a median LVEF of 29% (23–35). Ischemic and chagasic etiologies were the most prevalent, with 46 (23.5%) and 40 (20.4%) patients, respectively. Among the associated comorbidities, arterial hypertension (63.8%) and diabetes mellitus (30.6%) were the most observed. As for the medications in use, 93.9% of the patients used beta-blockers, 74% used ACEI/ARB, 13.3% used sacubitril/valsartan and 75% used spironolactone. After a 1-year follow-up, it was found that 25 (12.8%) patients died and 50 (25.5%) were hospitalized during the period. In the HF grading by the New York Heart Association (NYHA), 57 (34.5%) patients belonged to functional classes III-IV. Conclusion: The present study showed that the prevalence of HFrEF in the studied population reflects a considerable degree of hospitalization and death. It is expected that the results found can improve health policies that help in the care of patients with HFrEF, as well as in the prevention of unfavorable outcomes. 111788 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANNA LUIZA ALVES DE OLIVEIRA MIRANDA1, Matheus Vinícius Mourão Parente1, Daniel Chagas Barreto1, José Pedro da Silva Sousa1, Beatriz Siems Tholius1, José Wilker Gomes de Castro Júnior1, Cláudio Eduardo Corrêa Teixeira1, Fabíola de Carvalho Chaves de Siqueira Mendes1 (1) Centro Universitário do Estado do Pará (CESUPA) Introduction: CardioVascular Diseases (CVD) are a public health problem, representing more than 30% of all deaths worldwide. In this context, evidencing the prevalence of the main diseases associated with CVDs and the profile of these patients is necessary, in order to guide the management of public health services that receive this demand. Objectives: In the present study, we aimed to highlight the prevalence of diseases and factors associated with CVDs in the population fraction admitted, via the Brazilian Unified Health System (Serviço ùnico de Saúde, SUS), at the Medical Specialties Center of the Pará State University Center, in the city of Belém (Brazilian Amazon). Methods: A cross-sectional study was carried out based on the analysis of the medical records of patients seen at a Cardiology outpatient clinic for a period of 1 year. Results: Of 202 patients seen at the outpatient clinic, it was observed that 68.3% were female. Regarding the age group, the group between 55 and 64 years old predominated (29.2%), followed by those over 65 years old (28.7%). The main complaint reported by patients was chest pain (11.5%), followed by dyspnea (10.9%) and Systemic Arterial Hypertension (SAH, 10.45%). In addition, physical inactivity (23.8%), smoking (18.1%) and alcohol consumption (16%) were observed, which are important risk factors for CVD. Regarding family history, SAH (35%) stood out, followed by cancer (17.3%), Diabetes Mellitus (15.22%), Stroke (12.75%) and Acute Myocardial Infarction. (10.3%). Consistently and as expected from the results cited, the most common diagnoses among these patients were: SAH (31.8%), DM2 (7.7%) and Dyslipidemia (7.3%). The most prescribed drugs were Losartan (18.6%), Hydrochlorothiazide (13.3%) and Simvastatin (9.9%). The main tests requested were the echocardiogram (9.5%) and electrocardiogram (5.4%). Conclusion: We conclude that the observed frequency of diseases and factors associated with the development of CVDs is of significant prevalence, and such data should be valued and used as support tools in the management of public health services, especially in primary care. 111792 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GABRIEL RIBEIRO DE SOUZA1, Mayuri Akemi Rodrigues Higashi1, Rafaela Andrade Penalva Freitas2 (1) Universidade de Santo Amaro; (2) Instituto Dante Pazzanese de Cardiologia Introduction: In Brazil, arterial hypertension has a high prevalence and low control rate, being a risk factor for cardiovascular diseases, which implies large public health expenses related to hospitalizations resulting from complications. Objective: To quantitatively analyze the hospitalizations resulting from hypertensive syndromes in the city of São Paulo in the year 2021. Methodology: It consists of a descriptive, retrospective, longitudinal epidemiological study, based on the analysis of a econdary source of data, with the aim of the studies being hospital admissions in the Unified Health System of the city of São Paulo, in the year 2021, for causes related to diseases. Hypertensive as inclusion criteria, all hospital admissions were selected through the Hospital Information System (SIH), through DATASUS. The period studied was the year 2021. The morbidities were separated and selected according to the main hospitalization diagnosis and classified according to the International Disease Code 10 (ICD10), the following being selected: I10 – Essential hypertension, I11 – Hypertensive heart disease, I12 – Hypertensive kidney disease, I13 – Hypertensive heart and kidney disease, I15 – Secondary hypertension. Gender, race/color and age group were also taken into account. Results: During the study period, 2219 hospitalizations were performed for hypertensive diseases, of which 49.2% were male and 50.8% female. Among the target audience of the study, when considering race/color, 35.8% of patients consider themselves white, 8% black, 29.5% brown and 0.2% yellow. 26.5% of the registered admissions had no description of race/color. Regarding the age groups studied, there is a gradual increase in the number of hospitalizations as the observed age group also increases, with the group of patients over 80 years of age having the highest number of hospitalizations, corresponding to 14% of all the hospitalizations. Conclusions: Hospitalizations in the city of São Paulo due to hypertensive syndromes were slightly higher in males, most of whom were white and aged over 80 years. 111842 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING LARA TELES ALENCAR DUARTE1, Cláudia Bispo Martins-Santos1, José Icaro Nunes Cruz1, Allexa Gabriele Teixeira Feitosa1, Gabriela de Oliveira Salazar1, Cleovaldo Ribeiro Ferreira Júnior1, Danilo Brito Silva de Oliveira1, Victor Ravel Santos Macedo1, Eduardo Jose Pereira Ferreira1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe (UFS) Introduction: Considering the epidemiological relevance of Chronic Coronary Syndrome (CCS) and Acute Myocardial Infarction (AMI), studies that statistically associate the two groups with their main outcome, Myocardial Ischemia (MI), are still rare. Objective: To assess the occurrence of MI related to the presence or absence of previous AMI, among patients with CCS undergoing Exercise Stress Echocardiography (ESE). Methods: A cross-sectional study between January 2000 and January 2022 with individuals aged 40 years or older and with a positive history of CCS who underwent ESE at a cardiology referral service in Sergipe. A total of 1956 patients (X̅ = 62 ± 9.6 years) were categorized according to the presence or absence of a history of previous AMI. Student’s t test and chi-square test were used. A significance level of 5% was assumed. Analyzes were performed using SPSS Statistics software. Results: 735 patients (37.6%) with previous AMI and 1221 patients (62.4%) without the ischemic event were evaluated. There was no difference between the mean age of the groups. Male sex was associated with previous AMI (75.9% vs. 65.7%; p < 0.001), as well as diabetes (23.6% vs. 19.9%; p = 0.05) and dyslipidemia (76.7% vs. 71.2%; p = 0.008). On the other hand, statin use was positively associated with the group of patients without previous AMI (80.6% vs. 59.9%; p < 0.001). When the ESE results were analyzed, there was an association between MI (ischemia, fixed ischemia or fixed and induced ischemia) with the presence of previous AMI (71.8% vs. 41.4%; p < 0.001). However, an association was found between the result of new ischemia (ie, induced) and the group with no history of previous AMI (14.7% vs. 7.2%; p < 0.001). There was no significant difference between the distributions of: obesity (p = 0.207), arterial hypertension (p = 0.12), family history (p = 0.159), use of oral antidiabetics (p = 0.283) and the presence of symptoms (p = 0.261). Conclusions: MI in CCS patients was associated with patients with previous AMI in a fixed and fixed and induced manner, while new ischemia was associated with patients with no history of AMI. The relevance of statins as a protective factor for MI is also confirmed. 111802 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES SARA CRISTINE MARQUES DOS SANTOS1, Maria Luiza Silva Barbosa1, Alice Machado de Sales Silva1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: The hypertensive crisis is the rapid increase in systemic arterial pressure, which can occur in people with systemic arterial hypertension or those with normotension, potentially complicated with target organ damage. Divided into two categories, such as hypertensive urgency and hypertensive emergency. The present study aimed to analyze the current panorama of hypertensive crisis treatment procedures performed in the city of Vassouras for 10 years and to correlate the current epidemiology with the results obtained. Methods: A systematic review of the literature and an observational, descriptive and cross-sectional collection of hypertensive crisis treatment data, available at DATASUS – SUS Hospital Information System (SIH/SUS) for a period of ten years – December 2008 to December 2018 – evaluating the value of public spending, complexity, mortality rate, deaths, permanence, and character of care and articles available in Scielo, Lilacs, and PubMed. Results: In the analyzed period, there were 213 hospitalizations for the performance of hypertensive crisis treatment procedures, representing a total expenditure of R $ 82,593.96, 2009 being the year with the highest number of hospitalizations (66) and 2009 the year responsible for the highest amount spent during the period (R $ 28,385.62). Of the total procedures, 6 were performed on an elective basis and 207 on an urgent basis, with all 213 considered to be of medium complexity. The total mortality rate in the 10 years studied was 1.41, corresponding to 3 deaths, with the years 2008 and 2014 having the highest mortality rate, 9.09, while the year 2009 had the lowest rate, 1,52. The mortality rate for elective procedures was 0 compared to 1.45 for urgent procedures. Death cases covered only the years 2008, 2009, and 2014, with 1 death each. The average total hospital stay was 5.7 days. Conclusions: Low mortality was demonstrated, with seven cases in 10 years analyzed. It is worth noting the greater occurrence of hospitalizations on an urgent basis, highlighting the need for primary and secondary prevention, in addition to investing in early recognition by the patient. It is important to have correct notification of the procedures, aiming to improve the current epidemiological analysis. 111803 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION LARISSA REBECA DA SILVA TAVARES1, Larissa Rebeca da Silva Tavares1, Claudia Bispo Martis-Santos2, Irlaneide da Silva Tavares2, José Icaro Nunes Cruz2, Gabriela de Oliveira Salazar2, Silvia Regina Freire Oliveira Melo3, Lais Oliveira Melo1, Stephanie Macedo Andrade4, Antônio Carlos Sobral Sousa2, Joselina Luzia Menezes Oliveira2, Diego Henrique da Silva Tavares1 (1) Universidade Tiradentes – UNIT; (2) Universidade Federal de Sergipe – UFS; (3) Hospital Primavera; (4) Hospital São Lucas Introduction: The metabolic equivalent intensity levels (MET) are a way of expressing cardiorespiratory fitness (CRF). High risk cardiovascular profiles are related to low aerobic capacity. Objective: To evaluate low cardiorespiratory fitness under stress during Physiological Stress Echocardiography (PSE). Methods: Transversal study between January 20000 and January 2022 with individuals who have been through PSE at a cardiology reference service. 3894 Patients were categorised (57 ± 11 years) under low (MET < 7,9), intermediary (7,9 ≤ MET < 10,9) or high (MET ≥ 10,9) CRF. The chi-square test and multinomial logistic regression were used with the SPSS Statistics software version 22.0. Admitting a significance level of 5%. Results: In the sample, 56.5% (2202) were female; 25.7% (1002) had low CRF, 33.5% (1306) intermediate and 40.7% (1586) high CRF. The comparison between high and low CRF groups show that older age (OR = 1.126; 95%CI = 1.113–1.139; p < 0.001), arterial hypertension (OR = 1.886; 95%CI = 1.501–2.369; p < 0.001), diabetes mellitus (OR = 1.447; 95%CI = 1.043–2.007; p = 0.027), obesity (OR = 1.936; 95%CI = 1.496–2.506; p < 0.001), sedentary lifestyle (OR = 3.18; 95%CI = 2. 55–3.966; p < 0.001), alcoholism (OR = 1.781; 95%CI = 1.387–2.286; p < 0.001) and segmental alteration during exertion (OR = 2.127; 95%CI = 1.562–2.896; p < 0.001) were predictors of low CRF. When compared to the intermediate CRF group, older age were predictors of low CRF (OR = 1.069; 95%CI = 1.058–1.08; p < 0.001); sedentary lifestyle (OR = 1.614; 95%CI = 1.32–1.975; p < 0.001), alcoholism (OR = 1.499; 95%CI = 1.177–1.908; p = 0.001), obesity (OR = 1.35; 95%CI = 1.076–1.694; p = 0.009) and segmental alteration during exertion (OR = 1.526; 95%CI = 1.168–1.996; p = 0.002). Male sex protected against low CRF in comparisons to the intermediate (OR = 0.491; 95%CI = 0.390–0.618; p < 0.001) and high cardiorespiratory fitness (OR = 0.164; 95%CI = 0.128–0.209; p < 0.001) groups. Conclusion: When evaluating patients with low CRF, older age, sedentary lifestyle, obesity, alcohol consumption and segmental alteration on exertion were predictors. The predictive model also showed that physical inactivity increased the chance of low CRF by three times, being the main predictor. 111809 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY SARA CRISTINE MARQUES DOS SANTOS1, Maria Luiza Silva Barbosa1, Alice Machado de Sales Silva1, Ivana Picone Borges de Aragão1 (1) Universidade de Vassouras Introduction: The defibrillator cardioverter implantation is intended to diagnose and treat rhythmic heart changes, preventing death. It is a minimally invasive and fast recovery procedure. The objective was to analyze the current panorama of single-chamber and transvenous double-chamber defibrillator implant procedures performed in Brazil and correlate the current epidemiology with the results obtained. Methods: A literature review and observational, descriptive, and cross-sectional collection of single-chamber and transvenous double-chamber cardioverter implant data, available at DATASUS – SUS Hospital Information System (SIH/SUS) – December 2008 to December 2018. Results: In the period analyzed, there were 10,736 hospitalizations for performing single-chamber and transvenous double-chamber cardioverter implantation procedures, representing a total expense of R$ 428,781,939.15, 2018 being the year with the highest number of hospitalizations (1,238) and the highest amount spent (R$ 54,823,826.53). The total mortality rate in the 10 years studied was 0.46, corresponding to 49 deaths, with 2008 being the year with the highest mortality rate, 3.17, while 2018 had the lowest rate, 0.24. The region with the largest number of hospitalizations was the Southeast with 4,837 hospitalizations, followed by the South with 2,570, the Northeast with 1,728, the Midwest with 1,434, and, finally, the North with 167 hospitalizations. The region with the highest number of deaths was the Southeast with 22 cases, while the North region had the lowest number, with 3 registered deaths. The northern region had the highest mortality rate (1.80) and the southern region had the lowest rate, with a value of 0.35. Conclusions: It can be observed from the present study that this is a procedure with a considerably low mortality rate with a progressive reduction in the period of 10 years from 2008. It is worth noting that the northern region despite having the lowest incidence of performing the procedure has the highest mortality rate. 111813 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LUIZ FELIPE SILVA MARCIÃO1, Mateus Itiro Tamazawskas Otake2, Micaella Yanne Fender Lobato2, Leonardo Barbosa Duarte2, Nyara Rodrigues Conde de Almeida3, Victor Matheus Mendonça de Araújo2, Rafael Silva Lemos2, Lívia Guerreiro de Barros Bentes2, Danusa Neves Somensi2, Herick Pampolha Huet de Bacelar2, Edmundo Frota de Almeida Sobrinho2, Deivid Ramos dos Santos2 (1) Centro Universitário do Estado do Pará; (2) Universidade do Estado do Pará; (3) Universidade Federal do Pará Introduction: The most serious representative of acute coronary syndromes is acute myocardial infarction, which is an extremely serious complication caused by the interruption of blood flow to the myocardium. Therefore, the therapeutic objective is arterial clearance and cardiac reperfusion; for this, there is the use of fibrinolytic and/or angioplasty, the therapeutic methods can be used exclusively or associated, depending on the patient’s clinic and the time of Evolution. Objective: To analyze the mortality rate between Coronary Angioplasty and Catheter-Directed Thrombolysis with fibrinolytic procedures in Brazil in the period 2011–2021. Methods: It is characterized as descriptive, quantitative, and retrospective performed by collecting data from the Hospital Information System of the Unified Health System (SIH/SUS-DATASUS) during May 2022. Data were organized according to the procedure performed in the Microsoft Excel program. Results: In the period from 2011 to 2021, 33.867 Coronary Angioplasty (CA) surgeries were performed, with the Southeast region having the largest number of procedures performed (41.6%), in addition to an average annual growth rate (AAGR) of 3.4% between 2011–2016 and an annual decrease of 2.2% between 2016–2021. Furthermore, 2.179 deaths were associated with the procedure, and the mortality rate (MR) was 6.42 ± 1.32. For the Catheter-Directed Thrombolysis with fibrinolytic (CDT), 1168 were performed between 2011 and 2021, with an AAGR of 26.8% between 2011–2016 and 1.7% between 2016–2021, and the majority in the Southeast (51.7%). There were 49 deaths recorded in this period and an MR of 4.17 ± 2.11. Therefore, for CA, the number of deaths in 2011 was 126, in 2016 it was 205, and in 2021 it was 243, with an AAGR of 10.2% between 2011–2016 and 3.5% between 2016–2021. Regarding CDT, for the same variable, 3 records in 2011, 6 in 2016, and 2021 were 7 deaths, with an AAGR of 14.9% between 2011–2016 and 3.1% between 2016–2021. Both procedures showed an increase in deaths in the number of deaths vs a number of procedures, with emphasis on the correlation made in the CA, where the increase did not follow the decrease of the last five years, in addition to the fact that the MR was higher in the CA compared to CDT. Conclusion: The Southeast region had the highest number of CA and CDT procedures performed in the period evaluated. In addition, a higher MR was observed for CA when compared to CDT and a decreasing AAGR between 2016–2021. 111815 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR IMAGING LARA TELES ALENCAR DUARTE1, José Icaro Nunes Cruz1, Cláudia Bispo Martins-Santos1, Allexa Gabriele Teixeira Feitosa1, Gabriela de Oliveira Salazar1, Cleovaldo Ribeiro Ferreira Júnior1, André Pinheiro Zylberman1, Edvaldo Victor Gois Oliveira1, Eduardo Jose Pereira Ferreira1, Antônio Carlos Sobral Sousa1, Enaldo Vieira de Melo1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe (UFS) Introduction: Chronic Coronary Syndrome (CCS) appears frequently to clinical cardiologists, but the literature still lacks data on the evolution of its complex pathology. Objective: To analyze, by Exercise Stress Echocardiography, the behavior of myocardial ischemia in CCS patients with previous Acute Myocardial Infarction (AMI), according to the given treatment. Methods: A cross-sectional study from January 2000 to January 2022 with individuals aged ≥ 40 years (X = 62 ± 9.6 years) who underwent the exam at a cardiology referral service in Sergipe. They were grouped by the intervention performed after the AMI: surgical revascularization, Stent, or clinical treatment. Patients with CCS with no history of AMI were also considered. The behavior of the left ventricle was evaluated considering the Left Ventricular Mass Index (LVMI) and the Ejection Fraction (EF) while the behavior of the myocardial ischemia, considering the Wall Motion Score Index (WMSI). Chi-square test and one-way ANOVA analysis for multiple comparisons with Tukey’s post-hoc were used. The significance level was set to 5%. Analyzes were performed using SPSS Statistics. Results: 727 patients (37.3%) with previous AMI were studied: 142 (7.3%) underwent surgical revascularization, 284 (14.6%), Stent, and 301 (15.5%), clinical treatment. 1221 patients (62.7%) without previous AMI were evaluated. On average, the increase in LVMI was greater among those who received only clinical treatment, compared to those who had no previous AMI (108g vs. 91.3g; p = 0.010); the other groups showed no significant difference. Regarding EF, individuals treated with surgical revascularization (14.4% vs. 3.5%; p < 0.001), with Stent (10.8% vs. 3.5%; p < 0.001) and clinically (9.5% vs. 3.5%; p < 0.001) had a higher percentage of reduced EF compared to patients with CCS without previous AMI. As for resting and exercise WMSI, the surgical revascularization group (averages 1.21 and 1.22 respectively) and the clinically treated group (averages 1.17 and 1.20) were statistically different from the Stent group (averages 1.11 and 1.10) (p < 0.001) and the SCC group without previous AMI (averages 1.04 and 1.05) – those last also different among themselves. Conclusions: In the stratification of patients with CCS with previous AMI, treatment with the Stent showed better evolution in the behavior of the LVMI, EF and WMSI on the Exercise Stress Echocardiogram at rest and exercise, when compared to patients with CCS without previous infarction. 111819 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM VITÓRIA CALDAS GONÇALVES1, Beatriz Caldas Gonçalves2, Ana Elisa Caldas Gonçalves3, Gabriela Magalhães Bandeira Gomes1, Isadora Vilela Rodovalho1, Rodrigo Rodrigues Pache4, Ana Luiza Espíndula Rocha1, Paulo Henrique Rodrigues1 (1) Universidade Evangélica de Goiás – UniEvangélica; (2) Universidade Federal de Goiás – UFG; (3) Universidade Federal de Uberlândia – UFU; (4) Universidade Federal do Tocantins – UFT Introduction: The mRNA vaccines developed against the SARS-CoV-2 virus, despite being well tolerated in most cases, are related to serious and potentially fatal reactions, such as myocarditis. This is characterized as an inflammation of the heart muscle resulting from the action of toxins, through hypersensitivity reactions or by an autoimmune mechanism. In order to reduce clinical complications and improve functional prognosis, diagnosis and treatment must be performed quickly and accurately. Objective: To understand the development of myocarditis as an adverse effect of the vaccine against the SARS-CoV-2 virus, as well as its main prognostic aspects. Methods: This is an integrative review with a search in the databases PubMed and The Virtual Health Library (VHL) using the Health Sciences Descriptors “myocarditis”, “covid-19” and “vaccine”, combined with the boolean operator AND. We selected 10 original articles, whose inclusion criteria consisted of recent publication date (last 5 years) and languages in Portuguese and English. Results: With regard to immunization against COVID-19, myocarditis should be suspected when symptoms of heart failure such as progressive chest pain, dizziness, fever, nausea and vomiting begin about five days after vaccination. It is common the elevation of cardiac biomarkers in the complementary exams, such as troponin I, in addition to evidence of changes in ventricular repolarization on the electrocardiogram. Typically, an increase in the plasma concentration of N-terminal pro-B-type natriuretic peptide (NTproBNP) and C-reactive protein (CRP) is found. A possible pathophysiological justification for the relationship studied is the fact that cardiac myocytes express ACE2 receptors, which can bind to the spike glycoprotein, translated from the mRNA vaccine, and trigger cardiac complications. However, there is still not enough evidence to establish this causal association. Conclusion: Myocarditis after vaccination against COVID-19 usually follows a benign course; however, medical monitoring is necessary due to the high rate of morbidity and mortality, even though it is an infrequent and poorly understood adverse effect. It is important to note that the benefits of vaccination outweigh the risks of developing myocarditis since COVID-19 infection carries a much higher rate of acute and chronic cardiovascular sequelae, including arrhythmias and thromboembolic events. 111827 Modality: E-Poster Scientific Initiation – Non-case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION MARIANA GUIMARÃES SOUZA DE OLIVEIRA1, Ana Carolina Augusto Rocha1, Eduardo Souza Amancio da Costa1, Marina Miguel de Lucena1, Alexandre Anderson de Souza Munhoz Soares2, Luiz Sergio Fernandes de Carvalho3 (1) Universidade Católica de Brasília UCB; (2) Instituto Aramari Apo; (3) Clarity Healthcare Intelligence Objectives: We seek to compare the prevalence of diabetes (DM) and cardiovascular outcomes (CO) associated with groups of young individuals who presented their first acute coronary syndromes (ACS) with ages <55 y/o (Premature ACS, pACS). In parallel, we sought to comprehend the economic impact of the pACS and DM association. 2. Methods: We analyzed the registers retrospectively with the first ASC aged <55 y/o from 2013 to 2015 within the B-CaRE:QCO study, a registry that includes all the admissions to public hospitals of Federal District, Brazil. DM has been defined as hemoglobin A1c >6.5%, documented diagnostics, or the reception of diabetes treatment. Cause of death was adjudicated and all cause-specific hospitalizations were retrieved from digital records. The primary outcome was overall health care spending (sum of the cost of lost productivity [CLP], ambulatory costs, and the cost of recurrent hospitalizations [CRH] in international dollars [Int$] per year), and the secondary outcomes were the mortality due to all causes of the recurrent ACS. 3. Results: Among 6,341 individuals with ACS, 2.242 were pACS (mean age 47.9 ± 5.5 years), diabetes was present in 514 individuals (29.7%), of whom 137 (27%) used insulin. During median follow-up of 6.5 (IQR 1.9) years, DM was associated with higher all-cause mortality (HR 1.58[95%CI 1.3–1.9]; p < 0.001) and recurrent ACS (1.48[95%CI 1.3–1.7]; p < 0.001) relative to young and non-diabetic individuals. These associations persisted after the adjustment for baseline covariates (all-cause mortality: 1.69; p = 0.003; recurrent ACS: 1.72; p < 0.001). Compared to older individuals, the pASC group had lower all-cause death rates (p < 0.001), but higher rates of recurrent ACS (p = 0.012) during the monitoring, particularly in diabetics. Driven primarily by CRH and CLP, young people with diabetes spent the highest amounts (Int$16,350 [12,545–23,740]/year in pACS+DM+ vs Int$14,555[12,593–20,354]/year in prACS+DM- vs Int$12,183 [10,082–17,416]/year in older individuals, p < 0.001). 4. Conclusions: Diabetes was present in 29.7% of patients presenting ACS at age <55 y/o and was associated with higher all-cause mortality at long-term as well as a more recurrent ACS in comparison to young non-diabetic individuals, but otherwise lower when compared to older individuals. However, young diabetics have shown the highest overall healthcare expenditures. These findings highlight the need for implementation of heftier therapies aimed at preventing. 111834 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT MICHELLE BOZKO COLLINI1, Gustavo Sarot Pereira da Cunha1, Leonardo Henrique dos Santos de Melo1, Matheus Bissa Duarte1, Jorge Tadashi Daikubara Neto1, Rafael Moretti1, Carolina Ruschel Senger1, Karoline Cordeiro Vercka1, Jessica Tamires Reichert1, Lucas Muller Prado1, Jamilly Giuriatti Anziliero1, Miguel Morita Fernandes-Silva1 (1) Universidade Federal do Paraná Introduction: The universal classification of heart failure defined four classes according to the ejection fraction (EF). These classes have different characteristics and prognosis in chronic patients, but the information in the literature on acute heart failure is scarce. Objectives: To compare the prognosis of the EF classes in patients admitted for acute heart failure. Methods: We studied patients admitted for acute heart failure in a tertiary hospital in Curitiba, Brazil, from October 2019 to July 2021. EF classes were defined as: reduced EF (HFrEF): EF ≤40%, mildly reduced EF (HFmrEF): EF 41–49%, preserved EF (HFpEF): EF ≥50% and improved EF (HFimpEF): baseline EF ≤40%, with ≥10 point increase from baseline, and a second measurement of EF >40%. The outcome was all-cause mortality in a six-month follow-up. Results: From 153 patients (67.21 ± 14.86 years, 50.3% female, EF = 43.79 ± 17.62%), 52% had HFrEF, 33% HfpEF and 12% HFmrEF. The proportion of patients with HFimpEF was very small (3%). As expected, HFrEF patients were mostly male (66.2%) and more likely to have an ischemic etiology (42.5%); and HFpEF patients were more likely female (67.3%), and had hypertension (90.9%) and atrial fibrillation (49.1%), as compared with the other categories. The 6-month survival rate did not significantly differed among the EF classes (p = 0.55) (figure 1). Conclusions: In patients with acute heart failure, the proportion of HFimpEF was small and the 6-month all-cause mortality was similar among the EF classes. 111837 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION ANA CAROLINE LEITE GUEDES1, Maria Eduarda Brotto de Souza1, Fernanda Dias de Oliveira1, Roberta Cristiane Oliveira da Silva1, Gesanaje da Paz Carvalho1, Jade Gomes da Costa Medeiros1, Caroline de Souza Alovisi1, Ana Júlia Omodei Rodrigues Martim1, Renata Gonçalves Silva Santos1, Lucas Vieira Amorim1, Sofia dos Santos Souza1, Fernanda Gabry Scazuza Gomes de Souza1 (1) Centro Universitário São Lucas- UNISL Introduction: Systemic arterial hypertension (SAH) is a multifactorial condition, which depends on genetic/epigenetic, environmental and social factors, characterized by persistent elevation of blood pressure (BP). The diversity of causes for the development of SAH is extensive and, in most cases, it is associated with alcoholism, smoking and physical inactivity, in the entire population, with a special focus on remote communities. The health of the Amazon riverside population has been the focus of special attention, mainly related to factors such as changes in the level of physical activity in their daily lives (fewer active individuals) due to the so-called “facilities” of the modern world, and changes in their patterns of physical activity. In this way, the present work aims to analyze the prevalence of this comorbidity in riverine people and highlight the relevance of the problem. Objectives: To describe the prevalence of SAH in a riverside community on the Jamari River in the Amazon, Brazil. Methods: The present work is a cross-sectional and quantitative cohort study, through the application of a questionnaire to the population, between the ages of 1 to 87 years, residents of the region corresponding to the district of São Carlos do Jamari, in Rondônia. The collected data were described and tabulated for further analysis. Results: 269 individuals from the region in question were randomly and unintentionally selected. Of these, 78 people (29%) reported having Systemic arterial hypertension, among them the highest prevalence for women. Social habits include drinking (15.3%) and smoking (10%), as well as unspecified heart problems (10%). Conclusion: The aforementioned study brings the discussion about the prevalence of SAH in riverside populations in the Amazon, which presents an expressive percentage, with an association of multiple factors, proving the necessary detailed analysis of the situation aiming at reducing risks and improving the population’s quality of life. 111841 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION GUILHERME NUNES MIRANDA1, Daniel Italiano de Araujo Filho1, Francisco José de Almeida Cruz Júnior2, Hermano Rodrigues Pinheiro3, Maria Clara Almeida4, Carlos Eduardo Batista de Lima2 (1) Universidade Federal do Piauí; (2) Hospital Universitário do Piauí; (3) Centro de Pesquisa Cardiolima; (4) Instituto do Coração – FMUSP Introduction: Implantable devices increase the detection of silent atrial fibrillation in high-risk populations, being a useful tool in the early diagnosis of these patients, allowing monitoring for a longer time. The Smartwatch could be a useful, easy tool for the daily cardiological propaedeutics. However, there is just a little information on the limitations of analysis on the watch’s Electrocardiogram (ECG) in comparison to the conventional one. Objective: To analyze, in patients with cardiovascular disease, the electrocardiographic aspects detected from the device in comparison to the conventional ECG, identifying aspects of similarity between the tracings. Method: A cross-sectional observational study of diagnostic accuracy is currently being carried out in a Hospital since July 2021. Each patient did three ECGs, ECG 1 = 12-lead conventional; ECG 2 = rhythm strip DI single lead conventional; ECG 3 = DI single lead on the smartwatch. The ECGs were randomly delivered for 3 cardiologists, the 3 examiners were blinded. The data was analyzed comparatively between the 3 measures in each patient and between each other. Data were analyzed using the Statistical Package for the Social Science program (SPSS®, version 20.0). For statistical analysis, the Kappa statistics, ANOVA test, Turkey post-test were used, considering a significant p-value <0.05. Results: 19 patients were analyzed between July 2021 and April 2022. Fifty five ECGs were collected and the 3 examiners registered a total of 165 reports. The average age was 61,68 and male gender was more prevalent (78,9%), the most common diseases were angina (57,9%) and Atherosclerosis (57,9%). The results showed the sinusal rhythm detection Kappa between examiners 1, 2, 3 in the Smartwatch’s ECG were (0,799; 0,895; 0,712) and the sinusal rhythm detection Kappa in the conventional 12-lead ECG were (1,000; 1,000; 1,000). Some data showed that the Smartwatches ECG’s could detect abnormal rhythms, even when we could not specify, nonspecific changes in ventricular repolarization (AIRV) were detected. Conclusions: In this 19 patient analysis, the Smartwatch has shown a good accuracy to detect if the patient had a normal rhythm. Even though the wereables presented some data capture limitations but, as we educate patients on how to use the 1-lead ECG, add more data and new technologies in each patient analysis, it could be a useful tool for monitoring and possible early diagnosis of abnormalities, such as arrhythmia. 111848 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION ANA ELISA BARRETO CALIXTO1, Ana Elisa Barreto Calixto1, Guilherme Rufino Marques Pellegrin1, Nathalia Noyma Sampaio Magalhães1, Luiza Dahbar Rodrigues1, Matheus Pericles Belcavello1, Patrick Ribeiro Reis1, Lucas Nicolato Almada2 (1) Faculdade de Ciências Médicas e da Saúde de Juiz de Fora – Suprema; (2) Hospital e Maternidade Therezinha de Jesus – HMTJ Introduction: Cardiovascular diseases are the leading cause of death globally. They are preventable by changing risk behaviors, such as a sedentary lifestyle and poor sleep quality. The implementation of low-cost technology-based interventions can contribute not only to improve health outcomes, but also to reduce public health costs related to cardiovascular disease. Wearable devices (WD) are gadgets that can be worn anywhere on the body, allowing the generation of data on physical activity, heart rate and rhythm and, more recently, impedance and thoracic fluid data. The assessment of these data is an important challenge in the clinical setting. Objectives: To evaluate the impact of WD, associated with physical exercises, in reducing cardiovascular risk. Methods: Literature review was carried out in the MedLine database using the descriptors: “Wearable Devices”, “Cardiovascular Risk” and their variations according to MeSH. Controlled and randomized clinical trials carried out in humans and published in the English language in the last 10 years were included. The PRISMA scale was used to improve the reporting of this review. Results: Two studies analyzed the outcomes of WD in patients with elevated cardiovascular risk. In the first one, 144 patients with a mean age of 61 years were recruited. 70 patients participated in the experimental group (EG) and, as an intervention, they used WD, resulting in a significant increase in the number of steps and activity level over time (p < 0.001 and p = 0.01, respectively). No significant changes were observed in blood pressure, weight, and sleep quality. In the second study, 40 individuals with a mean age of 72 years were randomized into two groups. Both of them received instructions concerning physical activity practice, but the EG also received WD. 12 weeks after randomization, the EG presented a significant increase in the number of steps (p < 0,05) while in the control group (CG) this number actually decreased. In EG, there was a reduction of 4 mmHg in blood pressure levels and the CG suffered an increase in this parameter. Sedentary behavior did not suffer any alteration in either group. Conclusion: The results of the studies showed that the reduction of cardiovascular risk was optimized by the association of WD with regular practice of physical activities, which suggests that the use of these devices has the potential to help in the prevention and treatment of cardiovascular diseases. 111865 Modality: E-Poster Scientific Initiation – Non-case Report Category: NUTRITION GABRIEL JUNQUEIRA JÚLIO1, Giuliano Fernando da Silva Júlio2, Sofia Sousa Alexandre1, Katarina Masciano Pereira1, Sevana Valadão Naves1, Danilo Cezar Aguiar de Souza Filho1, Douglas Araujo Menezes Filho1, Renato Tassi de Lima1, Isadora Silva de Sousa1, Lorena Costa de Holanda1, Giovanna Cristina Silveira Corrêa1, Mariana Mello Menezes1 (1) Centro universitário euro americano – UNIEURO; (2) Prevencor Introduction: The increased consumption of energy drinks (ED) in recent years has encouraged studies with the purpose of identifying possible damage caused by indiscriminate and prolonged use. The increase in consumption is associated with the intention to improve performance in daily activities. The compounds present in ED show that the levels of caffeine and sugar ingested can generate cardiovascular, behavioral and metabolic repercussions when used for a long time. Objectives: Acknowledge the risks of indiscriminate consumption of abusive doses of ED and the effects caused by the chronicity of use. Methodology: A systematic literature review was performed. In data collection, a bibliographic and documentary search was carried out on the subject, using articles that discuss the cardiovascular repercussions associated with the use of energy drinks. The search platform was PubMed, using the terms “energy drink” and “cardiovascular” as descriptors. 16 articles were selected, and the selection criteria were free systematic review studies or clinical trials, with 5 years of publication, in English or Portuguese, the articles that didn’t match the theme were eliminated. For analysis, the different results of the studies were systematized and compared. Results: When comparing the studies, it was found that the abusive use of ED increases the probability of cardiovascular repercussions such as tachycardia, arrhythmia, ischemia, increased systemic blood pressure, changes in repolarization, conduction disturbances and prolongation of QT intervals on the electrocardiogram. In addition to behavioral changes, such as stress, increased anxiety attacks and insomnia. The metabolism is altered by elevated cortisol levels and increased sugar consumption, providing greater risk for developing type 2 diabetes mellitus. Caffeine is the main compound that influences the perceived changes and its association with alcoholic beverages generates more serious and faster-evolving repercussions. Conclusion: The effects of the use of ED are already present in the studies carried out and show consequences in the lives of users who often don’t have the necessary knowledge about the drink and its long-term damage remains uncertain. Thus, the cardiovascular and metabolic systems are affected, but also the psychological system undergoes negative changes, demonstrating the need for studies with longer follow-up. 111907 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT KARLA SANTOS PINTO1, Raissa Barreto Lima1, Ana Luísa de Aguiar Almeida Silva1, Eduarda Luciana Mendes Borges1, Elias Soares Roseira1, Livia Maria Goes Lemos1, Aurea Maria Lago Novais1, Beatriz Barbosa Viana1, João Pedro Martins Moreira Granja1, Karla Santos Pinto1, Larissa Xavier Gomes da Silva1, Clara Salles1 (1) Hospital Ana Nery Introduction: Chronic heart failure is a condition associated with high rates of morbidity and mortality. Despite recent advances in optimized drug therapy for patients with reduced ejection fraction (HFrEF), they still have a significant risk of death. Therefore, the objective of this study is to identify predictors of death among individuals with HFrEF in a referral hospital in the city of Salvador, Bahia. Methods: This is a prospective cohort of patients with HFrEF, treated at a heart failure outpatient clinic from September 2020 to July 2021. The outcome variables evaluated were mortality and hospitalization for any cause in one year. For data analysis, the Odds Ratio (OR) was used as a measure of association between categorical variables and 95% Confidence Intervals (CI), using the chi-square test in the bivariate analysis. Continuous variables with normal distribution were compared using Student’s t test. Multivariate analysis with logistic regression was used to assess independent predictors of mortality. A value of p < 0.05 was considered for statistical significance. Results: We included 196 patients with heart failure with a median age of 60.5 (49–68) years, most of them men (54.1%) and a median LVEF of 29% (23–35). Ischemic and chagasic etiologies were the most prevalent, with 46 (23.5%) and 40 (20.4%) patients, respectively. After 1 year of follow-up, it was found that 25 (12.8%) died, 50 (25.5%) were hospitalized and NYHA III-IV totaled 57 (34.5%) patients. Most patients who died were male (64%) and 49% had a history of hospitalization <1 year. In the multivariate logistic regression model, Chagas‘ etiology remained the only independent predictor of mortality (OR 1.36; 95% CI 1.52–10.11; p = 0.005). In the univariate analysis, the mortality predictors were: DM2, CAD, hospitalization <1 year and Chagas‘ etiology (Table 1). Conclusion: Chagas’ etiology was the only independent predictor of mortality in patients with HFrEF. 111875 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT EDUARDA CORRÊA MAIA1, Evandro Tinoco Mesquita1 (1) Universidade Federal Fluminense Fundamentals: Myocarditis is an inflammation of the myocardium that can affect cardiac function, reducing ejection fraction and causing arrhythmias, leading to heart failure syndrome, cardiogenic shock and sudden death. This condition can be caused by a viral infection, an immune-mediated response due a late viral response, inflammatory autoimmune diseases or an auto-inflammatory response, making myocarditis even more relevant within the COVID-19 pandemic. Colchicine is a low-cost anti-inflammatory, already known and used in rheumatologic diseases, and, now, used in cardiology for pericarditis and coronary disease. In cases of colchicine use for myocarditis there have been publications with positive results, however its effects in patients with acute myocarditis are not clearly demonstrated. Goal Systematically review the literature to assess the effects of colchicine use in patients with acute myocarditis. Methods: We performed a systematic review of the effects of colchicine in patients with myocarditis. Case series, randomized studies and case controls published on PubMed, SciELO and Google Scholar platforms were selected, from January 2010 to January 2022, in Portuguese, English, Spanish and French. Results: Initially, a total of 62 studies were found. After exclusions, 30 articles remained, with a total of 128 patients with acute myocarditis who used colchicine. Of this group, 10.93% (14 patients) had myocarditis in association with another disease, 3.9% (5 patients) in association with autoimmune diseases, and 16.4% (21 patients) with myopericarditis. In the only study using a control group consisting of 60 patients (didn’t use colchicine), 31.6% (19 patients) had complete resolution of symptoms. In the end, a positive association was demonstrated in the use of colchicine in the treatment of myocarditis, with 75.78% (97 of 128 patients) of patients presenting resolution of symptoms within 14 months after starting treatment. Conclusion: The results found in this systematic review, so far, suggest that the use of colchicine in the treatment of acute myocarditis has been effective. The study also showed the absence of randomized studies that analyze the effectiveness of colchicine in cases of acute myocarditis, with only one study using a control group and showing significant improvement in the clinical picture. In addition, the little evidence related to cases of post-covid myocarditis is even more relevant in the current pandemic context. 111897 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MANUELA FURTADO SACIOTO1, Thaís Lorenna Souza Sales2, João Victor Baroni Neves1, Vivian Costa Morais de Assis1, Maíra Viana Rego Souza-Silva3, Polianna Delfino-Pereira3, Amanda Rabello Conceição3, Anna Luiza Homan Araújo Carvalho1, Isabela Moraes Gomes3, Lucas Emanuel Ferreira Ramos3, Magda Carvalho Pires3, Milena Soriano Marcolino3 (1) Faculdade Ciências Médicas de Minas Gerais (FCMMG); (2) Universidade Federal de São João Del-Rei (UFSJ); (3) Universidade Federal de Minas Gerais (UFMG) Introduction: There is a lack of studies investigating the influence of HIV infection, on the cardiovascular outcomes, in COVID-19 patients in Latin America. Objective: To compare cardiovascular outcomes, between patients with COVID-19 with and without HIV infection, in two consecutive years. Methods: This is a substudy of a multicentric cohort, which included 27 Brazilian hospitals, comprising March to September 2020 and March to December 2021. Cardiovascular outcomes included acute myocardial infarction (AMI), pulmonary thromboembolism (PTE), deep vein thrombosis (DVT) and stroke. Patients with COVID-19 and HIV coinfection were matched for age, sex, number of comorbidities and hospital. Groups were compared using the Chi-Square Test for categorical variables and the Wilcoxon test for continuous variables. Results were considered statistically significant at a significance level of p < 0.05. Results: From 17,101 COVID-19 patients, 130 were people living with HIV (PLHIV) (0.76%). Of these, 86 were hospitalized in 2020 and 44 in 2021. The median age in 2020 and 2021 periods was 53 and 54 years, respectively, with no significant differences between groups. The most frequent COVID-19 symptoms in both groups were dyspnea (2020: 52 [60.5%] vs 227 [67.0%]; p = 0.314/2021: 30 [68.2%] vs 122 [69.7%]; p = 0.989) and fever (2020: 50 [58.1%] vs 207 [61.1%]; p = 0.710/2021: 20 [45.5%] vs 76 [43.4%]; p = 0.942). The main cardiovascular outcomes between PLHIV in 2020 were PTE (4.7%), DVT (2.3%), AIM (2.3%), and stroke (1.2%). Non-HIV infected patients showed similar prevalence for the outcomes under study, with no significant difference. PTE (13.6%) and DVT (2.3%) were also the most common cardiovascular outcomes in PLHIV in 2021, however, no cases of AIM and stroke were observed in this group. Comparison of outcomes with the controls also showed no significant difference in this period. When observing the years of 2020 and 2021, there was a total reduction of cardiovascular outcomes, except for PTE. Conclusion: There were no significant differences in the cardiovascular outcomes between PLHIV and their controls. However, the reduction of the cardiovascular outcomes between 2020 and 2021 indicates the importance of vaccination in the prognosis of COVID-19 in HIV coinfected patients. 111913 Modality: E-Poster Scientific Initiation – Non-case Report Category: DIGITAL HEALTH/INNOVATION LAÍS ARAÚJO CARNEIRO DE CAMPOS1, Laís Araújo Carneiro de Campos1, Marília Menezes Gusmão1, Marília Menezes Gusmão2, Júlia Schoucair Neves1 (1) Escola Bahiana de Medicina e Saúde Pública (EBMSP); (2) Escola Bahiana de Medicina e Saúde Pública (EBMSP) Introduction: Atrial fibrillation is a common cardiac arrhythmia that affects about 59 million people worldwide. In many cases its diagnosis occurs late, due investigation of an ischemic stroke. In other situations, it presents a paroxysmal behavior, which makes it difficult to detect this arrhythmia in conventional electrocardiography exams. Among recent technologies being used to monitor and detect cardiac arrhythmias, the smartwatches are becoming very popular. For that reason, it is important to verify if those devices are accurate to detect atrial fibrillation. Objective: To describe the diagnostic accuracy of smartwatch-like devices in detecting atrial fibrillation. Methods: A systematic review was conducted using PRISMA methodology, between June and September 2021, in the Databases of Pubmed/Medline and Virtual Health Library. The descriptors used included the terms “watch”, “wearable electronic devices”, “photopletysmography” and “atrial fibrillation”. After removal of duplicates, the results were screened for inclusion and exclusion criteria. Results: 409 articles were returned by the search, of which, 120 were duplicates. After screening the remaining 289 titles and abstracts for inclusion and exclusion criteria, 96 were selected for the second screening (full article read), of which 10 studies matched the criteria and were included in the review. The accuracy was measured on 10 different devices models produced by 5 companies. The sensitivity of the smartwatches included in the review varied between 67–100%, the specificity 67–99%, positive predictive value 80–90% and negative predictive value 85–100%. The accuracy was available in only two studies (70% and 97,5%). Conclusion: The smartwatches models reviewed by this study are accurate to detect atrial fibrillation. 111915 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH FERNANDA AMPARO RIBEIRO1, SUELLEN MOURÃO SILVA1, GILBERTO ANTONIO REIS1, GILBERTO ANTONIO REIS1, FERNANDA AMPARO RIBEIRO1, FERNANDA AMPARO RIBEIRO1 (1) PONTIFICIA UNIVERSIDADE CATOLICA DE MINAS GERAIS – PUC-MG; (2) Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG Hospitalizations for Primary care-sensitive conditions are preventable health complications through disease prevention, diagnosis and care of acute conditions, monitoring of chronic conditions. The use of this indicator is based on the principle that effective and effective primary health care can prevent hospitalizations. Thus, a high number of Hospitalizations for primary care-sensitive conditions in a population points to difficulties in accessing and operating health services. Different activities of primary health care and specialized outpatient and hospital care were interrupted or paralyzed due to the priority given to COVID-19. For different reasons, both professionals and primary care users have dangerously delayed non-COVID-19 procedures, which could interfere with the effectiveness and effectiveness of primary health care and which, in turn, would reflect on Hospitalizations for primary care-sensitive conditions. Thus, the hypothesis presented is that the incidence of Hospitalizations for Primary care-sensitive conditions care in the population over 34 years of age in the city of Betim-MG may vary between the periods before and during the COVID-19 pandemic. Objective: To measure the occurrence of hospitalizations for cardiovascular conditions sensitive to primary care in adults over 34 years of age living in the city of Betim – Minas Gerais and to estimate the variability between the previous period and during the COVID-19 pandemic. Methods: This is an ecological, descriptive, longitudinal study with a quantitative approach. Data were extracted from the Hospital Information System available on the DATASUS, portal of the Ministry of Health. Descriptive statistical measures of data were used to analyze the variables included in the study. Results: Among hospitalizations for cardiovascular conditions sensitive to primary care in the period between 2018–2021 in the city of Betim-MG, it was observed that the total number of hospitalizations behaved in a stationary way year after year. At the same time, there was a drop in the number of emergency admissions in 2021 compared to 2018, 2019 and 2020. The population most affected by cardiovascular and cerebrovascular causes were black and brown people and men. Stroke was the main cause of elective and emergency admissions. 111919 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LAURA COUTINHO VIANA1, Laura Coutinho Viana1, José Wilker Gomes de Castro Júnior1, Marcello Vieira dos Santos1, Pedro Arthur Rodrigues de Oliveira1, Daniely Maués Beliqui1 (1) Centro Universitário do Estado do Pará – Cesupa Introduction: Tetralogy of Fallot (TF) is a complex cyanotic congenital heart disease that involves changes in 4 cardiac segments in the newborn. They are: Right ventricular hypertrophy, aortic overriding of the interventricular septum, pulmonary artery stenosis, and sub-aortic interventricular communication (IVC). Its cause is multifactorial and affected pediatric patients may experience intense hypoxemic crises. Objective: This work aims to perform an evaluation of the epidemiological profile of live births with tetralogy of Fallot in Brazil in the period from 2016 to 2020. Methodology: A descriptive, retrospective and quantitative study was conducted based on secondary data provided by the Information System on Live Births (SINASC), SUS Department of Informatics (DATASUS). The collected information was stored and tabulated in Microsoft Office Excel™ program. Results: Among the 527 cases found after analyzing the evaluated period, higher incidence is found in the years 2018, with 136 cases (25.8%) and 2019, with 126 cases (23.9%). The Brazilian region with the highest number of those born with TF was the Southeast region with 358 cases (67.9%) with the highest rate in the state of São Paulo with 286 cases (54.2%). Regarding racial profiles, whites were the highest number, with 310 cases (58.8%), followed by mixed race, with 158 cases (29.9%). Regarding gestational age, there were 369 cases (70.1%) of term pregnancy (37–41 weeks) and 134 cases (25.4) of premature pregnancy (before 37 weeks). Conclusion: Therefore, we observed a higher incidence profile of TF in children born in the state of São Paulo, white and born at term. The possible cause would be success in screening and identification of the disease, which is indifferent to early birth. 111942 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH BRUNO ENÉAS ROLIM PAIVA1, Monique Benemérita Vilela Gomes1, Isabella Aparecida Abreu Garcia1, Isabela Rezende Costa1, Paulo Henrique Tenório Queiroz1, Renata Adélia Alves de Oliveira1, Giovanna Maria Lopes Magalhães1, Cléio Pereira dos Santos1, Paulo José da Silva1, Nisandra Pereira da Silva1, Ana Caroline Borges Lustosa1, Isabella de Almeida Nascimento1 (1) Universidade Federal do Piauí (Campus Senador Helvídio Nunes de Barros) Introduction: Chagas disease is a clinical condition caused by the protozoan Trypanosoma cruzi. This pathology is an important public health problem in Brazil, especially due to its cardiovascular complications. In average, between 10 and 30 years after the acute infection, 30% of the infected have chronic Chagas disease cardiomyopathy, which is the main cause of morbidity in this pathology, and sudden death is the main cause of mortality in those cases. Goals: To assemble an epidemiological overview of Chagas disease in Brazil. Methods: For this analysis, an epidemiological study was made using the data about acute Chagas disease in the Notifiable Diseases Information System (SINAN/DataSUS). The search considered the data from 2016 to 2020, grouping the number of confirmed cases by region of notification and sex. Hereafter, a data search of the number of deaths due to acute Chagas disease was performed using the same filters. Moreover, this study determined the Brazillian population by using the demographic census of 2010 from the Brazilian Institute of Geography and Statistic (IBGE). Results: According to DataSUS, between 2016 and 2020, there were 1662 notified cases of Chagas disease, affecting mostly and being more deadly in males. Between 2017 and 2019, an increase of 12.94% occurred, and 2019 was the year with most notifications, representing 23.1% of the cases between 2016 and 2020. The North region showed relatively more cases, representing 91.66% of deaths and 94.64% of the notified cases, with approximately 0.012% of the population being infected. Conclusion: The majority of the cases of Chagas disease occurs in the North region, therefore, the most cases of Chagas disease cardiomyopathy. Furthermore, it was observed that many regions do not notify new cases of Chagas disease, making it harder to paint a real epidemiological estimative and intervention necessity throughout the country. Besides that, in 2020 there was a decrease in notifications, explained by the Covid-19 pandemic, that caused a reduction in the notification of diseases non-related to respiratory syndromes. 111949 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY LARISSA DE OLIVEIRA BELTRÃO1, Beatriz Ximenes Bandeira de Morais1, Maria Letícia Garrote Bulhões2, Andreza Leite Marques de Sá Souza1, Luiza Dias Aguiar1, Leticia Ferreira Leal3, Isabela Torres Castro1, Juliana Mirelly Moura de Oliveira1, Catiane Kelly Schaefer4 (1) Faculdade Pernambucana de Saúde (FPS); (2) Universidade Estácio de Sá (UNESA); (3) Universidade de Pernambuco (UPE); (4) Universidade de Santa Cruz do Sul (UNISC) Background: Heart transplantation is the therapeutic option for refractory heart failure cases and is a procedure with limited access in many underdeveloped countries. With the COVID-19 pandemic, health resources were directed to contention plans and patient management, leading to a global decrease in transplantations. Objectives: This study aims to demonstrate the disparity in the impact of the COVID-19 pandemic on heart transplantation among the sociodemographic regions of Brazil. Methods: This is a retrospective descriptive study with data from the Department of Informatics of the Unified Health System from 2018 to 2021. A comparative analysis was performed between the average of the first two years of study and the two subsequent years, before and during the pandemic, respectively. Results: Between 2018 and 2021, 1,146 Brazilians underwent heart transplantation. Before the COVID-19 pandemic, 159.5 transplants were achieved in the Southeast region, the most developed in Brazil, 53.5 in the South, 31.5 in Midwest, and 80 transplants in the Northeast. With the COVID-19 pandemic, there was a drop to 41 procedures executed in the Northeast, 24.5 in the Midwest, 25.5 in the South, and 157.5 in the Southeast. Conclusion: There was a decrease of approximately 50% of heart transplants achieved in the Northeast and South, while there was maintenance in the numbers of other regions, which are two of the three most developed regions of Brazil. 111953 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ANA LUIZA DE ARAUJO GARCIA1, Ana Luiza de Araujo Garcia1, Maria Luiza Rangel de Vasconcellos Guerra1, Michelle Rabello da Cunha1, Samanta de Souza Mattos1, Mario Fritsch Toros Neves1 (1) Universidade do Estado do Rio de Janeiro (UERJ) Introduction: Acute infection caused by the SARS-CoV-2 can generate functional changes in the vascular endothelium but structural vascular alterations and long-term effects on the sympathovagal balance are still unknown. Objective: To compare central hemodynamic parameters, arterial stiffness, and autonomic activity between men and women with a history of symptomatic COVID-19. Materials and Methods: Cross-sectional study with patients aged between 30 and 70 years, both sexes, submitted to clinical evaluation, central hemodynamic parameters and pulse wave velocity (PWV) by Mobil-O-Graph, and sympathetic tone by heart rate variability (HRV). Patients were divided into two groups: Control (n = 25) with patients with no history of COVID-19, and post symptomatic COVID (n = 29), which included patients with positive RT-PCR in the last 12 months before enrollment in the study. An intragroup analysis was performed to verify the differences between men and women. Results: Patients were predominantly female in both groups (64 vs 62%, p = 0.883) and with similar ages (55 ± 6 vs 51 ± 10 years, p = 0.088). In the analysis by sex, the frequency of hypertension was similar between men and women in the control group (100 vs 81%, p = 0.243) and in the post-COVID group (60 vs 56%, p = 0.570). The post-COVID group presented, in females, higher peripheral systolic and diastolic pressures (114 ± 15 vs 131 ± 23 mmHg, p = 0.040; 72 ± 9 vs 85 ± 16 mmHg, p = 0.013), central systolic pressure (106 ± 15 vs 122 ± 23 mmHg, p = 0.037), augmentation pressure (5 ± 3 vs 11 ± 7 mmHg, p = 0.018), augmentation index (16 ± 7 vs 30 ± 12%, p = 0.006) and pulse wave velocity (6.7 ± 0.9 vs 7.8 ± 1.6 m/s, p = 0.033). The control group showed no significant difference between sexes in these parameters. HRV indices were similar for both sexes in both groups. The sympathovagal balance, represented by the low frequency/high frequency (LF/HF) ratio, was higher in men in the post-COVID group (4.7 ± 8.6 vs 1.2 ± 1.5, p = 0.185), although not reaching statistical significance. Conclusion: Early vascular aging, characterized by higher arterial stiffness and increased central pressure, was more evident in women who had symptomatic COVID-19 compared with men in the same group, even without differences in sympathovagal balance. 111963 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR PHARMACOLOGY ISRAEL FIGUEIRA LEMOS1, Alice Jacomini Barcos1, Gabriel do Nascimento Oliveira1, Ana Lucy Peixoto Rabelo1, Camila Namie Seki Garzon1, Edson Luis de Barros Siqueira1, Ana Luiza Nepomuceno Sampaio1, Caroline Hipólito Pires1, Luma Maria Favacho Bordalo1, Maria Eduarda Dantas da Veiga1, Juliana de Sousa Tavares1, Erick Clayton Gonçalves Feio1 (1) Universidade do Estado do Pará Introduction: Beta-blocker drugs are widely used in the treatment of systemic arterial hypertension, being able to be used in monotherapy treatments or combined with other medications. However, due to their complex pharmacological actions and adverse effects, the effectiveness of beta-blockers raises uncertainties. Objectives: Evaluate the effectiveness of beta-blockers in the control of hypertension. Methods: A systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations, using the PICO strategy to elaborate the guiding question “Have beta-blockers been effective in hypertensive people?”. The data collection was based on the use of the following keywords: “beta blockers‘‘ AND “effectiveness” AND “systemic arterial hypertension”, which were obtained in English and Portuguese on the Medical Subject Headings (DeCS/MeSH). The articles were searched on the PubMed and Biblioteca Virtual em Saúde (BVS) databases, including those that approach the treatment of beta-blockers in systemic arterial hypertension, indexed in the selected databases and published between 2012 and 2022, in Portuguese and English. All studies that are not randomized clinical trials were excluded. Results: A total of 20 articles were selected to integrate this study. Traditional beta-blockers (BB) are efficient agents to reduce BP, but they‘re not recommended as standard therapy to uncomplicated essential hypertension, for being less efficient in reducing cardiac events. However, new generation BB‘s, nebivolol for example, has auxiliary vasodilating properties. Compared to ACE inhibitor therapy, BB therapy was superior in hypertensive patients and required fewer antihypertensives to control BP. Furthermore, BB effects had different outcomes in regard to age, sex, ethnicity and daily habits. Conclusion: In general lines, a positive response was seen on the hypertensives’ treatment with beta-blockers, considering the singular effects of the medicine’s actions of vasodilation and complacency on hypertensive patients, according to the drug manipulation, different treatments and varied physiologies. 111971 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LARISSA MELO TARGINO1, Claudio Lucas Silva Cunha1, Larissa Melo Targino1, Helen de Araújo Alves2, Luiz Eduardo Fonteles Ritt2 (1) Escola Bahiana de Medicina e Saúde Pública (EBMSP); (2) Hospital Cárdio Pulmonar da Bahia (HCP) Introduction: The trend towards population aging and its direct association with the prevalence of heart disease has resulted in a higher incidence of Heart Failure (HF). However, extreme old patients are still underrepresented in clinical studies and few data regarding their profile and prognosis is present in the literature. Objective: To describe and compare clinical characteristics and clinical outcomes between extreme elderly (≥80 years-of-age) and their counterparts patients hospitalized for decompensated HF. Methods: Prospective cohort study in a single-center registry model. Patients hospitalized for HF at a private institution in Salvador, Bahia, Brazil, were included. Data were obtained from hospital records from 2019 to 2022. In addition, telephone follow-up was carried out 30 days after discharge, when the outcomes of death or readmission were measured. A p < 0.05 was set as a level of significance for all analyses. Results: We analyzed 356 patients hospitalized for HF, who were divided into groups of extreme elderly (n = 159) and non-extremely elderly (n = 197). Compared to the non-extremely elderly group, the extreme elderly group were more likely to be female, had hypertension and was less likely to have obesity, smoking, use of a pacemaker, had paroxysmal nocturnal dyspnea and third beat extra heart sounds. The group ≥80 years had higher values of Left Ventricular Ejection Fraction (mean = 48.56, 95% CI [46.16–50.96] versus mean = 41.43, 95% CI [38.9–43.9]; p < 0.001), as well as higher levels of urea and brain natriuretic peptide (BNP) at discharge. There was no difference in the rate of betablocker or ACE inhibitor/angiotensin receptor blocker usage between groups. During hospitalization and the 30-day follow-up, the mortality in the extreme elderly group was significantly higher (28.9% versus 14.4%; p = 0.047). Within 30 days after discharge, 7.9% of patients <80 years were readmitted, while 13.4% of patients ≥80 years-of-age, although without statistical significance. Conclusion: Extremely elderly patients with HF are susceptible to higher mortality rates and have a particular clinical profile. Future studies focusing on the treatment of these subgroup are warranted. 111974 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY KARLA SANTOS PINTO1, Beatriz Barbosa Viana1, Larissa Xavier Gomes da Silva1, João Pedro Martins Moreira Granja1, Elias Soares Roseira1, Tainá Viana1, Luiz Carlos Passos1, Pollianna Roriz1, Adriano Tamazato1, Raissa Barreto Lima1, Ana Luísa de Aguiar Almeida Silva1, Eduarda Luciana Mendes Borges1 (1) Hospital Ana Nery Introduction: Coronary Artery Diseases (CAD) are responsible for the highest mortality rate in the world. Acute ST-segment elevation myocardial infarction (STEMI) corresponds to one third of CAD and has a higher mortality among the disease subtypes. Early diagnosis and prompt reperfusion are effective ways to limit ischemia, reducing the risk of complications. Angioplasty is the main reperfusion strategy and, if it is not performed within 120 minutes, fibrinolysis therapy must be managed. Objective: To describe the epidemiological and therapeutic clinical profile and clinical consequences in a tertiary referral hospital in the city of Salvador – BA, which receives an average of 50 patients per month in this condition. Methodology: Descriptive study with 329 patients treated at health units in Salvador and the metropolitan region with suspected AMI, transferred through the AMI Protocol – SAMU for angioplasty proposal in a referral hospital, between January 2021 and April 2022. demographic variables, risk factors, first care, angiographic data, and outcomes. Categorical variables were presented as frequencies and continuous variables as mean or median. Results and Discussion: There were 329 patients with STEMI, 60% of whom were men. The mean age was 61 years. Previous diseases were SAH (64%), DM (32%) and obesity (15%). 19% were smokers and 16% were ex-smokers. Most with Killip I in the first evaluation (84.8%). The most affected wall was the anterior one (46%). The most performed reperfusion was primary angioplasty (71.4%). The average time for the balloon holder was 354 minutes. The most frequent culprit artery was the anterior descending artery (47.1%), followed by the right coronary artery (28.2%). There was occlusion in 43.4% and none in 12% of the cases. Most access was via the Radial artery (76%). 40% had an initial TIMI of 0 and 20% had a TIMI of 3. 65% of the patients had a final TIMI of 3 and only 4.3% had a final TIMI of 0. Angioplasty was successful in about 70% of cases. As an outcome, there was death of 6, 6% during hospitalization. Conclusion: Patients undergoing primary angioplasty were men with anterior wall AMI. Despite the long door-to-balloon time, a high success rate was obtained in restoring normal flow after the procedure. These results corroborate the generation of strategies to reduce injuries in patients undergoing angioplasty. 111985 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH FERNANDA AMPARO RIBEIRO1, FERNANDA AMPARO RIBEIRO1, SUELLEN MOURÃO SILVA1, GILBERTO ANTONIO REIS1 (1) PONTIFICIA UNIVERSIDADE CATOLICA DE MINAS GERAIS – PUC-MG; (2) Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG Hospitalizations for Primary care-sensitive conditions are preventable health complications through disease prevention, diagnosis and care of acute conditions, monitoring of chronic conditions. The use of this indicator is based on the principle that effective and effective primary health care can prevent hospitalizations. Different activities of primary health care and specialized outpatient and hospital care were interrupted or paralyzed due to the priority given to COVID-19. For different reasons, both professionals and primary care users have dangerously delayed non-COVID-19 procedures, which could interfere with the effectiveness and effectiveness of primary health care and which, in turn, would reflect on Hospitalizations for cardiovascular conditions sensitive to primary care. Patients with cardiovascular diseases are more vulnerable to hospitalizations due to adverse events related to poor symptom control. Thus, the hypothesis presented is that the incidence of Hospitalizations for cardiovascular conditions sensitive to primary care in the population over 34 years of age in the city of Betim-MG may vary between the periods before and during the COVID-19 pandemic. Objective: To measure the occurrence of hospitalizations for cardiovascular conditions sensitive to primary care in adults over 34 years of age living in the city of Betim – Minas Gerais and to estimate the variability between the previous period and during the COVID-19 pandemic. Methods: This is an ecological, descriptive, study with a quantitative approach. Data were extracted from the Hospital Information System available on the DATASUS, portal of the Ministry of Health. Descriptive statistical measures of data were used to analyze the variables included in the study. Results: Among hospitalizations for cardiovascular conditions sensitive to primary care in the period between 2018–2021 in the city of Betim-MG, it was observed that the total number of hospitalizations behaved in a stationary way year after year. At the same time, there was a drop in the number of emergency admissions in 2021 compared to 2018, 2019 and 2020. The population most affected by cardiovascular and cerebrovascular causes were black and brown people and men. Stroke was the main cause of elective and emergency admissions. The COVID-19 pandemic had an important impact on the care routine of all health service users. It is necessary to carefully investigate the long-term impacts of this health crisis. 111986 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIELLY NASCIMENTO1, Celina Maria de Carvalho Guimarães1, Silvia Marinho Martins1, Luis Eduardo Martins Alves1, Taciane Queiroz Medeiros Gomes1, Maria Elisa Lucena Sales de Melo1, Maria da Glória Aureliano de Melo1, Carolina de Araújo Medeiros1, Maria das Neves Dantas da Silveira Barros1, Maria da Piedade Costa Reis de Albuquerque1, Cristina de Fátima Velloso Carrazzone1, Wilson Alves de Oliveira Júnior1 (1) Pronto Socorro Cardiológico de Pernambuco – PROCAPE Introduction: Heart failure with improved ejection fraction (HFimpEF) is associated with improved clinical outcomes[1,2], however little is known about HFimpEF of chagasic etiology[3]. Objective: Identify predictors of progression to HFimpEF in Chagas‘ etiology. Methodology: Cohort study in a referral outpatient clinic for Chagas Disease admitted between march-2016 and august-2019. From the 862 included in database, 86 were selected, under two criteria from two different echocardiograms (echos), the first was the admission one, with left ventricular ejection fraction (LVEF) <40%, and the second one, the follow-up. The mean time interval between the echos was 3.13 years (1 ± 5 years). HFimpEF was defined as LVEF ≥40% on the follow-up echo. The variables analyzed were sex, age, presence of comorbities (diabetes mellitus, dyslipidemia and arterial hypertension), medications: spironolactone, beta-blockers, loop and thiazide diuretics, angiotensin II receptor blocker/angiotensin converting enzyme inhibitor and sacubitril valsartan, left ventricle end-diastolic diameter (LVEDD) and right ventricule function to the Echo, QRS duration, presence of right bundle branch block and left bundle branch block on the electrocardiogram. For data analysis the technique utilized was a logistic regression with variables that presented p-value < 0,15 in the univariate analyses. Results: On admission, mean age was 60.9 years (standard deviation 11.5, minimum 30 and maximum 80), 52% women, 77.7% had arterial hypertension, 19.7%: diabetes, 33.7%: dyslipidemia. On the electrocardiogram: 43% right bundle branch block, 37.2% left bundle branch block. On the admission echo, LVEF with median 33% (minimum 16%, maximum 39%), LVEDD average of 65(standard deviation: 1,25, minimum 45, maximum 81) and 25.5% with right ventricle dysfunction. On therapeutics: 95.3% beta blockers, 77.9% used loop and thiazide diuretics, 74.4% angiotensin II receptor blocker/angiotensin converting enzyme inhibitor, 67.4% spironolactone, 8.1% sacubitril valsartan. In the univariate analysis, use of spironolactone (p = 0.027) and LVEDD (p = 0.001) were shown to be factors for improving LVEF, but in the multivariate analysis, only LVEDD (Odds Ratio 0.87, 95% CI 0.80–0.95, P = 0.002) was an independent predictor associated with improvement in LVEF. Conclusion: In agreement with the literature when analyzing different etiologies, in Chagas‘ etiology, only LVEDD was able to predict progression to HFimpEF. 111990 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GIULIA VITORIA NASCIMENTO DA SILVA1, IGOR LUCAS FARIAS LIMA1, MARCOS AURÉLIO VIEIRA DA COSTA FILHO2, JÚLIA DE MOURA CARVALHO FARIA1, WANDA MARIA DE FRANÇA PIRES2, ARIANE LOBATO MORAES2, LARISSA DACIER LOBATO COMESANHA2, LUCIANO MOURA DE ASSUNÇÃO3 (1) Universidade do Estado do Pará (UEPA); (2) Universidade Federal do Pará (UFPA); (3) Fundação Santa Casa de Misericórdia do Pará Introduction: Rheumatic fever (RF) is a pathology that follows as an immune response to an infection from group A β-hemolytic Streptococcus. Its prevalence is higher in sub-evolved countries, being that around 15 million cases in the world are linked to valve damage, a characteristic of chronic rheumatic heart disease – most serious manifestation of the disease. Objective: To analyze the epidemiological profile of hospital admissions, mortality and cost per hospitalization resulting from acute RF with cardiac in the northern region of Brazil, from 2017 to 2021. Methods: Observational, cross-sectional, analytical-descriptive and quantitative study based on data from Sistema de Informações Hospitalares do SUS (SIH/SUS) – DATASUS. The number of hospitalizations, age, sex, average hospital stay, average cost per hospitalization and mortality rate were analyzed, referring to hospitalizations due to acute RF in the North region of Brazil, from 2017 to 2021. Results: In the period described, 868 hospitalizations for acute RF were reported in the Northern region of Brazil. The lowest number occurred in 2021, with 99 admissions and the highest in 2018 and 2019, with 219 and 218 cases, respectively. The average number of hospitalizations per year was 173.6. Females represented 53.6% (465) of the hospitalizations and males 46.4% (405). The most affected age groups were 5 to 14 years old, with 263 (30.2%), and 40–49 years old, with 101 cases (11.6%) of the total. Children under 5 years had 44 admissions and adults over 20 years old had 515 cases. The average length of hospitalizations during 2017 and 2021 was 6.1 days and the standard deviation was 0.67 between the annual values. The shortest admission time was in 2018, with an average of 5.2 days, and the longest was in 2020 with 7.2 days. The mortality rate was 1.14% with the lowest in 2019 (0.46%) and the highest in 2021 (3.03%). R$328,658.56 was spent on hospitalizations with an average cost of R$373.04. Conclusion: The number of hospitalizations gradually decreased during the study period. Among these hospitalizations, most were women and the main age group affected was children between 5 to 14 years old. The mortality rate followed a pattern contrary to the hospitalization rate, increasing gradually until 2021. It is notorious for the necessity of affirmative actions in public health to prevent the disease and reduce its impact on the brazilian health and economic system. 111996 Modality: E-Poster Scientific Initiation – Non-case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS JÚLIA SCHOUCAIR NEVES1, Pollianna de Souza Roriz1, Fabiana Benevides Pontes1, Mariana Mendonça de Almeida1, Frederico Gesteira de Viveiros Júnior1, João Gabriel Batista Simon Viana1, Franciele Mascarenhas Alves Luz1, Marianne Soglia Calixto Costa1, Luiz Ricardo Cerqueira Freitas Junior1, Victoria Kelly Lima de Castilho1 (1) Serviço de Atendimento Móvel de Urgência – SAMU Introduction: Social determinants of health –such as the level of one’s education – as risk factors for cardiovascular diseases have been highlightened by several studies. These determinants are directly related to the prognosis, and may impact the patient’s identification of the disease itself. In this context, the present study aims to evaluate the influence of the level of education on the self-perception of disease in patients with ST-segment elevation Acute Myocardial Infarction (STEMI) treated in the public health system of the city of Salvador – BA. Methods: A cross-sectional study, carried out with data collected from the STEMI survey from Salvador (PERSISST), which analyzed the association between suspected AMI in patients who had a heart attack between January/2019 and December/2021 and their corresponding level of education. The sample of 859 people was divided into Group I – patients who did not suspect AMI – and Group II – patients who suspected AMI –. Each group was subdivided into 5 different levels of education. Such division of groups aimed to possibly identify an association between the lowest level of education with a lower suspicion of the condition in question. Results: 859 patients were included in the survey (590 in Group I and 269 in Group II). In both groups there was a predominance of male patients, with no difference between the two groups in terms of gender and age. In Group I, 26.8% (158) had gone to school until the 4th year of elementary school; 27.1% (160) had finished elementary school (up to 5th grade); 18.6% (110) had finished middle school (up to 9th grade); 21.2% (125) finished high school and 6.3% (37) had graduated from college. While in Group II, the following distribution was observed in terms of education: 23.8% (64) up to 4th grade; 20.4% (51) finished elementary school, 18.2% (49) finished middle school; 27,9% (75) finished high school and 9.7% (26) finished college. After comparing the groups, it was found that patients with a lower level of education had a lower degree of suspicion, pointing to a statistically significant difference of p = 0.034. Conclusion: In our sample, composed by STEMI patients treated by the public health system of Salvador, the low level of education was related to the patient’s lower self-suspicion of heart attack. This analysis reinforces the need for educational measures, which could positively influence the early recognition of time-dependent diseases. 112013 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ARIANE LOBATO MORAES1, Ariane Lobato Moraes1, Icaro Jose Araújo de Souza1, Cassio Kenzo Câmara Yamada2, Jayssa Leite Freitas3, Bruna Araujo Smith3, Giovana Fonseca Pontes3, Rayane Calandrini de Azevedo3, Luciano Moura de Assunção4 (1) Universidade Federal do Pará (UFPA); (2) Universidade do Estado do Pará (UEPA); (3) Centro Universitário do Pará (CESUPA); (4) Fundação Santa Casa de Misericórdia do Pará (FSCMPA) Introduction: Chagas disease or American trypanosomiasis is a parasitic infection caused by the protozoan Trypanosoma cruzi. Transmission occurs from the contact of the injured skin or mucous membranes with contaminated feces, mainly through the ingestion of contaminated food, such as açaí. Affected individuals may develop Chagas myocarditis, with possible electrocardiographic changes. Objectives: To analyze the epidemiology of Chagas disease cases in the state of Pará, from 2010 to 2020, analyzing the following variables: sex, race, age group, area of residence, probable mode of infection and disease evolution. Methods: This is an epidemiological, descriptive and analytical study, whose data were collected in the Notifiable Diseases Information System (SINAN) made available by the Department of Informatics of the Unified Health System (DATASUS), referring to reported cases of Acute Chagas, in the state of Pará, from 2010 to 2020, totaling 2,221 cases. For the statistical analysis, the Chi-Square test of equal expected proportions was used, through the Bioestat 5.3 program, in order to calculate the p-value of the data found. Data with p value < 0.05 were considered significant. There was no need for submission to the research ethics committee. Results: This study showed that, of the 2.221 reported cases, there was a higher frequency of Acute Chagas Disease in brown patients (80.10%), male (55.16%) and in adults between 20 and 59 years old (58, 89%). The most frequent route of transmission was oral (79.78%). The significant number of unreported data prevents a more detailed analysis. Finally, the predominant evolution was the survival of 86% of the total cases in the analyzed period. Conclusion: The study reports that the epidemiological profile of the patient with Chagas cardiomyopathy between the years 2010 to 2020 is a male patient, brown and aged between 20 and 59 years. In addition, underreporting and missing data are relevant features. Thus, we identified the demand for new studies in order to generate more data and promote public policies aimed at reducing the incidence of Chagas Disease in the region. 112017 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ROBERTA GABRIELA DA SILVA1, Laís Vitória de Andrade Miranda1, Roberto Vieira Botelho4, Rodrigo Alves da Silva4, Marcelo Vieira Damião5, Marcio Sommer Bittencourt3, Miguel Morita Fernandes da Silva2, Odilson Marcos Silvestre1 (1) Universidade Federal do Acre; (2) Universidade Federal do Paraná; (3) University of Pittsburgh; (4) Instituto do Coração do Triângulo; (5) ITMS Telemedicina Brasil Introduction: Alcohol consumption can lead to tachyarrhythmias, particularly atrial fibrillation. Aim: We evaluated the association between alcohol consumption at the population level and the incidence of atrial fibrillation/flutter (AF) in Brazilian capitals. Methods: We performed an ecological time-series study using electrocardiogram (EKG) reports in all Brazilian capitals from 2008 to 2013. EKG data were provided by ITMS Telemedicine Network, and the diagnosis of AF was defined according to the EKG medical report. Abusive alcohol consumption at each capital, defined as the estimated proportion of individuals who binge drinking (≥4 doses for women and ≥5 doses for men, on an occasion), was estimated from the Surveillance of Risk and Protective Factors for Chronic Diseases by Telephone Survey (VIGITEL) in the respective year and divided in terciles: 1st tercile (10.6–14.6%); 2nd tercile (14.7–16.9%); 3rd tercile (17.1–26.6%). A stratified two-stage weighted survey analysis was performed to estimate the incidence of AF at each tercile of alcohol consumption. Results: We evaluated 474,357 EKGs (48.8 ± 18.5 years old, 50% women) performed in all 27 Brazilian capitals. The incidence of AF was significantly higher in the 3rd tercile (5.3%, 95% Confidence interval [CI] 1.6, 9.0%) as compared with the 2nd (1.5%, 95%CI 1.2, 1.7%) and 1st (2.1%, 95%CI 1.7, 2.6%, p < 0.001, figure) terciles of alcohol consumption, respectively. Conclusion: In this population-level study of Brazilian capitals, higher alcohol consumption was associated with increased incidence of AF. 112027 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH HENRIQUE GUIMARÃES AIRES E SILVA1, Pedro Ermel Martins1, Márcia Barbosa de Freitas2, Chaiana Esmeraldino Mendes Marcon1 (1) Universidade do Sul de Santa Catarina; (2) Instituto Nacional de Cardiologia Background: The 12-lead electrocardiogram (ECG) is one of the most used tests in the evaluation of cardiovascular diseases (CVD) in primary health care (PHC) because it is cheap and available in the Sistema Único de Saúde (Brazil’s health care model). However, particularities in the frequency of electrocardiographic findings are observed when population characteristics are considered. Objective: To identify the main electrocardiographic abnormalities and the clinical and epidemiological factors associated with their occurrence in primary health care. Methods: This study analyzed all ECG and application forms of patients submitted to primary care examination over 20 years of age in a municipality of Santa Catarina – Brazil – in 2020. Descriptive analysis of the data and statistical inference was performed. The level of significance adopted was 5%. Results: ECGs of 1907 patients were analyzed, 57.37% of them abnormal. The mean age of the patients was 54,8 ± 15,0 years-old. The abnormalities found varied according to age group, however the most common were nonspecific changes in ventricular repolarization (52.93%), right branch conduction block (28.52%), left ventricular overload (12.07%), electrically inactive areas (10.42%) and divisional blocks of the left branch (8.96%). The main clinical and epidemiological factors related to the presence of an abnormal ECG were age over 60 years (OR 2.17 [CI 95% 1,79–2,62]), systemic arterial hypertension (OR 1.76 [CI 95% 1,46–2,11]), diabetes mellitus (OR 1.78 [CI 95% 1,38–2,30]), dyslipidemia (OR 1.87 [CI 95% 1,49–2,34]), previous acute myocardial infarction (OR 5.63 [CI 95% 1,28–24,72]), previous myocardial revascularization surgery (OR 8.24 [CI 95% 1,06–64,01]), or the use of any medication (OR 1.55 [CI 95% 1,27–1,88]). The report of the electrocardiogram by the cardiologist was made available with less than two days in more than 70% of the tests. Conclusion: The evaluation of the prevalence of electrocardiographic findings and associated factors allows the construction of pre-test probability, which is essential in the context of public health for the indication of the examination rationally and without burdening Brazil’s health care system. The use of telediagnostic resources in this setting allowed to minimize the effects of the large geographic dimentions and social disparitys that exist in Brazil. 112030 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY DAVI GABRIEL BARBOSA1, Paola Bitar de Mesquita Abinader2, Ana Paula Correa de Lima2, Ana Cláudia Reis Guilhon2, Camila Rodrigues Maciel2, Fernando Gabriel Rodrigues Pereira1, Isabella Soares Souza3, Luiz Fernando Leite da Silva Neto1, Fernando Tavares Brasil Teixeira1, Luis Eduardo Werneck de Carvalho4 (1) Universidade do Estado do Pará – UEPA; (2) Centro Universitário do Pará – CESUPA; (3) Universidade Federal do Pará – UFPA; (4) Sociedade Brasileira de Cancerologia – SBC Introduction: Immunotherapies have demonstrated a relevant effect on the prognosis of neoplasms over the years. However, antineoplastic agents increase the risk of cardiovascular disease by inducing cardiotoxicity. Objective: To analyze the induction of cardiovascular diseases by cardiotoxicity caused by antineoplastic drugs in cancer patients. Methods: Systematic review about cardiotoxicity due to antineoplastic treatment, whose data were obtained through the Biblioteca Virtual da Saúde (BVS) from the last 5 years, using the PICO with PRISMA strategy, excluding case reports. The descriptors used were antineoplastic, cardiotoxicity, and anthracycline. Results: On the clinical aspects, no deaths were reported due to cardiotoxicity, i.e., anthracyclines did not cause any effect on mortality, and cardiotoxicity with the occurrence of heart failure was not statistically significant in treatment with anthracyclines. But 4 studies have analyzed the occurrence of arrhythmias, presenting statistically significant numbers, occurring in 317 patients. 3 studies have addressed the side effects of cardioprotective drugs: hypotension, acute pulmonary edema (enalapril), and bradycardia (metropol). About cardiac markers the changes in CK-MB, Troponin I and NT-proBNP were statistically significant along with markers of inflammation and oxidative stress having increased IL-6 and ROS1 with anthracycline treatment. On LVEF The cardioprotective drugs showed a statistically significant benefit in preventing LVEF reduction compared to the control group (MD 3.57, 95% CI 93%). Also, analyzing the cardioprotective drugs, a positive trend was found with the studies involving beta-blockers. The SRAA blockers as a cardioprotective drug also had positive results regarding LVEF. The cardioprotective effects were positive and similar in the various types of chemotherapeutic drugs: doxorubicin and epirubicin, especially in a study addressing breast cancer patients where LVEF obtained a considerabldifference, The cardioprotective drugs showed a possible benefit in preventing the reduction of Mitral Peak E/A Wave Velocity. Conclusion: In summary, there was an increase in inflammatory and cardiac markers and the presence of cardiotoxicity with the occurrence of arrhythmias by the use of anthracyclines. Moreover, the use of drugs with cardioprotective potential has efficacy in preventing the reduction of left ventricular ejection fraction, such as angiotensin-converting enzyme inhibitors. 112031 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT RAFAEL MORETTI1, Michelle Bozko Collini1, Karoline Cordeiro Vercka1, Carolina Ruschel Senger1, Jorge Tadashi Daikubara Neto1, Matheus Bissa Duarte Ferreira1, Jessica Tamires Reichert1, Lucas Müller Prado1, Gustavo Sarot Pereira da Cunha1, Leonardo Henrique dos Santos Melo1, José Renan de Matos Pain1, Miguel Morita Fernandes da Silva1 (1) Complexo Hospital de Clínicas da Universidade Federal do Paraná (CHC-UFPR) Background: Initiating or uptitrating doses of modifying-disease drugs for heart failure (HF) is recommended during hospitalization for decompensated HF. However, data on the impact of HF medical therapy optimization (MTO) during hospitalization on long-term prognosis is scarce. Objectives: This study aims to evaluate the association of MTO during hospitalization and the composite outcome of re-hospitalization and death in patients with decompensated HF. Methods: Prospective cohort study that included patients >18 years old admitted for decompensated HF in a tertiary hospital from October 2019 to February 2022. Patients with ejection fraction >50% or who died during hospitalization were excluded. MTO was defined as any of the following during hospitalization: 1) addition of one of the following classes: ACE-I (Angiotensin-Converting Enzyme Inhibitors)/ARB (Angiotensin II Receptor Blockers)/ARNI (Angiotensin Receptor-Neprilysin Inhibitor), beta-blocker, spironolactone and SGLT2 inhibitor; 2) exchange of ACE-I/ARB for ARNI; or 3) reaching the target dose of one of these medications already in use. The outcome was the composite of re-hospitalization or death from any cause during a follow-up of up to 180 days. Survival analysis was performed by COX regression adjusted for confounding factors. Results: 109 patients were analyzed (66 ± 14 years old, 40% women, ejection fraction: 33 ± 9%), with 36 (33%) presenting the composite outcome. Compared to patients without MTO (n = 44), those with MTO (n = 65) had lower serum creatinine values [1.1 (0.8–1.5) vs 1.4 (0.9–2.0) mg/Dl; p = 0.014) and higher systolic blood pressure [129.7 ± 23.9 vs 119.8 ± 22.8 mmHg; p = 0.035] at hospital admission, and were less likely to have decompensated due to infectious causes (7.7% vs 20.5%; p = 0.05) or non-adherence to sodium and water restriction (1.5% vs. 18.2%, p = 0.002), respectively. After adjusting for these confounders, therapeutic optimization was associated with a lower incidence of the composite outcome (Hazard Ratio: 0.42; 95% Confidence Interval: 0.18–0.96, p = 0.039). Conclusion: In patients with decompensated HF, therapeutic optimization during hospitalization was associated with a lower incidence of the composite outcome of death or re-hospitalization in the 6-month follow-up. 112040 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ALICE ZANETTI DUSSIN1, Anna Paula Tscheika2, Luiz Claudio Danzmann3, Marcus Vinicius Simões4, Andrielle Dias Pinheiro2, Luiz Carlos Bodanese1 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS); (2) Hospital São Lucas da PUCRS (HSL); (3) Universidade Luterana do Brasil (ULBRA); (4) Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) Background: Congestion detected by lung ultrasound (LUS) through B-lines is related to prognosis in HF (heart failure) patients; performing it after a physical stress test can detect congestion even earlier. The 6-minute walk test (6MWT) is a submaximal stress test used to assess response after treatment, functional capacity, and prognosis in HF patients. There are no studies evaluating the use of lung US after the 6MWT. Aim: To evaluate the correlation of the number of B-lines on LUS before and after the 6MWT with the distance covered in the stress test in outpatients with heart failure with reduced ejection fraction (HFrEF). Methods: It is a cross-sectional analytical study with prospective inclusion of patients from three HF outpatient clinics. Inclusion criteria were: age ≥18 years, to have the diagnosis of HF for at least 6 months and to have echocardiogram with ejection fraction <40%. Patients were submitted to the LUS before and after 6MWT. Results: From September 15, 2020 to November 30, 2021, 188 patients were included in the study. The mean age of the patients was 61.83 years (±12.13 years); most patients were male (63.8%); the median left ventricular ejection fraction was 31.73% (interquartile range 25–75% [IQR] 28–37%); the most prevalent etiology of HF was ischemic (52.7%); the median number of B-lines was 3 (IQR 1–9) at rest and 6 (IQR 2–13) after submaximal stress; the median distance covered on 6MWT was 363.5 m (IQR 270–436). The distance covered on the 6MWT was inversely correlated with the total number of B-lines after submaximal stress (r = –0.235; p = 0.001) and at rest (r = –0.253; p = 0.001). Conclusion: The distance covered on the 6MWT was inversely correlated with the total number of B-lines after submaximal stress and at rest in outpatients with HFrEF. There was no significant difference between stress and rest B-lines. More studies, with prognostic follow-up, are needed to assess whether LUS associated with the 6MWT adds more benefit in the evaluation of patients with HF. 112049 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH ANNA LUIZA ALVES DE OLIVEIRA MIRANDA1, Maria Eduarda dos Santos Lopes Vasconcelos1, José Pedro da Silva Sousa1, José Wilker Gomes de Castro Júnior1, Beatriz Siems Tholius1, Evaldo da Costa Sá Borges de Rezende2, Matheus Rocha Maia1 (1) Centro Universitário do Estado do Pará (CESUPA); (2) Universidade Federal do Pará (UFPA) Introduction: Chagas disease (CD) is a zoonosis caused by the protozoan Trypanosoma cruzi, whose vector is a hematophagous triatomine. The transmission mechanisms are vectorial, oral, vertical, accidental, by blood transfusion or organ transplantation. Objective: To analyze the behavior of the annual number of cases of CD in Brazilian regions, between 2016 and 2020. Methods: Descriptive, retrospective, and quantitative study, from 2016 to 2020. Data were extracted from the Notifiable Diseases Information System (SINAN) from the Department of Informatics of the SUS (DATASUS), with the variables: sex, probable mode of infection and confirmation criteria. Results: From 2016 to 2020, were 1,662 new cases of CD in Brazil, 1,573 (94%) in the North, 75 (9%) in the Northeast, 7 (0.5%) in the Southeast and 7 (0.5%) Midwest. Regarding the year of detection, 359 (22%) occurred in 2016, 338 (21%) 2017, 384 (23.5%) 2018, 385 (23.5%) 2019 and 157 (10%) 2020. Gender, 897 (54%) were men and 765 (46%) were women. According to the probable mode of infection, 97 (6%) were: vectorial, 5 (0.3%) vertical, 5 (0.3%) accidental, 1,405 (84.4%) oral and 150 (9%) ignored. According to the confirmation criteria, 1,579 (95%) were performed by laboratory, 45 (2.5%) clinical-epidemiological, 7 (0.5%) remained under investigation, and 31 (2%) were ignored. Conclusion: There was an increase in the annual number of CD cases in Brazil in the years studied, with a predominance in the North region, men, browns and oral mode of infection, the diagnosis being confirmed through laboratory tests, they are the research of the parasite and the identification of antibodies in the serum. In recent years, the average worldwide incidence of CD per year has remained around 30,000 cases/year, with Brazil being an endemic area, especially in the state of Pará, corresponding to 74% of cases in the five years evaluated, a state that has culture of consumption of açaí and sugarcane juice, which are the main sources of food contamination by deposition of feces of infected triatomines or crushing of the same during processing. In this way, it is evident that acting in the awareness of the population about the consumption and treatment of food, as well as greater participation of inspection in the places where potentially contaminated food is sold, contributes to the prevention of Chagas disease, a serious pathology that can lead to cardiomyopathy. and progress to heart failure. 112052 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION GABRIEL IRISMAR RODRIGUES SCHWAMBACK1, RAIMUNDO BENÍCIO DE VASCONCELOS NETO1, Rebecca Shaiane Soares Nunes Rivoredo1, Ana Caroline Leite Guedes1, Jade Gomes da Costa Medeiros1, Lucas Vieira Amorim1, Archimedes Fernandes Alves de Santana1, Byron Maia Feitosa1, Maria Eduarda Brotto de Souza1, Renata Gonçalves Silva Santos1, Orisman Martins de Souza Rocha Filho1, Fernanda Gabry Scazuza Gomes1 (1) Centro Universitário São Lucas – UNISL Introduction: Systemic Arterial Hypertension (SAH) is considered a chronic non-communicable disease (NCD) of insidious onset and that, if not diagnosed and treated, evolves as a precursor to other cardiovascular pathologies. With the aging process, it is common for this CNCD to become more frequent in the population, given the physiological processes of the heart that are affected by time and harmful lifestyle habits. Thus, tracking the number of pre-elderly people (between 55 and 64 years old) and elderly people (over 65 years old) with SAH contributes to highlighting the situation in which a given population finds itself, in addition to providing data for later health interventions. Objectives: To estimate the prevalence of SAH in pre-elderly and elderly people residing in a riverside community in the Western Amazon, Brazil. Methods: This is a cross-sectional, quantitative study carried out with individuals aged between 55 and 87 years, regardless of gender, residing in a riverside community in Porto Velho, Rondônia, Brazil. Data collection was made possible by the application of an individual epidemiological questionnaire during home visits. The analysis was performed by applying the prevalence ratio and the Mantel-Haenszel chi-square test, with statistical significance p < 5%. Results: Data from 58 individuals were analyzed, of which 30 (52%) were male and 28 (48%) were female. We found 35 (60.3%) individuals with an informed prior diagnosis of SAH, with a mean age of 69 years, ranging from 55 to 87 years. As a risk factor for SAH, females have a prevalence ratio equal to 1.4 (variation from 0.93 to 2.19) compared to males (p = 4.9%). Conclusion: SAH is a highly relevant disease to be studied in elderly populations, especially with regard to tracking this CNCD in remote communities, given the strong association between cardiovascular diseases and advanced age. The data point to a prevalence of more than 60% of respondents, an expected value for pre-elderly and elderly people, reinforcing the importance of monitoring the cardiovascular health of this population. No associations were found between sex and SAH in this study. 112054 Modality: E-Poster Scientific Initiation – Non-case Report Category: PSYCHOLOGY LUMA MARIA FAVACHO BORDALO1, Yasmin Cavalleiro de Macedo Maranhão1, Paula Cordeiro Aguiar de Almeida1, Letícia Mariana Gomes Santiago Freire1, Glendse Giovanna Costa Pinheiro1, Luma Fleury de Figueiredo1, Ana Júlia Farache Cabral1, Heullem Uyhara da Silva Amorim1, Israel Figueira Lemos1, Maria Eduarda Dantas da Veiga1, Juliana Tavares de Sousa1, Erick Clayton Gonçalves Feio1 (1) Universidade do Estado do Pará Introduction: In analyzing heart diseases, multiple factors are considered, such as the presence of Major Depressive Disorder (MDD) since pathologies can establish, among themselves, a syndemic relationship capable of aggravating the patients‘ condition. Therefore, the investigation of the impacts of this scenario on the quality of life of those affected is essential to establish improved management measures. Objective: To verify the influence of depression in the treatment of people with heart disease. Methods: The methodology was based on the PRISMA protocol and searches were conducted in the Virtual Health Library (VHL) database using the descriptors “treatment”, “heart diseases” and “depression”; observational studies, published between 2017 and 2022 in Portuguese and English were included. Those that do not relate to the central question, do not have full text available on the platform, or were duplicated were excluded. Results: 6042 articles were obtained with the descriptors. After applying the filters 134 were selected and after deep reading, 4. The first literature indicated that among patients with cardiovascular disease (CVD), there is a prevalence of major depressive disorder (MDD) two or three times greater than the general population and that the possible mechanisms that justify this syndemic relationship between MDD and CVD include dysfunctional neurohormonal and autonomic nervous system effects, brain-derived neurotrophic factor and behavioral factors, as well as inflammation and increased platelet aggregation. Confirming the existence of this relationship, another article points out that individuals with MDD are more likely to develop CVD and associated cumulative diseases, in addition to being more vulnerable to subsequent mortality. Two articles show that patients with CVD and MDD represent a significantly higher financial cost for follow-up than patients without MDD. The fourth study indicates that motivational interviews and interventions based on the transtheoretical model are shown to be effective in improving depressive symptoms associated with coronary atherosclerosis. Conclusion: Patients with CVD are more likely to have MDD and both health conditions have an imbricated relationship, in which the worsened manifestation of one tends to cause the same scenario in the other. In addition, TDM and CVD, when together, result in more financially expensive treatment. 112068 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT VINÍCIUS MACIEL VILHENA1, Aurea Nathallia Gomes de Souza1, Bianca Paula Miranda Martins1, Camila Silva de Oliveira1, Cecília Rodrigues Viana1, Larissa Silva Ferreira1, Luiz Felipe Façanha Ramos1, Marcos Roberto Marques da Silva Júnior1, Reny Wane Vieira dos Santos1 (1) Universidade Federal do Amapá Introduction: Acute myocardial infarction is an ischemic lesion of the heart muscle due to a severe alteration in the functioning of the coronary arteries, leading to decreased blood flow to the myocardium and consequent tissue necrosis. This event can lead to death if not diagnosed urgently. In addition, this disease represents one of the main causes of death in Brazil and in the world, with serious consequences for mortality, morbidity and cost to society. Objective: To analyze the epidemiological profile of deaths caused by acute myocardial infarction in Brazil during 2020. Methods: Epidemiological study with a cross-sectional design of mortality from acute myocardial infarction in Brazil during 2020, divided by regions. The data presented were extracted from the DATASUS database, operated by the Health Department. Results: According to the data collected, in Brazil in 2020 there were 90.465 deaths caused by acute myocardial infarction. With a mortality rate of approximately 42.72 per 100.000 inhabitants. Among the Brazilian regions, the South region had the highest mortality rate, approximately 46.85 per 100,000 inhabitants, followed by the Northeast region with a rate of 43.67. The northern region had the lowest mortality rate, approximately 29.78. Regarding the states, those with the highest rate were: Mato Grosso do Sul (65.67), Rio de Janeiro (61.68) and Piauí (56.83). Those with the lowest rate were: Amazonas (19.03), Amapá (22.28) and Distrito Federal (23.63). According to sex, 53.925 men and 36.534 women died from acute myocardial infarction in 2020. Conclusions: It was observed that Brazil presents different situations in relation to mortality from acute myocardial infarction in each region and state. It is possible to reflect on the quality of life in each region and the possible risk factors related to this disease and how this can change the data, which should be further studied. In addition, there is the possibility of underreporting in regions with lower infrastructure conditions for the collection of these data, which can make the management of this disease difficult. 112088 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT VINICYUS EDUARDO MELO AMORIM1, Ana Cecília Araújo Cabral2, Letícia Fagundes do Nascimento Silva3 (1) Faculdade Pernambucana de Saúde; (2) Faculdade Pernambucana de Saúde; (3) Faculdade Pernambucana de Saúde Background: The coronavirus (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 has negatively affected millions worldwide. There is an urgent need to advance vaccine campaigns, however, the adverse effects of these vaccines remain unclear. Some patients have reported chest pains and atypical symptoms characteristic of Takotsubo cardiomyopathy (TCM) shortly after receiving the vaccine. TCM is often triggered by intense emotional stress. It characteristically presents with features that mimic acute myocardial infarction. Objectives: To assess the relationship between Takotsubo Cardiomyopathy and the Covid-19 vaccine through a systematic review. Methods: The bibliographic search was performed, using the PRISMA protocol, from articles published in PubMed from January 2021 to April 2022 associating the key terms: Takotsubo Cardiomyopathy, Vaccination and COVID-19. From this, 154 articles were found. The articles included fulfilled the criteria for discussing the arising of TCM after vaccination for covid-19. Articles that only approach the increase in the incidence of TCM during the pandemic were excluded. Results: After selection, nine case reports and one case series were identified through this search process totalling eleven patients. The median age of the 11 TCM patients was 62,27 years, with most being female [81,8%]. Vaccines received by patients were Pfizer-BioNTech Covid-19, mRNA-1273 and ChadOX1 nCOV-19. Ten patients developed atypical symptoms within the first 10 days post-vaccination. The most common symptoms were: body pain, chest pain, chest pressure, dyspnea, palpitation, malaise, nausea, fatigue, sweating, vomiting and hypotension. Eight patients had comorbidities, but only 1 had a previous history of Covid-19 infection. All patients had positive troponin and electrocardiogram changes, with a negative T wave being the most common finding. Coronary angiography and X-ray were used to rule out other possible diagnoses. Cardiac magnetic resonance, transthoracic echocardiography and ventriculography were used to diagnose TCM and the most common findings were left ventricular akinesia and hypokinesia, apical ballooning and ejection fraction reduction of less than 50%. Conclusion: Despite the similarity between the cases, the causality of TCM triggered by vaccination cannot be proven, due to the small sample size; however, it appears to be the most likely cause, as the onset of symptoms occurred very soon after vaccination. 112100 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY LETÍCIA RIBEIRO DO VALE1, Elias Pereira de Lisboa2, Pedro Guido Rocha de Almeida3, Vitor Hugo Soares Rosa4, Maria Isabel Fortunato Cavalcante5, Felipe Eduardo campos da Silva6, Lucas Amaral da Silveira7, Elany Maria Ferreira Portela8, Helvecio Teixeira Mazon Junior9, Giovanna Vinhal Reis10, Brenna Pinheiro Zuttion11, Letícia Ribeiro do Vale1 (1) Letícia Ribeiro do Vale; (2) Elias Pereira de Lisboa; (3) Pedro Guido Rocha de Almeida; (4) Vitor Hugo Soares Rosa; (5) Maria Isabel Fortunato Cavalcante; (6) Felipe Eduardo campos da Silva; (7) Lucas Amaral da Silveira; (8) Elany Maria Ferreira Portela; (9) Helvecio Teixeira Mazon Junior; (10) Giovanna Vinhal Reis; (11) Brenna Pinheiro Zuttion Introduction: Atrial fibrillation (AF) results from abnormalities in electrical conduction. The left atrial appendage (LAA) is the most recurrent site for thrombus formation and its occlusion is an intervention that reduces the risk of stroke. Objective: To compare the results of LAA occlusion during cardiac surgery in patients with AF and the development of Ischemic Stroke. Method: Systematic review using the PRISMA methodology and the PICO criteria in the MEDLINE, COCHRANE and LILACS databases, using the keywords “Atrial fibrillation”, “Left Atrial Appendix” and “Thromboembolism” to identify relevant articles. Five publications were considered for the meta-analysis, involving 15,595 patients with AF who underwent surgical cardiac interventions. The odds ratio (OR) values and their respective 95% confidence intervals (95% CI) were calculated using RStudio. Results: Of the five studies, one presented an OR value greater than 1, rejecting the hypothesis. The overall OR was 0.68 [95% CI 0.57; 0.82]. The I² heterogeneity measure was 0%, a value considered as low heterogeneity. Publication bias was assessed using the funnel plot. Conclusion: Patients with AF who underwent LAA occlusion reduced the chances of developing ischemic stroke when compared with patients with AF who did not undergo the procedure. This study presents evidence in favor of performing the LAA occlusion procedure as an effective alternative to reduce the risk of stroke. 112094 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY DAVI GABRIEL BARBOSA1, Marcella Oliveira Monte Santo2, Paola Bitar de Mesquita Abinader3, Daniel Oliveira da Costa1, Afonso Moraes Melo Junior3, Camila Rodrigues Maciel3, João Lucas Watrin Braga3, Bruna Freitas Vinagre3, Igor Lucas Farias Lima1, Luig Matias Barreiros Pires3, Wadilla Fiuza da Silva2, Luis Eduardo Werneck de Carvalho4 (1) Universidade do Estado do Pará – UEPA; (2) Universidade Federal do Pará – UFPA; (3) Centro Universitário do Pará – CESUPA; (4) Sociedade Brasileira de Cancerologia – SBC Introduction: Antineoplastic chemotherapy consists in the administration of drugs that act at the cellular level, destroying tumor cells. Because they act without specificity, they can generate undesirable effects, such as cardiotoxicity; presenting as main damages the higher frequency of moderate or severe heart failure associated with systolic or diastolic ventricular dysfunction. Objectives: To analyze the induction of heart failure in patients on antineoplastic treatment by searching for scientific evidence in the database literature. Methods: A systematic review was conducted through a bibliographic search of articles, using the Virtual Health Library database, whose filters were: articles published in the last 5 years, published freely and in full, and randomized clinical trials. The descriptors used comprised (Heart Failure) AND (Antineoplastics) AND (Cardiotoxicity) and the exclusion criteria were case reports and other systematic reviews. PICO and PRISMA methodology was employed. Results: The use of trastuzumab can lead to decreased left ventricular function and moderate or severe HF. There is a decline in LVEF with improved survival with the use of ACEI and beta-blockers, reducing mortality and patient hospitalization. Crizotinib showed few events associated with HF. Anthracycline, used in first-line lymphoma treatment increases the risk of CHF in patients with no prior history of heart disease, particularly when treatment courses with more than 6 cycles of R-CHOP/CHOEP. The expression of TLR2 and TLR3 biomarkers is up- and down-regulated, respectively, in patients treated with doxorubin, causing cardiac dysfunction. ACE inhibitors in anthracycline-induced cardiotoxicity, did not develop arrhythmia or heart failure, and enalapril acted in reducing cardiac toxicity after anthracycline administration. Conclusion: It is inferred that the antineoplastic drug with the highest relation to HF is anthracycline. However, the group of ACE inhibitors and enalapril decrease the cardiotoxicity of anthracycline. Moreover, trastuzumab is related to the onset of moderate or severe HF, and the ACE inhibitor drug groups IECA and beta-blockers decrease mortality in these patients. Doxorubin has predictive markers: TLR2, up-regulated, and TLR3, down-regulated. 112099 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH GABRIELLY CARVALHO LEÃO1, Gabrielly Carvalho Leão1, Saul Rassy Carneiro1, Vando Delgado de Souza Santos1, Bruno Patricio dos Santos Oliveira1 (1) Universidade Federal do Pará Introduction: Cerebrovascular Disorders encompass several diseases with clinical manifestations resulting from the interruption of brain blood flow, which can manifest in an ischemic way, usually linked to circulation obstruction, or in a hemorrhagic way, when there is vascular rupture. In view of the wide occurrence of deaths and permanent sequelae that such diseases generate, investigation and understanding of them become indispensable. Objectives: To identify and analyze the epidemiological profile linked to the mortality of female individuals due to Cerebrovascular Disorders in the northern region of Brazil, from 2010 to 2020, as well as to associate it with regional factors of a social and economic nature. Methods: This is an observational and cross-sectional study, which used mortality records in cases of cerebrovascular diseases in northern women, between January 2010 and December 2020, obtained through the Departamento de Informática do Sistema Único de Saúde (DATASUS), using the variables age group, color, schooling and place of occurrence. Results: The data gathered showed the occurrence of 30,490 deaths of women from cerebrovascular diseases in the region in question during the period considered, a value that corresponds to 45.94% of the total deaths of this category in the north of Brazil. Regarding the profile of these northern women, it should be noted that 79.96% of them were aged over 60 years. In addition, 33.47% of these women did not have any complete levels of education and 66.35% declared themselves to be brown. Finally, regarding the place of death, 74.55% took place in hospital settings. Conclusion: Thus, it is evident that cerebrovascular disorders predominate in the female population in disadvantaged socioeconomic situations, especially elderly brown women, without a degree of education, with low income and possibly without access to health surveillance and primary prevention mechanisms (such as encouraging and subsidizing healthy eating and physical activity). In addition, the high death rates in the hospital context indicate the contribution of the diseases analyzed to the burden of the health system, reinforcing their severity and the need for preventive actions, in order to guarantee the well-being and longevity of women northerners. 112104 Modality: E-Poster Scientific Initiation – Non-case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BIANCA VASCONCELLOS RODRIGUES LOPES1, Luisa de Oliveira Pereira Vila Nova Ramalho1, Laura Cordeiro Gomes3, Philip Brainin2, Anna Engell Holm2, Karine Oliveira Lima1, Isabelle Victória Martins Vieira1, Marliton Vinicius Pedrosa Evangelhista1, Luan Oliveira Matos1, Iara Fernanda Vasconcelos de Oliveira e Silva1, Tor Biering-Sørensen2, Odilson Marcos Silvestre1 (1) Federal University of Acre; (2) Herley-Gentofte University Hospital; (3) University of São Paulo Introduction: Neglected diseases, less access to health services and socioeconomic factors makes the Amazon Basin a vulnerable area for heart disease. There is no data concerning the risk for developing heart failure in this setting. Aim: To assess the population at risk for developing heart failure in the Amazon Basin. Methods: We performed a cross-sectional study in the Brazilian Amazon Basin. Patients were classified as at risk for HF according to the American College/American Heart Association staging system. For stage A, we considered the presence of at least one of the following factors: hypertension, diabetes, BMI > 30 kg/m2, hypercholesterolemia, moderate or intense alcohol use, smoking and history of rheumatic fever. We compared those at stage A with those with no risk factors (table). Results: The mean age was 39 years and 47% were male. 70% of all patients were in stage A for HF. Among those, 29% had hypertension, 4% had diabetes, 24% were obese, 38% had hypercholesterolemia, 11% consumed alcohol at moderate or intense level, 11% smoked and 6% had a history of rheumatic fever. HF risk participants had lower educational levels, 214 (27.7%) participants did not complete elementary school vs. 43 (13.4%) in out of risk population (p < 0.001). A total of 385 (49.9%) stage A patients have lived in rural areas for the most part of their lives against 121 (37.7%) at non-risk group (p < 0.001). Median income was lower in stage A [median (IQR) R$ 360.0 (175 to 700.0)] when compared to the other group [median (IQR) R$ 400.0 (240.0 to 800)] (p < 0.004). Conclusion: Most of Amazon Basin population is at risk for developing heart failure. Patients at stage A are older, have lower income and educational levels and have lived in rural areas for the most part of their lives. 112112 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LÍVIA FARIAS DE HOLANDA FURTADO1, Patrícia Oliveira Lima de Macedo1, Lais Vieira Araujo1, João Victor Bezerra Nunes1, Abel Belarmino de Amorim Neto2, Lara Andrade Dantas2, Ranise Nunes Pereira Moura2, Daniel Marcelo Silva Magalhaes2, Carlos Marximiliano Alves de Oliveira2, Juliana Sousa Soares de Araujo1, Cláudio Teixeira Regis3, Fabrício Leite Pereira2 (1) UFPB – Universidade Federal da Paraíba; (2) Hospital Metropolitano Dom José Maria Pires; (3) Complexo Pediátrico Arlinda Marques Introduction: Coarctation of the aorta is a congenital heart disease in which there is narrowing of the aortic isthmus. Stenting for treatment is well established in adults and in children over 25 kg. However, in younger children the usual treatment is surgery or balloon recoarctation. Currently, this technique has been used in selected cases. Objective: To demonstrate the early results of stent implantation for the treatment of carotid coarctation in infants. Methods: This is a cross-sectional, observational, retrospective study of a case series of stenting in carotid coarctation in a public hospital in Paraíba. Results: 4 infants underwent carotid stenting in infants with a mean age of 110 days (22 days to 12 months), mean weight 5.3 kg (2.9 to 10.6 kg) all male, and with another cardiac alteration. In the first case, the coarcted area increased from 2.6 to 7 mm; the second increased from 1.3 to 6 mm; the third increased from 3 to 8 mm, and the fourth increased from 3.4 to 8.5 mm. Thus, the pre-treatment mean was 2.58 mm and became 7.38 mm, with the size of the balloon chosen by measuring the abdominal aorta and the aortic arch. Only 1 patient presented as a complication a small aortic dissection, of expectant management. All patients presented normalization of the abdominal aortic flow in the control echo. Conclusion: Stent implantation proved to be effective and safe in the short term, and may be a treatment option also at this age. However, further long-term studies are needed to monitor the effectiveness of the technique in terms of reinterventions required due to somatic growth of patients. 112116 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIO-ONCOLOGY PAOLA BITAR DE MESQUITA ABINADER3, Marcella Oliveira Monte Santo1, Ana Josefina Gonçalves Salomão3, Davi Gabriel Barbosa2, Joao Lucas Watrin Braga3, Leandro Lourenço Silva Monteiro2, Manuela pedreira da Cruz Rocha3, Vinicius Queiroz Silva2, Alexandre D‘Annibale Cartágenes2, Luis Eduardo Werneck de Carvalho4, Luiz Fernando Leite da Silva Neto2 (1) UNIVERSIDADE FEDERAL DO PARÁ (UFPA); (2) UNIVERSIDADE ESTADUAL DO PARÁ (UEPA0; (3) CENTRO UNIVERSITÁRIO DO PARÁ (CESUPA); (4) ONCOLÓGICA DO BRASIL Introduction: Antineoplastic protocols guarantee standards and criteria to treat different neoplasms, based on effectiveness and scientific evidence. Henceforth, it is possible to define the degree of validity in therapeutic category. In the cardio-oncology field, adopting these protocols is essential, since some pharmacological interventions can be cardiotoxic, such as the use of anthracyclines, becoming necessary the regulation of therapies for these pathologies. Objective: Analyzing antineoplastic protocols in cardio-oncology searching for scientific evidence and database updates. Methods: This is a systematic literature review with descriptive collection and a qualitative approach to data on antineoplastic protocols in cardio-oncology, available in the BVS database. The search formula used was: Antineoplastic Protocols AND Cardiac Neoplasms. Studies that presented a 5 years maximum gap since their publication, and published in completion and in English, were selected. Exclusion criteria were: other types of studies. The data extraction and synthesis strategy followed the PICO and PRISMA guidelines. Results: 795 studies were found on the search platform with the descriptors, 113 remained after initial filtering, 40 of these were applicable in the inclusion criteria, 4 studies were selected for analysis. In the case of cardiac rhabdomyomas related to tuberous sclerosis, surgery is indicated, but it has a high mortality rate. Regarding pharmacological treatment for them, there are studies indicating that prolonged treatment with sirolimus has shown positive results and has been shown to be more beneficial in male patients. In the case of ventricular fibromas and cardiac myxomas, surgical resection with cardiopulmonary bypass showed benefits and a high survival rate with few complications. Conclusion: In this viewpoint, it is noted a beneficial relationship in the use of antineoplastic drugs in patients affected by cardiac tumors, especially in male subjects, with the treatment being faster; aged over 4 years and fewer cardiovascular surgeries. Thus, even though the number of studies on this condition is scarce, there is an important possibility of pharmacological intervention in cardio-oncology services that needs further studies. 112123 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY GUSTAVO GUIMARÃES OLIVEIRA3, Gustavo Guimarães Oliveira3, Carolina Maria Vianna Guimarães2, Jackson Brandão Lopes2, Tainá Teixeira Viana1, Diêgo Moreira Arruda1 (1) Hospital Ana Nery – HAN; (2) Universidade Federal da Bahia – UFBA; (3) Escola Bahiana de Medicina e Saúde Pública – EBMSP Background: Posterior pericardiotomy (PP) is a surgical technique in which a 4 cm incision is made in the pericardium in order to avoid complications in the postoperative period (PO) of cardiac surgeries, such as pericardial effusion (PE) and atrial fibrillation (AF). There are, however, few studies addressing the presence of electrocardiographic findings of pericarditis in patients undergoing this procedure. Objective: Compare the presence of electrocardiographic alterations compatible with pericarditis, AF and PE in the PO period of myocardial revascularization between the groups. Methods: A double-blinded, single center, randomized study, conducted by Hospital Ana Nery – Bahia, with a sample size of 30 patients. Patients were randomized into two groups: intervention group (submitted to PP) and control group (CG), which did not undergo PP. Data collected for the study included pre-, intra- and postoperative data. Results: In the sample, 62.5% are men, the PP group was composed by 66% male and the CG 80%, while the mean age of the study was 62.4 ± 8.9 years. The patient’s EUROSCORE II had a median of 0.76 with a maximum of 2.5 and a minimum of 0.5. All the patients analyzed used the internal mammary artery in the surgery, while 26.7% of the CG used double mammary against 6.7% in the PP group. The radial artery was used in 33.3% of patients in the PP group and 53.3% of the CG. In the PP group, a frequency of 6.7% of AF was observed in the PO, while in the CG 0.0% (p = 0.30). Regarding PE, there was a 26.7% incidence in the PP group and 40.0% in the CG (p = 0.43). Electrocardiographic findings of pericarditis were identified in 6.7% of patients in the CG and 20.0% of the PP group (p = 0.28). There was one death in the study that occurred in the CG on the 20th postoperative day (p = 0.30). Conclusion: Although a lower incidence of PE was found in the PP group, it was not statistically significant. It was also not possible to identify a statistically significant difference in the occurrence of AF between the groups or of electrocardiographic changes compatible with pericarditis. Due to the low incidence of events in this study, the N was not powerful enough to identify the statistical differences already reported in the literature, which makes us believe that a larger sample is needed to corroborate or disprove previously published data. 112124 Modality: E-Poster Scientific Initiation – Non-case Report Category: DYSLIPIDEMIA AMANDA PEREIRA MATOS1, Rogério Krakauer2, Renato Jorge Alves2 (1) Faculdade de Ciências Médicas da Santa Casa de São Paulo; (2) Santa Casa de Misericórdia de São Paulo Background: Familial Hypercholesterolemia (FH) is a disorder characterised by an increased serum LDL-cholesterol level (LDL-c > 190 mg/dL). It represents a high cardiovascular risk and leads to complications that can compromise the ventricular myocardium function. There are no studies on left ventricular (LV) diastolic function in patients with FH. The early detection of subclinical morphological changes could contribute to a more effective prevention strategy. Purpose: This study aimed to obtain the prevalence of diastolic dysfunction among patients with FH under primary prevention (PP) and to epidemiologically compare PP and secondary prevention (SP) in patients with FH. Methods: We performed a cross-sectional study with 42 patients with FH over 18 years old of a dyslipidemia outpatient clinic of a tertiary hospital. The patients were divided into two groups: PP and PS. We excluded those who, at the time of the echocardiographic assessment, presented: ejection fraction less than 50%, LV regional wall motion abnormality, significant valve dysfunction, cardiac arrhythmia, pericardial effusion or unavailable information to determine LV diastolic function. Each exam was performed on the same machine and analysed by the same physician. Results: Out of 42 patients, 9 were excluded. Of a total remaining 33, 12 were included in the PP group and 21 in the SP. Therefore, 36,4% (12/33) underwent the transthoracic echocardiogram exam for diastolic function assessment. After exams‘ analysis: 75% (9/12) of the patients had normal diastolic function, 8.3% (1/12) indeterminate function and 16.7% (2/12) presented grade I diastolic dysfunction. Of those with normal function, one had diabetes and one, grade I obesity. Of those with the dysfunction, one had diabetes and one, diabetes and grade III obesity, both elderly. By comparing the two groups: PP had an average age of 60.5 years old and SP 67 years old; PP 91.7% women and SP 71.4%; PP 25% and SP 38.1% diabetes; PP 50% and SP 90.5% arterial hypertension (p = 0,015). Conclusions: Patients with FH under PP did not show significant changes in LV diastolic function (low prevalence of grade I dysfunction only). Furthermore, patients under SP showed a higher frequency of arterial hypertension. However, new studies must be carried out, with a higher number of patients, for better clarification. 112125 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY DAVID CESARINO DE SOUSA1, Fábio Antônio Serra de Lima Junior1, Renan Furtado de Almeida Mendes1, Andre Loureiro Fernandes1, Ana Beatriz Venancio de Paula Bezerra1, Raphael Patrik Borges da Costa1, Tiago Lucena de Brito Pereira2, João Bosco Ferreira Gadelha2, Isaac Newton Guimarães de Andrade1, André Telis de Vilela Araújo1 (1) Universidade Federal da Paraíba; (2) Clínica Dom Rodrigo Introduction: Postoperative atrial fibrillation is responsible for important postoperative morbidity and mortality, and the ability to predict it could allow prophylactic measures and intensive monitoring in patients at higher risk. Objectives: To relate preoperative ECG findings of patients undergoing cardiac surgery with the occurrence of postoperative arrhythmias, and compare the results with those found in national and international studies. Methods: A longitudinal observational study was conducted with patients undergoing cardiac surgery between 2019 and 2021 in a cardiology referral hospital in João Pessoa. Pre- and postoperative 12-lead electrocardiogram data were collected and processed, in addition to patients‘ constitutive and surgical data. Results: A total of 105 patients were observed, with the presence of supraventricular extrasystole (2 vs. 0 cases, p = 0.017), P-wave amplitude (0.993 ± 0.8426 mV vs. 0.318 ± 1.1467 mV, p = 0.015), the Morris index (–94.286 ± 78.2849 mV.ms vs. –36.951 ± 94.7297 mV.ms, p = 0.023) and the presence of left ventricular overload (6 vs. 12 cases, p = 0.015). Conclusion: The presence of supraventricular extrasystole, P wave amplitude, Morris index, and the presence of electrocardiographic criteria of left ventricular overload were associated with a higher risk of developing postoperative atrial fibrillation. Larger samples may reveal yet other significant predictor variables. 112127 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION ISRAEL FIGUEIRA LEMOS1, Alice Marcely dos Santos Tuñas1, Rafael Malcher Meira Rocha1, Giovanna Lopes Evangelista2, Ronaldo Monteiro Veras3, Guísela Gabriela Silveira de Souza1, Maria Giovanna Trindade Rocha1, Lucas Matheus Marinho de Miranda1, Maria Eduarda Dantas da Veiga1, Juliana de Sousa Tavares1, Erick Clayton Gonçalves Feio1, Luma Maria Favacho Bordalo1 (1) Universidade do Estado do Pará; (2) Centro Universitário do Pará; (3) Universidade Federal do Pará Introduction: According to the new Brazilian Hypertension Guidelines (2020), the main reason for inadequate control of hypertension is noncompliance with long-term treatment. Some factors such as the absence of symptomatology and the requirement for continuous treatment, as well as changes in habits, are important in this statistic. Depression, however, can make the patient reduce his health care for a psychic issue, by the symptoms of discouragement, easy fatigue, and need for greater effort to do things. Objectives: To evaluate the relationship between adherence to treatment for hypertension and depressive symptoms. Methodology: This is a systematic literature review, it was used as a database the Virtual Health Library (VHL), with the descriptors “Hypertension”, “Depression” and “Treatment Adherence” between the years 2017 and 2022, in English and Portuguese, published free and fully, selected through the PRISMA method. Where articles that did not address the relationship to be evaluated and literature reviews were excluded. Results: 283 articles were obtained with the use of the descriptors, 87 with the application of search filters, after detailed reading, 8 met the objectives of the study. From the literature, it is observed that depression negatively influences adherence to hypertension treatment. The chance of a patient being at the target pressure increases significantly with increased adherence to treatment for depression. In one study, with women only, depression was more reported by patients with worse adherence to AH treatment (p = 0.026). However, no differences in depression symptoms were observed between adherent and non-adherent patients seen in an emergency room. Hypertensives with depressive symptoms have a significantly higher relative risk of treatment noncompliance. In this sense, the negative impact of depression on the capacity for self-care and continuation of the prescribed drug therapy is noted, as well as the vulnerability and need for support. Conclusion: In this article, we sought to evaluate adherence and continuity of pharmacological and non-pharmacological treatment for AH in patients with depressive symptoms. Therefore, expanding studies that propose and evaluate strategies for adherence to drug treatment is essential to ensure that individuals remain normotensive, with the potential to reduce the pressure on the health system and represent simultaneous gains in the quality of life of hypertensive individuals. 112134 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM RAFAEL BRACCIO ZAWISLAK1, Mariana Saadi de Azevedo1, Alice Zanetti Dussin1, Thomás Ranquetat Andrade1, Mario Wiehe1 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) Background: Coronavirus disease (COVID-19) is caused by SARS-CoV-2, in which the patient affected has different clinical manifestations. The infection may cause different cardiovascular complications, such as sudden death and cardiac arrest. Besides the pathophysiologic relation of the infection with cardiovascular diseases, these different bad outcomes may also have a social and political component, such as saturation of the health system and loss of patient follow-up. Aims: To evaluate the impact of the Covid-19 pandemic on cases of cardiac arrest with death outcome in Brazil and verify the frequency according to in or out-hospital events. Methods: The data of the last 4 years (2018 to 2021) of deaths due to cardiac arrest were collected using the Mortality Monitoring Panel of the Health Surveillance Department of Brazil. The number of deaths were separated into tables comparing the year of the deaths to location of event. This was conceived as a descriptive study. Results: Pre-pandemic numbers (2018 and 2019) were very similar, averaging them for comparison. The number of deaths of the 2 years before the Covid-19 pandemic were quite similar in age groups and sex. The average number of deaths in the 2018–19 period was 1384, with an increase of 56.21% in 2020 and 130.41% in 2021. The cause of the cardiac arrest was described as sudden death in all periods analyzed. The average number of cardiac arrests in-hospital in the 2018–19 period was 383.5, with an increase of 32.72% in 2020 and 87.48% in 2021. In the 2018–19 period, out-hospital cardiac arrests were at an average number of 1000.5, with an increase of 65.21% in 2020 and 146.87% in 2021. Conclusion: The number of cardiac arrests during the COVID-19 pandemic increased overall, in-hospital as well as out-hospital. This may be related to pathophysiological and social-political components. Events that occurred at home had a significant growth, which may be associated with lockdown protocols and difficulty in obtaining basic access to healthcare, possibly related to a hesitation of a possible acquisition of SARS-CoV-2 infection and the overload of hospitals. 112144 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MARIANA SAADI DE AZEVEDO1, Rafael Braccio Zawislak1, Alice Zanetti Dussin1, Rafael Vianna Behr1, Mário Wiehe1 (1) Pontifícia Universidade Católica do Rio Grande do Sul Background: Covid-19, disease caused by Sars-CoV-2, is responsible for the pandemic that generated instability in health systems across the globe and over 66,0000 deaths in Brazil since 2020. There is evidence that arterial hypertension is associated with a more severe course and higher death rates. In addition, control of chronic diseases has been a challenge due to loss of patient follow up and failure to seek medical help in an acute condition. Aims: To convey epidemiological data of the last 5 years of death by hypertensive disease in Brazil and to elucidate its possible relation to Sars-CoV-2 infection and social aspects of the Covid-19 pandemic. Methods: This was conceived as a descriptive study. The Mortality Monitoring Panel of the Health Surveillance Department of Brazil was used to collect the data of the last 5 years (2017 to 2021) of deaths due to hypertensive disease. The data was organized in a table comparing the years pre and post Covid-19 advent. Results: There has been a 19.98% increase in the average of death rates by hypertensive disease in Brazil post covid-19 (years 2020 and 2021) in relation to the years previous to the pandemic that were analyzed (2017–2019). In the years 2017–2019, the average of yearly deaths at home was 21440, while in the years 2020 and 2021 the average escalated to 30146 cases. This shows an increase of 40.6% in the average of deaths at home by hypertensive disease in Brazil post Covid-19. Concerning deaths in hospital, there was an increase of 0.72% in the yearly average post Covid-19. Conclusion: There has been an increase of death by hypertensive disease in Brazil in the years of 2020 and 2021, which coincides with the Covid-19 pandemic. This can be attributed to a significant rise in domicile death rates. Future studies can evaluate factors involved in this increase and, consequently, the forms of preventing it in similar situations. In addition, it must be observed if, in the years following the pandemic, the number of out hospital deaths will return to that of previous years, or if they will still be affected by the pandemic period. 112152 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH VANDO DELGADO DE SOUZA SANTOS1, Saul Rassy Carneiro1 (1) Universidade Federal do Pará – UFPA Introduction: Diseases of the circulatory system (DCS) are diseases that affect the heart and blood vessels. Since 1980, they have been the leading cause of death in the world. The analysis of the association between DCS and financial transfers becomes, therefore, essential for the implementation of health policies. Objectives: To analyze the relationship between hospital admissions for DCS and transfer of financial resources via the National Health Fund (NHF) to the northern region of Brazil from 2011 to 2021. Methods: This is a longitudinal epidemiological study carried out with secondary data from the Department of Informatics of the Unified Health System on the number of hospitalizations for diseases of the cardiocirculatory system and the transfer of resources via NHF to the states of the northern region between January 2011 to December 2021. Results: In the analyzed period, a linear regression model was performed, where the transfer of funds via NHF was the independent predictor to analyze hospitalizations for DCS, under the formula y = 69–2.57e–9x. The allocation of resources to the states of the northern region resulted in a significant reduction in the number of hospitalizations (R = 0.748; p < 0.001). A model with the number of family health strategies was used, but it proved to be less adjusted than the transfer of resources, perhaps because the increase in liquidity in the states had a direct impact on the increase in family health teams. Conclusion: There was an inversely proportional relationship between hospitalizations for DCS and financial transfers via NHF. It is important to emphasize the aspect of primary care performance in controlling these indicators, because, with efficient primary care integrated with reference services, the number of hospitalizations for CAD can be reduced. 112170 Modality: E-Poster Scientific Initiation – Non-case Report Category: ANTICOAGULATION REGINA MARIA ALEXANDRE FERNANDES DE OLIVEIRA1, Amanda Pereira Matos1, Ronaldo Fernandes Rosa2, Milton Luiz Gorzoni2, Renato Jorge Alves2 (1) Faculdade de Ciências Médicas da Santa Casa de São Paulo; (2) Hospital da Santa Casa de Misericórdia de São Paulo Background: Coronavirus disease 2019 has been associated with a prothrombotic state, which puts into question whether a prophylactic anticoagulant therapy could be used to prevent bad outcomes in hospitalised patients since they represent the most severe cases of the disease. However, substantial studies have reported divergent results regarding its benefits and applicability in prevention strategies. Purpose: To explore the incidence of bad outcomes in COVID-19 hospitalised patients that have received Anticoagulants or not. Methods: This retrospective observational study analysed electronic medical records from hospitalised patients with laboratory-confirmed COVID-19 in a large tertiary hospital, between March 2020 and July 2020. The study sample was divided between those who received any dose of anticoagulation (low molecular weight or unfractionated heparin) and those who did not (control group). Our primary outcome was data on mortality, hospital length of stay, need for Intensive Care Unit admission and use of vasoactive medications between the two groups. Results: Of a total 317 patients, 259 (37.8% females, 62.2% males) received Anticoagulants and 58 (37.9% females, 62.1% males) did not. The mortality rate was 49.4% in the Anticoagulant recipient group, and 58.6% in the control group. However, no association could be made between Anticoagulant use and hospital survival (odds ratio [OR] 1.4; 95% confidence interval [CI], 0.8–2.6). The length of hospital stay showed to be similar between the two groups, with an average of 17.8 days (95% CI, 15.3–20.2) for the Anticoagulant group and 16.2 days (95% CI, 9.5–22.8) for the control one. Additionally, lesser need of intensive care could not be associated with blood thinners use (OR 1.6; 95% CI, 0.8–3.1), whereas utilisation of vasoactive medications could (OR 6.5; 95% CI, 3.2–13.3). Conclusion: The findings of our study suggest that the use of Anticoagulants in COVID-19 hospitalised patients is associated with reduced need for vasoactive drugs, with no impact, however, in mortality, hospital length of stay or need for Intensive Care Unit admission. We intend to reexamine the matter with a larger sample of patients. 112160 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION LETICIA FAGUNDES DO NASCIMENTO SILVA1, Ana Cecília Araújo Cabral2, Vinicyus Eduardo Melo Amorim3 (1) Faculdade Pernambucana de Saúde – FPS; (2) Faculdade Pernambucana de Saúde – FPS; (3) Faculdade Pernambucana de Saúde – FPS Background: Systemic arterial hypertension (SAH) is a highly prevalent disease that can be both the cause and the consequence of chronic kidney disease (CKD). Chronic kidney disease is defined when the glomerular filtration rate (GFR) is less than 60 mL/min/1.73 m2 for three or more months. In this way, the treatment objective to reduce the progression of kidney disease and cardiovascular risk. Given this, percutaneous sympathetic denervation emerges as a promising new therapy for patients with resistant hypertension when drug therapy fails. In this procedure, ablation of sympathetic fibers is performed through the application of radiofrequency transluminally in the renal arteries. Methods: The study is designed as a systematic review and the articles used were collected through the “PUBMED” and “BVS” platforms and selected according to the PRISMA 2020 protocol. Using the descriptors “Renal denervation”, “Hypertension” and “Chronic kidney disease”, 105 articles were found. Those with full texts were selected while duplicate articles and that did not directly address the research topic were excluded, leaving 18 articles for review. Results: 18 articles included in the review, including randomized clinical trials, clinical trials, meta-analyses, prognostic study, diagnostic study and etiology study. It was observed an improvement in central pressures and pulse wave velocity, a reduction in systolic pressure was observed in most patients after 3 years of treatment, in addition to renal protection in most of the studies performed. Changes in renal sensory function participate in the maintenance of high vasomotor activity and cardiorenal changes; as such, renal sensory fibers may be a potential therapeutic target for the treatment of cardiorenal diseases. However, results from some studies do not show a reduction in the degree of kidney injury. Furthermore, the change in aldosterone level evidenced reaffirms the effect of denervation on the renin-angiotensin-aldosterone system. It is necessary to point out that more clinical trials are needed to assess the safety and reproducibility of the method. Conclusion: Renal denervation is safe for patients with resistant SAH and CKD, with a significant reduction in systolic pressure. However, further studies are needed to assess safety, application advantages and clinical impact. In addition, the mechanisms involved in cardiorenal protection by denervation have not yet been fully clarified. 112171 Modality: E-Poster Scientific Initiation – Non-case Report Category: PSYCHOLOGY MARIA EDUARDA DANTAS DA VEIGA1, Juliana de Sousa Tavares1, Erick Clayton Gonçalves Feio1, Luma Maria Favacho Bordalo1, Israel Figueira Lemos1, Amanda de Queiroz Andrade1, Bruno Kauê Rodrigues Vilhena1, Chan David Ribeiro1, Greta Évelin da Silva Mota1, Luis Arthur Moreira Ferreira1, Victor Leno Silva Paes1, Alice Marcely dos Santos Tuñas1 (1) Universidade do Estado do Pará – UEPA Introduction: Acute Myocardial Infarction (AMI) is a fatal condition, and survivors can present multiple complications related to mental health and quality of life. Thus, the psychological consequences in the individual who suffered AMI interfere with the prognosis of the disease, as there is an association between psychological and biological factors, such as depression and anxiety. Furthermore, these pathologies affect the patient’s recovery and life expectancy, increasing the risk of AMI recurrence. Objectives: Identify the psychological impacts on patients after AMI. Methods: Systematic literature review with searches in the Virtual Health Library (BVS) using the descriptors “Psychological distress”, “Psychological stress”, “Myocardial Infarction” and “Post-traumatic stress disorder”. The filters used were original articles, complete and open to public consultation from the last 10 years in Portuguese, English and Spanish. Case reports, notices, editorials, comments and literature reviews were excluded. The PRISMA protocol and the question formulated by the PICO methodology were used. Results: 158 articles were obtained with the descriptors. After applying the filters and detailed reading of the data, 17 articles were included in the present study. The literature pointed to the occurrence of several psychological disorders in patients who suffered from AMI, which made recovery even more difficult. It was pointed out that individuals with Post Traumatic Stress Disorder (PTSD) after infarction had a greater stress response, suggesting an association between PTSD with or without cardiovascular outcomes. Based on this logic, it was found that patients with emotional distress are more likely to die after the AMI. Other results, in addition, indicated heterogeneity of emotional suffering, showing a higher incidence of stress related to youth, poverty and the female gender. Furthermore, high levels of anxiety and depression were noted as predictive factors for mortality and readmission to patients with symptoms of ischemic heart disease. Moreover, professional psychological support is also related to a reduction in the mortality rate and decrease in the recurrence of the cardiac condition. Conclusion: It was identified that the psychological impacts, especially depression and anxiety, are evident in people who have suffered from AMI. In this sense, a more in-depth study of the topic and its relationship with the quality of life of these patients is necessary. 112176 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY KARLA SANTOS PINTO1, Elias Soares Roseira1, Rafaela Ferreira dos Santos1, José Victor de Sá Santos1, GABRIEL SAPUCAIA DA SILVA DE QUEIROZ1, Pollianna Roriz1, Luiz Carlos Passos1, William de Carvalho1 (1) Hospital Ana Nery Introduction: Chagas disease (CD) is the fourth disease with the greatest social impact in Latin America. It is associated with ventricular tachyarrhythmias – an important cause of sudden death (SD). Thus, the implantable cardioverter-defibrillator (ICD) appears as a treatment option for being able to reverse these arrhythmias. Despite this, there are gaps in the indication of the device with a focus on this population, especially in the context of primary prevention. Objective: To describe the profile of CD patients with ICD implantation, prevalence of triggered therapies and post-implantation outcomes. Methods: Descriptive study including patients treated at a tertiary hospital in Salvador-BA diagnosed with Chagas Disease, between January/2021 to April/2022. The variables described were age, sex, NYHA functional class, previous comorbidities, medications, Rassi score (in primary prevention), ICD indication for primary or secondary prophylaxis, number of ICD electrodes, Maggic ICD therapy score and deaths. Results: Sample (n) included 234 CD patients, with a mean age of 60 years, 63.7% of whom were men. The most prevalent previous comorbidities were systemic arterial hypertension (61.1%) and type II diabetes mellitus (14.5%). 27.8% had atrial fibrillation, 15.4% and 9.4% had a history of AMI and CVA, respectively. 62% were classified as NYHA II. 90.2% implanted an ICD for secondary prophylaxis. 76.5% implanted a bicameral ICD. 13.7% had a history of arrhythmic syncope. 40.6% had episodes of unstable ventricular tachycardia (VT). 20.1% suffered cardiorespiratory arrest due to ventricular fibrillation or VT. 85, 8% did not present therapies applied by the ICD, in 13.8% appropriate therapies were applied; 0.4% took inappropriate therapies. 62.7% had high mortality by the Rassi Score. The average Maggic score was 13 points for 1 year and 29 points for 3 years. There was death in 19.7%. Conclusion: Most patients who underwent ICD implantation were for secondary prevention, but there is room for primary prevention. This pathology continues to add high morbidity and cost to its treatment (like ICD), and it is important to act at all levels of care to minimize deaths from CD. 112179 Modality: E-Poster Scientific Initiation – Non-case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIELLA DE SOUSA CARBALLOSA GONZÁLEZ1, Elisabeth Uchoa de Melo1, Érika Thienne Lopes da Silva1, Luciana Andrade Tavares1, José Kaellyson Barbosa dos Santos Oliveira1, Carolline Araujo1 (1) Faculdade de Medicina de Olinda The COVID-19 pandemic, responsible for the biggest health crisis today, has caused more than 6 million deaths worldwide, constituting a serious public health problem. Cardiovascular diseases, such as hypertension and dyslipidemia, associated with diabetes mellitus and obesity, characterizing the metabolic syndrome, have been associated with severe forms of the disease and death. We investigated mortality in patients with SARS-COV-2 infection and its association with cardiometabolic diseases. This is a systematic review and meta-analysis following the PRISMA recommendation. Cohort, case-control and cross-sectional studies on the investigated topic were considered. The MEDLINE/EBSCO, Cochrane Library, Pubmed and SciELO databases were consulted using strategies limited to English, Spanish and Portuguese, using the descriptors available in MeSH (Medical Subject Headings) and DeCS (Health Sciencies Descriptors) as keywords. The meta-analysis involved all studies that presented a measure of association (Odds ratio, relative risk and others) in their results. Heterogeneity was assessed using Pearson’s chi-square test. A p-value was adopted, at a significance level of 5%. Data analysis was performed using Stata software. From this search, 24 articles were included. The median number of people among the analyzed works was 382 (P25 = 154/P75 = 894) and involved older individuals with a mean age of 56 years (dP = 11.4), predominantly male (63.6%). Covid-19 infection was confirmed using the RT-PCR molecular test. The mortality rate in this population was 18.5%. Among the cardiometabolic comorbidities that were significantly associated with mortality (p < 0.005), hypertension was more prevalent (25%), followed by diabetes (19.9%), obesity (18.7%) and dyslipidemia (9.35%). The presence of cardiometabolic comorbidities constituted factors associated with mortality in patients with SARS-COV2 infection. Future studies are needed to accurately determine the pathogenic mechanism involving these patients, especially hypertensive, diabetic and obese men, and the development of severe forms of COVID-19 infection. 112194 Modality: E-Poster Scientific Initiation – Non-case Report Category: CARDIOVASCULAR SURGERY ANA BEATRIZ VENANCIO DE PAULA BEZERRA1, Fabio Antonio Serra de Lima Junior1, David Cesarino de Sousa1, Renan Furtado de Almeida Mendes1, Raphael Patrik Borges da Costa1, Ana Gabriela Venancio de Paula Bezerra2, Joao Bosco Ferreira Gadelha3, Tiago Lucena de Brito Pereira3, Isaac Newton Guimarães Andrade1, Andre Telis de Vilela Araujo1 (1) Universidade Federal da Paraíba; (2) Faculdade de Medicina Nova Esperança; (3) Clínica Dom Rodrigo Introduction: Pain is one of the main morbidity factors associated with heart surgery, affecting about 50% of patients. Sternotomy can cause intense pain in the postoperative period, most commonly in the first 7 days, and can lead to significant short and long term consequences. Among the complications we can mention postoperative delirium, poor prognosis, increased length of stay in hospital and in the intensive care unit or in the post-anesthesia recovery unit, increased treatment costs and increased mortality. Few controlled and randomized clinical trials have addressed the efficacy of pregabalin administration in the perioperative period for pain control or recovery after surgery, obtaining variable results. Objectives: This study aims to evaluate the partial results if the preemptive use of pregabalin reduces the perception of pain in the patient undergoing heart surgery in the first 72 hours. Results: 23 patients submitted to cardiac surgery were randomized into two groups: 11 of them used preemptively 150 mg pregabalin starting 1 hour before surgery until the 3° postoperative day, the other 12 used a placebo for the same period. After extubation they were evaluated in 4 moments for the visual analog scale (VAS) and clinical parameters, at the 2° postoperative day the Qor-40 was applied. After analisis 70% were men, 78,2% had elevated blood-pressure, 39,1% had coronary arterial disease and 30,4% were diabetic. In relation to the clinical parameters studied, we verified that none showed statistically relevant differences between the use of pregabalin or placebo, but the pCO2 was the only one that was able to show a certain tendency that the pregabalin group accumulated less CO2 in the postoperative period (p = 0,074), which could mean a better thoracic expansion and therefore, less pain. The intervention group presented a mean QoR-40 of 180,17 + 17,88, while the control, 183,80 + 9,17 (p = 0,692). Revealing a non statistically significant better post-anesthesia recovery in the control group, which may be explained by an attrition bias while questioning, aimed to be ratified after team training in the remainder of this research. Conclusion: This partial analysis suggests that there may be a difference in the preemptive use of pregabalin, but it may be better verified in a bigger sample of patients. 112195 Modality: E-Poster Scientific Initiation – Non-case Report Category: EPIDEMIOLOGY AND HEALTH POLICIES/GLOBAL HEALTH YURI MAGALHÃES FERNANDES1, Bárbara Bernardes Magalhães1, Maria Inês Alves Brasil1, Rafaela Frota Malheiro2 (1) Universidade Estadual do Sudoeste da Bahia – Campus de Vitória da Conquista (Bahia); (2) Faculdade Santo Agostinho – Campus Vitória da Conquista (Bahia) Introduction: The gestational hypertensive syndrome (defined as systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg between 20 weeks of gestation and eight weeks postpartum) as a quite prevalent condition in the Brazilian population, affecting about 10% of pregnant women in the country. Furthermore, data from the Ministry of Health shows that maternal mortality in the country related to Pregnancy-Specific Hypertensive Disease (DHGE) amounts to about 35% of all maternal deaths while perinatal mortality reaches about 150 for every 1000 deliveries performed. For this, it is necessary to know the prevalence of deaths and hospitalizations related to the pregnancy-specific hypertensive disease (DHGE) in the last decade (2011–2020) in the unified health system (SUS), in order to know a little more about these comorbidities that affects many women during their pregnancy. Objectives: To analyze the prevalence of deaths and hospitalizations due to pregnancy-specific hypertensive disease in Brazil in the last decade (2011–2020) in the single health system (SUS). Methods: This is a retrospective cross-sectional study of secondary data made available by the Department of Informatics of the SUS (DATASUS) about mortality and hospitalization for pregnancy-specific hypertensive disease (DHGE) In Brazil in the period of years 2011 to 2020. Results: During this period (2011 to 2020) there were 1198 deaths across the country from pregnancy-specific hypertensive disease. The most recent data show the northeast and southeast regions with the largest number of deaths (420 and 421, respectively). It is worth mentioning the increase in the number of cases in 2011 (116) compared to 2020 (146) and the upward trend in the 2 previous years analyzed (2019, 2018), with, respectively, 118 and 112 deaths. Conclusions: The analysis of the data collected and the epidemiological knowledge about DHGE that the rate of women who still die as a result of this pathology is still high in our country. However, the most recent rates (2020 and 2019) show a great increase in relation to the previous years analyzed, leading to speculations about interrupted care or little assistance in this period, which coincides with the COVID-19 pandemic. In this sense, the analysis of these data is an important tool for understanding the national reality about care and deaths from the DHGE, as well as stimulates intervention formulation to reduce mortality in these patients. 112208 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LÍVIA FARIAS DE HOLANDA FURTADO1, João Victor Bezerra Ramos1, Patrícia Oliveira Lima de Macedo1, Ingrid Gabriele de Souza1, Maria Gabriela Medeiros Cunha de Araújo1, Dionarte Dantas Araújo3, Lara Andrade Dantas3, Fabrício Leite Pereira2, Cláudio Teixeira Regis4, Juliana Sousa Soares de Araújo1 (1) UFPB – Universidade Federal da Paraíba; (2) Hospital Metropolitano Dom José Maria Pires; (3) Rede Cuidar; (4) Complexo Pediátrico Arlinda Marques Introduction: The Rede Cuidar is a health care strategy developed in Paraíba since 2018 that has as one of its lines to ensure the therapeutic care and follow-up of children with congenital heart disease in the state. The network is a primary strategy for the early identification, correct care, and follow-up of this population in the state. For this it uses three strategies: pulse oximetry test, screening echocardiogram, and active search through the caravan in 13 cities in the state, for early identification of congenital heart disease and thus reducing mortality. Objective: To describe the incidence of cardiac alterations through pulse oximetry, echocardiogram and active search of patients in Paraíba. Method: This is a retrospective epidemiological study, of observational nature, with patients screened by the Rede Cuidar between the years 2020 and 2021 through pulse oximetry, echocardiogram and active search of patients in Paraíba. Results: Over one year (November/2020 to October/2021), in 18 hospitals and maternity hospitals in Paraíba, 34978 pulse oximetries were performed, identifying 150 patients with indication for echocardiogram, which represents an incidence of 4.2 for every 1000 live births. Of those, 67 were female (44.7%), 82 (54.7%) male, and 1 (0.6%) intersex. Still, 110 (73.3%) were adequate, 19 (12.7%) large and 12 (8%) small for gestational age, 9 missing information. Throughout 2021, 379 echocardiograms were performed, of which 253 (66.7%) were abnormal, 4 (1.1%) had an inconclusive report, and 122 (32.2%) were normal. In addition, by May 2020, 596 heart patients were followed in the 13 Heart Rooms, of these 12.6% had complex type heart disease and 10.4% had obstructive type heart disease. Conclusions: One realizes, then, the importance of the strategy created in the state of Paraíba for early detection of children with heart disease, not only by traditional methods, but by actively searching in areas with difficulty in accessing a specialized health service. 112217 Modality: E-Poster Scientific Initiation – Non-case Report Category: HYPERTENSION/RENAL DENERVATION FABIO ANTONIO SERRA DE LIMA JUNIOR1, Ana Beatriz Venancio de Paula Bezerra1, Ana Beatriz Torres Figueiredo de Lacerda1, Isaac Newton Guimarães Andrade1, André Telis de Vilela Araújo1 (1) Universidade Federal da Paraiba Introduction: In puerperal women who had gestational hypertension, there is uncertainty regarding the choice of maintaining methyldopa or replacing it with other drugs. Objectives: This review seeks to evaluate the efficacy of different antihypertensive agents tested during the puerperium on pressure control and their safety. Methodology: MEDLINE, Embase, LILACS, Web of Science, CENTRAL and ClinicalTrials were searched for articles with terms associated with postpartum period and hypertension, eclampsia or pre-eclampsia. Randomized clinical trials that evaluated puerperal women who used antihypertensive agents during the puerperium and had their blood pressure monitored during the period were included. The primary endpoint was blood pressure control, and the secondary endpoints were incidence of hypertensive spikes and medication-associated adverse events. Results: Initially, 2470 studies were found, which were filtered by title and abstract and full text. After screening by the PRISMA method, 11 studies were included. In patients with severe preeclampsia, the most studied antihypertensive was nifedipine, which showed benefits in mean arterial pressure control and renal perfusion compared to placebo, was similar to methyldopa in duration of treatment and in need for other agents, but inferior to diltiazem and labetalol in both effectiveness and hypertensive crisis profile. Furosemide monotherapy may reduce the risk of postpartum hypertension and its faster resolution in puerperae who had non-severe gestational hypertension, while in those who had severe preeclampsia, its association with nifedipine may reduce the need for other doses of antihypertensive agents. Protocols with different durations of magnesium sulfate in the postpartum period have been evaluated, and maintaining it for 6 hours may be as effective as 24 hours, but reducing the time for bladder catheterization, the beginning of ambulation, and contact with the newborn. Clonidine and captopril had similar efficacy and safety in puerperae who had severe preeclampsia. Labetalol and nifedipine are equally effective, although the former is associated with fewer hypertensive peaks. Studies comparing methyldopa to captopril and magnesium sulfate are ongoing. Conclusion: Antihypertensives in the puerperium of gestational hypertension should be chosen according to the patient’s clinical pattern, service availability, and desired profile of pressure control and side effects. 112219 Modality: E-Poster Scientific Initiation – Non-case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIA GABRIELA MEDEIROS CUNHA DE ARAUJO1, João Victor Bezerra Ramos1, Ana Quezia Bezerra de Holanda Sousa1, Júlia de Melo Nunes1, Lucas Emmanuel Freitas Mendes1, Pedro Nascimento Araujo Brito1, Patrícia Oliveira Lima de Macedo1, Dionarte Dantas Araujo2, Lara Andrade Dantas2, Fabricio Leite Pereira3, Juliana Sousa Soares de Araújo2 (1) Universidade Federal da Paraíba; (2) REDE CUIDAR; (3) Hospital Metropolitano Dom José Rodrigues Introduction: In Brazil, 28.9 thousand children are born with congenital heart disease per year (1% of the total births). Of these, about 80% (23.8 thousand) need heart surgery, and half need surgery in the first year of life. In the context of the state of Paraíba, until 2010, there was no qualified team in the area of cardiopediatrics. Knowing this, pediatric cardiology network of services was created to work in heart disease screening programs, in order to reduce late diagnosis. To this end, the Caravan of the Caring Network is held annually, a screening program held in conjunction with the Secretary of Health of the State, which actively searches for children with heart diseases in 13 cities in the state, and refers these patients for specialized monitoring. Objective: To analyze the active search for children with congenital heart disease in the state of Paraíba in the year 2021. Methods: This is a retrospective descriptive epidemiological study referring to the records obtained by the Caravan in the year 2021. Between November and December 2021, the action traveled through the cities of Paraíba: Monteiro; Princesa Isabel; Itaporanga; Cajazeiras; Sousa; Catolé do Rocha; Pombal; Patos; Queimadas; Picuí; Guarabira; Mamanguape; Itabaiana. In these records, information about the echocardiogram performed on patients and the diagnosis defined by professionals were observed, in order to allow the categorizations: shunt heart disease; complex heart disease; obstructive heart disease; acquired heart disease, and arrhythmias. Results: In 2021, the total number of visits was 6291. About 92 children (1.4%) were diagnosed with some heart disease. Analyzing the diagnosis of those children, 56 (60.86%) have shunt heart disease, 17 (18.47%) have obstructive heart disease, 8 (8.69%) have acquired heart disease, 6 (6.52%) have complex heart disease and 5 (5.43%) arrhythmia. Conclusion: Therefore, the relevance of the Caravan of the Caring Network as a mechanism for the detection and diagnosis of congenital heart diseases in Paraíba was evidenced, being important for the epidemiological monitoring of the State and for ensuring early care for children with heart disease. From this perspective, a higher incidence of shunt-type heart diseases was observed in the 2021 cut-off, included in the study, representing about 60% of the diagnoses performed. 107873 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES MONIREH ZIMMERMANN RAMEZANALI1, Amanda Cristina dos Santos1, Gabrielle Purnhagen1, Natasha Silva Constancio2, Caroline de Oliveira Fischer Bacca1 (1) UNIDAVI – Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí; (2) Hospital Regional Alto Vale Introduction: Methotrexate (MTX) is a folic acid analogue that inhibits DNA synthesis by causing an acute intracellular deficiency of folate coenzymes. It is a pillar in the treatment of various immune-mediated diseases. Among the side effects of the MTX therapy, mucositis and hematological symptoms are common. Besides, serositis is also a major adverse effect, presenting as pneumonia and pleural effusion. Pericarditis and pericardial effusion (PE) are uncommon ones. We present the first clinical case in Brazil of MTX-induced pericarditis with PE. Case Report: 67-year-old man, with systemic atherosclerosis, started a dermatologic treatment for cutaneous psoriasis with MTX 15 mg/week. After 30 days, he complained of chest pain radiating to the right shoulder, dyspnea and hypotension. Echocardiography (ECHO) showed a moderate PE (14 millimeters), without cardiac tamponade signals. Computerized tomography revealed PE around the cardiac area, with no evidence of lynphonodes, neoplastic lesions nor infections, including tuberculosis. During hospitalization, PE was aspirated and a pericardial biopsy was made. The fluid did not reveal any abnormalities and biopsy showed a chronic inflammatory infiltrate. All the laboratory tests for rheumatoid diseases were within normal limits. The suspension of MTX therapy and use of Prednisone 40 mg daily produced improvement in symptoms within 14 days and follow-up ECHO showed complete resolution of the PE. Conclusion: Although extremely rare, pericarditis and PE might be consequences of MTX treatment. For the right diagnosis, a careful analysis should be undertaken to discontinue the drug therapy. The use of ECHO provides important information on the severity of the clinical condition. The patient was treated with MTX withdrawal and steroid therapy. 108354 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR IMAGING LARISSA ARAÚJO DE LUCENA1, Felipe Leite Guedes2, Fabiano César de Medeiros Júnior1, Márcio Vilar de Freitas2, Rodrigo Azevedo de Oliveira1 (1) Universidade Federal do Rio Grande do Norte; (2) Hospital Universitário Onofre Lopes Renovascular disease (RVD) is responsible for 5.8% of secondary hypertension cases in young adults, caused mainly by obstructive lesions due to either atherosclerotic renal artery stenosis or fibromuscular dysplasia. Although the renal arteries’ supradiaphragmatic origin is exceedingly rare, up to date, three cases of secondary hypertension due to single ectopic renal arteries originating from the thoracic aorta have been reported in patients with customarily positioned kidneys. Herein we describe a case of a 21-year-old man with resistant hypertension whose investigation showed an ectopic right renal artery originated from the internal thoracic artery. Although both renal arteries were free of obstructive lesions, the right one was very long and tortuous, causing kidney hypoperfusion. A bypass surgery between the right renal artery and the aorta was performed uneventfully, leading to better blood press control. To the best of our knowledge, there are no previous reports of secondary hypertension due to the renal artery arising from the internal thoracic artery. 108454 Modality: E-Poster Scientific Initiation – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM DAVI SALES PEREIRA GONDIM3, Maria Eduarda Quidute Arrais Rocha3, Rodrigo Carvalho Paiva2, Marcela Albuquerque de Holanda2, Eduardo Arrais Rocha4 (1) Universidade Federal do Ceará-UFC; (2) Universidade Unichristus; (3) Universidade de Fortaleza-UNIFOR; (4) Centro de Arritmia do Ceará-CACE Introduction: Implantable cardiac defibrillators (ICDs) represent the most effective therapy in the treatment of severe ventricular arrhythmias. Routine follow-up with periodic scheduled office visits may not be sufficient due to the complexity of the pathologies. The COVID-19 pandemic determined more limitations in this follow-up. In this work, we report a series of cases in which remote monitoring (RM) of ICDs were of great importance in treatment. Case 1: Male patient, 65 years old, with ischemic cardiomyopathy, sustained ventricular tachycardia (SVT), with ICD implantation after reversed cardiac arrest. After 7 years, changes in the impedance of the shock electrode were detected, information transmitted remotely, requiring reprogramming in the shock vector. After 6 months, informations of changes in impedance of stimulation was received by e-mail as a red alert. Implantation of a new electrode was necessary. During the follow-up, he presented several SVTs and atrial fibrillation (AF), needing pharmacological adjustment and just one office visit for reprogramming. Case 2: Female patient, 91 years old, with ICD with multisite pacemaker due to dilated cardiomyopathy. During acupuncture, there was an ICD shock. The professional had inadvertently used electro-acupuncture. Due to portable RM, the information was transmitted to the physician, who detected an inappropriate therapy and advised the professional not to use electrical stimulation during acupuncture. Case 3: Female patient, 72 years old, with ischemic and valvar cardiomyopathy, hospitalized with SVT, when was implanted an ICD. After starting antiarrhythmics drugs, no new arrhythmias occurred in the short term. After months, she presented with several SVTs, some with high rate, reversed with anti-tachycardia pacing (ATP) and others SVTs with low rates, outside the therapy zones. AF was also detected, inappropriately recognized by the ICD as SVT and treated with inappropriate ATP therapies. Therapeutic adjustments were made and on 2 occasions there was a need for immediate reprogramming of the ICD. Discussion and Conclusion: RM of patients with ICD during the COVID-19 pandemic allowed therapeutic optimization, reduction of scheduled office visits, hospitalizations and inappropriate shocks, with an improvement in the treatment and in the quality of life. 108936 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY MIKE VINICIUS CANTO DE ANDRADE1, Natália Barreira Silva1, Rodrigo Penha de Almeida1, Paulo Cesar Santos1, João Lucas O‘Connell1 (1) Universidade Federal de Uberlândia Introduction: Left ventricular aneurysm (LVA) can be defined as a thin and dilated portion of the left ventricle (LV) due to the predominance of fibrosis and scar area. This phenomenon generates contractile disorders and, consequently, akinetic or dyskinetic ventricular segments observed during systole. They may develop after myocardial infarctions and secondary to some cardiomyopathies, such as chronic Chagas cardiomyopathy. Case Description: Female patient, 61 years old, with Chagas cardiomyopathy and heart failure (HF) functional class III (NYHA) with low tolerance to drug therapy optimization due to arterial hypotension. A transthoracic echocardiogram and cardiac catheterization were performed. The exams identified significant left ventricular dysfunction with an image of a large aneurysm associated with a thrombus at the LV apex. Pointing out the refractoriness to clinical treatment, along with complex ventricular arrhythmias on Holter and the presence of a well-defined base, it was decided to perform an LV aneurysmectomy. A significant improvement was observed in the patient’s clinical state in the subsequent months. Currently, 2 years after the surgical treatment, he is in functional class I (NYHA), using Warfarin 5 mg/day, Enalapril 10 mg/day, Carvedilol 25 mg/day and Spironolactone 25 mg/day. There was a significant reduction in the size of the left ventricular cavity and a significant improvement in ventricular function (currently with normal LVEF). Not only pre- and post-surgical morphofunctional data from echocardiograms and holter will be presented, but also preoperative ventriculography and postoperative magnetic resonance imaging. Conclusion: The treatment of patients with LV aneurysms is eminently clinical, through the optimization of drug therapy for HF. However, the surgical indication of aneurysmectomy should be considered for cases that develop refractory HF, angina, complex ventricular arrhythmia and systemic embolization of intraventricular thrombi. The reported case shows that the performance of aneurysmectomy, with preservation of viable myocardium in the basal and middle portions of the LV, is capable of significantly reducing the ventricular cavity and promoting a significant improvement in the global contractile function of the LV in selected cases. Surgical treatment can also provide an important improvement in the patient’s clinical condition. 108550 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY BERNARDO MUSSI SOARES1, Andressa Mussi Soares2, Renata de Backer Pacífico2, Resi Apolinário2, Paulo José Ferreira Soares2 (1) Fundação Técnico Educacional Souza Marques – FTESM; (2) Hospital Evangélico de Cachoeiro de Itapemirim – HECI Pulmonary artery sling – rare diagnosis in a wheezing baby. Introduction: Pulmonary artery (PA) sling is a rare congenital vascular disease (59 cases per million children) in which the left pulmonary artery (LPA) derives from the right pulmonary artery (RPA), forming a ring that passes anteriorly to the esophagus and posteriorly to the trachea, which may compress mediastinal structures and cause severe upper airway symptoms in children. Case Description: 2 years old male child, with respiratory distress at birth, evolved with frequent wheezing and tachydyspnea, without improvement. He presented several hospitalizations for respiratory symptoms, being diagnosed with allergy to cow’s milk protein. After evaluation by the immunoallergy, he was referred for echocardiographic evaluation. The echocardiogram (Echo) detected the APE emerging from the posterior wall of the RPA that originates from the pulmonary trunk and has a usual course, and the diagnosis of AP sling was made. Therefore, CT angiography of the chest was performed to better evaluate possible compressions in the trachea and esophagus. APE emerging from the dorsal surface of the RPA was detected, passing inferiorly to the origin of the upper lobar bronchus that exits directly from the trachea and over the right main bronchus, forming the vascular ring that causes a moderate degree of stenosis of the distal trachea. In addition, the APD presents an anatomical variation with anomalous segmentation at the hilum, where it trifurcates forming three main arterial trunks. This data is especially important at the time of the surgical approach to avoid incidental complications. CT angiography was performed with reconstruction focused on the air phase, allowing an excellent assessment of tracheal stenosis and thoracic esophageal distention. The child has just been diagnosed and will undergo surgery. Conclusions: Due to the non-specific symptoms, the clinical diagnosis can be difficult and delayed, compromising the general condition and in some cases leading to death in the first months of life. Echo performed in detail is a robust tool for diagnosing PA sling. The other exams offer a better assessment of the relationships with neighboring structures. With an early diagnosis and surgical correction, the prognosis is generally good, but the anatomy of the structures must be very well evaluated because the risk of death during surgery increases when there is significant bronchial or tracheal stenosis. 108571 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY RODRIGO BATISTA WARPECHOWSKI1, Emmanuela Flavia Alves Pinto2, Fabiana Rodrigues Philippsen2, Paulo Warpechowski2, Tiago Luz Leiria1 (1) Instituto de Cardiologia do Rio Grande do Sul; (2) Sociedade de Anestesiologia (SANE) In cardiopulmonary bypass (CPB) surgeries, adverse events such as desaturation, hypoflow, hypoxia and brain injury may occur. Such situations may lead to complications such as cognitive decline (CO), observed in up to 50% of the patients. Herein, we present a case of hypoxia during cardiac surgery with CPB identified using the INVOS™ 5100 C Cerebral/Somatic Oximeter. Male patient, 77 years old, 80 kg, hypertensive, with root and ascending aortic aneurysm and aortic valve insufficiency. Scheduled aneurysm repair and valve replacement. No previous neurological deficits. Regional saturation (INVOS™) and anesthetic depth (Bispectral Index-BIS) monitors were installed. Regional brain O2 saturation (rSO2) was between 65–80 and BIS between 30–60 throughout the process. At thirty minutes of CPB, there was an abrupt drop in rSO2 (Image 1), reaching critical values <40, a drop in mean arterial pressure and a BIS reduction, with high suppression rates. Arterial blood gases revealed severe hypoxemia (PO2 of 40 mmHg and SO2 of 62%). A failure in the CPB gas mixer was identified by the perfusionist and it was replaced. Subsequently, rSO2 returns to values >55. The CPB time was 138 min, and aortic aneurysm repair was performed using the Bentall de Bono procedure. The patient showed no neurological damage and progressed satisfactorily, being discharged from the ICU after 3 days and discharged from the hospital 12 days after surgery. Studies such as those by Murkin et al and Slater et al, which evaluated the effectiveness of rSO2 monitoring by INVOS™, demonstrate that intraoperative cerebral O2 desaturation is significantly associated with an increased risk of CD and prolonged hospital stay after CPB, and its treatment was associated with a shorter length of stay in the ICU and a significantly reduced incidence of morbidity and mortality. Despite performing preventive maintenance procedures, equipment failure may occur. Thus, it is imperative that the anesthesiologist is aware and uses all available monitoring means to recognize possible failures and so that management can be carried out in the best way as possible. 108581 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ISADORA BUELONI GHIORZI1, Adriano Louro Moreira1, Juliana Bergmann1, Mateus dos Santos Taiarol1, Marina da Rocha Besson1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre Introduction: Valproic acid is a medication frequently used in the treatment of epilepsy, and, among anticonvulsants, it has the highest incidence of major malformations. Our aim was to report a patient with fetal valproate syndrome (FVS) presenting congenital heart disease. Case Description: The patient was the third child of a mother diagnosed with epilepsy. She used valproic acid (up to the third month) and phenobarbital (from the third month onwards) during pregnancy. She reported episodes of frequent seizures until the end of the third month of pregnancy. The female child was born by vaginal delivery, premature at 35 weeks, weighing 2450 grams and with Apgar scores of 8 at first minute and 9 at fifth minute. She had heart failure after birth. Echocardiography identified perimembranous ventricular septal defect (VSD) with a wide outflow tract, mild peripheral pulmonary stenosis, and left superior vena cava persistence draining into the coronary sinus. The evaluation also showed growth retardation, keeled skull, epicanthal folds, bilateral tear duct obstruction, well-marked infraorbital crease, anteverted nostrils, long philtrum, thin upper lip, retroverted and low-set ears, and accessory nipples to the right. Head computed tomography confirmed the diagnosis of trigonocephaly. The karyotype was normal. The child underwent surgery to correct the VSD at 8 months of age. Conclusions: The findings presented by the patient were compatible with the diagnosis of FVS. Congenital heart defects may be part of the picture and they have been described in about 25% of patients. The VSDs were less common malformations. 108591 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY MARIA EDUARDA QUIDUTE ARRAIS ROCHA1, Pedro Sales Pereira Gondim3, Arthur Holanda Dantas3, Marcela Albuquerque de Holanda3, Camila Pinto Cavalcante Miná2 (1) Universidade de Fortaleza (Unifor); (2) Universidade Federal do Ceará (UFC); (3) Centro Universitário Christus (UNICHRISTUS) The Tilt Test in Munchhausen Syndrome Introduction. The Tilt Test (TT) is a complementary method widely used to investigate the susceptibility to vasovagal phenomena or late orthostatic hypotension. Reproduction of clinical symptoms is essential to the true positivity of the test and its good accuracy. Patients may present with several symptoms during the test, without concomitant hemodynamic changes, called psychogenic response. We described a remarkable case of a patient with a suspected diagnosis of Munchhausen Syndrome, a more severe form of factitious disorder self-imposed, confirmed during the TT. Case: Female patient, 22 years old, with a history of more than ten years of multiple symptoms of loss of consciousness, intensification in recent years, descriptions and videos showing tonic movements, with deviation of looking up, pallor, falls, palpitations, causing a worsening in her quality of life. She had a history of childhood sexual abuse, dependence on the use of morphine and psychotropic drugs for the treatment of fibromyalgia, herniated disk and depression. There were several hospitalizations, with suspected diagnoses of pheochromocytoma, hyperthyroidism, adrenal insufficiency, autoimmune diseases and dysautonomia, when she was referred to syncope unit for investigation. There was a hypothesis of her psychiatrist of the syndrome of post traumatic disorder. Laboratory tests showed nonspecific findings. The cardiologic tests: sinus tachycardia 120–151 bpm, even when lying down; normal 24-hour Holter with RR variability showing preserved autonomic balance. Neurologic tests were normal. In 2020, she underwent TT with a report of pseudo syncope. In 2021, during a recorded TT, there was full reproduction of the symptoms, without any correlation with hemodynamic changes. The diagnosis was made according to the criteria of the manual diagnostic for mental disorders (DSM-5), and she was forwarded to the psychiatrist. In the evolution, the patient no longer returned for revaluation, a situation described in this syndrome after confirmation of the diagnosis, remaining meanwhile with evaluations by other professionals. Discussion and Conclusion: This work highlights the importance of knowledge of Munchhausen Syndrome due to the wealth and multiple symptoms presented and the role of the Tilt Test in excluding other etiologies, since the diagnosis is rarely done by the psychiatrist, but mainly by the specialist. 109134 Modality: E-Poster Scientific Initiation – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM CAROLINA TISOTT BURTET1, Amanda Hillesheim Schuck1, Maria Constanza Cé Erig1, Rafaella Magni Berthier1, Olga Sergueevna Tairova1 (1) Universidade de Caxias do Sul (UCS) Introduction: Coronavirus disease (COVID-19) is an infectious disease which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients with previously established comorbidities, such as heart failure, are at particularly high risk of morbidity and mortality from this infection – they generally have a worse prognosis and a mortality rate of more than 10%. Studies suggest that viral infections can exacerbate a preexisting cardiac insufficiency, with multiple mechanisms responsible for triggering and aggravating this process. Case Description: Female patient, 34 years old, without comorbidities. History of mild blood pressure increase and left ventricular enlargement since 2015. Progression of loss of ejection fraction (EF) up to 20% after COVID-19 infection in December 2020. Evolved with worsening of the general condition, acute heart failure and need for sequential dialysis. After the condition, the patient became a candidate to receive a heart transplant, which was performed in February 2021, with good postoperative clinical evolution. Complementary tests after transplant: Electrocardiogram with regular rhythm, HR 95, without alterations; Echocardiogram with improvement in EF (69%), anomalous movement of the interventricular septum and left atrial enlargement. Stress Test: Distance covered 0.86 km; Duration 11 minutes; maximum HR 129; Max Vo2 15.44; Maximum power: 29.9 W; NYHA III functional group; Also showing a very low cardiorespiratory fitness (AHA). The patient has been followed up on a weekly basis for physical and cardiopulmonary rehabilitation at the Sports Medicine outpatient clinic, with good evolution. Conclusion: Acute infections result in an inflammatory cascade, leading to a severe inflammatory storm that can exaggerate the initial injury. Furthermore, it is already documented that increased metabolic demand can lead to cardiac depression and new-onset heart failure or acute decompensation of chronic cardiac insufficiency. The case above describes a severe acute decompensation in a young patient, without associated comorbidities with the need for immediate heart transplantation. Thus, we can conclude that it is essential to understand the interactions between heart disease and the virus to prevent unfavorable outcomes like this one and improve the management of these patients. In addition, there are yet few reports in the literature that demonstrate the need for heart transplantation after COVID-19. 108771 Modality: E-Poster Scientific Initiation – Case Report Category: HYPERTENSION/RENAL DENERVATION DANIELLE CAMPOS DE ALMEIDA1, Benara Otoni de Siqueira1, Ebes Kléofas da Silva Magalhães1, Gabriel Alves Meneses1, João Lucas O’Connell1 (1) Faculdade de Medicina, Universidade Federal de Uberlândia Introduction: Renal Artery Stenosis (RAS) in elderly is, in most cases, a consequence of a progressive atherosclerosis, that may be associated with other blood vessels atherosclerosis. The most common symptoms are related to tough control of blood pressure (an important cause of secondary hypertension) and renal insufficiency. It’s described a patient’s case with multiple comorbidities and systemic atherosclerosis, evolving with refractory arterial hypertension, low back pain and progressive renal disease, even as relevant right renal artery stenosis. Case Description: 75-years-old woman, smoker, hipertensive and dyslipidemic, with previous transitory ischemic attack, bilateral carotid stenosis, carotid endarterectomies, acute myocardial infarction, coronary angioplasties and stents implantation, mesenteric artery angioplasty with two stent implantation. Presenting for six months with worsening of blood pressure control (maintaining levels higher than 170 mmHg), right low back colic-like pain episodes, and recent worsening of creatinine levels (from 1,6 to 2,5 mg/dL). It was requested an abdominal aorta angiotomography (and renal arteriography later) that identified important right renal artery stenosis. The Patient was subjected to a angioplasty and successful stent implantation in the right renal artery without any complications. There was improvement of low back pain, blood pressure control and creatinine levels (returning to normal values). Performed new abdominal aorta angiotomography that detected blood vessels permeability and absence of stenosis within previously implanted prosthesis. Conclusion: Renal Artery Stenosis diagnosis can be done by renal artery doppler, renal scintigraphy and abdominal aorta angiotomography. Concerning treatment, literatura still is controversial about drug treatment and percutaneous modalities effectiveness. For some cases, as described here, interventionist approach, renal artery angioplasty and stent implantation, may be mandatory for blood pressure control, pain control, due to a renal ischemia and to avoid renal insufficiency. 108778 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES DANIELLE CAMPOS DE ALMEIDA1, Karina Baltor Cabral1, Mike Vinicius Canto de Andrade1, Paulo Cesar Santos1, João Lucas O’Connell1 (1) Faculdade de Medicina, Universidade Federal de Uberlândia Introduction: Cardiac perforations usually are due to stab wounds or bladed weapons, firearms or eventually due to chest trauma followed by penetration of foreign bodies inside the chest wall. Although most of cardiac perforations are life threatening, minor lacerations may evolve to tamponade and cardiac surgery can avoid death. Cardiac perforations because of sharp perforation material are rare. Even more uncommon when surgical material was used on procedures outside the chest. Case Description: A female patient of 60-years-old woman had orthognathic surgery 36 year ago and post-surgery identification of a suture needle “forgotten” on cervical region. Comes into the Emergency Room complaining about a progressive chest pain ventilator-dependent, associated with dyspnea. Physical exams evidence pericardial friction rub. Electrocardiograms and myocardial necrosis markers had no major changes. Coronary angiography showed absence of coronary obstruction. However, a suture needle was identified in the topography of the right ventricle (RV). A chest tomography was performed and revealed pericardial effusion and thickening, as well as micro-perforations of RV free wall associated with needle suture image. After two days, the patient evolved with an increase in pericardial effusion and tamponade cardiac signs, requiring emergency surgery aiming to remove the foreign body, followed by ventricule suture and pericardiocentesis. Patient is asymptomatic six months after the event. Conclusion: Cardiac perforations caused by “forgotten” foreign bodies in previous surgeries are rare. Furthermore, if associated with surgical material used in surgeries outside the chest. The needle displacement from cervical region to chest cavity was very likely provoked by shoulders and knees magnetic resonance imaging (MRI) performed four months before. The magnetic field produced by MRI equipaments is capable of shifting ferromagnetic objects first implanted, that may result in tissue injury. The fast and efficient cardiac surgery was essential to manage cardiac tamponade, foreign body removal, RV suture and to save the patient’s live. 108783 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES DANIELLE CAMPOS DE ALMEIDA1, Alice Mirane Malta Carrijo1, Marcela Gomes de Souza1, Gabriel Alves Meneses1, João Lucas O’Connell1 (1) Faculdade de Medicina, Universidade Federal de Uberlândia Introduction: Coronary Artery Anomalies (ACA) are characterized by changes on these vessels origin, course or structure. Compose a rare condition: less than 1% of general population. Within these anomalies, there are anomalous origin of coronary arteries from aorta which can have retroaortic, subpulmonary, pre-pulmonary and interarterial courses. At large, these abnormalities remains asymptomatic until adult age and are incidental findings on complementary exams or necropsy. Yet, it can cause angina, syncope, ischemia, arrhythmia or sudden death, especially in young athletes. Case Description: 56-years-old men, hypertensive, family history of coronary disease, dyslipidemic, pre-diabetic, sedentary and obese. Was admitted in the hospital for evaluation of mild unstable angina. Electrocardiogram (ECG): Sinus rhythm and left ventricular overload signs. There wasn’t any increase on cardiac enzymes. Coronary angiography highlighted the origin of the right coronary from the left coronary sinus, slit ostium and moderate ostial stenosis (for probable extrinsic compression). Left coronary without stenosis. Left ventricules‘ global and segmental systolic function were preserved. Coronary artery angiotomography confirmed inappropriate RCA origin from left valsalva sinus, interarterial course (between aorta and pulmonary artery) and proximal moderate luminal reduction (“slit-like orifice” course). The possibility of surgical treatment has been considered, however it was opted for myocardial scintigraphy which showed normal myocardial perfusion. The conduct taken was drug therapy maintenance. The Patient remains asymptomatic even after two years passed from initial diagnosis. Conclusions: Although anomaly’s interarterial course is associated to higher incidence of sudden death, especially after vigorous exercises, low physical efforts diaries, associated with absence of detectable ischemia on appropriate exam, reduce risk for fatal events, as well corroborates the therapeutic option chosen on this case. Moreover, the absence of angina symptoms after drug treatment also reinforces the initial non-interventionist option for the case in discussion. 108789 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISABELA MARTINS RODRIGUES1, Joaquim Adélio de Oliveira Neto1, Ana Júlia Carvalho Paulinelli1, Gustavo Nahuel Leyes Ontivero1, João Lucas O‘Connell1 (1) Faculdade de Medicina da Universidade Federal de Uberlândia Introduction: Spontaneous Coronary-Artery Dissection (SCAD) is defined as the rupture of the intima and media layer of an epicardial coronary-artery wall by an intramural hemorrhage and is not secondary to atherosclerotic disease, aortic dissection, or intravascular trauma. SCAD occurs primarily in women, and the predisposing factors include intense emotional stress, intense physical activities (especially isometric exercises), hormone therapy, use of corticosteroids, use of cocaine, and smoking. Case Description: A 39-year-old man, smoker, and cocaine user was referred to our hospital with intense chest pain at rest, associated with dyspnea, for more than 24 hours. He maintained mild chest discomfort at the time of admission and was hemodynamically stable. The electrocardiogram showed sinus rhythm, mild and diffuse ST-segment elevation. Coronary angiography revealed dissection in the left coronary trunk, involving the entire left anterior descending artery and the entire first left marginal branch with distal occlusion of these two vessels. Left ventriculography showed a significantly decreased global left ventricular systolic function, dyskinesia of the anterolateral wall, and akinesia of the apical and inferoapical walls. An attempt was made for coronary angioplasty in marginal branch, without success, because access to the true lumen of the vessel was not possible. Clinical treatment was performed and reassessment was scheduled with a new coronary angiography in 30 days or an emergency cardiac surgery in case of instability of the condition. The patient remained stable, and drug treatment was maintained for Coronary Artery Disease and Heart Failure. He is asymptomatic with partial recovery of ventricular function one year after the initial condition. Conclusion: SCAD is a dramatic clinical and angiographic situation that can lead to death in young individuals without risk factors for coronary artery disease. There is a current trend towards adopting a conservative strategy, especially in those patients without recurrent ischemia. When attempting angioplasty, verification of the correct positioning of the guidewire in the true vessel lumen is mandatory. Stent angioplasty has failure rates that can reach 35.0%. The case reported is a SCAD, probably due to cocaine use, with spiral dissection involving multiple vessels. Despite the severity of the lesions, the conservative approach was successful, and the patient presented a satisfactory clinical response. 108790 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MIKE VINICIUS CANTO DE ANDRADE1, Letícia Guimarães Mendonça1, Natália Barreira Silva1, Eduardo Henrique Costa Vitor1, João Lucas O‘Connell1 (1) Universidade Federal de Uberlândia Introduction: Often, even patients with a favorable evolution in the first days after an Acute Myocardial Infarction (AMI) can develop important mechanical complications. These complications can be sudden and lethal, such as, for example, left ventricular (LV) free wall rupture. Another serious mechanical complication of patients with AMI is LV pseudoaneurysm, which develops due to rupture of the left ventricular (LV) wall contained by pericardial adhesions. When not diagnosed and treated early, the pseudoaneurysm can also have a catastrophic course. Case Description: Male, 65 years old, presented with non-thrombolyzed inferolateral wall AMI. Coronary angiography, still in the hospital phase: three-vessel obstructive pattern with important and proximal stenosis in the anterior descending and circumflex arteries, in addition to total occlusion of the right coronary artery. Ventriculography showed a large extension of akinetic area in the inferior wall and “image suggestive of aneurysm in the inferior wall of the LV”. He was discharged from hospital on the sixth day; he was advised to look for a cardiac surgeon to schedule coronary artery bypass graft surgery (and possible LV aneurysmectomy). Outpatient echocardiogram (performed on the tenth day after AMI) identified the presence of a pseudoaneurysm of the inferior wall of the LV. Still in the echocardiography room, the patient had a cardiorespiratory arrest in a pulseless electrical activity (PEA) rhythm, which was not reversed after cardiopulmonary resuscitation (CPR) or puncture pericardiocentesis maneuvers. A new echocardiogram during cardiac arrest confirmed an image suggestive of significant pericardial effusion with signs of cardiac tamponade, not identified in the exam performed a few minutes earlier. Conclusion: Left ventricular aneurysms and pseudoaneurysms are types of mechanical complications associated with AMI and the differential diagnosis between one situation and another is still a challenge in clinical practice. Unlike true aneurysms, false aneurysms have a high tendency to rupture and should be operated on soon after diagnosis. This report reinforces the importance of careful analysis of risk factors for the occurrence of mechanical complications after AMI and also of careful analysis of echocardiography and ventriculography images that allow early treatment of this potentially fatal clinical situation when the diagnosis is not made. 108799 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS ISABELA MARTINS RODRIGUES1, Rodrigo Penha de Almeida1, Dennis Miguel Lemos da Silva1, Fabiane Mian de Souza1, João Lucas O‘Connell1 (1) Faculdade de Medicina da Universidade Federal de Uberlândia Introduction: The occurrence of concomitant association between acute coronary syndrome and stroke is rare. Although several cases have been described in the literature, we did not find any case description of a patient with concomitant cerebrovascular accident (CVA) associated with Spontaneous Coronary-Artery Dissection. Case Description: A 39-year-old man, tennis coach, and player without morbid history or classic risk factors for coronary artery disease presented to our hospital with sudden mental confusion, disorientation, and mild dysarthria, with partial reversal during the 7-day hospital stay. Magnetic Nuclear Brain Resonance showed alterations compatible with stroke. Electrocardiographic and echocardiographic alterations were seen suggestive of acute inferior wall myocardial infarction and led to the performance of coronary angiography, which revealed proximal occlusion of the right coronary artery, receiving grade II coronary collaterals left, without stenosis in other coronary arteries. Left ventriculography showed significant hypokinesia of the inferior wall with preserved global systolic function. We decided not to approach the right coronary artery at that time and the possibility of coronary dissection was considered a pathophysiological mechanism to explain the acute coronary syndrome and mobilization of the coronary thrombus to explain the consequent cerebral embolism. He returned 30 days after the initial condition and identified a recanalized right coronary artery, with subocclusive stenosis in the proximal segment and an image compatible with a long line of coronary dissection. Angioplasty of the right coronary artery was performed with successful implantation of two stents. The patient remains well and without further events two years after the initial event. Conclusion: Several pathophysiological mechanisms can explain the occurrence of simultaneous ischemia in cardiac and cerebral territories. Firstly, the same traditional risk factors act for the genesis and rupture of obstructive atherosclerotic plaques in both territories. Secondly, one can also lead the other which justifies the simultaneous manifestation of the conditions. The initial percutaneous approach to a coronary vessel suspected of having dissection can be postponed to a second moment, in case of clinical stability of the patient. In the case reported, this initial clinical management proved to be very important. 108802 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS LETÍCIA GUIMARÃES MENDONÇA1, Gustavo Nahuel Leyes Ontivero1, Victor Custódio Ribeiro1, Sthephany Yamaguchi de Melo1, João Lucas O‘Connell1 (1) Universidade Federal Uberlândia Introduction: Coronary aneurysm (CA) is defined by the presence of a vessel segmental dilatation 1,5 times larger than the adjacent segment or as the largest vessel’s segment with the larger diameter of the vessel. Aneurysms incidence varies between 1,5% a 5% in adults and is more frequent in males. Also, this condition is related to Takayasu, Kawasaki arteritis, Polyarteritis Nodosa, and syphilitic, or previous exposure to trauma, dissection, angioplasty, atherectomy, and herbicides. The role of the classical risk factors in the genesis of aneurysms is controversial. Case Description: Male, 49 years, class 3 obesity, hypertensive. Appeared with Non-ST segment elevation myocardial infarction (NSTEMI) of the inferior wall. Heart catheterization showed ectasias along the entire length of the right coronary, an aneurysm (vessel maximum diameter 12 mm), and an occlusive thrombus in the middle third. The exam also showed coronary ectasia, without significant stenosis in the anterior interventricular and circumflex branches. An attempt of primary balloon transluminal coronary angioplasty was made, it was associated with intracoronary thrombolysis and venous Alteplase maintenance, obtaining TIMI II coronary flow. A coronary stent wasn’t possible due to de excessive dilatation of the vessel. The patient evolved well until his hospital discharge and did not present new cardiological problems in the last 4 years. The patient took Aspirin for the first 28 days. Currently, he takes Atorvastatin (80 mg/day), Metoprolol (50 mg/day), Clopidogrel (75 mg/day), and Rivaroxaban (15 mg/day). Conclusions: The pathophysiology of CA is related to multifactorial endothelial damage, which activates inflammatory mediators and vasodilator substances that lead to degeneration of the vessel layer and progressive coronary dilatation. Percutaneous revascularization can be associated with distal thrombus embolization, no-reflow phenomenon, malposition of the stent, dissection, and flow compromise. There are no specific guidelines for the management of CA in acute coronary syndrome and there is no data in the literature about the efficacy of thrombolytic therapy in this case. Finally, this case suggests that balloon angioplasty and the use of thrombolytic, followed by the clinical treatment optimized with high doses of statin, and antiplatelet aggregation associated with anticoagulation in the long term, can be good therapeutic choices for some selected cases of NSTEMI with CA. 108805 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY CAROLINE DA SILVA TEIXEIRA1, Lucca Ziravello Elias Coelho1, Gabriela Rodrigues de Oliveira2, Ricardo Santiago Ferreira Coelho2, Juliano Novaes Cardoso2 (1) Faculdade Santa Marcelina; (2) Hospital Santa Marcelina Coarctation of the aorta (CoA) is not only considered a narrowing of the aorta but part of a generalized arteriopathy. The incidence corresponds to 3 cases per 10.000 births. The associated lesions include a bicuspid aortic valve (BAV) in up to 85% of the cases. Patients with mild CoA may not manifest until adulthood, which can be accidentally diagnosed through clinical examinations as arterial hypertension. This case discusses a 36-year-old male who presented to the emergency department with a 1-hour history of sudden, tearing and severe (10/10 intensity) retrosternal pain radiating to his back. Initial electrocardiogram revealed first-degree AV block and left anterior superior divisional block. Blood pressure was different between the upper and lower limbs. His past medical history includes a non-investigated and non-treated asymptomatic arterial hypertension since he was seven years old. Aortic dissection was suspected and further investigation with angiotomography was realized. The image revealed an aortic coarctation after the emergence of the subclavian artery, associated with an aortic fusiform aneurysm after the stenotic region, parietal thrombus, aortic ulcer, and dissection flap. A transesophageal echocardiogram was performed, which detected a bicuspid valve with significant insufficiency. The patient was hospitalized, medicated and awaiting a surgical procedure. A medical history of re-coarctation, descending aortic aneurysms, hypertension, and recurrent procedures associated with congenital heart disease are related to higher cardiovascular morbidity. The diagnosis of CoA is the most commonly missed, being detected at the prenatal screening with less than one-third of the cases. In conclusion, early diagnosis is very important to reduce disease morbidity. 108816 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR PHARMACOLOGY HELENA MARCON BISCHOFF1, Helena Marcon Bischoff1, Sérgio Ferreira de Ferreira Filho2, Raphael Boesche Guimaraes2, Mathias Silvestre de Brida2 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (2) Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia (IC-FUC) Introduction: Amiodarone is a widely used drug in the treatment of arrhythmias, however, its metabolites are responsible for numerous side effects. We report a rare dermopathy secondary to the chronic use of amiodarone. Case Description: Female patient, 57 years old, hypertensive and with a history of paroxysmal atrial fibrillation and mechanical mitral valve prosthesis, on chronic use of warfarin and amiodarone, sought consultation due to the appearance of bluish spots on her face and upper limbs in the last three months. She reported daily use of 400 mg of amiodarone for the last four years. Patient had lost medical follow-up, with no consultation in the last 4 years. On physical examination, areas of blue-gray hyperpigmentation were found in photoexposed areas, predominantly on the face and upper limbs. Chest X-ray and laboratory tests to evaluate other adverse effects were unremarkable. Considering the characteristic clinical findings, the hypothesis of amiodarone-induced skin disease, known as Blue-Gray Syndrome was raised. The patient was then instructed on the discontinuation of the drug, protection against sun exposure and the need for ophthalmological evaluation. After four months, she showed significant improvement in the lesions. Conclusions: Blue-Gray Syndrome is an adverse effect that occurs in 1 to 7% of patients on chronic use of amiodarone. Differential diagnosis is made with other situations that cause facial discoloration, especially with the use of chlorpromazine and tricyclic antidepressants. Treatment is based on photoprotection and drug suspension, with usual resolution of the discoloration in 6 to 12 months. In conclusion, side effects of amiodarone should be widely known to the prescriber and follow-up is necessary in order to avoid unwanted events. 108822 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS VICTOR CUSTÓDIO RIBEIRO1, Huggo Santana Machado1, Rodrigo Penha de Almeida1, Dennis Miguel Lemos da Silva1, João Lucas O‘Connell1 (1) Universidade Federal Uberlândia Introduction: Complications after myocardial infarction (MI) includes mechanical, arrhythmogenic, and hemorrhagic causes. In general, it happens in the first 5 days after the MI, which justifies the orientation of keeping the patients hospitalized during this period. However, the risk of complications remains high until the 14th day. The mechanical causes are more frequently associated with MI with ST-segment elevation, being responsible for 15% of the deaths by MI. There is a higher risk associated with a larger extension of the necrosis area, the longer period of time until the myocardial reperfusion, and the treatment with fibrinolytic (when compared to primary angioplasty). Case Description: Female, 74 years, obese, smoker, family history of coronary heart disease. Presented with typical chest pain, being admitted 36 hours after the beginning of the symptoms, already asymptomatic. The electrocardiogram showed an ST-segment positive deflection in the inferolateral-dorsal wall and right ventricle (RV). The patient evolved with decompensated heart failure and improved after clinical support. The echocardiogram showed biventricular dysfunction with a left ventricular ejection fraction of around 50%, inferior akinesia, and inferolateral hypokinesia. The cineangiocoronariography indicated a multivessel obstructive pattern, and surgical treatment was chosen. On the fifth day of hospitalization, the patient presented with abdominal pain followed by a sudden loss of consciousness without pulse. Cardiopulmonary resuscitation and Marfan puncture were done, with a moderate volume of bloody secretion expelled. Unresponsive, the patient evolved to death. At necropsy, a rupture of the posterior wall of the left ventricle with hemopericardium was identified. Conclusion: The most severe mechanical complication after MI is the rupture of the left ventricle. This condition is 7 times more common than RV rupture, with an incidence of up to 10%, especially when it affects territories supplied by the anterior descending artery. The acute form, with the presence of hemopericardium, progresses fast to death. The chronic form can be asymptomatic with the formation of pseudoaneurysms. Our case reinforces the importance of effective and early revascularization treatment. The persistence of ST-segment elevation after 72 hours attests to the risk of serious complications. Pericardiocentesis is an interim measure until clinical stabilization and referral for cardiac surgery. 108828 Modality: E-Poster Scientific Initiation – Case Report Category: ATHEROSCLEROSIS/CARDIOVASCULAR RISK FACTORS/CARDIOVASCULAR PREVENTION RIZIA CARLA DA SILVA LEOPOLDINO1, Karina Baltor Cabral1, Fernanda Fernandes Gonçalves Ribeiro1, Caio Araújo da Cunha1, João Lucas O‘Connell1 (1) Universidade Federal Uberlândia Introduction: Coronary artery anomalies have an incidence ranging from 0.1% to 1.4% in studies of necropsy and coronary angiography series. These anomalies are mostly benign and asymptomatic. However, they can cause symptoms when there are repercussions on coronary flow. The diagnosis of double Anterior Descending Artery (AD) occurs when identifying a short anterior descending artery that runs in the interventricular groove but does not reach the cardiac apex, and another long anterior descending artery, which has a variable proximal course and returns to the distal interventricular groove. The incidence of this type of anomaly is low: 0.017%. There is little data in the literature on the incidence of severe obstructive disease related to double AD. Case Description: A 68-year-old female patient presenting myocardial ischemia in angina investigation. Coronary angiography was submitted that identified double AD and the presence of obstructive coronary artery disease associated with both the proximal segment of the AD (originated from the left coronary trunk) and the anomalous AD ostium (bifurcation stenosis with the right coronary artery (DC), which also presented important stenosis). The patient underwent Coronary Angioplasty for both branches of AD and DC, with good clinical and angiographic evolution. Images of coronary angiography will be presented before and after angioplasty treatment. The patient progressed well clinically and is asymptomatic, 2 years after angioplasties. Conclusion: The diagnosis of double AD is given by coronary angiography or, more recently, by coronary tomography. Double AD should be suspected when there is a discrepancy between the apparent coronary distribution pattern and the motility of the ventricular walls. Dual Type IV AD is characterized by the origin of long AD in the right Valsalva sinus or right coronary artery. This anomaly is rare: 0.004% – 0.006%. Double AD can also be confused with occlusion of the middle third of AD, especially if only left coronary angiography is performed and long anomalous AD does not undergo selective catheterization. In addition, this anomaly may be associated with coronary obstructive atherosclerotic disease and the clinical aspects of the patient, angiographic complexity, and discussion among the members of the heart team should be considered in order to offer the best therapeutic proposal to patients with this pathology. 109111 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY INGRID SAGRILLO1, Júlia Carvalho Brasil Malaquias1, Cézar Ladeira Macedo Junior2, Juan Dias de Lima2 (1) Faculdade de Minas – Muriaé (FAMINAS – Muriaé); (2) Casa de Caridade Hospital São Paulo (HSP – Muriaé) Introduction: The intraventricular communication (IVC) is among the main mechanical complications of acute myocardial infarction (AMI), with an incidence of 0.2%, next to free-wall papillary muscle rupture, all generally associated with hemodynamic instability and high mortality. Case Report: J.H.V., a 59-year-old male, without previous comorbidities, came to the emergency room with typical chest pain for 20 hours, and electrocardiogram showed supra-ST-segment elevation in the inferior wall. He was immediately transferred to the hemodynamics department where primary angioplasty of the occlusive lesion of the right coronary artery was performed. On the third day after the onset of symptoms, the patient developed anginal pain, sweating, hypotension, and transthoracic echocardiogram showed IVC and Left Ventricular pseudoaneurysm (LVP). Thus, the patient was referred to a new hemodynamic procedure for percutaneous closure of the two IVC holes together with the LVP. Conclusion: The main symptom of AMI is sudden and constant chest pain due to reduced blood flow. Diagnosis is through the electrocardiogram, which shows a ST-segment elevation. Left ventricular pseudoaneurysm is a rare complication after AMI that results in ventricular wall rupture without cardiac tamponade. It is diagnosed by echocardiography or nuclear magnetic resonance, but most are done in the chronic phase, which often evolves asymptomatically. However, once diagnosed, it must be treated surgically. Echocardiography is also the gold standard for the diagnosis of IVC, in which it causes significant occlusions, which with advancing age may arise adjuvant diseases. The treatment of choice is surgical, usually -pericardial patch-, with low operative mortality and good long-term results. 108846 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES VALENTINA BRATTI DE NADAL1, Pedro Boustany Escobar1, Isabelle Gambin Antonini1, Leonardo Mussoi1, Rafael Coimbra Ferreira Beltrame2 (1) Universidade do Vale do Rio dos Sinos (UNISINOS); (2) Hospital Dom João Becker Introduction: Pickwick syndrome (PS) is defined by obesity, chronic hypoventilation and hypercapnia in absence of other secondary causes. Pulmonary hypertension with cor pulmonale is one of the consequences of severe untreated forms. Although well described, PS is often underdiagnosed. The following case illustrates the history of the untreated disease and the current challenges in its diagnosis. Case Description: A 43-year-old male with grade 3 obesity, hypertension and obstructive sleep apnea syndrome with CPAP indication, was admitted due to generalized peripheral edema with spontaneous transudation through lower limbs skin, and dyspnea at rest initiated within the last 3 months. He was on continuous and multiprofissional care regarding his obesity for 8 years, being in pre-op evaluation for bariatric surgery. At admission he weighed 230 kg, oxygen saturation was 85% on room air, and cyanosis, jugular turgor, bilateral pulmonary crackles were present. Echocardiogram showed biventricular dysfunction (Figure 1). Abdominal tomography showed congestive hepatomegaly and splenomegaly. Managed with arterial and pulmonary vasodilators during hospitalization, CPAP and high dose diuretics, with complete improvement of congestion and 67 kg of fluid loss – discharge with 163 kg. Arterial blood gas analysis on room air after compensation showed a pCO2 of 63.3 mmHg and a pO2 of 52.6 mmHg, confirming the diagnosis of PS. Discharged with nutritional plan designed for obesity care, treatment for pulmonary hypertension and biventricular dysfunction, CPAP and home oxygen therapy. Conclusions: Obesity is a public health epidemic. The patient above received long standing medical follow-up for obesity, without ever receiving specific evaluation for PS and its consequences, resulting in extremely advanced cardiopulmonary disease. Although a well-described entity, PS is rarely remembered and treated early by health care teams. The reasons are multifactorial, ranging from negative health care team biases related to the treatment of obese patients to lack of focus on the particularities of care for this group of patients. 108892 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY ELISA ROCHA NONEMACHER2, Elisa Rocha Nonemacher2, Gabriel Almeida Krul2, Rafaela de Oliveira Leite2, Marcelo Sabedotti1 (1) Hospital Geral; (2) Fundação Universidade de Caxias do Sul Introduction: Cocaine is one of the most used illicit drugs worldwide. Its use can lead to acute or chronic cardiovascular intoxication – among them, the Acute Coronary Syndromes (ACS), aortic dissection and rupture, arrhythmias and cardiomyopathies stand out. Its effect is related to blocking adrenergic reuptake in the synaptic cleft in addition to blocking sodium channels. The acute myocardial infarction is present in 6% of the population with recent cocaine use. Clinic History: 44-year-old woman, with a history of systemic arterial hypertension, smoker and cocaine user for 8 years. She sought emergency for medical care complaining of typical chest pain, associated with nausea, in 90 minutes of evolution. She was referred to the Coronary Intervention Unit of reference in the region, 4 hours after the onset of pain. On examination, she was with signs of hypertension and tachycardia. Electrocardiogram (ECG) with sinus tachycardia, ST elevated myocardial infarction (STEMI) in anteroseptal-lateral wall, being referred for early intervention. Coronary Coronary angiography demonstrated occlusion of the anterior descending artery. The thrombus burdens were aspirated resulting in restoration of coronary flow. Right after there was insertion of a drug-eluting stent in the affected artery, with clinical and hemodynamic improvement. Cocaine is a substance that, from a cardiological point of view, generates a cascade of events that is conducive to acute coronary syndromes, as it is a potent platelet activator, in addition to increasing heart rate and promoting vasoconstriction (mediated by alpha receptors). The blockade of sodium channels also contributes to a tendency to ventricular arrhythmias. Regarding drug therapy, it is prudent not to prescribe beta-blockers, especially in the acute phase, due to the risk of diverting all adrenergics from the synaptic cleft to alpha receptors, worsening the deleterious effects of cocaine. Conclusion: Considering that ACS are an important cardiovascular consequence of cocaine use, proper investigation and early treatment are necessary. Even though protocol treatment is the most appropriate alternative, there are some important differences in relation to conventional drug treatment that should be considered. 108893 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY PEDRO RAFAEL VIEIRA DE OLIVEIRA SALERNO1, Santiago Raul Arrieta2, Mario Henrique Hattori2, Willian Yoshinori Kawakami3 (1) Faculdade Pernambucana de Saúde; (2) INCOR-SP; (3) Hospital das clínicas da faculdade de medicina da universidade de são paulo Introduction: In univentricular patients, lymphatic complications, such as CPE, represent an important cause of morbidity and mortality.1 Identification of the source of the leak and management remains a challenge. ILG provides visualization of the lymphatic system and may represent a therapeutic alternative to surgical ligation of the TD2. Case Report: 10-month-old patient, 6,6 kg, born full-term, with an intrauterine diagnosis of double-inlet single ventricle with aortic atresia and hypoplasia of the aortic arch presented with irritability. Patients‘ medical history included stage 1 hybrid palliative surgery with 7 days of life and Norwood-Glenn, atrioseptostomy, and stent placement in the left pulmonary artery at 8 months of life. CPE on the fifth postoperative day was reported. Physical examination: cyanotic child, dyspnea, and no fever. Gasometry with a Sat02 83,8%, lactate 18 mg/dL. Chest X-ray showed bilateral pleural effusion. Tube thoracostomy (TT) was performed, pleural fluid analysis: triglycerides 515 mg/dl and cholesterol <50 mg/dl. With a high bilateral TT drainage (46,66 ml/kg) and failure of clinical management, ILG was done. Guided by ultrasound, an inguinal lymph node was punctured bilaterally with 25G needles and ethiodized oil was injected. After visualization of the cisterna chyli, a 22G Chiba Needle was used to access it. Due to its reduced size, TD cannulation was not feasible. Therefore, further injection of ethiodized oil was performed and the lymphatic leak was observed. After 48 hours, TT drainage reduced (5 ml/kg) and computerized tomography showed a reduction of CPE. 1 week after the procedure, clinical and radiological improvement was observed. Conclusion: CPE in univentricular patients results from iatrogenic lesions during palliative procedures or as an outcome of the increased lymph flow due to increased central venous pressure in patients with lymphatic anatomic variants.1 ILG is a low-risk procedure capable of identifying and treating the source of leakage.2 In this case, due to incapacity of TD cannulation, further use of ethiodized oil was implemented and successfully controlled the effusion. 108944 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT CAROLINA TISOTT BURTET1, Amanda Hillesheim Schuck1, Luísa Gailhard Brito1, Rafaella Magni Berthier1, Olga Sergueevna Tairova1 (1) Universidade de Caxias do Sul (UCS) Introduction: Pompe disease or glycogen storage disease type II (GSD II) is a rare hereditary autosomal disorder, whose incidence reaches 1:50.000. It’s caused by gene mutations that encodes the acid alpha-glucosidase enzyme that is responsible for the glycogen degradation – mainly in the muscles. Therefore, this hereditary disease may cause symptoms such as muscle weakness, unsteady gait and cardiorespiratory failure. It can be categorized as early onset Pompe disease (less than 12 months age) or late onset Pompe disease (more than 12 months age), and it is usually known based on the onset symptoms and on the presence or absence of cardiomyopathy. Case Description: 53 year-old female patient with a heart failure history. The patient has been diagnosed with Pompe disease at 34 years of age, and has the family history of two brothers with the same disease. In the last 20 years, it progressed with dyspnea during physical exercise, global strength reduction with predominance in the right hemibody, associated with degenerative anatomical changes. In addition, it presented significant worsening in the lower limbs in the last 5 years. Physical examination: altered gait phases, decreased balance, altered cognitive and verbal level and decreased coordination of selectivity. Complementary Tests: Echocardiogram with ejection fraction (EF) of 37%, eccentric hypertrophy, diastolic dysfunction and systolic dysfunction with a pattern of relaxation deficit; normal catheterization. Patient has no conditions of motor physiotherapy due to the lack of physical fitness. Conclusion: Late-onset Pompe disease usually progresses with a less aggressive presentation and without major cardiac involvement. Despite that, the patient evolved into heart failure with reduced EF. Then, it is possible to propose an association between this condition and late Pompe disease. Although there are few reports in the literature that demonstrate this association, we can conclude that, even if it is rare, it may be possible to find this condition not only in early onset disease. 108971 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY RAFAELA OLIVEIRA LEITE1, Bibiana Guimarães Maggi2, Marcelo Sabedotti2, Rafael Massuti2, Leandro Gazziero Rech2 (1) Fundação Universidade de Caxias do Sul; (2) Hospital Geral de Caxias do Sul Introduction: The no-reflow phenomenon is defined by the lack of myocardial perfusion despite the opening of the coronary vessel in the context of primary percutaneous coronary intervention (PPCI) after stenting, with no presence of dissection, mechanical obstruction, significant residual stenosis, or spasm. Angiographic evaluation is done by assessing the contrast flow beyond the PPCI location to the distal vessel and is related to an increased risk of major cardiovascular events. To solve this phenomenon, intracoronary infusion of adenosine distal to the stent, by microcatheter, can improve the angiographic pattern. However, in the Brazilian public health care system, also known as Sistema ùnico de Saúde (SUS), PPCI doesn‘t contemplate microcatheter due to its high cost, making alternative techniques necessary. Case Report: A 70-years-old male, with no previous comorbidity, was hospitalized due to typical chest pain without signs of hemodynamic instability. Complementary tests demonstrated an electrocardiogram with ST-segment elevation on the anterior-septal-lateral and high lateral walls, glomerular filtration rate of 43 mL/min/1,73 m2 and elevated troponin levels (3,9, for a reference of 0,014). Cardiac catheterization, performed through a puncture of the right radial artery, demonstrated ostial occlusion of the anterior descending artery. The left main coronary artery was cannulated with an EBU 4 (6F) catheter, followed by access of a 0,014 guidewire, with the thrombus burden aspiration with an Export® Medtronic aspiration catheter and circulation returned. When implanting the drug-eluting stent, the no-reflow phenomenon occurred. For its management, we positioned an aspiration catheter distal to the stent, and administered 1,2 mg of adenosine intracoronary, reestablishing the vessel flow (TIMI 3, BLUSH 3). The patient was transferred to the ward, where he remained asymptomatic until discharged after 6 days. Conclusion: The technique of distal infusion of adenosine through a thrombus aspiration catheter is effective and can be used on the SUS as an alternative to the microcatheter. 108972 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY RAFAELA OLIVEIRA LEITE 1, Elisa Rocha Nonemacher1, Gabriel Almeida Krul1, Marcelo Sabedotti2 (1) Fundação Universidade de Caxias do Sul; (2) Hospital Geral de Caxias do Sul Introduction: Temporary short – and intermediate-term mechanical circulatory support devices (MCSD) may be used as an escalation strategy to manage situations like cardiogenic shock (CS). The selection of an MCSD depends on the specific indication for hemodynamic support, the urgency of need, costs, and patient-related or device-related risks. The main etiology of CS is acute myocardial infarction (AMI), with seven percent incidence and evolving with loss of up to forty percent of left ventricular function. This study aims to report and discuss a case of acute myocardial infarction with ST-segment elevation associated with occlusion of the left main coronary artery evolving to CC and the use of an Intraaortic balloon pump (IABP). Case Report: A 40-years-old male, previously diagnosed with systemic arterial hypertension arrive at the hospital because of an acute myocardial infarction with ST-segment elevation. His physical examination demonstrated signs of hemodynamic instability, a Killip score of four (suggesting CS), and preserved neurologic status. High-sensitive troponin of the admission was 0,084 (reference of 0,014) and an electrocardiogram demonstrated sinus tachycardia, ST-segment elevation of 7 mm in aVR, and ST-segment depression in anterior-septal-lateral and inferior walls. The patient was promptly referred for early intervention. We performed coronary angiography, which demonstrated left main coronary artery occlusion. The thrombus burdens were aspirated with restauration the coronary flow, and then there was insertion a drug-eluting stent on the left main coronary artery towards the anterior descending artery. We opted for the implantation of an IABP after the procedure, for 24-hour duration support. The patient underwent intensive care for 72-hours. He was successfully discharged after ten days, asymptomatic, with orientation for optimized therapy, request for outpatient care, and cardiac rehabilitation. Conclusion: Knowing that CS increases the morbidity and mortality of patients, it is necessary to evaluate the use of MCDS by the Heart Team. However, the available evidence does not support the routine use of IABP in most patients with AMI. There is evidence that IABP can be an extremely important adjunct in the treatment of these patients, reducing hospital mortality and myocardial injury. Therefore, the technique must be proposed in cases of AMI with rapid clinical deterioration. 108974 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY LEO CHRISTYAN ALVES DE LIMA1, Leo Christyan Alves de Lima1, Hildeman Dias da Costa2, Laura Jane França Lacerda1, Milena Stephanie Alves Matos3 (1) Centro Universitário São Lucas (UniSL); (2) Universidade Federal de Rondônia (UNIR); (3) Secretaria de Estado da Saúde de Rondônia Introduction: Primary cardiac tumors are extremely rare, with an incidence of less than 0.1%. In adults, most benign lesions of the heart are myxomas, accounting for more than 75% of cases. Case Description: Male, 48 years old. A magnetic resonance imaging and transthoracic echocardiogram were performed for diagnostic investigation due to chest pain, which showed an enlarged right atrium (RA) with a mass pedunculated to the interatrial septum, irregular, mobile, invading the right ventricle (RV) in atrial systole, measuring 44 × 35 mm. In the left atrium (LA), oval mass, adhered to the interatrial septum, regular contours, measuring 15 × 11 mm. In RV, oval, subvalvular mass, measuring 19 × 14 mm. The images suggested myxoma in the right atrium and thrombus in the RV and LA, and surgical treatment was proposed. Intraoperatively, excision of the tumor that invaded the entire right atrium was performed, in addition to resection of the mass in the LA and RV, with resection of the atrial septum where the pedicle was located. The septal defect was closed with a continuous suture pericardium patch. He was taken to the ICU and discharged after 4 days. The anatomopathological result was positive for myxoma. Discussion: Differently from the preoperative imaging exams, where two thrombi and one myxoma were suspected, they were confirmed and the three masses were confirmed for myxoma. The vast majority of myxomas are located in the left atrium (LA), the biatrial ones being infrequent and even rarer when associated with the LV location. Conclusion: Due to the location of multicavitary myxoma and less prevalent prevalence, this report shows a rare and atypical morphological presentation of myxomas, with a classic diagnostic method, satisfactory recent postoperative surgical result and good prognosis. 109045 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES MIKE VINICIUS CANTO DE ANDRADE1, Karina Baltor Cabral1, Rizia Carla da Silva Leopoldino1, Sthephany Yamaguchi de Melo1, João Lucas O‘Connell1 (1) Universidade Federal de Uberlândia Introduction: Pulmonary thromboembolism (PTE) has variable clinical conditions. They may be completely asymptomatic or there may even be situations in which massive emboli lead the patient to death, and the diagnosis only through necropsy is not uncommon. Fibrinolytic treatment is indicated for patients who develop clinical instability. However, the establishment of this therapy for hemodynamically stable patients with pulmonary hypertension and/or right ventricular dysfunction is controversial. Case Report: Female, 34 years old, obese, no previous history, taking oral contraception. Reports an episode, 12 days before, of intense chest pain which radiated to her back along with pre-syncope and malaise. Since then, she has maintained dyspnea and fatigue on minor exertion. Physical examination: tachycardia and tachypnea, although hemodynamically stable; normal respiratory and cardiac auscultation. Electrocardiogram: sinus tachycardia (120 bpm) and S1Q3T3 pattern. Chest radiography: no changes. Arterial blood gases without hypoxemia. Elevated D-dimer, Troponin T and BNP. Transthoracic echocardiogram: right chamber dilation, significant right ventricular (RV) dysfunction, good left ventricular (LV) function. Pulmonary artery systolic pressure: 68 mmHg. Normal lower limb venous Doppler. Chest CT angiography: Signs of bilateral submassive thromboembolism. Full dose Enoxaparin was started on the 12th day and chemical thrombolysis was performed by an infusion of 100 mg of thrombolytic recombinant human tissue plasminogen activator (rTPA) in 2 hours (on the 14th day). She improved the symptoms, being discharged. Oral Rivaroxaban was prescribed. A transthoracic echocardiogram performed 3 days later revealed normalization of the right chambers, RV systolic function, and pulmonary artery pressure. Conclusion: Patients with hemodynamically unstable PTE should receive thrombolytic treatment as early as possible; preferably within the first 48 hours. However, if the clinical picture persists, it can be performed up to 14 days after the acute event. In hemodynamically stable patients with signs of RV dysfunction on echocardiography, the use of fibrinolytics is controversial. The use of thrombolytic in this case allowed a significant improvement in the patient’s clinical condition, even though it was performed 14 days after the initial diagnosis. The use of the new oral anticoagulants also allows effective, safe anticoagulation and early hospital discharge. 109079 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIO-ONCOLOGY NÍCOLAS HENRIQUE BORGES1, Ana Paula Guimarães Feuerschuette1, Jamylle Araújo Dias dos Santos2, Paulo André Bispo Machado-Júnior1, Gustavo Gavazzoni Blume1 (1) Pontifícia Universidade Católica do Paraná (PUC-PR); (2) Hospital Santa Casa de Misericórdia de Curitiba Introduction: Renal cell carcinoma (RCC) is a solid lesion responsible for 90% of neoplasms of renal origin. It is classified as an aggressive, lethal tumor with high metastatic power in rare and uncommon locations. Tumor growth can extend to the intravascular environment through the renal veins (RVs) to the inferior vena cava (IVC) with an incidence of 15% in patients diagnosed with CRC, in addition, there is an additional extension of the tumor, which can reach the right atrium (RA), being considered rare and present in 1% of cases. Clinical Description: Female patient, 68 years old, with a previous history of hypertension using hydrochlorothiazide 25 mg/day and hyperthyroidism using tapazole 10 mg/day, sought medical attention with complaints of palpitations and asthenia on minor exertion. In the investigation, exercise stress test was limited by fatigue, indicating functional class III (NYHA), and transthoracic echocardiogram showed a diastolic dysfunction with concentric remodeling of the left ventricle, in addition to the presence of a mobile mass inside the right cavities. On admission, she was dyspneic (RR: 20 mrpm), hypertensive (BP: 140 × 80) and presence of a murmur in the tricuspid focus. MR Angiography report an expansive cortico-medullary lesion in the lower 2/3 of the right kidney, with a central area of necrosis. There was involvement of the ipsilateral renal vein, which showed a filling, suggesting a tumor thrombus that extends to the inferior vena cava and into the right chambers. Cardiac MRI revealed dumbbell-shaped intracavitary mobile mass with protrusion through the tricuspid ring. The patient underwent a total nephrectomy, venous system thrombectomy and retroperitoneal lymphadenectomy. She remained under ICU for 3 days and stay under observation before, being discharged. Biopsy confirming a unifocal, histological grade G3, clear cell renal cell carcinoma of Fuhrmann. The patient presented an excellent evolution only taking medications. Conclusion: Cardiac metastasis to the right atrium is rare in patients with renal cell carcinoma. In our case report, surgery was effective for clinical improvement. 109175 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS PEDRO HENRIQUE ROSA E SILVA2, Pedro Henrique Rosa e Silva2, Jairo Rosa e Silva Junior3, Thiago Quinaglia4, Maria A Delbin1 (1) Department of Structural and Functional Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas-SP; (2) School of Medical Sciences, University of Campinas (UNICAMP), Campinas-SP; (3) Climecárdio, Centro Integrado de Clínica Médica e Cardiologia, Ribeirão Preto-SP; (4) Discipline of Cardiology, School of Medical Sciences, University of Campinas (UNICAMP), Campinas-SP Introduction: Takotsubo Syndrome (TTS) is an acute cardiac syndrome characterized by typical regional wall motion abnormalities that reflect impairment of myocardial contractility. TTS predominantly affects postmenopausal women and is often preceded by emotional stress. The case report was approved by the Ethics Committee (CAAE 45476521.9.0000.5404). Results: A 69-year-old postmenopausal woman with hypothyroidism, type 2 diabetes mellitus, systemic hypertension, dyslipidemia and history of chronic anxiety had in 2/6/2013 an intermittent chest pain accompanied by dyspnea and triggered by emotional stress. The ECG showed an inverted and peaked T wave in the anterolateral and inferior wall, ECHO showed apical segments dyskinesia with aneurysmatic dilatation and coronary angiogram revealed no evidence of coronary artery disease, the patient was diagnosed with TTS. On 10/14/2015, the 71-year-old patient presented sudden symptoms of typical chest pain, accompanied by malaise and diaphoresis, also triggered by emotional stress. The ECG showed an inverted symmetrical and spiked T wave in anterior wall, the left ventricle (LV) ECHO showed the wall entire apex and basal portion hypokinesis, with global contractile function moderately depressed, and coronary angiogram revealed normal coronary arteries and alteration of LV segmental contractility with mild global systolic dysfunction, being diagnosed with TTS. At 74-year-old, on 11/25/2018 the patient presented typical chest pain, dyspnea and diaphoresis, triggered by emotional stress. The ECG showed an inverted and spiked T wave in the septal wall and ECHO showed alteration of segmental mobility in the septal wall associated with mitral regurgitation. Due to the previous medical history of TTS and to the similarity of the clinical conditions with previous events the coronary angiogram wat not performed, being diagnosed with TTS recurrence. In all events, the patient was admitted to the intensive therapy unit for five days with treatment for acute chest pain protocol and hemodynamic support. The ECG and ECHO changes were completely reversed. Years later a myocardial scintigraphy was performed showing no sign of sequels from the events. Conclusion: The report presented a case of recurrent TTS triggered by emotionally stressful events in postmenopausal woman, two events with a differential diagnosis with coronary angiogram. 109176 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY FRANCIELLE GODOI SANTOS1, ENZO OKU MARTINAZZO1, RÔMULO FRANCISCO DE ALMEIDA TORRES2 (1) Pontifícia Universidade Católica do Paraná; (2) Hospital Marcelino Champagnat Introduction: Coronary obstruction (CO) during the transcatheter aortic valve implantation (TAVI) procedure remains an important complication due to a high mortality rate. Its main risk predictors are low origin of coronary ostia, narrow sinus of valsalva and valve-in-valve procedures. Coronary protection (CP) with guidewire and eventual stent implantation is used in patients at high risk for CO in order to increase the safety of the procedure. This study reports the case of a patient with symptomatic severe aortic stenosis who underwent TAVI with the CP technique. In the pre-intervention evaluation, she presented high risk predictors for CO and a rare anomaly of coronary origin. Case Description: An 82-year-old patient was admitted to a tertiary hospital with heart failure. The transthoracic echocardiogram showed the presence of low-gradient normoflow aortic stenosis. Aortic valve calcium score was performed, which reinforced the presence of severe aortic disease. The evaluation with CT angiography showed the presence of a single ostium-like anomaly of the coronary artery originating in the left sinus of valsalva with a low origin in relation to the plane of the annulus. After evaluation by the institutional Heart Team, she underwent TAVI with the CP technique. Conclusions: This report demonstrates the feasibility and safety of the coronary CP technique during the TAVI procedure in a patient with a rare coronary anomaly and at high risk for CO. 109237 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES NICHOLAS VINCENT LEE1, Nicolas Henrique Borges1, Rosane Carolina Paes de Lira2, Paulo André Bispo Machado Junior1, Gustavo Gavazzoni Blume1 (1) PONTIFICIA UNIVERSIDADE CATÓLICA DO PARANÁ – PUC/PR; (2) HOSPITAL SANTA CASA DE CURITIBA – HSC Introduction: Infective Endocarditis (IE) is an uncommon disease with high morbidity and mortality, being commonly associated with gram-positive microorganisms. The IE by Proteus mirabilis, gram-negative, it’s rare, difficult to diagnose and the treatment is controversial, being necessary a fast intervention to avoid severe complications. Case Report: Healthy young patient, come to the emergency room with symptoms of asthenia and progressive dyspnea for the last 2 months. On physical examination, the patient had a systolic murmur in aortic focus with irradiation to the carotid arteries. The echocardiogram shows us a severe double aortic valvar lesion and an importante systolic disfunction (Simpson’s Ejection Fraction of 29%), being indicated aortic valve replacement surgery. While waiting for the surgery, the patient developed bacteremia secondary to phlebitis in peripheric venous access, hemoculture with Staphylococcus epidermidis was positive, initiated a guided antibiotic regimen (oxacillin 500 mg 4/4 h IV). However, the patient remained feverish with high inflammatory markers, evolving to sepsis, and a new antibiotic regimen was started (vancomycin 1000 mg 12/12 h IV). Eight days after the diagnosis of the infeccion, news hemocultures revealed the presence of Proteus mirabilis in blood, being administrated a new antibiotic treatment (cefepime 2g 8/8 h IV) but without a good response. After multiples antibiotics and roled out other infectious foci, a new transesophageal ecocardiogram identify a laminar image in the aortic root suggestive of abscess without vegetations. Opted of emergency surgery, the diagnosis was confirmed of abscess valvar, but despite the intervention, the patient died. Conclusion: The present report case demonstrated a rare and defiant association of IE by Proteus mirabilis with an atypical presentation in echocardiogram (abscess without vegetation) and even with the combined antimicrobial management and surgery, it did not present a favorable outcome. 109261 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS SARA AYRES SOARES DE SOUZA1, Danielle Caiado de Castro Dragalzew2, Felipe Augusto Lima Rodrigues2 (1) Universidade Católica de Brasília – UCB; (2) Instituto Hospital de Base do Distrito Federal – IHBDF Introduction: Coronary anomalies (CA) are a group of cardiac malformations that encompass variations as anomalous origin of the left coronary (AOLC) and coronaries fistulas. Those abnormalities are rare (0.2–1.2%) and difficult to diagnose, usually needing an imaging test. Patients with this condition are asymptomatic at onset, however they have a high risk of sudden death. Latest findings include valvular complications, myocardial ischemia, angina and congestive heart failure. As spontaneous resolutions are rare, it is essential to diagnose and treat these cases. Case Report: JRSB 44-years-old woman, hypertensive, under treatment for squamous cell carcinoma of the cervix IIIB. Myocardial Scintigraphy (2018) shows preserved ejection fraction (EF), evidencing high degree of persistent hypoperfusion of the anterior wall and apex of the left ventricle, indicative of fibrosis/hibernating myocardium and apex myocardial ischemia. No follow-up was performed. She presented with acute coronary syndrome (ACS) in 2021. In the same year a coronary angiography showed right coronary artery (RCA) without obstruction, AOLC without possibility of selective catheterization and one fistula communication from the RCA to the pulmonary artery. Transthoracic echocardiogram with an EF of 65%, mild mitral, tricuspid and pulmonary valves insufficiency and septal dyskinesia. Electrocardiogram: diffuse repolarization abnormality and extrasystole. In 2022 patient presented with chest pain and tachycardia in moderate to mild intensity exercises, no findings on physical examination. The patient uses daily metoprolol 100 mg, enalapril 10 mg, simvastatin 20 mg, trimetazidine 80 mg. At the found diagnostic stage, the patient presented with symptomatic cardiac morphological change, and waits for surgical approach. Conclusion: The diagnosis of CA is rare, but important as differential in cases of chest pain. In the present case, this is showed by the imaging tests demonstrating the presence of AOLC and coronary fistula, abnormalities that can evolve with ACS, myocardial ischemia and valve insufficiency as happened. This evolution was favored by the absence of follow-up and proper treatment. The patient is still waiting for surgical approach which will help to avoid unfavorable outcomes. 109297 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ERYCLAUDIA CHRYSTIAN BRASILEIRO AGRIPINO1, Matheus Henrique Soares Neves da Silva1, Hanna da Costa Avelar Domingues1, Raquel Travassos Oliveira1, Estefane Ribeiro Melo1 (1) Universidade de Pernambuco – Campus Garanhuns (UPE) Introduction: Eisenmenger syndrome (ES) represents the triad of pulmonary arterial disease, reversal of the central shunt’s flow and cyanosis resulting from systemic-to-pulmonary congenital cardiovascular communication. The key to decide management is the detection and treatment of the cardiac defect before development of pulmonary hypertension, preferably during early childhood. In this case-report, it’s described the diagnosis and following management of an ES secondary to intracardiac communication and a right-to-left shunt in a 6 months old child. Case Report: A 6 months old girl presented with respiratory discomfort since birth, developmental delay and low weight gain was admitted to a tertiary hospital to undergo clinical evaluation. On examination, the patient presented a pansystolic murmur (5+/6+) and signs of respiratory failure. The remainder of the examination was normal. On the third day of hospitalization, it was ordered a transthoracic echocardiogram (TTE), that demonstrated interatrial communication with bidirectional shunt (right-to-left predominance), interventricular communication with bidirectional shunt (left-to-right predominance), moderate pulmonary hypertension (PAH), patent ductus arteriosus (PDA) with left-to-right shunt and relative mitral valve stenosis. On the fourth day, it was initiated therapy with spironolactone 1 mg/kg/day, digoxin 0,01 mg/kg/day and captopril 1 mg/kg/day. She was referred for a cardiological surgical service on the sixteenth hospital day. Atrial septoplasty and ventricular septoplasty with ligation of the PDA were performed. In the last week of hospitalization, a new TTE was ordered, which demonstrated intact atrioventricular septums, preserved biventricular systolic function and pulmonary pressure gradient unsuggestive of PAH. She was discharged after 43 days of hospitalization with a long-term therapy plan with furosemide 1 mg/kg/day, spironolactone 1 mg/kg/day and referral to ambulatory care. Conclusion: ES is a truly devastating condition, mainly without the early diagnosis and adequate treatment. Most of the patients tend to suffer an early death due to heart failure, arrhythmias and thromboembolic cerebrovascular disease. In this case-report, the early diagnosis associated with clinical and surgical optimized intervention were decisives for the patient’s outcome to be modified following the literature recommendations regarding management in such conditions. 109327 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT FILIPE EVERTON SILVA RODRIGUES 1, João Gabriel Oliveira de Souza1, Ana Carolina Sampaio Freire1, Victor Oliveira da Cruz1, Alexandre Anderson de Sousa Munhoz Soares1 (1) Universidade de Brasília – UnB Introduction: Takotsubo cardiomyopathy (TTC) is a reversible left ventricular dysfunction. The initial symptoms mimic acute coronary disease, as patients present with dyspnea, hypotension, syncope, elevated troponin levels and suggestive electrocardiographic changes. Predisposing factors include emotional stress, endocrine disease and adrenal insufficiency. Case Report: A diabetic, hypertensive 50 year old woman with a history of chronic pericarditis and TTC seeked the emergency department presenting chest pain, sweating, hypotension and dyspnea. Transthoracic Echocardiogram showed regional changes in the left ventricle contractile function, suggesting TTC with a left ventricular ejection fraction of 47,6% and cardiac magnetic resonance imaging revealed pericarditis. After hospitalization, she had improvement in the symptoms, being discharged with further ambulatorial investigation scheduled. A week later, she presented with worsening symptoms, with high troponin and D-dimer levels. She developed an episode of unstable polymorphic ventricular tachycardia with QT prolongation, corrected QT (QTc) of 518 ms, needing electrical cardioversion. Upon investigation and questioning the patient revealed a visual impairment that began 6 months prior. With that information and imaging it was diagnosed a brain tumor (very likely a glioma) compressing the pituitary gland. After starting hormonal replacement therapy (HRT), there were no new episodes of arrhythmia. The patient was discharged waiting to begin tumor treatment. Conclusion: Although the evolution of TTC in the majority of cases is favorable, prolonged QTc should be evaluated due to the potential of death. QT prolongation serves as a substrate for ventricular tachycardia and sudden cardiac death, but the occurrence of ventricular fibrillation and its predictors in the disease are still unknown. The exact pathophysiology of TTC is not fully understood. In this case, a panhypopituitarism due to tumor compression might of caused a secondary adrenal insufficiency, resulting in glucocorticoid deficiency, hyponatremia and related hypoglycemia, which may have induced a catecholamine release resulting arrhythmia and the recurrence of abnormal wall motion. To guide clinical decision-making, patients with cardiac abnormalities should be checked for hormonal alterations, knowing the possible connection between endocrine disorder in TTC and torsades de pointes, resulting in a potential therapeutic opportunity with HRT. 109329 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY CAROLINA GUIMARÃES HERZOG1, Marco Antônio Vinciprova Dall’Agnese1, Pedro Dutra Batista1, Gabriel de Paula Alves1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA Introduction: The 22q11 deletion syndrome (22q11DS) is considered the second most known genetic cause of congenital heart disease (CHD). Here we reported two patients with different types of CHD presenting 22q11DS. Cases Descriptions: The first patient was a three-year-old girl, who was referred to the service due to a diagnosis of type A aortic arch interruption. She underwent cardiac surgery shortly afterwards, and developed hypoparathyroidism. Abdominal ultrasound revealed a left renal agenesis. The patient also presented intermittent strabismus, high palate, micrognathia, posteriorly rotated right ear, left ear in faun and umbilical hernia. Karyotypic analysis was normal. However, a posterior evaluation through the use of fluorescence in situ hybridization (FISH) technique confirmed the diagnosis of 22q11DS. The second patient was a two-day-old girl who presented an echocardiography with evidence of a type I truncus arteriosus. She had already been submitted to karyotypic evaluation during the prenatal period – due to an increased nuchal translucency measurement -, which was normal. The patient presented in her clinical evaluation blepharophimosis, posteriorly rotated and low-set ears, and clubfoot on the right. The use of the FISH technique verified the diagnosis of 22q11DS. Conclusion: The 22q11DS presents an important association with CHDs, especially with the conotruncal ones. About half of the patients presenting type A interrupted aortic arch and one third of those with truncus arteriosus disclosed 22q11DS. As a consequence, patients with those CHDs should always be carefully evaluated regarding the existence of this syndrome. 109373 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MARIA CLARA SERAPIÃO FERREIRA1, Maria Clara Serapião Ferreira1, Alessandro da Costa Machado2, Fernanda Snovarski Mota3, Alberto Acosta Hermida3 (1) Universidade Federal da Integração Latino-Americana (UNILA); (2) Hospital Municipal Padre Germano Lauck (HMPGL); (3) Hospital Ministro Costa Cavalcanti (HMCC) Introduction: Syphilis is a sexually transmitted infection (STI) caused by the bacterium Treponema pallidum; when not properly treated, it can progress to tertiary syphilis and produce cardiovascular lesions, mostly affecting the aorta. Syphilitic aortic aneurysm, although rare, is still a possibility. We report the case of a young patient with an aortic arch aneurysm susceptible to rupture of syphilitic origin. Case Description: Male, 37 years old, with progressive dyspnea for 5 days, dry cough, mainly during inspiration, evolving with hoarseness for about 1 day. Hypertensive without treatment and smoker, associated with history of multiple sexual partners, not using protection. There is no history of STI or recent trauma. On physical examination, a hypertensive patient 140 × 80 mmHg (left upper limb) and 160 × 90 mmHg (right upper limb), normocardic, eupneic on room air. On cardiac auscultation, he had intense diastolic murmur (+4/+6) associated with thrill in aortic focus and hyperphonetic heart sounds. Chest angiotomography showed an aortic arch aneurysm, with an extension of 11,5 cm, with a mural thrombus in the right lateral portion, associated with tracheal deviation due to mass effect. Coronary computed tomography angiography showed no significant coronary lesions. Therefore, it was indicated a surgical approach for aneurysm repair, with aortic reconstruction and implantation of a 28 mm Dacron tube, without complications. Afterwards, considering the patient’s history and the aneurysm characteristics, especially its location, nontreponemal test was requested, being positive, with a VDRL of 1:128. For that reason, the treatment for tertiary syphilis with benzathine penicillin was initiated. The patient evolved uneventfully, was discharged six days after the operation, with outpatient follow-up. Conclusions: Although the treatment of syphilis with benzathine penicillin is effective and widely available, and the manifestations of tertiary syphilis are uncommon, the diagnosis of syphilitic aortitis should be considered. We must consider that there is a direct relationship between the sociocultural level of the patient and the development of the disease. Thus, the case demonstrates that the late diagnosis of syphilis causes important cardiovascular repercussions, which require immediate recognition and surgical approach to obtain a favorable prognosis. 109420 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY KESLLY KRAUSPENHAR CUCHINSKI1, Eduardo Bartholomay1, Liara Eickhoff Coppetti1, Melissa Nadal Duarte1, Tilae Steinmetz Soares1 (1) Universidade Luterana do Brasil – ULBRA Introduction: Pacemaker implantation is a safe procedure and widely used in clinical practice. Intercurrences are infrequent, but a serious complication can occur, such as vessel perforation. The present study aims to report a case of cardiac pacemaker insertion with unfavorable outcome due to ascending thoracic aorta perforation and to discuss ways to avoid this rare complication. Case Description: Male patient, 71 years old, submitted to pacemaker implantation due to sinus dysfunction and Wenckebach type 2 atrioventricular block with a narrow QRS. The device chosen was a DDD pacemaker with ventricular stimulation through the conduction system by the bundle of His. The implantation was performed by placing the atrial lead in the anteroseptal region through posteroanterior radiographic visualization and the ventricular lead by radiological orientation and by the His bundle electrogram in the septal region, close to the membranous septum. The procedure was performed with a duration of 1.5 hours with 7 min of radioscopy. After arriving in the intensive care unit (ICU), the patient showed cardiac tamponade detected by echocardiography. Pericardial drainage of 100 ml of blood was performed with resolution of the hemodynamic condition. The patient was referred for atrial lead replacement, since the His bundle lead was in the septum. During anesthetic preparation, the patient presented cough and sudden hemodynamic collapse, evolving to cardiorespiratory arrest. With the opening of the pericardium, a large amount of arterial blood was detected. It was opted for emergency sternotomy and direct cardiopulmonary resuscitation (CPR). A small screw tip of the atrial electrode was visualized, which had perforated the atrium and “clamped” the aorta. The bleeding was manually stopped and the spontaneous circulation returned after 60 minutes of CPR. The atrial electrode was repositioned in the anterior region of the right atrial appendage and the sternum was sutured. The patient was referred to the ICU, but evolved with hemodynamic shock due to systemic inflammatory response syndrome, passing away 4 days after the procedure. Conclusion: Aortic perforation by atrial lead is a rare event described in few cases in literature; however, it is of extreme clinical relevance due to its high mortality. Therefore, we suwe suggest that the right oblique view should always be performed to certify the location of the anterior lead, aiming to avoid this rare, yet tragic complication. 109425 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PEDRO GUSTAVO STEVANATO DE OLIVEIRA1, Georgia Marques Jardim1, Nadine Edda Corrêa2, Gustavo Gianesini1, Neivis Cubillos1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre; (2) Universidade Federal de Santa Catarina, Campus Araranguá Introduction: Congenital complete heart block (CHB) occurs in approximately 1/20,000 live births and is associated with low ventricular rate and reduced cardiac output, leading to heart failure and fetal hydrops. Congenital CAVB is associated with a significant increase in mortality and morbidity and may or may not be secondary to immunological processes. The transplacental passage of maternal anti-RO and anti-La antibodies, which causes damage to the fetal conduction system, is responsible for congenital CHB of immunological etiology. Case Description: Female patient, 31 years old, Sjogren’s syndrome with idiopathic thrombocytopenic purpura (remission for four years) and depression. The fetus was diagnosed with CHB during the prenatal follow-up of her fifth pregnancy. She followed up with fetal cardiology through echocardiograms that showed bradycardia around 42 bpm, ventricular rate around 47 and no signs of hydrops. The newborn (NB) was born full-term with high levels of anti-RO and anti-La, with a heart rate of 45 bpm without clinical hemodynamic repercussions. A postnatal echocardiogram showed ventricular chambers‘ enlargement and mild mitral and tricuspid regurgitation. A 24-hour holter monitoring confirmed CHB and QTC interval increase. After nine days, the patient underwent surgery to implant a pacemaker (Median Sternotomy: Implantation of an epicardial bipolar electrode in the left ventricular tip). He was presented with mild metabolic acidosis in the immediate postoperative period and progressed well, with a heart rate of 100 bpm, being extubated the next day, with hospital discharge one week after the procedure. Conclusion: Congenital CABG is of significant risk to the newborn and may be secondary to immunological mechanisms, such as Sjogren’s syndrome, as seen in this case by the high anti-RO and anti-La levels in the NB. It can be treated by implanting a pacemaker with good evolution. 109438 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PEDRO GUSTAVO STEVANATO DE OLIVEIRA1, Nadine Edda Corrêa2, Georgia Marques Jardim1, Gustavo Gianesini1, Bárbara Zottis1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre; (2) Universidade Federal de Santa Catarina, Campus Araranguá Introduction: Atrial flutter (AF) is a supraventricular tachyarrhythmia, rare in neonates, marked on the Electrocardiogram (ECG) by irregular, rapid atrial activity with 280–500 beats per minute (bpm), generally associated with 2:1 atrioventricular (AV) conduction. It usually affects children with cardiac malformations, but it can also develop in newborns with normal cardiac anatomy. Sinus rhythm can be restored with antiarrhythmic drugs, electrical cardioversion, or transesophageal stimulation. Case Description: 8-day-old male newborn, cesarean delivery due to maternal desire, at term, without complications, with 24-hour Holter showing AF with ventricular response ranging between 105 and 235 bpm, with an average of 168 bpm, in addition to AV conduction in a 2:1 ratio. After five days of finding AF in the exam, propranolol was started, with no adequate response. The patient was transferred to the Pediatric Intensive Care Unit of the reference hospital in the city, where he arrived hemodynamically stable, without vasoactive drugs, on room air, and without the need for oxygen therapy. ECG and echocardiogram were requested, continued monitoring and anticoagulation with heparin 29IU/kg/h was initiated in case of intra-atrial thrombus. Echocardiogram performed the following day showed mild enlargement of the cardiac chambers, biventricular systolic function in the lower limits of normality, mild mitral regurgitation, and absence of intracardiac thrombi. Electrical cardioversion was chosen. Pre-procedure orotracheal intubation was performed with a rapid intubation sequence, an uneventful procedure. Two cardioversion attempts were performed with 2 Joules (0.5 joules/kg) each, successfully after the second. It was documented an electrocardiogram after the procedure with sinus rhythm. The patient was extubated throughout the day without complications. Anticoagulation was suspended 96 hours after cardioversion. He remained hospitalized, stable, without new arrhythmias for seven days after the procedure. He was discharged with a referral for cardiological follow-up within 30 days. Conclusion: Despite being potentially fatal, atrial flutter in neonates has a good prognosis when diagnosed early, with electrical cardioversion representing an excellent therapeutic option, as evidenced in the above case. 109444 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY ISABELLA CAROLINE SOUZA DANTAS1, Marco Arthur Queiroz Passos1, Mary Anny Silva Fonseca1, Tarcisio Esdras Araujo Moura2, Jose Carlos Pachon Mateos2 (1) Faculdade Ages de Medicina; (2) Associação Beneficente Síria HCOR Introduction: Bradyarrhythmias are rare in children, the main, of significance is the complete atrioventricular block (CAVB), whose reported incidence is one case for every 25,000 live births, being more predominent in females. Children with a normally structured heart, in general, have a congenital etiology and are often linked to the presence of maternal systemic lupus erythematosus (SLE). Case Report: M.E.C.A.A.P., 01 year and 8 months old, presented dyspnea between feedings, low weight gain and syncope for +/–02 months. An electrocardiogram was performed, which showed a CAVB and a transthoracic echocardiogram (ECOTT) with a dilation of the left chambers. In the family history, mother with SLE. On 07/04/2018, an implantation of a definitive unicameral pacemaker by puncture of the left subclavian vein was performed, the electrode was positioned in the middle septum of the right ventricle and atrial loop was performed for growth curve. After implantation of the device, the patient evolved with significant improvement in dyspnea, with no recurrence of any syncope episodes and with the last ECOTT, performed four years after implantation, within normal limits. Conclusion: Congenital CAVB is a rare disorder and occurs due to the pathological changes in the AV conduction system that can be associated or not with congenital heart disease. In 50% of cases, it occurs due to malformation of the conduction system and is associated with structural diseases, the most frequent being left atrial isomerism and corrected transposition of the great arteries. Autoimmune CAVB, associated with maternal anti-Ro and Anti-La antibodies, represents approximately 40% of cases. This pathology can evolve with ventricular dysfunction, severe heart failure and even fetal or neonatal death, justifying the importance of awakening the academic community for early diagnosis and adequate therapy. In the case described above, the patient had signs and symptoms of low cardiac output, syncope and growth issues. In addition, maternal autoantibodies (Anti-Ro and Anti-La) and heart rate lower than 70 bpm were still present with structural heart disease or ventricular dysfunction. For these reasons, a pacemaker was indicated, whose endovascular implant was performed with the atrial loop aiming at the child’s growth curve. After implantation of the device, the patient’s s symptoms and heart disease improved, in a four-year follow-up. 109474 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY WALDEMIR FERRARI JUNIOR1, Mariana Castro Pires1, Júlio Pasquali Andrade1, Leonardo Nunes Sanson1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre Introduction: Cat eye syndrome (CES) is a rare disease caused by a partial tetrasomy of chromosome 22. We aim to report a patient with CES presenting a total anomalous pulmonary venous return (TAPVR). Case Description: The patient was the fourth child of a couple with no previous history of similar cases in the family. The child was a non-identical twin and was born by cesarean section, premature, weighing 2,240 g, with a length of 46 cm and a 6/8 Apgar score. The child needed oxygen therapy and mechanical ventilation immediately after birth, when a cardiac murmur was discovered. Due to an anal atresia with a vaginal fistula, the child required a colostomy surgery. Echocardiography revealed a non-obstructive TAPVR. The child underwent a cardiac surgery with 15 days of life, and kept the sternum open in the post-operative period. In the newborn evaluation, a right iris coloboma was also observed, in addition to both bilateral preauricular appendages and pits, micrognathia and a cutaneous appendage at the anus’ topology. The karyotype test revealed a partial tetrasomy of chromosome 22, resulting from a dicentric supernumerary marker chromosome: inv dup (22) (pter->q11.2::q11.2->pter). The child evolved with a persistent chylothorax, requiring thoracic drainage and pleurodesis, and died with 2 months old. Conclusion: Congenital heart defects are observed in more than half of patients with CES, especially TAPVR. When patients present extracardiac manifestations, as coloboma, preauricular pits/tags and anal atresia, TAPVR should be investigated. 109582 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JOÃO VICTOR ANDRADE PIMENTEL1, Júlia Souza Diniz1, Beatriz Luduvice Soares1, João Paulo Dias Costa1, Emerson Santana Santos1 (1) Universidade Federal de Sergipe Introduction: Alpha-protein kinase 3 (ALPK3) is one of the genes related to the development of cardiomyopathy. Patients with pathogenic variants in ALPK3 may present dilated cardiomyopathy that can progress to hypertrophic cardiomyopathy with poor systolic function. Biallelic pathogenic variants can cause several clinical features, with early-onset cardiomyopathy and extracardiac manifestations. Case Report: A 16-year old male was referred to our cardiogenetics outpatient clinic due to the diagnosis of non-obstructive concentric hypertrophic cardiomyopathy (HCM) at the age of 10. Born from healthy and non-consanguineous parents. He has four siblings without any cardiac conditions. His maternal and paternal grandparents had sudden cardiac deaths. Extracardiac features were also observed (facial dysmorphism, downslanting ears, short neck). Patient has also depression and has been assisted by a psychiatrist. He has been using beta-blocker and fluoxetine. A HCM gene panel found two different pathogenic variants on ALPK3 gene. Genetic studies confirmed that both parents were carriers. Cardiac magnetic resonance showed a maximal wall thickness of 33 millimeters. Conclusion: This case report highlights the importance of searching for the etiology of HCM and the genetic counseling for proper management of patients and their families. 109604 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES DIEGO ARAÚJO SILVA1, Thaísa Moreira Cenção1, Gabriela Rodrigues de Oliveira2, Ricardo Santiago Ferreira Coelho2, Juliano Novaes Cardoso2 (1) Faculdade Santa Marcelina; (2) Hospital Santa Marcelina Myocardial calcification, for the most part, has an etiology related to chronic pericardial diseases of multiple causes (viral, metabolic, rheumatologic). 67% of the patients with cardiac calcification have symptoms of heart failure; 8% have chest pain; 6%, abdominal pain; 4%, atrial arrhythmias; and 5%, symptoms suggestive of cardiac tamponade¹. The present study aims at reporting a case considered unprecedented in the literature because although most cases of cardiac calcification are related to constrictive pericarditis, the case in question is a calcification secondary to a subendocardial infarction, not correlated with any identified etiology. A 69-year-old female patient, seen due to complaint of acute vertigo, which evolved with unstable ventricular tachyarrhythmia and in need of electrical cardioversion. After cardiological evaluation, coronary cineangiography was submitted without alterations; However, ventriculography calcifications were identified leading to myocardial resonance imaging for differential diagnosis, which showed: subendocardial calcifications in inferolateral, basal, middle, and apical segments, compatible with subendocardial infarction. Due to the impossibility of surgical removal of the calcifications and the occurrence of ventricular tachycardia, implantation of an implantable cardioverter defibrillator was indicated. The patient presented stabilization of the condition and remains under outpatient follow-up. Unlike what is presented in the literature as the most common, the patient evolved with subendocardial calcifications of unspecified etiology, without a positive clinical history, nor infectious, metabolic, or rheumatological markers identified during the etymological evaluation performed. Subendocardial calcification is very rare, with few studies on this subject being found. Adequate planning and outpatient follow-up should be performed, considering subendocardial calcification, ICD, and the team’s experience. 109652 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY MARIA GABRIELA PIMENTA DOS SANTOS1, David Ferreira de Lima Duarte1, Maria Carolina Terra Cola2, Fabio Akio Nishijuka2, Thaíssa Santos Monteiro2 (1) Universidade Estacio de Sá/IDOMED; (2) Instituto Nacional de Cardiologia (INC) Introduction: Aortic coarctation (AC) and bicuspid aortic valve (BAV) are commonly associated. Diagnosed of AC must be early. Case Report: A 23 year-old patient diagnosed with AC and BAV after he had been hospitalized due to heart failure. Had many suggestive signs of AC in the physical exam, such as reduced pulse in the lower limbs, significant gradient of blood pressure between upper and lower limbs, and even a disproportion of upper and lower limbs development and signs of a significant aortic regurgitation as well. Echocardiogram (ECHO) identified BAV with severe regurgitation, left ventricle (LV) enlargement with moderate systolic dysfunction, monophasic blood flow in the distal descending aorta and abdominal aorta. Chest x-ray showed enlargement of the heart and erosions of the inferior border of the costal arches. Angiotomography (AT) showed a focal stenosis after the left subclavian artery, multiple collateral vessels and brachiocephalic trunk aneurysm. The initial strategy was to stent the AC and then treat the BAV surgically. The patient was submitted to an attempt of percutaneous treatment, but there was no success in passing the wire guide through the stenosis. It was then performed surgery with extra-anatomical tube implantation and aortic valve plasty with successful results and recovery. Conclusion: AC associated with BAV with significant regurgitation can evolve with significant impairment of the LV, impacting the morbidity and mortality of a young patient. The physical exam can be very helpful in the suspicion of these diagnoses, highlighting the importance of a thorough physical exam. Confirmation with non invasive tests are required, such as ECHO and AT, but catheterization will evaluate the gradient of the AC and often treat the lesion in the same procedure. 110116 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY RODRIGO BATISTA WARPECHOWSKI1, Thomás Ranquetat Andrade3, Nicholas Travi Dornelles3, Iuri Schwaab1, Paulo Warpechowski2 (1) Instituto de Cardiologia do Rio Grande do Sul; (2) Sociedade de Anestesiologia (SANE); (3) Escola de Medicina da PUCRS Introduction: Heyde’s syndrome (HS) is described as a multisystem disorder, characterized by a triad composed of aortic stenosis (AS), angiodysplasia (AD) of the gastrointestinal tract and acquired von Willebrand syndrome (AVWS) type IIA. In this case report, we present the evolution of a patient with HS presenting symptoms of congestive heart failure (CHF) and anemia. Case Report: Female patient, 73 years old, hypertensive, diabetic, dyslipidemic, smoker, with a history of iron-deficiency anemia and atrial fibrillation, admitted for aortic valve replacement. Complains of dyspnea, chest pain and symptoms of CHF. Echocardiogram showed severe aortic valvular stenosis (0,6 cm²), calcified valve leaflets, biatrial overload, left ventricular concentric hipertrofy, and pulmonary hypertension. The patient presented anemia, (Hb 6.9 g/dL), and received packed red blood cells treatment. After 3 days, there was an improvement in her blood count, with acceptable haemoglobin levels. No coronary lesions. Blood transfusion and CHF therapy were performed, with correction of congestion and dyspnea. A valve replacement procedure with biological prosthesis was performed without intercurrences. A new episode of anemia (Hb 6,1 g/dL) due to gastrointestinal bleeding occurred, presenting melena and requiring a new blood transfusion. The patient was discharged with general improvement, with a request for colonoscopy in search of AD. Conclusion: The most accepted AVWS mechanism proposes that in a situation in which shear forces are increased, as in AS, conformational changes occur in the multimers of von Willebrand Factor as it passes through the turbulent flow of a stenotic valve, causing interference in the control of angiogenesis and in the distribution of clotting factor VIII throughout the body. This association provides an important pathophysiological basis that supports valve replacement as a key strategy for long-term resolution of recurrent bleeding. Currently, most patients with HS after valve replacement have a reduction or disappearance of bleeding episodes. Furthermore, transcatheter aortic valve replacement (TAVR) has shown excellent results as well, with fewer perioperatory complications. Despite TAVR reduced risk, there are no differences in outcomes in terms of all-cause mortality in patients with HS undergoing these procedures. In addition, in patients who received bioprosthesis, the risk of recurrence of bleeding was 45% lower than in the mechanical ones group. 109729 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY WALDEMIR FERRARI JUNIOR1, Capitulino Camargo Junior1, Júlio Pasquali Andrade1, Vitor Reis de Souza1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre Introduction: Russell-Silver Syndrome (RSS) is a genetically heterogeneous condition characterized by the fault of the primordial growth. Our goal was to report a case of RSS presenting transposition of great vessels. Case Description: The patient is the fifth child of a non-consanguineous couple, without history of similar cases in the family. The child was born of vaginal delivery, with 37 weeks of gestation, weighing 1,785 grams and with Apgar scores of 7/8. He was hospitalized in the neonatal intensive care unit requiring oxygen therapy. In this period, there was suspicion of a congenital heart disease. Echocardiography showed transposition of great vessels, persistence of the ductus arteriosus, ventricular septal defect and atrial septal defect of patent oval foramen type. The child underwent cardiac surgery and resection of the ductus arteriosus, as well as recovery and atrial septal defect repair on the thirteenth day of life. In the physical examination, with 11 months old, growth retardation was observed, upslanting palpebral fissures, epicanthic folds, wide and low nasal root, high arched palate, micrognathia, bilateral single palmar crease, fifth fingers clinodactyly, bilateral camptodactyly from the second to the fifth hand fingers, lower lombo asymmetry (the left member was smaller than the right), overlapping toes and hypotonia. He evolved with neuropsychomotor delay and feeding difficulties. His karyotype test was normal. Conclusion: The sum of the presenting findings our patient was compatible with the diagnosis of RSS. The pre and postnatal growth retardation and limb asymmetry are considered major criteria for diagnosis. Congenital heart diseases are rarely described among these individuals. 109770 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY DAVID FERREIRA DE LIMA DUARTE1, Maria Gabriela Pimenta dos Santos1, Maria Carolina Terra Cola2, Fábio Akio Nishijuka2, Thaíssa Santos Monteiro2 (1) Universidade Estacio de Sá/IDOMED, Campus Città e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (2) Instituto Nacional de Cardiologia Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease. It is associated with multisystem organ damage mediated by autoantibodies and immune complexes. Lupus Aortitis is an uncommon and potentially fatal complication of SLE. Case Description: Female 37-year-old, known to have SLE 15 years ago and Sjögren’s syndrome. Has had, lupus nephritis type IV, sensory motor polyneuropathy, idiopathic thrombocytopenic purpura and depression. Reports irregular use of azathioprine and hydroxychloroquine when presented a chest pain radiating to the neck and jaw after emotional stress, which lasted 15 minutes. Transesophageal echocardiographym identified an aneurysm of the ascending aorta measuring 4.9 cm with dissection 1.6 cm above the aortic valve, and severe tricuspid regurgitation with overload of the right ventricle. Angiotomography confirmed the diagnosis of aortic dissection Stanford A, Debakey I. was performed with replacement of the ascending aorta and arch, resection of vegetations in the tricuspid valve (TV) that was identified through histopatological analysis as thrombus, and plastic repair of TV. Controlled angiotomography after surgery identified several saccular aneurysms in the aortic root. FDG pet scan was performed to assess the SLE activity in the aorta, which identified inflammatory activity. Adjustments of the medical treatment were made in order to control lupus activity before the patient can be submitted to another surgery. Conclusions: Lupus aortitis is a rare and severe complication of SLE, and surgical treatment as well as controlled of the rheumatological disease are required. The FDG pet scan can help in assessing the inflammatory activity of the disease, determining adjustments of the medical treatment and the best timing for surgery. 109783 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT BRUNA DE ALMEIDA FREIXEDELO1, Mateus de Sousa Cavalcante1, Dara Medeiros Mendes1, Jefferson Luís Vieira2, Leandro Cordeiro Portela1 (1) Universidade Federal do Ceará (UFC), campus Sobral, Sobral/CE-Brazil; (2) Hospital de Messejana Dr. Carlos Alberto Studart Gomes, Fortaleza/CE-Brazil Introduction: Heart transplant (HT) recipients are at an increased risk of complications from COVID-19 due to chronic immunosuppression and comorbidities. Yet, no immunological link has been proven to explain an association between COVID-19 infection and late post-HT complications, including graft vascular disease (GVD) or late rejection. Case Description: Male patient, 63 years old, with a history of HT for 18 years, admitted for HF and LVEF 35% one month after COVID-19 and change of immunosuppressive regimen due to unavailability of sirolimus in the health system. The pharmacological stress echocardiogram with dobutamine had been normal in the previous year. Diagnostic screening with endomyocardial biopsy (EMB) and panel of antibodies (including CD3, CD68, CD20 and C4D) ruled out cellular and/or humoral rejection, showing extensive interstitial and endocardial fibrosis, with myocytic hypertrophy and ischemic pattern; coronary angiography revealed significant multivessel coronary disease, with ectasias and rosary bead-like occlusions, confirming the clinical suspicion of GVD. Subsequent intracoronary ultrasound identified a minimum luminal area of 5 mm2 and the Heart Team opted for conservative management of the LVD. Conclusion: GVD consists of the narrowing of the arterial lumen, due to the thickening and pathological remodeling of the tunica intima, due to the infiltration of mononuclear inflammatory cells. This condition has multiple possible etiologies, with immunological and non-immunological factors involved in the pathophysiology. The disease may result from a local and systemic inflammatory response initiated by alloantigen-dependent factors and non-immunological stress agents. The patient reported presented a clinical picture compatible with HFrEF due to decompensated GVD one month after infection by COVID-19 and change of the immunosuppressive regimen, without anatomopathological evidence of cellular or humoral rejection. In rare cases, there is evidence of accelerated progression of GVD from grade 0 (angiographically undetectable) to grade 3 (graft failure) in less than 4 months. Consequently, we believe that the systemic inflammatory response to SARS-Cov-2, with widespread cytokine production, direct myocytic damage, and vascular inflammation, may have played a key role in GVD progression. More studies with adequate methodology are needed to elucidate this potential association between GVD, myocardial damage and COVID-19 in HT recipients. 109816 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MATHEUS RODRIGUES TEIXEIRA NETTO1, Eduardo Comazzeto dos Reis2, Selma Rodrigues Chaves2, Alessandra Teixeira de Oliveira2, Valter Correia de Lima2 (1) Universidade Federal de Ciências da Saúde de Porto Alegre; (2) Santa Casa de Misericórdia de Porto Alegre Introduction: Spontaneous coronary artery dissection (SCAD) is an important cause of myocardial infarction. The increase in its occurrence is related to the widespread use of coronary angiography in recent years. The etiology of SCAD is poorly understood. Its pathophysiology consists of separation of the arterial wall layers by intimal rupture or intraparietal hemorrhage, so that luminal narrowing and consequent coronary syndrome with elevation of biomarkers occur. Angiography shows the appearance of extraluminal contrast, multiple radiolucent lumens, and intraluminal filling defects. Conservative treatment is recommended, given the spontaneous healing of most lesions, while thrombolysis is contraindicated. Revascularization should be considered in case of ongoing ischemia, hemodynamic instability, arrhythmias or involvement of the left main coronary artery. Case Report: Female patient, 54 years old, hypertensive and diabetic, presented an episode of oppressive retrosternal pain. In the first evaluation, electrocardiogram showed a current from a subepicardial lesion in the lower-dorsal wall. At the origin hospital, loading doses of ASA and Clopidogrel were administered, followed by thrombolysis with Alteplase and Enoxaparin. Due to the absence of reperfusion criteria, she was referred for rescue catheterization, which showed a left main coronary artery (LMCA) with 98% stenosis and occlusion of the circumflex (LCX) and anterior descending (LAD) branches. After LMCA predilation, LMCA and LAD dissection were diagnosed. Implantation of 3 drug-eluting stents was performed in LMCA, LAD and LCX, with good angiographic results. Follow-up was given with Aspirin, Clopidogrel, Simvastatin and Metoprolol Succinate. Conclusion: The increase in the occurrence of SCAD brings with it the need for new studies to facilitate the understanding of how to manage it. Patients‘ inadvertent thrombolysis can worsen the condition and add to the need for new interventions. Therefore, whenever possible, all approaches should be based on the findings of coronary angiography. 109825 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANA OLÍVIA DANTAS1, KELSON KEMUEL CONFESSOR DE SOUSA2, THALINY BATISTA SARMENTO DE OLIVEIRA1, KELTON DANTAS PEREIRA2, FABIO MASTROCOLA2 (1) UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE; (2) HOSPITAL UNIVERSITÁRIO ONOFRE LOPES Introdução: Heyde’s Syndrome (HS) is classically described as an association between aortic stenosis and gastrointestinal bleeding from angiodysplastic lesions. Acquired von Willebrand factor (fVW) deficiency is related to its pathogenesis and results from hemodynamic stress on the stenotic valve. It is believed that valve replacement reduces the risk of bleeding, with success in 80% of cases. Relato De Caso: Female patient, 60 years old, with chronic intestinal bleeding due to vascular ectasias in the gastrointestinal tract. She had clinical decompensation with major bleeding. Upper digestive endoscopy showed ectasia in the bulb and in the second and third duodenal portions without bleeding. Colonoscopy showed two angiectasia of the cecum and colon. On physical examination, aortic and mitral ejection systolic murmur was observed. Patient was unaware of previous heart disease. The ECG showed signs of left ventricular overload and the echocardiogram revealed asymmetric septal hypertrophy with left ventricular outflow tract (LVOT) obstruction – septum 26 mm and maximum systolic gradient 230 mmHg – and significant mitral regurgitation. Thus, the hypothesis of hypertrophic cardiomyopathy (HCM) with possible associated Heyde syndrome was raised, because, similarly to aortic stenosis, HCM in its obstructive asymmetric septal form could lead to the same mechanism of degradation of von Willebrand multimers of high molecular weight by the ADAMST13 enzyme due to the high shear stress generated by the interventricular septum in the LVOT. However, the dosage of Von Willebrand factor antigen (fVW) and ristocetin cofactor were normal. Conclusão: We describe a case of probable SH in a patient with bleeding from angiodysplasias in the gastrointestinal tract associated with hypertrophic cardiomyopathy. Despite the normality of the VWf antigen, this data does not invalidate a possible association between MCH and SH, since the isolated dosage of the von Willebrand factor antigen may be normal in patients with acquired von Willebrand disease. The patient was discharged after stabilization of intestinal bleeding and correction of anemia, using a beta-blocker with reduced LVOT gradient. If bleeding recurs, specific therapies can be tried to reduce the diameter of the interventricular septum, such as alcohol ablation, myectomy and radiofrequency ablation, aiming to correct the pathophysiological substrate responsible for gastrointestinal bleeding. 109833 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT JOÃO CLEITON MARTINS RODRIGUES2, João Cleiton Martins Rodrigues2, Talles Levi Pereira Nogueira2, Márcio César Ribeiro Marvão3, João Maria Silva Rodrigues1 (1) Instituto Clinicárdio; (2) Universidade Federal do Pará (UFPA); (3) Instituto Evandro Chagas (IEC) Introduction: Chagas heart disease (ChHD) is caused by the protozoan Trypanossoma cruzi, was discovered in 1909 by Carlos Chagas and to this day is among the main endemic diseases of the Amazonic region. In the chronic phase the symptoms begin, resulting from the replication of protozoan amastigotes in myocytes, which causes mechanical cell’s rupture and release of pro-inflammatory cytokines, resulting in loss of tissue function and fibrosis. From this there is the process of cardiac remodeling and heart failure due to chagas disease. In 2014, the PARADIGM-HF study reported that the use of sacubitril/valsartan (neprilysin inhibitor and angiotensin II receptor blocker) significantly reduced mortality and hospitalization for heart failure with reduced ejection fraction (EF) compared to the use of enalapril. Another research was conducted with ChHD with reduced EF doing sacubitril/valsartan treatment for 6 months, which showed an improvement in the functional classification of NYHA of patients, without significant improvement of the EF on doppler echocardiography (DEC). Description: A male patient M.J.P.S., 46 years old, 2 years ago sought clinical cardiology complaining of asthenia, epigastralgia, weight loss and insomnia. She had systemic arterial hypertension; heart failure (NYHA III); IgG indirect hemagglutination test and bioelisa reagents for chagas, cardiomegaly (+++) on chest x-ray PA; DEC showing diffuse involvement of an important degree in the left ventricle (LV), a image suggestive of thrombi at LV apex (approximately 20 × 21 mm²), EF of 29%, LV diastolic dimension of 69 mm (05/25/2020). After addicting sacubitril/valsartan in the treatment with carvedilol 6,25 mg/day, furosemide 20 mg/day, spironolactone 12,5 mg/day and sodic levothyroxine 50 mcg/day for approximately 6 months, a significant clinical improvement was noticed and DEC reported an enhancement in EF: preserved cardiac chambers, EF of 67%, diastolic dimension of LV of 53 mm (02/26/2022). Conclusion: Therefore, the sacubitril/valsartan, associated with carvedilol, spironolactone and furosemide improved the clinical outcome of a ChHD patient, reverting a condition of reduced EF. Then, that medication eschedule is effective as a treatment for chagas cardiomyopathy. 109841 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES HENRIQUE KRICHENKO LEDRA1, Marcelo Vier Gambetta1, Franciani Rodrigues da Rocha1, Caroline de Oliveira Fischer Bacca1 (1) UNIDAVI – Centro Universitário para o Desenvolvimento do Alto Vale do Itajaí Introducion: Adult-Onset Still’s Disease is a rare condition, with a prevalence of 1–34 cases per million people, classified as a multigenic auto-inflammatory disorder, negatively affecting the patient’s cardiovascular system through pericardial and myocardial damage mainly, considered an important risk factor for the development of cardiovascular diseases. Case Report: We reported ten years of evolution of a male patient, 30 years old, hospitalized due to presence of low blood pressure, jugular veins distention, and muffled heart sounds on cardiac auscultation, diagnosed with cardiac tamponade. A successful emergency pericardial drainage was performed. A month later he was hospitalized again because of chest pain, fever and dyspnea. Diagnosed as aortic valve endocarditis with important aortic regurgitation after echocardiological evaluation. Aortic valve replacement surgery with metallic valve implant was performed and the vegetation biopsy showed chronic inflammatory lesion with no infeccion associated. He was referred to outpatient evaluation with a rheumatologist doctor, who diagnosed him with Adult-Onset Still’s Disease. Ten years after his first cardiovascular disability, the patient presented an important hemodynamic disturbance being hospitalized due to acute coronary syndrome because of the presence of thrombus in the descending and diagonal coronary arteries. He underwent successful balloon catheter angioplasty. Conclusion: Our hypothesis for the patient’s cardiac involvement is based on the complex pathophysiology of the disease and it’s possible complications. Pericarditis is well documented as a quite common Still’s Disease complication. However the same does not apply to aortic valve vegetation since just a few reports bring the same association between those conditions. As far as we know, based on the literature, the occurrence of an intracoronary thrombus in a patient with Still’s Disease is unprecedented and we consider the coagulation disorder the best pathophysiological hypothesis. 109856 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS DAVID FERREIRA DE LIMA DUARTE1, Maria Gabriela Pimenta dos Santos1, Julia Resende de Oliveira1, Gabriela Gama Zagni Jardim3, Lílian Soares da Costa4 (1) Universidade Estacio de Sá/IDOMED, Campus Città e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (2) Universidade Estacio de Sá/IDOMED, Campus Città e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (3) Universidade Estacio de Sá/IDOMED, Campus Città e Presidente Vargas e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC; (4) Universidade Estacio de Sá/IDOMED, Campus Vista Carioca e Instituto Estadual de Cardiologia Aloysio de Castro/IECAC Introduction: The analysis of arterial stiffness through the measurement of pulse wave velocity (PWV) has been listed as one of the oscillometric parameters among the best predictors of cardiovascular (CV) risk, when compared to the use of traditional scores and brachial artery pressure analysis, which, although consecrated in the literature, have numerous limitations and complexity in their evaluation. Different studies have shown that increasing PWV doubles the risk of CV events, CV mortality and death from all causes. In individuals at high CV risk, regardless their vascular age, this correlation is not entirely clear and defined. Case Report: We report a cohort study of 163 individuals at high CV risk, object of a scientific initiation project at our institution. Male, 58 years old, white, hypertensive, obese, diabetic, submitted to percutaneous revascularization after acute coronary syndrome. Although with optimized prescription, it maintains irregular drug treatment and low adherence to lifestyle modifications. On examination, baseline blood pressure was 135 × 87 mmHg; FC 63 bpm; pulse pressure 46 mmHg. Vascular parameters showed central aortic pressure 120 mmHg and PWV 8.4 m/s, highlighting that the standards adopted for the medians adjusted for sex, age and presence of risk factor are 124 mmHg and 7.9 m/s, respectively. Remaining symptomatic in his outpatient risk stratification, he was submitted to a hemodynamic study, demonstrating severe coronary lesions (>80%) proximal in 4 vessels, and referred for surgical revascularization. Conclusion: The analysis of PWV has been important as an additive value in the functional analysis of individuals with coronary artery disease (CAD), in different suggestions of new risk models. In this context, we believe that arterial stiffness, in addition to the possible early manifestation of atherosclerosis, as established in national and international guidelines, can be a CV prognostic marker of individual relevance in high CV risk, as demonstrated in this case, where the presence of high PWV could even be an additional factor in the severity and extent of CAD. Whether this finding could predict a higher risk of CAD evolution, its therapeutic response or even infer prognosis, so far there are no answers, but we hope to be able to contribute to future analyzes with our cohort study. 109919 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY GUILHERME RODRIGUES VIANA1, Guilherme Taioqui Fioruci1, Helena Guedes da Rocha1, Rafael Fabiano Machado Rosa1 (1) UNIVERSIDADE FEDERAL DE CIÊNCIAS DA SAÚDE DE PORTO ALEGRE – UFCSPA Introduction: Oral facial digital syndrome (OFDS) represents a heterogeneous group of disorders of genetic cause, characterized by oral, facial and limb anomalies with or without associated visceral malformations. Our aim was to describe a patient diagnosed with OFDS presenting an atrioventricular septal defect (AVSD) associated with truncus arteriosus. Case Description: The pregnant, a 25-year-old woman, was in her third pregnancy and was referred at 20 weeks of pregnancy for presenting fetal ultrasound with description of congenital heart disease and polydactyly. The patient had a description of a previous pregnancy with a fetus with a history of hard palate and tongue abnormalities, polydactyly, syndactyly of fingers and toes, and suspected congenital heart disease. The child died on the first day of life. In the second trimester ultrasound performed in the current pregnancy at 20 weeks, the interatrial septum and the right ventricular outlet were not visualized. In addition, polydactyly of hands and feet was observed. Fetal echocardiography revealed a complete AVSD associated with pulmonary atresia. The fetal karyotype was normal (46, XY). The child was born by cesarean section at 38 weeks, weighing 3,200 g, with Apgar scores of 8 and 9. Physical examination revealed facial dysmorphia and tongue alteration indicative of the presence of hamartoma. There was also polydactyly of both hands, with the presence of 7 fingers, and syndactyly between the fourth and fifth fingers. There was also polydactyly of both feet, with partial syndactyly between the first two toes. Echocardiography performed shortly after birth revealed a complete type C AVSD and a ventriculo-atrial communication with a single right ventricular outflow valve. Computed angiotomography performed on the same day also showed the presence of a truncus arteriosus. Conclusion: The clinical findings herein observed, including the cardiac ones, are compatible with the OFDS type II diagnosis, also known as Mohr syndrome. Cardiac malformations, overall ASD and VSD, have been reported in patients with this subtype of OFDS. Nevertheless, the truncus arteriosus, as observed in our patient, is considered to be a quite rare finding. 109982 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY CAIO RESENDE DA COSTA PAIVA1, Brenna Pinheiro Zuttion2, Pedro Pimentel Rocha Faria2, Verônica Homem de Carvalho e Silva1, Ana Carolina Bezerra Goes1 (1) Universidade de Brasília; (2) Hospital Universitário de Brasília Introduction: Among the most common congenital anomalies of the aortic arch, the retroesophageal right subclavian artery (RSA) (prevalence 0.5–1%) and Truncus bovis (prevalence 20–32%) stands out. These changes, mostly asymptomatic, are the result of defects in embryogenesis. Aortic aneurysm (AA) is an acquired anomaly and consists of a pathological dilation of this vessel. The case in question presents a patient in which these 3 alterations are present, without evidence of the presence of genetic syndrome (GS). Case Report: Patient, female, 59 years old, complaining of paresthesia in the right upper limb and neck pain for 3 days. He has a history of arterial hypertension, surgical repair of ascending AA and aortic trunk in 2012, with a Dacron tube installed, without valve replacement. In use of atenolol, ASA, metoprolol, olmersartan + amlodipine. Physical examination without changes. He was admitted to the Hospital de Base de Brasília for investigation. An angiotomography of the chest and abdomen was performed, which showed type 1 thoracoabdominal AA, with a greater diameter of 75 mm in the descending segment, and intramural hematoma in this portion. Tortuous aorta except for the ascending portion, truncus bovis and retroesophageal ASD. Echocardiogram showed mild aortic insufficiency and coronary cineangiography showed stenosis of 60% in the anterior descending artery. SG were discarded. In view of this, we opted for a right carotid-subclavian bypass and, later, open surgery for thoracoabdominal AA, with cardiopulmonary bypass. Endovascular procedure was not performed due to impossibility due to aortic tortuosity. During the second operation, the patient evolved with myocardial infarction, refractory cardiogenic shock and death. Conclusion: Anatomical changes of the aorta and its branches in combination are uncommon and poorly reported. When present, they occur mainly in patients with some genetic syndrome. Although usually asymptomatic, the congenital changes seen can lead to symptoms resulting from local compression, aneurysmal degeneration or atherosclerosis, causing dysphagia and paresthesias. In these cases, surgical intervention is the option of choice as well as for AA depending on its diameter and complications. Due to the high aortic tortuosity, percutaneous treatment was discarded, opting for open surgical repair of the vessel. The large scale of the surgery and the patient’s hemodynamic instability may have contributed to the outcome of the case. 110028 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES BRENO VINICIUS DIAS DE SOUZA1, Humberto Cabral de Oliveira Filho1, Heron Alves Vale1, Gerson Barbosa do Nascimento1 (1) Universidade Federal do Rio Grande do Norte (UFRN) Introduction: Infective endocarditis (IE) is characterized by inflammation of the endocardium, heart valves, or even an intracardiac device, such as pacemaker leads and prostheses. It is mainly by bacteria, and more, by fungi. Acute IE is usually caused by Staphylococcus aureus, and there is the subacute form, with slow evolution from weeks to months, with mild toxic, chronic fever and weight loss. A heart murmur may be present, as well as immunological and vascular phenomena. Case Report: Male patient, 80-years-old, retired, is supported in a referral hospital with a weight loss of 12 kilograms in 5 months. During that time, he had sporadic low-grade fever, which gave way to the use of simple analgesia, followed by sweating. Still, had severe arthritis, with limited range of motion due to pain. He had anemia in this temporal cut. The patient is hypertensive and has a history of exchange cancer treated with radiotherapy and valve surgery by aortic operation 10 years ago. It evolved with persistence of the joint, with characteristics of the lower limbs. Still, general condition, suffered with feverish brand, without compromising the general condition. On examination, calculated oral without teeth, with unsatisfactory hygiene. There was a murmur in the aortic focus. It presented three changes persistently, with an echocardiogram showing vegetation in the aortic valve. He received antibiotic therapy with ceftriaxone and oxacillin, evolving with improvement. Conclusions: The insidious course of the disease brought definitive treatment to establish a definitive diagnosis. The important echocardiogram image and the patient’s predisposition to valvular heart disease were used to guide the management, but it is noticed that there is a limitation for some times, since fever is poorly measured and that immunological manifestations are often not confirmed. Therefore, attention should be paid to non-positive germs such as blood cultures, such as Coxiella burnetii and fungi, which have a prolonged course. 111770 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES MATEUS DOS SANTOS BANDEIRA1, Ana Luísa Guedes de França-e-Silva2, Maria Luiza Novaes de Souza1, Eduarda Corrêa Maia1, Claudio Tinoco Mesquita1 (1) Universidade Federal Fluminense – UFF; (2) Universidade Federal de Goiás – UFG Introduction: 3D printing is revolutionizing medical imaging. In cardiology, 3D printing is increasingly used by surgeons and health professionals, such as cardiovascular surgeons, in procedures with cardiac involvement, including mitral stenosis. Description: A 57-year-old female patient reported dyspnea on moderate exertion, which began gradually and progressed to progressive worsening, being associated with orthopnea. She complained of chest discomfort. Patient had a history of interatrial communication corrected in 1987 associated with cleft repair of the anterior mitral leaflet, severe mitral regurgitation, tricuspid regurgitation, permanent atrial fibrillation, in addition to having systemic lupus erythematosus. A transthoracic echocardiogram demonstrated a cleft of the anterior leaflet of the mitral valve associated with severe regurgitation. 3D impression of the heart was performed with atrial and ventricular sections through preoperative CT images. The objective was to better understand the extent of the disease and assist in surgical planning, seeking to understand the potential benefit of the surgical approach. After 3D printing the selected approach was a median sternotomy with resection of the leaflets and implantation of a metallic mitral valvar prosthesis (numb er 3), associated with closure of the interatrial septum with a bovine pericardium patch and tricuspid annuloplasty with a number 28 ring. Unfortunately, patient died in 22nd postoperative day due to central venous catheter infection. Conclusion: We describe the use of 3D printing to anticipate a realistic cardiac surgery, to guarantee a safe surgery, as well as to promote a reduction in the duration of the surgery. Due to the size of the IAC dimension, it is possible to allow a joint visualization of the defect size in the middle of a wide 3D impression of the target organ. This case reinforces the future promise of modeling for surgical planning purposes, in addition to the future of such modeling for education. 110385 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES GABRIEL ALVES MENESES1, Tauã Richel Belchior da Costa1, Kaio Saramago Mendonça1, Marcela Souza Carneiro1, João Lucas O‘Connell1 (1) Universidade Federal de Uberlândia Introduction: Usually, the main causes of maternal mortality related or aggravated by pregnancy are hypertension, hemorrhage, puerperal infections, and miscarriage. However, other cardiovascular causes have contributed to increased pregnancy-related morbidity and mortality. In this scenario, arterial dissections (mainly of the aorta and coronary arteries) have an important role and also different therapeutic possibilities. Case Report: Second-time pregnant, 34 years old, 35 weeks and 5 days, obese, without other comorbidities or gestational complications, presented with intense precordialgia with irradiation to the dorsum and left upper limb, lasting more than one hour after a light walk. Referred to the tertiary service with positive myocardial injury markers, dynamic T-wave in anterior wall on electrocardiogram (ECG), but without ST segment depression. Started propedeutics of Non-ST Segment Elevation Myocardial Infarction (NSTEMI). The hypothesis of coronary dissection was considered and initial drug treatment was chosen. Patient remained hospitalized and underwent cinecoronariography at 37 weeks of gestation, which showed dissection of the left main coronary artery involving the anterior descending and circumflex arteries with TIMI III coronary flow. Clinical treatment was maintained and caesarean section was performed at 38 weeks under general anesthesia. She presented, in the puerperium, a new presentation of chest pain, with dynamic ECG, without enzymatic elevation. Coronary angiography was repeated and improvement of the angiographic aspect of the coronary dissection was identified, and the expectant management was maintained. The binomial was discharged from hospital in good general condition and remained well for the last 6 months. Conclusion: Due to hormonal changes and the hyperdynamic state, pregnancy alone increases the risk of acute myocardial infarction (AMI). Commonly, because we deal with young women, atherosclerotic diseases are not the main cause of AMI during pregnancy, but arterial dissections, which normally receive a differentiated therapeutic strategy. In addition, these dissections are associated with the occurrence of death or a reduction in maternal ejection fraction, which is directly related to maternal-fetal complications. 110398 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANTONIO PEDRO LIMA COSTA PEREIRA1, Giovanna Lucieri Alonso Costa1, Ana Paula Cassetta dos Santos Nucera1, Maria Angelica de Faria Domingues de Lima2, Fábio de Souza1 (1) Universidade Federal do Estado do Rio de Janeiro (UNIRIO); (2) Hospital Universitário Gaffrée e Guinle (HUGG) Introduction: Peripartum cardiomyopathy (PPCM) can be an initial manifestation of familial dilated cardiomyopathy (DCM). The BAG3 gene encodes a protein that is highly expressed in cardiac muscle and is involved in the anti-apoptosis effect. BAG3 pathogenic variant is associated with autosomal dominant DCM and has been reported in PPCM and in families undergoing sudden death. Case Description: A 36-year-old black woman presented to the emergency department with palpitations while breastfeeding her 2-week-old daughter. She denied any other symptoms. Medical history included diabetes mellitus type 1 and preeclampsia. Electrocardiogram (ECG) exhibited ST segment deviation and inverted T-waves in the precordial leads. Echocardiogram revealed left ventricular enlargement with diffuse hypokinesis. There were no records of raised troponin levels. Weeks later, magnetic resonance (MRI) showed reduced left ventricular ejection fraction (29%) and late gadolinium enhancement detecting presence of fibrosis. In the patient’s family history, her father, brother and paternal half-brother died suddenly at 42, 28, and 26 years old, respectively. During a further investigation, a cardiomyopathy gene panel identified a heterozygous BAG3 pathogenic variant with a cytosine deletion at position 824 (c.824del). The result was consistent with the diagnosis of, BAG3-related conditions, including autosomal dominant DCM, and this variant has not been reported in the literature. Regarding her medical history, coronary artery disease was ruled out, and her coronary calcium score was 0. It was considered to place an implantable cardioverter-defibrillator, but myocardial findings on MRI were completely reverted as were ECG and echocardiogram repeated after three years. At present, the patient has no complaints. She is under clinical cardiology care, and genetic counseling will be provided for her relatives. Conclusion: This case report highlights a rare occurrence of peripartum cardiomyopathy in a patient in which a novel pathogenic variant in the BAG3 gene was identified and related to a strong family history of sudden death. 110547 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ANA GABRIELLA MEDEIROS1, Caroline Bittar Braune2, Marcio Aloysio Freitas Siqueira Junior3, Ana Paula Cassetta dos Santos Nucera1, Fábio de Souza1 (1) Universidade Federal do Estado do Rio de Janeiro; (2) Hospital Universitário Gaffrée e Guinle; (3) Hospital São Lucas Introduction: Transthyretin cardiac amyloidosis (TTR-CA) is currently being discussed as a possible underlying cause of heart failure with preserved ejection fraction (HFpEF). Despite advances in cardiac imaging, monitoring electrocardiograms (ECGs) remains imperative for reducing TTR-CA misdiagnoses. Case Presentation: A 78-year-old black man presented with breathlessness and progressive exercise intolerance. He had a medical history of mild hypertension controlled with losartan 50 mg once daily. Physical examination showed blood pressure of 120/80 mmHg, heart rate 66 bpm, and no edema or pulmonary congestion. Initial ECG was described as nonspecific and indicated a possible previous infarction. Two-dimensional echocardiogram (2D-Echo) revealed ejection fraction 55%, left ventricular (LV) hypertrophy (septum 13 mm), diastolic dysfunction grade 3, and no segmental changes. HFpEF was considered and a loop diuretic was prescribed. In fact, ECG demonstrated sinus rhythm, PR interval 220 ms, narrow QRS complex with left axis deviation, disproportional amplitude of QRS complex related to assumed LV hypertrophy by 2D-Echo, and absence of R-wave progression in the anterior leads, which were interpreted as a pseudo-infarction. A reduced global longitudinal strain (–13%) measurement was obtained with an “apical sparing” pattern. Scintigraphy with technetium pyrophosphate confirmed TTR-CA (Perugini grading scale = 3). Genetic testing detected a pathogenic variant in TTR gene (genotype V142I) in which cardiac involvement is predominant. Conclusion: This report highlights ECG findings in a case of TTR-CA. Pseudo-infarction pattern and/or discordant amplitude of QRS compared to LV hypertrophy can be subtle clues indicating TTR-CA. Attention to the ECG is crucial to enable this challenging diagnosis. 110896 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR PHARMACOLOGY GEORGIA MARQUES JARDIM 1, Rafael Fabiano Machado Rosa1, Marco Antônio Vinciprova Dall Agnese1, Rafaella Aléssio Naibo2, Alexandre Perin Decol1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA; (2) Universidade Federal do Rio Grande do Sul – UFRGS Introduction: Marfan syndrome (MS), is a systemic connective tissue disorder with a high degree of clinical variability. The main manifestations consist of alterations involving the ocular, skeletal and cardiovascular systems. The short-term segment of a patient with MS, emphasize the importance of using propranolol in preventing the development of aortic aneurysm. Case Report: A 13 years old female patient who had a paternal family history of cardiopathy in a cousin and grandparents. Both grandparents passed away due to heart disease. Patient full-term newborn, with 2,9 kg weight and 51 cm of height. The patient was forwarded for investigation of fainting and right facial paralysis within one week of evolution. The examination identified oblique palpebral slits, low ear implantation, important scoliosis, arachnodactyly in both hands and feet, joint hypermobility and Marfan’s habitus. In cardiac auscultation, was identified a systolic murmur 3+/6+. The electrocardiogram had a precordial repolarization alteration. The echocardiography showed normal systolic biventricular function, mild left ventricular enlargement, systolic prolapse of the mitral valve in both leaflets, moderate valve regurgitation, ectasia in the region of the sinuses of Valsalva, aortics and ascending aorta, and aortic regurgitation. Therefore, she started using propranolol 10 mg, 3 times a day. During his echocardiographic follow-up, no dilatation or appearance of aortic aneurysm was observed, in the period of three years. The spine radiography showed marked dextroconvex dorsal and left convex lumbar scoliosis. The ophthalmologic evaluation revealed bilateral iridodonesis, Bell’s palsy, alternating exotropia, and superior lens subluxation. All these findings led to the diagnosis of Marfan syndrome. Conclusions: Drugs that decrease hemodynamic stress on the aortic wall, such as the use of β-blockers or angiotensin receptor blockers, are usually initiated at diagnosis or upon evidence of significant and/or progressive aortic dilatation in patients with MS. This therapy aims to prevent the development of aneurysms and the possible occurrence of aortic dissection, an important cause of death among these patients. 110607 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY GRASIELE DO AMARAL MARTINS1, Carolina Guimarães Herzog1, Emanuella Lara Tarzo de Medina Coeli1, Rafaella Aléssio Naibo2, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (2) Universidade Federal do Rio Grande do Sul Introduction: Insulin-dependent diabetes mellitus (DM1) can lead to the presence of a spectrum of malformations known as diabetic embryopathy. The aim of this report was to discuss the findings of a fetus whose gestation course was characterized by diabetic embryopathy, highlighting its relationship with congenital heart disease (CHD). Case Description: A 24-year-old, primiparous pregnant woman with a past medical history of DM1. Fetal ultrasound was performed outside the hospital at 22 weeks of pregnancy, the report described holoprosencephaly, congenital heart disease and single umbilical artery. Fetal echocardiography showed dextrocardia; double inflow tract atrioventricular connection to the left ventricle; single outflow tract ventriculoarterial connection and truncus arteriosus. Morphological ultrasound also showed increased amniotic fluid, single umbilical artery, and dilation of the third and lateral ventricles. The complementary examination also showed no visualization of the right kidney, the left kidney was small, and an abnormality was observed in the fetal spine. Fetal magnetic resonance imaging confirmed the finding of supratentorial hydrocephalus, in addition to the other malformations described on the ultrasound. The fetal karyotype was normal. The child was born by c-section at 33 weeks gestation, with a head circumference of 45 cm and Apgar scores of 3/7. Abdominal ultrasound showed that the right kidney was pelvic and the left kidney was multicystic dysplastic. Spinal x-ray showed hemivertebrae and butterfly vertebrae. Conclusion: In the literature, there are reports that there is a relationship between diabetic embryopathy and fetal findings of VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities) association, as observed in the present case report. CHD are part of the clinical spectrum of both conditions, with emphasis on those of the conotruncal type, i.e., involving the outflow tracts of the heart, as in the case of truncus arteriosus. 110855 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES MIKE VINICIUS CANTO DE ANDRADE1, Danielle Campos de Almeida1, Gabriel Alves Meneses1, Fábio Vieira Fernandes1, João Lucas O’Connell1 (1) Universidade Federal de Uberlândia Introduction: The cardiologic syndrome called “Takotsubo”, also called broken heart syndrome, is a type of stress-induced cardiomyopathy characterized by transient apical bulging and left ventricular dysfunction. The clinical picture usually mimics the same one of an acute myocardial infarction. In the absence of significant coronary obstruction, it courses with transient left ventricular dysfunction (usually apical). Characteristically, the clinical picture is preceded by intense emotional and/or physical stress. There is little data in the literature on fear or phobia reactions to insects or other potentially repulsive animals that lead to this condition. Case Report: Female patient, 58 years old, previously healthy, asymptomatic. Started with typical intense and tight chest pain for 2 hours. She describes similar, less intense episodes in the 2 days prior to arrival at the hospital. Given a history of catsaridaphobia, she reported significant acute stress after contact with a cockroach 12 hours before the initial episode. Physical examination without changes. Electrocardiogram: sinus rhythm, anteroseptal inactive electrical zone and slight ST-segment elevation in the anterior wall. There were typical enzymatic alterations. Cardiac catheterization did not identify coronary stenoses, but a pattern of left ventricular apical akinesia characteristic of Takotsubo Syndrome. Echocardiogram confirmed apical akinesia. Cardiac Magnetic Nuclear Resonance, 5 days later, showed basal anterior myocardial edema and medial septum, without fibrosis and with improvement in ventricular function. The patient had a good clinical evolution and was discharged from the hospital on the 7th day, with normal global contractile function. She is asymptomatic one year after the initial event. Conclusion: The reported clinical picture simulated an acute myocardial infarction, once absence of coronary obstruction was established, along with apical systolic dysfunction of the left ventricle and hyperkinesia of the basal portions. Transient acute ventricular dysfunction was clearly related to intense emotional stress, fulfilling current criteria for the diagnosis of stress-induced cardiomyopathy syndrome. The inducing stress of the condition was physical contact with an insect to which he has a phobia, whose occurrence may be the stressor mechanism for a part of the cases that develop Takotsubo syndrome. 110928 Modality: E-Poster Scientific Initiation – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM ALINE PETRACCO PETZOLD1, Bibiana Liberman Thomé1, Fernanda Serratte Warlet2, Larissa Arruda Ferreira2, Andrea Mabilde Petracco2 (1) Pontifícia Universidade Católica do Rio Grande do Sul; (2) Hospital Materno Infantil Presidente Vargas Introduction: Kawasaki disease (KD) is one of the most common primary systemic vasculitis in childhood, being the main cause of acquired heart disease in children. Throughout the COVID-19 pandemic, cases with manifestations similar to KD were reported, being classified as Multisystem Inflammatory Syndrome (MIS-C) following SARS-CoV-2 infection. Most findings for the disease are nonspecific, such as high, persistent fever, and inflammatory findings without an infectious explanation. Despite usually being a self-limited disease, prompt diagnosis and management are fundamental to prevent cardiac sequelae, since coronary artery aneurysms occur in 25% of untreated or poorly treated cases. Case Report: Male, 2-year-old patient, had contact with SARS-CoV-2 in January 2022. Admitted to the emergency for persistent fever for 20 days, with peaks every 2 days, associated with greenish coryza, rhinorrhea and eye discharge. A week before, was treated for otitis media with 3 doses of ceftriaxone, having momentary improvement, but further worsening. In the hospital, he presented fever, upper airway infection symptoms, conjunctival hyperemia with bilateral discharge, exsudate in oroscopy, opacity in the right tympanic membrane, hemoglobin of 10.2, and negative hemocultures and PCR for COVID-19. Echocardiography revealed patent foramen ovale and dilation of the anterior descending coronary artery, raising the hypothesis of post-COVID-19 MIS-C. The patient was started on acetilsalicylic acid (AAS) and methylprednisolone, persisting with mild fever. Since it was not available previously, he received intravenous immunoglobulin (IVIg) only 12 days after the diagnosis, showing clinical improvement. He was discharged with AAS and a scheduled echocardiogram in 15 days. Conclusion: Since the findings for MIS-C are nonspecific and, in the absence of specific tests for the disease, its diagnosis relies on clinical manifestations, it is important to maintain a high degree of suspicion for the syndrome. Children with unremitting fever, an epidemiologic link to SARS-CoV-2 and suggestive clinical symptoms should be thoroughly evaluated. Prompt recognition and management are crucial to control the hyperinflammatory state and prevent severe organ dysfunction and potentially fatal complications, such as coronary aneurysms. Supportive care, IVIg, corticosteroids, biological immunomodulators, antiplatelet therapy, and prophylatic anticoagulation are key in the management of these childre. 110997 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR INTENSIVE CARE/CARDIOVASCULAR EMERGENCIES LUANA DIAS XAVIER1, João Paulo Dias Costa1, Julia Sousa Diniz1, Beatriz Luduvice Soares1, Emerson De Santana Santos1 (1) Universidade Federal de Sergipe Introduction: Aortic root dilatation/dissection is one of the cardinal features of Marfan syndrome (MS), a heritable disorder of the fibrillin 1 gene (FBN1), which leads to a systemic disorder of connective tissue, with a high degree of clinical variability manifestations and involves the ocular, skeletal, and cardiovascular systems. Abnormalities of the aortic wall are quite frequent in this population, causing progressive aortic dilatation, thus increasing the risk of acute aortic dissection. Case reports: Two adult patients were referred from cardiothoracic surgery service to cardiogenetics evaluation after recovering from the successful intervention for acute aortic root dissection. Both patients were middle-aged males, not related, who fulfilled the Ghent criteria for MS. Patient 1 had no other affected member in the family and an FBN1 pathogenic variant was found. Patient 2 had an affected son and no molecular exam was performed. Other clinical data are summarized in the table. Conclusion: Although other MS typical features were previously present, the two reported cases had the diagnosis confirmed only after cardiogenetics evaluation. The delay in diagnosis is quite harmful once, with proper management, the life expectancy of someone with Marfan syndrome approximates that of the general population. Genetic counseling was provided for both families and the patients have been followed by a multidisciplinary team. 111085 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY LUÍSA RIGO LISE1, Luiza Fernandes Xavier1, Sabrina Comin Bizotto1, Júlia Helena Wegner1, Adriana Chassot Bresolin2 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS); (2) Universidade Estadual do Oeste do Paraná Introduction: The Complex Cyanotic Congenital Heart Disease is a deformation in the embryonic development of cardiac structures, causing an altered blood flow which may result in failures in the development of the circulatory system and systemic arterial oxygen desaturation. Its prevalence is aproximadely 1.45 per 1000 children. Description: Female, 8 years old, diagnosed in intrauterine life by fetal echocardiography with situs inversus and cyanotic congenital heart disease. Father, 35 years old, and mother, 24 years old, both healthy with family history of second cousin with aortic coarctation. Full-term cesarean delivery with premature placental detachment. The echocardiography postnatal showed: situs inversus with levocardia; single left ventricle; hypoplastic right ventricle with double-outlet right ventricle; transposition of great arteries; total anomalous pulmonary venous connection (TAPVC); ostium primum; interventricular communication. At 1 year and 5 months, Glenn procedure and correction of TAPVC were performed without complications. At 6 years old, she developed upper extremity deep vein thrombosis, involving brachiocephalic, internal jugular and right subclavian. A computed tomography showed agenesis of inferior vena cava and overload of azygos-hemiazygos system that drains to right brachiocephalic vein. The patient progressed with more cyanosis, hepatomegaly and increased intensity of second sound and initiated Furosemide and Enoxaparin. Carvedilol, Enalapril, Dildenafil and Spironolactone were associated because of worsening signs of heart failure. 6 months after this episode, Enoxaparin was changed by Warfarin. Currently, at 8 years and 3 months, thrombosis of brachiocephalic and right subclavian veins are already resolved and thrombosis of internal jugular vein is in regression. In addition, she has diastolic dysfunction of ventricles, moderate insufficiency of atrioventricular valve and, due to the difficulty of controlling anticoagulation, warfarin was suspended and dabigatran was prescribed. Conclusion: In this case, the association between anomalous venous return, blood hyperviscosity and hyperglobulinemia due to chronic hypoxia is observed as the cause of thromboembolism. Besides that, the Fontan Surgery was contraindicated due to agenesis of inferior vena. Therefore, early diagnosis, through fetal echocardiography and a careful medical follow-up, guarantees a better prognosis and lower morbidity and mortality in cases like this. 111168 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES LEONARDO PESSANHA CORDEIRO1, Juliana Fraga Soares1, Mellyssa Dias de Oliveira2, Pedro Hissa Monteiro1, Daniel J.M. Medeiros Lima1 (1) Faculdade de Medicina de Campos – FMC; (2) Centro Universitário Redentor – UniRedentor Carcinoid tumors are tumors that originate most commonly in the enterochromaffin cells of the gastrointestinal tract, especially in the small intestine. In the presence of liver metastases or a production that exceeds the metabolism capacity, substances such as serotonin fall directly into the systemic circulation, causing the carcinoid syndrome. The prognosis is worse in patients with carcinoid syndrome and carcinoid heart. Here we report an atypical presentation of carcinoid syndrome with early involvement of the right heart chambers, without flushing or diarrhea: a 49-year-old woman with complaints of nausea and vomiting with one-month evolution, lower limb edema associated with increased abdominal volume, and an involuntary weight loss of 10% in 6 months. Physical examination showed important ascites with mobile dullness and positive Piparote sign, bilateral lower limb edema 2+/4, pathological jugular swelling, and systolic murmur in tricuspid focus 2+/6 with reinforcement after the Riveiro Carvalho maneuver. A magnetic resonance imaging revealed numerous hypervascular nodular lesions, suggestive of metastasis. A liver biopsy, immunohistochemistry, scintigraphy with a somatostatin analogue (Octreoscan), Chromogranin A and 5-hydroxy indole acetic acid levels were compatible with carcinoid syndrome resulting from a carcinoid tumor of the gastrointestinal tract. The echocardiogram showed an increase in the volume of the right atrium and ventricle with severe tricuspid regurgitation, the valve was thickened, with failure to coaptation of its leaflets. The main therapy for the carcinoid heart currently used is the somatostatin analogues. Surgical intervention as valvar replacement is also a possibility for a sectioned patient who is more likely to benefit from an invasive approach. 111275 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOLOGY OF SPORTS, EXERCISE, ERGOMETRY AND CARDIOVASCULAR REHABILITATION LUÍSA DE LAMARE DOS SANTOS PAULA 1, Gustavo Henrique de Oliveira1, Thiago Honório Dutra da Silva2, Jaqueline Lyrio Bermudes Okawa2, Rogério Toshiro Passos Okawa1 (1) Universidade Estadual de Maringá; (2) Avancor Cardiologia R.A.F., male, 35 years old, businessman, born in Japurá-Paraná, from Santa Monica-Parana. Patient had a Covid-19 infection in March 2021, being admitted to an intensive care unit, for supplemental oxygen therapy in a high-flow mask. Thorax tomography showed 75% of pulmonary involvement. He had elevation of troponin and BNP during hospitalization. He was discharged with dyspnea on exertion, NYHA functional class II, being evaluated and requested complementary exams 30 days after hospital discharge, with echocardiogram with left ventricular ejection fraction with slight reduction: 42%, and reduced global longitudinal strain: – 12%. Cardiopulmonary stress test demonstrated Vo2: 24 mL/kg·min (72% of predicted), aerobic threshold 38% of predicted, with drop in oxygen pulse. He was medicated with Sacubitril/valsartan 49/51 mg every 12 hours, Bisoprolol 2.5 mg daily and Dapagliflozin 10 mg daily. Referred to cardiovascular rehabilitation, with 3 sessions per week, lasting 1 hour in each session. After 3 months of cardiovascular rehabilitation and pharmacological treatment, the symptoms improved, with a functional class I NYHA. The echocardiogram was repeated with improvement in left ventricular ejection fraction: 52%, improvement in global longitudinal strain: –16%, and improvement in ergospirometry parameters: Vo2: 37 mL/kg·min (90% of predicted), aerobic threshold 55% of predicted, oxygen pulse normalization. The present case report demonstrates a post-covid myocarditis, with left ventricular systolic dysfunction and the importance of pharmacological treatment with cardiovascular rehabilitation in this case. 111281 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MARIA GABRIELA PERERA1, Éveny Moraes Prola1, Helena Fussiger1 (1) Universidade Feevale Introduction: Ischemic stroke (IS) is the leading cause of morbidity in the world. In 30–40% of cases, TOAST classification is from a cryptogenic etiology. In these cases, the patent oval foramen (POF) may be the etiologic agent through paradoxical embolism. In young patients (18–60 years), 10% of cases are associated with POF, in elderly, however, attributing the cause of the vascular event to FOP is still a major challenge. Case: A previously healthy 70-year-old male presented with sudden onset of vertigo, nausea and vomiting, ataxia, hypoesthesia, and paresthesia of the left hemiface and left upper limb, lasting for 2 hours. His MRI scan showed no acute changes; only malacia in the left cerebellar hemisphere, but previous clinical history of focal neurological deficits. Then he was diagnosed with a transient ischemic attack (TIA). In the etiological investigation, transcranial Doppler identified passage of >20 HITs at rest, and after Valsalva maneuver >60 HITs, within the first 10 seconds of the infusion, considered a large shunt. Transesophageal echocardiography revealed interatrial septal aneurysm and POF with passage of a large amount of bubbles during the Valsalva maneuver. The rest of the investigation (with cervical Doppler, ECG, and laboratory tests) showed no changes that would justify the cerebral vascular event. RoPE score was 3 and PASCAL classified as a vascular event possibly related to POF. Treatment with Apixaban was initiated and a percutaneous endovascular closure procedure was performed for correction of POF. Discussion: The association of POF with IS in young is already established. However, in case of elderly with cryptogenic event, especially in cases of TIA, where we don‘t have the imaging to indicate event suggestive of embolism, the connection becomes even more difficult. According to a systematic review of 2021, in patients with possible PASCAL classification, the absolute risk of ischemia recurrence in 2 years was 3.6 and 1.5 for those treated conservatively versus invasively, respectively. But the data are valid mainly for patients between 8–60 years. In elderly, more studies are needed to inform the therapeutic decision, which is still a challenge today. References: Saver JL, et al. POF closure versus medical therapy for cryptogenic IS: topical review. 2018. Kent DM, et al. Heterogeneity of treatment effects in an analysis of pooled individual patient data from randomized trials of device closure of POF after stroke. JAMA. 2021. 111282 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY ARNÓBIO ÂNGELO DE MARIZ NETO1, Carolline Araújo2, Luan Martins de Sousa3, Mateus Macena Correia de Lima3, Arnóbio A. Mariz Jr3 (1) UNINASSAU; (2) Faculdade de Medicina de Olinda – FMO; (3) Pronto Socorro Cardiológico Universitário de Pernambuco Prof. Luiz Tavares, PROCAPE/UPE Percutaneous coronary angioplasty in patients with non-ST elevation acute myocardial infarction (NSTEMI) is a relatively common procedure and severe acute aortic regurgitation is a rare complication. We report a case of acute reversible aortic regurgitation during percutaneous coronary angioplasty in a 67-year-old woman, diabetic and hypertensive, with chronic coronary atheromatous disease who was admitted to hospital with NSTEMI. Subocclusive lesion in the circumflex artery and severe lesion in the anterior descending artery were evidenced in cardiac catheterization. During anterior descending angioplasty, a slowing of the flow through the vessel was observed (Figure 1), proceeded with the direct implantation of a drug-eluting stent with Sirolimus. At this moment, the patient developed oppressive chest pain, radiating to the upper limbs, profuse sweating, vomiting, sinus bradycardia and cardiogenic shock (BP = 64 × 42 mmHg). The images revealed significant acute aortic insufficiency (Figure 2) caused by the guiding catheter (Amplatz AL1) and reversed with the removal of the catheter. A new angiographic control revealed stents implanted with good results and a final TIMI III arterial flow (Figure 3). No dissection of the left main coronary artery was observed. Acute aortic regurgitation caused by coronary catheterization can be avoided through careful handling of the catheter, especially in challenging scenarios or in patients with an anomalous coronary origin. 111785 Modality: E-Poster Scientific Initiation – Case Report Category: HYPERTENSION/RENAL DENERVATION PEDRO HENRIQUE TORRES TIETZ1, Laís Bettoni1, Carolina Feijó Bombana1, Guilherme Rodrigues Viana1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) Introduction: Trisomy 13 or Patau Syndrome (PS) is considered a chromosomal disease characterized by multiple malformations and a limited prognosis. The following reports indicate that pregnancies with trisomy 13 have higher risk of developing preeclampsia. This association is related to the placental abnormalities which are frequently found in pregnancies of PS fetuses, including reduced placental volume and vascularization and placental mesenchymal dysplasia. Case Description: The first case reported a 30-year-old pregnant woman in her fifth pregnancy. She had a past medical history of one pregnancy loss. She was monitored by prenatal care and at the final stage of pregnancy she developed preeclampsia. She wasn’t submitted to fetal ultrasound at anytime of the pregnancy. The child was born prematurely at 36 weeks, weighing 2550 grams, by cesarean section. The Apgar score was 3/9. On examination, multiple alterations were observed, which included microcephaly, aplasia cutis on the scalp, cleft lip, micrognathia and hypertonia. Her karyotype indicated mosaic PS (47, XX, +13/46, XX). The child evolved with clinical worsening and died at 26 days of life. The pregnant woman in case 2 was 27 years old and in her second pregnancy. The pregnancy was accompanied by prenatal care, and it evolved with an episode of vaginal bleeding at 13 weeks. In addition, there was a description of an episode of urinary tract infection and preeclampsia. Due to the latter, the child was born by induced vaginal delivery. The other ultrasound exams performed during pregnancy were described as normal. The child was born at 37 weeks‘ gestation, weighing 2445 grams and with Apgar scores of 6 and 8. In her examination, there were alterations such as microcephaly, trigonocephaly, aplasia cutis on the scalp, microphthalmia, micrognathia, micropenis, empty scrotum, and polydactyly of hands and feet. The karyotype was compatible with PS (47, XY, +13). The child died at 12 days old. Conclusion: Pregnant women with PS fetuses have a higher risk of developing preeclampsia, considered one of the main causes of maternal death during pregnancy. This may have important implications for the management and prognosis of these patients. 111333 Modality: E-Poster Scientific Initiation – Case Report Category: HYPERTENSION/RENAL DENERVATION IUGO ALVES DE SOUSA1, Filipe Batista de Brito3, Gabriela Menezes Gonçalves de Brito2, Matheus Araújo de Medeiros1, Renner Cassio Nunes de Lucena1 (1) Universidade Federal do Rio Grande do Norte; (2) Universidade Tiradentes-SE; (3) Hospital de Cirurgia-SE Introduction: Adenomas are among the causes of increased aldosterone production by the adrenal gland. The prevalence in hypertensive patients varies from 3% to 22%, being higher in hypertensive patients who are difficult to control. In general, patients have stage 2 or 3 hypertension, which may be refractory to treatment. Currently, it is known that the prevalence of hypokalemia in primary hyperaldosteronism varies from 9% to 37% of cases. In the diagnosis of this condition, the differentiation between hyperplasia and adenoma is essential for proper treatment. From a clinical and laboratory point of view, patients with adenoma are, in general, younger, have more severe hypokalemia and higher aldosterone concentrations (> 25 ng/dl). Case Report: Female, 32 years old, asymptomatic, with grade III arterial hypertension despite the use of three antihypertensive drugs of different classes. When performing abdominal ultrasound for ovarian evaluation, a random adenoma was observed in the left adrenal measuring approximately 3.5 × 2.9 × 2.5 cm with a volume of 19.2 cm², which was later confirmed by on total abdominal tomography. Aldosterone and renin levels were then requested and the results showed an aldosterone overproduction (37.2 ng/dL) not accompanied by a renin suppression (4.7 uIU/mL). A potassium dosage was also requested, which allowed the identification of hypokalemia (2.2 mEq/L). After confirmation, the patient followed for evaluation of cardiovascular alterations. Echocardiography showed eccentric left ventricular hypertrophy with mild mitral regurgitation, which suggests a functional impairment of the heart. Then, the patient was referred for unilateral adrenalectomy. Conclusion: The case in question presents the diagnosis of a unilateral aldosterone-producing adenoma, in a young woman with a history of resistant arterial hypertension, accidentally identified in an imaging exam. The report aims to alert professionals about the need to screen this cause of secondary hypertension in all hypertensive patients with spontaneous or diuretic-induced hypokalemia and in hypertensive patients resistant to usual treatments, so that the disease is diagnosed early and treated according to guidelines. 111441 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIAC ARRHYTHMIAS/ELECTROPHYSIOLOGY/ELECTROCARDIOGRAPHY ANTONIO MAYCON DA SILVA SOUSA1, Carlos Eduardo Batista de Lima2, Patryck Araujo Dantas da Silva1, Marcelo Madeira Pinheiro Silva2 (1) Universidade Federal do Piauí (UFPI); (2) Hospital Universitário da Universidade Federal do Piauí – (HU-UFPI). Introduction: Atrial fibrillation (AF) is the most frequent arrhythmia in clinical practice with a higher occurrence in the elderly and rarely occurs in pediatric patients with a structurally normal heart. Bayes syndrome has been described as the occurrence of AF in the elderly associated with electrocardiogram (ECG) findings of interatrial block by the Bachmann bundle, and there are no reports of cases in pediatrics so far. The reduced atrial myocardium mass in children makes the occurrence of AF difficult, reinforcing the mechanism triggered by the interatrial delay. Case Report: S.R.C., male, 5 months old, in December 2018, admitted to a public hospital in Teresina-PI due to respiratory distress reported by the mother, which had been frequent since birth. Born at term, with no pathological antecedents. On physical examination, he had a 2+/4+ systolic murmur in the mitral area, irregular heart rhythm, high heart rate (HR), normal vesicular murmur, without adventitious sounds or signs of pulmonary edema. On admission, an echocardiogram (ECHO) was performed, which showed dilated left chambers, left ventricular ejection fraction of 55% and mild mitral and tricuspid regurgitation. The admission ECG showed AF with HR around 180 bpm and episodes characteristic of Ashman’s phenomenon. After reversal, in sinus rhythm, morphological alterations of the p wave characteristic of interatrial delay by Bachmann’s bundle were evidenced, a fixed alteration associated with a greater predisposition to the occurrence of AF. The patient used antiarrhythmic medications, with complete normalization of the ECHO and maintaining long-term sinus rhythm on an outpatient basis. Final Considerations: We report a rare case of Bayes syndrome in an infant with AF and mild tachycardiomyopathy associated with an electrocardiographic alteration characteristic of a fixed conduction disturbance in the Bachmann fascicle due to interatrial delay. The presence of Bachmann’s bundle block on the electrocardiogram justifies the pathophysiological mechanism of the arrhythmia as a probable etiology, as it is a known risk factor for AF and no other clinical alteration was evidenced that would justify the occurrence of arrhythmia in this patient. 111450 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY PEDRO HENRIQUE TORRES TIETZ1, Vitor Agne Magnus1, Marco Antônio Vinciprova Dall‘Agnese1, Gabriel de Paula Alves1, Jorge Alberto Bianchi Telles2 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA); (2) Hospital Materno Infantil Presidente Vargas (HMIPV) Introduction: A twin pregnancy with an acardiac fetus is a rare event, with a low number of cases in the literature describing conservative treatments and parameters to be assessed. Case Description: 29 years old female with a 14 week pregnancy presented to fetal medicine service due to an ultrasound evidencing a monochorionic and diamniotic twin pregnancy. One of the fetuses had a malformation in the superior part of the body, suggestive of an acardiac fetus. The patient was submitted to fetal ultrasound which confirmed this diagnosis of twin reversed arterial perfusion (TRAP). The patient was assessed for possible laser ablation of the umbilical cord. There weren‘t any signs of hemodynamic decompensation on the pump twin and a blood flow almost restricted to the umbilical cord on the acardiac fetus, without surgical indication at that moment. The 22 week ultrasound showed no morphologic alterations on the pump twin, which weighed 560 g, and a dysmorphic mass of 160 g representing the acardiac fetus. There was evidence of vascularization in its interior shown by a doppler ultrasound. The doppler echocardiogram and a magnetic resonance imaging were also normal for the pump fetus. The acardiac fetus still presented to be a misshapen mass with significantly peripheral edema. Ultrasounds were regularly made as of the 26 weeks of gestation, aiming at the early diagnosis of any sign of cardiac decompensation of the pump fetus. The evaluated parameters consisted on the relation between the weight of the acardiac and pump fetuses, cardiothoracic ratio, visualization of tricuspid regurgitation, presence of polyhydramnios and doppler ultrasound of the venous duct in the pump fetus. These parameters stayed normal and it was decided to hospitalize the patient with 30 weeks of gestation for an ultrasound follow-up and for daily cardiotocography (CTG). The CTGs were normal. The pump fetus, a girl, was born with 33 weeks of pregnancy and weighing 2.125 g, presenting an Apgar score of 8/9. The acardiac fetus weighed 1.090 g, being a misshapen mass, with more developed inferior limbs. The pump twin presented a respiratory failure. However, she progressed without major complications, being discharged 14 days after birth. Conclusion: Although there is not a well defined pattern established for the management of twin pregnancy with an acardiac fetus, some factors like early diagnosis and hemodynamic assessment can help future cases to end successfully. 111476 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY GABRIEL SILVA DE CARVALHO PONCIANO1, Amanda Metsa da Silva Cardoso1, Henrique Lobo Saraiva Barros2, Rafaela Marrocos Bezerra2, Sônia Maria Cavalcante da Rocha2 (1) Universidade do Estado do Rio Grande do Norte – UERN; (2) Policlínica dr. Luiz Carlos Fontenele – SPDM Introduction: Brugada Syndrome (BrS) is a genetic disease of low worldwide prevalence and male preponderance, which can lead to severe arrhythmias. The diagnosis includes an electrocardiogram (EKG) with the presence of spontaneous or drug-induced ST elevation, followed by a symmetrical negative T wave in the right precordial leads and clinical features. Brugada phenocopies present an EKG pattern equivalent to BrS, but may arise from a previus ventricular repolarization alteration or associated with factors like hydroelectrolytic disorders. Such changes might generate a transmural myocardial gradient or delay the conduction around the right ventricular outflow tract, inducing the EKG pattern. This diagnosis includes EKG pattern, low pretest probability for the syndrome, drug and genetic testing. Case Description: A 6 years old male child with psychomotor agitation was sent to cardiological evaluation in order to use methylfnidate. There were no heart complaints and a negative family history of sudden death or arrhythmic syncope. Short PR interval and delta wave were seen in all EKG leads. A 24H Holter monitoring was requested, which showed a short PR and delta wave throughout the tracing and no documented arrhythmias. Ventricular pre-excitation became the main diagnostic hypothesis. It was indicated a careful follow-up for the child. In early 2022, during a routine appointment, the child showed no heart complaints, but he was in a recovery from gastroenteritis. The EKG showed a pattern of type 1 BrS. The EKG was repeated after 2 days with no more BrS pattern. The possibility of BrS was ruled out due to the absence of clinical features, normalization of the EKG and negative family for BrS. For that ocasion the diagnosis of phenocopies seemed more likely to be suitable. Conclusion: Considering phenocopies as a differential diagnosis of Brugada syndrome is important. A complete investigation of the patient is necessary for the diagnostic suspicion, considering the possible related factors for the condition. A careful follow-up along with a complete explanation of this entity is the best way of deal with this case. 111594 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT STHEPHANY YAMAGUCHI DE MELO1, Sthephany Yamaguchi de Melo1, Isabela Souza Cruvinel Borges1, Danielle Campos de Almeida1, João Lucas O‘Connell1 (1) Universidade Federal de Uberlândia – UFU Introduction: Takotsubo cardiomyopathy (or broken heart syndrome) is characterized by transient left ventricular systolic and diastolic deficits, mimicking acute coronary syndrome (ACS). It is more common in women (9:1), elderly, with a history of psychiatric disorder and physical or emotional stress. Classically, the ventricular abnormality is analogous to the Japanese pot used to fish for octopus, which resembles the apical ballooning of the typical form of the disease. The description of this case highlights the possibility of other regional abnormalities (less common) that have also been described in the literature: the one that evolves with midventricular akinesia, with severe hypokinesia restricted to the middle ventricle. Case Description: Female patient, 54 years old, hypertensive, smoker and anxious. She presented with syncope at home after emotional stress and being taken to the emergency room. Electrocardiogram: discrete infra-ST on the anterior wall. Elevation of myocardial necrosis markers. Coronary angiography: absence of obstructive coronary lesions; Significant hypokinesia of the middle segments with moderate global systolic dysfunction suggestive of Takotsubo cardiomyopathy of the midventricular form. He evolved well and was discharged with optimized clinical treatment for ventricular dysfunction. She is asymptomatic and with full recovery of contractility and ventricular function one year after the initial condition. Conclusion: Although most cases are benign, Takotsubo heart disease is a potentially fatal disease, with a severe and irreversible course. Diagnostic criteria aim to identify acute left ventricular dysfunction and treatment is directed towards heart failure that sets in. Despite not being a diagnostic criterion, cardiac catheterization is commonly performed to rule out ACS. When performed, the absence of coronary artery disease is expected. Echocardiography and myocardial resonance assist in the diagnosis and identification of ventricular abnormalities that are sudden and without a defined time for reversibility. The mid-ventricular form represents a minority of cases (15%). Its benign character has been questioned in recent reviews due to the number of underestimated cases that may evolve unfavorably without a well-established prognosis. 111597 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY GUSTAVO LUIZ MONTENEGRO DA COSTA1, Dany David Kruczan2, Lilian Soares da Costa2, Edgard Freitas Quintella2, Márcio José Montenegro da Costa2 (1) Faculdade Técnico-Educacional Souza Marques; (2) Instituto Estadual de Cardiologia Aloysio de Castro Introduction: Valvular heart disease is an important contributor to morbimortality worldwide. This case report proposes to switch a redo operation on a valvular aortic regurgitation, in a 60-year-old man post David procedure for a transcatheter aortic valve replacement (TAVR-in-David). Case Description: A 60-year-old man, after almost a 10-year outpatient follow-up due to a prior David procedure, presents onset of dyspnea with minimal exertion and chest pain in July 2021. Until March 2022, this patient was managed even optimal medical treatment has kept non-responsiveness and then was admitted in our hospital. The first choice of treatment during this admission was the conventional surgery for aortic valve replacement, but the surgical team denied the procedure due to the high risk of complications. Then we have implanted a balloon expandable valve with no leak and good final result. TAVR should be contemplate in the role of procedures as a final solution for severe aortic regurgitation in case of high surgical risk or inoperable patients, especially in centers of excellence. Conclusion: The decision to treat a patient with a TAVR-in-David procedure is off label but we believe that with permission of the patient and, a multidisciplinary approach to get the best decision must be a rule inside Cardiology. In conclusion, we need a series of cases and a follow-up to validate this new proposal to switch a redo David procedure for a V-i-David transcatheter. 111600 Modality: E-Poster Scientific Initiation – Case Report Category: ACUTE AND CHRONIC CORONARY DISEASE/THROMBOLYSIS RODRIGO LACERDA GERVOU1, Daniel Luiz Messias Pereira1, Miguel Ângelo Ribeiro2, Natália Dominguez Paes Leme2, Nathalia Duarte Camisão2 (1) Universidade Federal do Estado do Rio de Janeiro UNIRIO; (2) Hospital Norte D’or Introduction: Spontaneous coronary artery dissection (SCAD) is a rare cause of acute coronary syndrome (ACS), myocardial infarction and sudden death, but becomes more relevant in younger women, with multiple pregnancies, especially in the puerperium phase, which consists of 2–18% of all cases of SCAD. It has few association with atherosclerotical risk factors but emotional stress preceded in 50% of the cases, SCAD consists in a non-traumatic, non-iatrogenic separation of the coronary arterial wall resulting in an ischemic event and myocardium injury, that may represent up to 1–4% of total ACS events. Case Report: A 39 years-old female patient arrives in the Emergency Room, reporting typical chest pain, started after the news that her newborn child had been transferred to the neonatal UCI, she referred of a similar pain one week ago, but with spontaneous cure. She has a history of arterial systemic hypertension but no other risk factors for atherosclerotic disease. An Electrocardiogram showed repolarization alteration in the Inferior wall and a small elevation of the ST segment in V2–V4. Echocardiogram stated a moderate dysfunction of Left Ventricular function (LVEF) and akinesis of apical segments. The patient was forwarded to coronary angiography that revealed SCAD of the left anterior descending artery, after diagonals branch. Due to the etiology of the coronary disease, opted for optical medical treatment, and no angioplasty was performed. Magnetic resonance Imaging showed poor LVEF of 40%, 11% infarcted area with disproportion between the ventricular dysfunction and the size of the ischemic area, suggesting a large stunned area that could become functional again. As the literature states, because of the risks of coronary angioplasty, especially without arterial oclusion, patient was discharged from hospital with optical medical treatment for heart failure. Conclusions: SCAD is not a common cause of ACS, but is associated with great mortality and morbidity. It is an important etiology and deserves our attention so we can have it in mind, identify and to have the best approach to this pathology. 111608 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY FABIO ANTONIO SERRA DE LIMA JUNIOR1, André Loureiro Fernandes1, Isaac Newton Guimarães de Andrade1, Petrúcio Abrantes Sarmento1, André Telis de Vilela Araújo1 (1) Universidade Federal da Paraíba Introduction: Tuberculous lymphadenitis is a rare cause of superior vena cava syndrome (SVCS). All of literature cases report a conservative or an endovascular approach to treat these cases. Case Description: We aim to report a 42 years old female case who was allergic to two components of both standard and alternative tuberculosis treatment, and required a surgical approach. However, the same patient had a large and excessively calcified tuberculous node exponentially decreasing the preview success rate of a possible endovascular approach, due to the risk of drift. On the case, the surgical team chose to execute a spiral saphenous vein graft, a method previously reported to treat SVCS, but never on association to tuberculous lymphadenitis. Conclusion: When treating superior vena cava syndrome, etiologies that induce intense vein fibrosis, such as mediastinal tuberculosis, may be unsuitable for endovascular treatment. In these cases, open surgical bypass using a spiral venous graft is an achievable and effective option for symptoms relief, with satisfactory long-term patency. 111758 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY MONIQUE DE SOUSA LOPES1, Débora dos Santos Silva1, Bruno Gonçalves Machado1, Aline Viana Alves1, Ewerton de Souza Abreu2 (1) Centro Universitário Faminas Muriaé (FAMINAS); (2) Hospital Prontocor de Muriaé (PRONTOCOR) The main indication for chronic total occlusion (CTO) percutaneous coronary intervention (PCI) is to improve anginal symptoms, being considerable when there is resistant angina or ischemia compatible with the territory of the occluded artery, however, there is no routine evidence of this approach for correction of ischemic mitral regurgitation (IMR). This study analyzed a 51-year-old patient, without previous comorbidities, with stage B Congestive Heart Failure. Transesophageal Echocardiogram showed severe Mitral Insufficiency, regurgitant jet and incomplete coaptation due to wide prolapse of the posterior leaflet, raising the hypothesis of IMR due to dysfunction of the posteromedial papillary muscle. Invasive coronary stratification revealed CTO in the middle third of the right coronary artery (RCA). Due to the persistence of symptoms despite optimized clinical treatment, a percutaneous approach to the RCA CTO was decided. With failure of an antegrade angioplasty, a retrograde approach was performed using the dissection and reentry technique, which was successful with positive results, including improvement of heart failure symptoms and immediate papillary dysfunction, confirming the diagnostic hypothesis. At ten-month follow-up after the procedure, the patient remains asymptomatic, with fully preserved physical and functional capacities and mild mitral regurgitation. Therefore, despite the technical limitations associated with the procedure and the need for further quality studies to better define the indications, specifically regarding the IMR, it is suggested that the approach for such purpose, when well indicated, may be a promising possibility within the interventional cardiology, impacting on the increase of survival and expanding the prospects of treatment. 111779 Modality: E-Poster Scientific Initiation – Case Report Category: HEMODYNAMICS AND INTERVENTIONAL CARDIOLOGY YURY PIFANO VARELA1, Marina Coelho Feitosa1, Caio Pessoa Cruz2, Isabelly Crysthynne Moreira da Luz3, Polianna Lemos Moura Moreira Albuquerque4 (1) Acadêmico do Curso de Medicina da Universidade de Fortaleza – UNIFOR; (2) Acadêmico do Curso de Medicina da Universidade Estadual do Ceará; (3) Acadêmico do Curso de Farmácia da Universidade Federal do Ceará; (4) Médica Nefrologista do Instituto Dr. José Frota e Coordenadora do Centro de Informação e Assistência Toxicológica (CIATox/IJF). Doutora em Ciências Médicas pela Universidade Federal do Ceará – UFC Introduction: Intoxication by selective α–1 and β–1 adrenorreceptor blockers may cause serious repercussions in the cardiovascular system, that can progress to Cardiogenic Shock, which can lead to death in 80% to 90% of patients. There are successful reports using 20% lipid emulsion (20% ILE) as treatment after cardiovascular collapse caused by lipophilic agents such as tricyclic antidepressants, propranolol, verapamil, bupropion and barbiturates, although the literature still does not assign a standard protocol. This report case demonstrates the emergency use of 20% ILE in acute toxicity from attempted suicide through ingestion of doxazosin and metoprolol. Case Description: A.H.M.L, 61 years old, male, admitted by a tertiary care hospital, reference in toxicological assistance, in Fortaleza/CE, in critical condition, 8 hours after intentional and unquantified ingestion of metoprolol and doxazosin. At admission, patient on mechanical ventilation, without sedoanalgesia, Glasgow Coma Scale 3, using high doses of vasoactive drugs (noradrenaline 40 ml/h + vasopressin 4 ml/h) (Table 1). With no response to the standard protocols for hemodynamic shock, it was suggested a therapeutic regimen with 20% ILE in 3 doses of 1 ml/kg (60 ml), implemented at intervals of 10 min, resulting in immediate hemodynamic upturn – distressed awakening and improvement in ventilatory parameters (Table 1). After 5 hours, it was necessary to repeat the 20% ILE regimen due to clinical instability. Over the 28 days of hospital stay, the initial high doses of vasoactive drugs were reduced without clinical complications. In addition, renal replacement therapy was performed (two sessions). Conclusion: This is the first successful experience in the state of Ceará for the treatment of Cardiogenic Shock resulting from intoxication by selective α–1 and β–1 adrenorreceptor blockers agents. The study provides evidence on the use of 20% ILE as a relevant therapy in patients after cardiogenic shock due to high ingestion of alpha and beta blockers. 111794 Modality: E-Poster Scientific Initiation – Case Report Category: PERICARDIUM/ENDOCARDIUM/VALVOPATHIES RAIMUNDO BENÍCIO DE VASCONCELOS NETO1, Ana Caroline Leite Guedes1, Rebecca Shaiane Soares Nunes Rivoredo1, Fernanda Gabry Scazuza Gomes de Souza1 (1) Centro Universitário São Lucas-UNISL Introdução: Dengue is a viral infectious process transmitted by Aedes aegypti and most of it presents with a limited high condition, however, the endemic outbreaks of the disease open the way for the emergence of serious cases with complications. Cardiac involvement associated with dengue is a rare and broad-spectrum process, in which pericarditis and pericardial effusion have a lower occurrence. The pathophysiology of such involvement is still uncertain, but it is believed that viral infection in the pericardium can generate the release of inflammatory cytokines that cause dysfunction in the cardiac endothelium, and that promote increased vascular permeability, culminating in fluid leakage to the third space. and generating the process of pericardial effusion, which in most cases does not constitute an accentuated process. This article aims to report a case of a patient with pericarditis and mild pericardial effusion as a complication of dengue. Relato: Female patient, 81 years old, hypertensive, with advanced stage of Alzheimer’s disease, was taken to the emergency room by family members who reported a decline in general condition, unmeasured fever and lowered level of consciousness. On admission, the patient presented Glasgow Coma Scale 14, was febrile, and diffuse rashes associated with significant swelling in the upper and lower limbs were observed. She was mildly dyspneic and hypoxemic, requiring the use of complementary oxygen therapy. In view of the symptoms presented, complementary exams and transthoracic echocardiography (TTE) were requested due to the suspicion of cardiac insufficiency. The hemogram results showed significant thrombocytopenia and leukometry within the normal range, serology was requested for dengue, which showed positive IgG and IgM for the viral infection. The TTE showed hyperrefringence of the pericardium and small effusion, concluding the diagnosis of viral pericarditis. The patient remained in the intensive care department under infectious, neurological and hemodynamic surveillance, with subsequent clinical improvement. Conclusão: The present report points to the unusual association between pericarditis and dengue, which demonstrates the importance of a thorough clinical management and an accurate look at the individual, since the signs can vary from mild to severe, including pericardial effusion. If treated satisfactorily, they potentially increase the chances of restoring the patient to health. 111800 Modality: E-Poster Scientific Initiation – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM MARIANA FURTADO DE SÁ1, Vanessa Ortega1, Vinicius Eiji Kameoka2, Bianca Altrão Ratti Paglia1 (1) Universidade Cesumar – Unicesumar; (2) Hospital Paraná Introduction: SARS-COV2 has been shown to be a virus that affects multiple organs, including the heart. This case report shows a severe cardiologic complication in a patient post mild COVID-19. Case Report: L.M.B, 37 years old, female, former smoker (10 pack-years) and with no medical conditions, reports mild COVID-19 two months before an episode of intense retrosternal burning associated with diaphoresis. Initial electrocardiogram (ECG) without ischemic alterations, however, due to the recurrence of the pain, she returned to the emergency room after two days, in which diffuse ST elevation was evidenced in new ECG, associated with pleuritic chest pain. The patient was referred for coronary angiography (CA) and later cardiac magnetic resonance imaging (CMR). CA: Absence of atherosclerotic process. Distal occlusion of the left anterior descending. Residual thrombus in marginal branch. CMR: Apical transmural infarct. Intracavitary thrombus in apex. Acute Pericarditis. Conclusion: Inflammation and hypercoagulability generated by COVID-19, increase the chances of cardiovascular events, such as the myocardial infarction evidenced in the case, even in the absence of significant risk factors. Therefore, the importance of studies is emphasized, to identify groups that will benefit from prophylactic therapies, avoiding similar outcomes. 111825 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES JOYCE FERNANDES COSTA1, Thaíssa Santos Monteiro2, Fabio Akio Nishijuka2, Maria Carolina Terra Cola2 (1) Universidade Estácio de Sá – UNESA; (2) Instituto Nacional de Cardiologia – INC Introduction: Cardiac tumors are rare, the majority being benign and atrial myxoma (AM) is the most common type. Most are symptomatic and may present Goodwin’s triad, with intracardiac obstruction, embolic manifestations and constitutional symptoms. The diagnostic method is echocardiography (ECO), assessing size and location of the tumor. Surgical resection is recommended. Anticoagulants may be indicated if embolic signs are present, although evidence points not to be an alternative to surgery, as half develop cerebral ischemia despite the anticoagulation. Local recurrence is uncommon and annual ECO follow-up is recommended for 4 years after tumor resection. Case Report: Female, 35 year-old, hypertensive, smoker, obese grade I and family history of coronary artery disease. Admitted with severe chest pain, blood pressure of 220 × 100 mmHg, right bundle branch conduction disorder and positive myocardial necrosis markers, diagnosed with acute myocardial infarction type II. Recent history of transient focal deficit in the left upper limb. Presented cutaneous lesions resembling Janeway lesions in hands and feet, but tender. ECO observed an heterogeneous and irregular mass in the left atrium adhered to the atrial septum, measuring 5.2 × 3.6 cm, projecting to the mitral valve orifice, without significant obstruction, suggestive of AM. The patient was then transferred to our institution, and the screening for COVID-19 was positive, but with normal chest tomography (CT) and no related symptoms. Coronary angiotomography performed later showed no coronary obstructions, and head CT showed no signs of ischemia. Full anticoagulation was started due to the possible cardioembolic nature although there were no CT evidence for ischemia, while waiting for surgery. She underwent resection of the mass with atrial septoplasty. Postoperative ECO showed no shunt through the atrial septal and absence of intracavitary mass. There were no surgical complications on follow-up. Conclusion: AM manifests itself in different ways and with imminent risks. Immediate surgical intervention is important, due to the risk of embolism, and in cases of previous embolic events, anticoagulation may also be considered, due to the risk of associated thrombi with the tumor. 111867 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY HALAN VITOR CORREIA EVANGELISTA VIEIRA1, Catarina Vasconcelos Cavalcanti1, Paulo Ernando Ferraz Cavalcanti1, Ricardo Felipe de Albuquerque Lins1, Monica Cristina Rezende Fiore1 (1) Pronto Socorro Cardiológico de Pernambuco (PROCAPE); (2) Universidade de Pernambuco (UPE); (3) Faculdade de Ciências Médicas da Universidade de Pernambuco (FCM-UPE) Introduction: Correlation between clinical findings and study with transthoracic echocardiography and angiotomography allow diagnosis of rare congenital heart disease in adult patient. Case Description: A 42-year-old female patient with a long-standing history of cyanosis and abdominal discomfort, presenting worsening of symptoms in the last months was admitted at our center. The physical examination revealed cyanosis and digital clubbing with outstanding difference between upper and lower limbs, since the right hand was quite normal. Placing oximeter probe on her fingers and toes when breathing room air, differential peripheral oxygen saturation (SpO2) preserving the right hand was noted (Right fingers 99%, left fingers 94% and toes 88%). A loud second cardiac sound with fixed splitting was heard, as well as a systolic murmur in the left sternal border. Two-dimensional transthoracic echocardiography revealed a ventricular septal defect (12.1 mm) and the color Doppler showed bidirectional flow across the ventricular septal defect (VSD). The estimated systolic pulmonary artery pressure was 105 mmHg. An angiotomography was performed due to difficulties in the analysis of the aortic arch, revealing a type B aortic arch interruption associated with VSD and a patent ductus arteriosus as well as signs of severe pulmonary artery hypertension. Severe kyphoscoliosis for her age as well as incidental finding of ectopic kidney in the pelvis were also revealed. Deoxygenated blood from right ventricle flows into the descending aorta and left subclavian artery through the patent ductus arteriosus preserving the right hand from the long-term effects of deoxygenated blood. A hypothesis of VACTERL association was also formulated in view of the demonstrated features: V, vertebral anomaly by kyphoscoliosis; C, cardiac anomaly due to type B aortic arch interruption with interventricular communication; and R, renal anomaly with the left kidney in pelvic location. Conclusion: The clinical and anatomical findings described and the rarity of the pathology in this adult patient (Not surgically treated) make this case report unique. 111876 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR SURGERY REBECCA SHAIANE SOARES NUNES RIVOREDO 1, Raimundo Benício de Vasconcelos Neto1, Ana Caroline Leite Guedes1, Fernanda Gabry Scazuza Gomes de Souza1 (1) Centro Universitário São Lucas/Afya UNISL Coronary fistulas are rare congenital anomalies that constitute connections between the coronary artery and the heart chambers (coronary-cameral fistula) or with the venous system of the myocardium (coronary arteriovenous fistula). This fact is due to the malformation of the individual’s cardiac vascular system, which presents continuity of the embryonic sinusoids of the myocardium, which are responsible for the formation of the perforating arteries and the right and left coronary trunks, and should close during the gestational period. Coronary arteriovenous fistulas are one of the rarest cases of this pathology, usually causing symptoms only after the 4th decade of life, which course with heart failure, angina, arrhythmias or endocarditis. The intervention of the pathological condition is established according to the patient’s condition, consisting of factors such as cardiovascular symptoms, fistula drainage volume and myocardial ischemia. This is a case report of a patient with an arteriovenous fistula in the right and circumflex coronary artery leading to the coronary sinus. Report: Female patient, 47 years old, hypertensive, diabetic and dyslipidemic. She sought medical care due to exertion angina, which started 3 months ago, of a progressive nature, having intensified in the week preceding hospitalization, which motivated her to seek care. She was received with type A chest pain, at rest, with an electrocardiogram showing anteroseptal subendocardial ischemia. At no time did she present positive myocardial necrosis markers, and she was treated as high-risk unstable angina and underwent coronary angiography that showed coronary arteriovenous fistulas (CAF) in the paths from the right coronary artery to the coronary vein and from the circumflex artery to the coronary vein. After diagnosis, in-hospital clinical treatment was optimized and the patient was referred to the cardiovascular surgery service for fistulization correction. Conclusion: The aforementioned case brings to light a discussion about coronary arteriovenous fistula associated with anginal cause, which, although rare, when detected early and managed clinically and surgically in an adequate and satisfactory manner, evidences the relief of symptoms and improvement in the patient’s quality of life. 111929 Modality: E-Poster Scientific Initiation – Case Report Category: NEGLECTED CARDIOVASCULAR DISEASES PEDRO LUCAS CARDOZO BARROS1, Ana Flávia Silveira de Souza1, Joana Raquel Cardoso dos Santos1, Hellen Dutra Passos2, José Augusto Soares Barreto Filho1 (1) Hospital Universitário da Universidade Federal de Sergipe (HU-UFS); (2) Hospital São Lucas, Rede D’or São Luiz (HSL) Takayasu’s arteritis (TA) is a chronic inflammatory disease of unknown etiology. It is a rare vasculitis involving the aorta and its main branches in which transmural granulomatous inflammation causes multiple arterial lesions. The present report is about a 32-year-old female which physical exam exposed asymmetric radial pulses and significant pressure difference between upper limbs. She was subjected to a tomography of thoracic and abdominal aorta for the investigation of an abdominal murmur and possible secondary arterial hypertension. Angiography then revealed diffuse arterial injuries, including dissection of right renal artery, stenosis of left renal artery and at infrarenal portion of abdominal aorta and fusiform aneurysm upstream. Outpatient follow-up almost 6 years after the diagnosis of TA reveals a completely asymptomatic individual without prior hospitalization or any other intervention besides conservative treatment. The woman has a history of two pregnancies among one abortion, practices regular physical activity and maintains all work performance. Her blood pressure is controlled; thoracic radiography, electrocardiogram and laboratory exams are all normal; and both transthoracic echocardiogram and Holter show discrete and unspecific cardiac alterations. Studies have demonstrated that few patients with symptomatic TA require surgery. The procedures indicated for occlusive disease are endovascular stent and arterial bypass; the aneurysmal form of TA may require arterial reconstruction by open surgery or endovascular approach. However, current literature lacks significant evidence to indicate most invasive treatments for asymptomatic individuals. Our patient’s clinical presentation is stable, thus not requiring invasive procedures despite of diffuse arterial injuries. 111939 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT GABRIELLA GOMES FERNANDES MAIA1, Rafael Souza Conceição da Silva2, Claudia Regina de Oliveira Cantanheda1, Dany David Kruczan1, Marcio Montenegro1 (1) Instituto Estadual de Cardiologia Aloysio de Castro – IECAC; (2) Unigranrio Barra da Tijuca Marfan syndrome is an autosomal dominant multisystem connective tissue disorder caused by mutations in fibrillin. HCM is a inherited disease caused by mutations in genes encoding sarcomeric proteins in the heart cells. The estimated incidence rate for Marfan syndrome is 1:5000, while for HCM is 1:500. Both association has no incidence, which explains the motivation of this case report. A 39-year-old female patient, reporting dyspnea on exertion for 2 years and palpitations for 18 months, presents with HTN and a murmur, which progressed to dyspnea on moderate exertion, diaphoresis and lipothymia. After 6 months, she presented a dry cough and chest discomfort such as tingling, radiating to the left arm and jaw. She uses antihypertensive drugs. History of descending aortic endoprosthesis in 2016. On physical examination, normal HR and BP. Abnormal kyphoscoliosis. Ogival palate and Steinberg’s sign. Regular and wide pulses. Palpable Ictus without deviation. On auscultation, mitral focus with hypophonetic sounds, holosystolic murmur and S3. In the supine position, the systolic murmur increases with expiration. In left lateral decubitus, it increases after inspiration. Mesocardium with hypophonetic sounds, systolic murmur and gallop. Accessory aortic focus with systolic murmur radiating to clavicles, carotids, and suprasternal notch, and an diastolic murmur. Pulmonary focus with a rough protosystolic murmur, well audible diastolic murmur and split S2. The X-ray shows slight cardiac enlargement and aortic calcification. ECG shows sinus rhythm, cardiac axis at –30° and alteration in ventricular repolarization. ECHO shows increased of LA and RA, asymmetric LV hypertrophy with reduced contractility but preserved global systolic function; MR with intense anterior systolic motion, AR with failure of central coaptation; big dilation of the ascending aorta plus descending aortic dissection. Diagnosed with Marfan syndrome, severe AR and MR, dilated ascending aorta and HCM. Surgical correction with Myectomy, placement of Aortic Valve Tube and mitral valve replacement was indicated. However, due to technical problems, the responsible institution chose to perform only the aortic valve replacement with a biological prosthesis. Currently, the patient remains symptomatic and Percutaneous Septal Alcoholization is indicated, due to a safer and more effective conclusion for the case. It was preferred a less invasive technique, since the main outcome is to provide quality life. 111982 Modality: E-Poster Scientific Initiation – Case Report Category: COVID-19 AND CARDIOVASCULAR SYSTEM GABRIELA ZIMMERMANN1, Melissa Dorneles de Carvalho1, Thamara Andressa Fagundes1, Fernando Caritas de Souza1, Giolana Mascarenhas da Cunha1 (1) Universidade Estadual do Oeste do Paraná (UNIOESTE) Pediatric presentation of coronavirus disease 2019 (Covid-19) rarely includes severe complications in acute phase; however, characteristics of the condition among children are not yet fully established. We report a case of a scholar who presented developed acute pericarditis, tested positive for Covid-19, progressed to myopericarditis and had completely recovery after the therapeutic management. Male, 11 years old, previously healthy, referred chest pain, for over one hour, no radiating, that started suddenly in prone position and presented spontaneous remission. Fourteen hours later he felt the same symptoms, so he was taken to the emergency service. An electrocardiogram (EKG) showed alterations that could suggest infarction, nonetheless, considering the patient’s symptoms and age, the heart attack was rejected, and pericarditis was the most likely hypothesis. The patient denied having fever or dyspnea, but a flu episode about twenty-five days earlier. Physical exam had no irregular findings. Laboratory evaluation included leukocytes 12.820/mm3, erythrocyte sedimentation rate 20 mm/h, troponin I 19.572 ng/mL, pro B-type natriuretic peptide 790 pg/mL, C-reactive protein 5,9 mg/L, lactate dehydrogenase 335 mg/dL, D-dimer 222 ng/mL and COVID-19 serology results: IgG positive and IgM negative. With the orientation to make EKG’s series, another exam was performed (Figure 1), which presented ST-segment elevation in D2, D3, avF and precordial leads. Echocardiogram was conclusive with pericarditis. The treatment included nonsteroidal anti-inflammatory drugs (ibuprofen and high-dose aspirin) for two weeks. The hospital length of stay was 4 days, the scholar turned asymptomatic, and EKG was normal. The patient was not vaccinated against SARS-Cov-2. This is a case of pericarditis after SARS-Cov-2 infection that might be a useful reference to other clinicians caring for pediatric patients affected by COVID-19 and contribute to the differential diagnosis of chest pain. Despite the rapidly growing research on management of COVID-19 and its complications, there are many unanswered questions and areas to explore. 111984 Modality: E-Poster Scientific Initiation – Case Report Category: CARDIOVASCULAR IMAGING ALICE ZANETTI DUSSIN1, Alice Einsfeld Britz1, Juliana Menezes Zacher1, Bárbara Dewes Silva1, João Carlos Vieira da Costa Guaragna1 (1) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) Background: Transthyretin amyloidosis (ATTR) is the main cause of cardiac amyloidosis and it is presented by clinical manifestations such as heart failure, diastolic dysfunction, left ventricular hypertrophy, atrial fibrillation (AF) and aortic stenosis. Technetium-pyrophosphate (Tc-99m-PYP) scintigraphy has a fundamental diagnostic role in differentiating types of amyloidosis, being able to detect the presence of transthyretin deposits. Case Report: A 85-year-old diabetic female, with a history of multiple hospitalizations due to decompensated heart failure and chronic obstructive pulmonary disease since 2009. Patient brought a 2009 transthoracic echocardiogram (TTE) – ejection fraction (EF) of 74%, all cavities had normal dimensions – and two myocardial scintigraphy from the same period with low probability of ischemia. Five years ago, was presented with acute chest pain and underwent coronary angiography that showed coronary arteries without obstructions. Soon after, she started having syncopal episodes. Three years ago, started experiencing dyspnea on moderate efforts, and was diagnosed with AF; cardioversion was successfully performed. Thirty days later, she once again presented AF with worsening dyspnea, lower limb edema, jugular swelling and hepatomegaly. At the time, a chest tomography (cardiomegaly, right pleural effusion and enlargement of the pulmonary trunk) and a TTE (EF of 68%, left ventricle with normal diameters and biatrial overload) were executed. A cardiac magnetic resonance also showed biatrial enlargement. A Tc-99m-PYP scintigraphy was then requested, which diagnosed grade 2–3 ATTR. Conclusion: Cardiac amyloidosis is a rare, progressive and lethal pathology; and its incidence has increased due to advances in cardiovascular imaging. A Tc-99m-PYP scintigraphy, for example, has a sensitivity of 90% and specificity of 97% for the diagnosis of ATTR. Therefore, it is extremely important for the cardiologist to be acquainted with this method, since this can allow an early diagnosis of ATTR without the need for invasive tests, such as biopsy, which is still considered the gold standard for diagnosis. 112065 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY JULIANA ROSSI CATAO1, Guilherme Girardi May1, Eric Schwellberger Barbosa1, Henrique Perez Filik1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) Introduction: Imperfect twinning occurs in 1 in 100 monozygotic twins. Our aim was to report the prenatal findings of a case of conjoined thoracopagus twins. Case Presentation: The patient was a 31-year-old pregnant woman in her fourth pregnancy. She was referred to the fetal medicine service due to an ultrasound at 12 weeks gestation, with a description of twins united in the abdomen. The examination at 14 weeks revealed that the twins were united at the chest and upper abdomen. There was apparent sharing of heart and liver. A single umbilical cord emerged caudally to the union of the fetuses. The emergence of the umbilical arteries was observed laterally to the bladders. The pregnancy was monochorionic and monoamniotic. Echocardiography revealed a normal fetus on the left and a fetus on the right with dextrocardia. Second trimester ultrasound, performed at 22 weeks of pregnancy, showed no morphological changes in the fetuses. Two venous ducts were visualized. The echocardiography performed soon after revealed an apparent communication of the hearts through the right atrium of the fetus located on the right with the left atrium of the fetus located on the left. The fetus on the left had a tetralogy of Fallot with pulmonary atresia. There was a large infundibular perimembranous ventricular septal defect with a large caliber aorta riding the trabecular septum in 50% of its annulus. Magnetic resonance imaging revealed fetuses fused through the thorax and upper abdomen, above the umbilical cord insertion. The right lobe of the liver of fetus one was related to the left lobe of the liver of fetus two. There were two stomachs and two gallbladders, as well as four kidneys. Close to the lower margin of the livers, small bowel loops were observed, which were closely related, making it difficult to characterize whether there was a crossing between them from one fetus to another. Discussion: Although imperfect twinning is a rare condition, estimated at 1 in 75,000 births, its prenatal diagnosis is very important. It makes it possible to assess the fetal fusion site and its complexity to help define management and prognosis. Final Comments: Thus, the evaluation of fetuses with imperfect twinning is usually multidisciplinary, involving mainly radiologists, obstetricians, pediatricians, and pediatric surgeons, intending to start birth planning during pregnancy, including a place that has adequate conditions for receiving and properly handling these babies. 112083 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY HELENA GUEDES DA ROCHA1, Estefany Karenine Rodriguez Casanova1, Caroline Engster da Silva1, Laís Bettoni1, Rafael Fabiano Machado Rosa1 (1) Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA Background: Jacobsen syndrome is a chromosomal abnormality characterized by a deletion involving the long arm of chromosome 11 and is associated with different clinical findings. Objective: To report the case of a patient with Jacobsen syndrome secondary to a translocation of paternal origin, highlighting its relationship with congenital heart diseases. Results/Case Report: The patient, who is the second child of young parents, was born by cesarean delivery weighing 3500 g and with an Apgar score of 10 at the fifth minute. He had a history of surgery for correction of pyloric stenosis at 54 days of life. On physical examination, palpebral ptosis, right inguinal hernia, bilateral hallux valgus, clinodactyly of the 5th finger and syndactyly between the 2nd and 3rd pododactiles were observed. Echocardiography revealed an interventricular communication. The patient also had a history of epistaxis, ecchymosis and coagulation problems. He had a neuropsychomotor developmental delay that affected his school performance. In addition, the neurological evaluation found an attention deficit hyperactivity disorder. Computed tomography showed an epidermoid cyst in the skull. Audiometric evaluation revealed moderate sensorineural hearing loss in both ears. Karyotype showed a deletion of part of chromosome 11 (46, XY, del (11) (q23.1q25) [28]), compatible with the diagnosis of Jacobsen syndrome. The complementary examination of the father revealed the presence of a translocation between chromosomes 6 and 11. Conclusions: Patients with Jacobsen syndrome may present a multisystemic involvement, which demands a multidisciplinary approach. Special attention should be dedicated to congenital heart diseases, since they are one of the main causes of death in these patients. Heart defects are observed in 56% of the cases and usually require drug treatment and/or surgical correction. The interventricular communications, such as the one observed in our patient, are the most common cardiac malformations. All this is of great importance, since hematological alterations are also common, which may corroborate to an increase in morbidity and mortality of the patients. 112102 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT LUANA DIAS XAVIER1, João Paulo Dias Costa1, Julia Souza Diniz1, Emerson De Santana Santos1, Joselina Luzia Menezes Oliveira1 (1) Universidade Federal de Sergipe Introduction: Val142Ile, predominantly found among African descents, is the most common transthyretin (TTR) amyloidosis variant and predominantly develops a cardiomyopathy phenotype. Individuals with two variants in the TTR gene are extremely rare. They tend to manifest symptoms a mean of 10 years earlier than heterozygotes and it can be more severe. We report a Northeast Brazilian family with recurrence of homozygotes for Val142Ile due to consanguineous marriages. Case Report: A 77-year-old male was referred to cardiogenetics evaluation due to progressive heart failure whose symptoms started at the age of 70 and cardiac amyloidosis was suspected. He had asymmetrical septal hypertrophy and arrhythmia. Holter showed sporadic ventricular ectopia and supraventricular ectopia. An echocardiogram revealed restrictive cardiomyopathy and left ventricle hypertrophy. 99mTc-Pyrophosphate scintigraphy was also suggestive of TTR cardiac amyloidosis. His cardiac magnetic resonance imaging (MRI) showed ventricular septal hypertrophy, perfusion deficit in stress, and delayed myocardial enhancement. A molecular exam detected Val142Ile in heterozygosity. His ex-wife (who is also his cousin), a 74-year-old lady who was born from a consanguineous marriage (her parents were first-grade cousins too), is also affected. Cardiac MRI showed asymmetric apical hypertrophy, stress echocardiogram was negative for ischemia, and the genetic test showed she was homozygous for the Val142Ile variant. Their older son had sudden cardiac death at the age of 52. Their younger daughter, a 42-year-old female, has no cardiac involvement yet and she is also homozygous for the Val142Ile variant. All reported patients had bilateral carpal tunnel syndrome and no other neurological manifestation. Conclusion: The frequency of consanguineous marriages in Northeast Brazil is higher than in other regions, with rates as high as 6 to 12% in rural areas. Inbreeding has certainly contributed to the homozygosity in Val142Ile detected in two members of the family. Genetic counseling was provided to the family since early diagnosis may benefit affected members with the available treatments. 112204 Modality: E-Poster Scientific Initiation – Case Report Category: CONGENITAL AND PEDIATRIC CARDIOLOGY FELIPE SALIM HABIB BUHAMARA ALVES NASSER GURJÃO1, Felipe Salim Habib Buhamara Alves Nasser Gurjão1, Bruna de Almeida Freixedelo1, Mateus Sousa Cavalcante1, Leandro Cordeiro Portela1, Dara Medeiros Mendes1, Vicente Lopes Montes Neto1 (1) Universidade Federal do Ceará – Campus Sobral Introduction: Ebstein’s anomaly corresponds to a set of malformations centered around the tricuspid valve. This syndrome corresponds to the most common congenital heart disease of the tricuspid valve. The severity of symptoms depends primarily on age and the degree of insufficiency presented. The clinical spectrum is variable and can range from death in 50% of cases in newborn patients to a silent disease with symptoms appearing only after decades. Case Description: Male patient, 68 years, diagnosed with systemic arterial hypertension, type 2 diabetes and heart failure, since 2017, sought medical care at the Senador Carlos Jereissati Municipal Hospital on 04/18/2022 due to respiratory distress with progressive worsening for two days. He had BP: 70 × 40 mmHg, Capillary blood glucose: 180, RR: 30, HR: 111, T: 35.1°C and oxygen saturation: 85% in oxygenation of 15 L/min. Thus, due to the patient’s serious condition and the lack of stabilization in the hospital of origin, a transfer to the North Regional Hospital was requested, where the patient was admitted in the early hours of the following day. At the time of admission, the patient was tachypneic, diffusely diminished universal vesicular murmurs with diffuse crackles. An electrocardiogram was performed which reported low voltage and atrial fibrillation. The patient was then hospitalized under the action of vasoactive drugs and oxygen therapy for better hemodynamic stabilization and search for the cause of heart failure decompensation. Laboratory tests and a transthoracic echocardiogram were requested. Among these tests, the echocardiogram, performed on 04/21/2022, showed a significant increase in the right chambers, with atrialization of the right ventricle and 31 mm inferior detachment of the tricuspid valve septal cusp of the tricuspid valve, adhered to the contractile right ventricular wall e decreased right ventricle contractile function, which indicates Ebstein Syndrome. Conclusion: Ebstein’s anomaly corresponds to a complex and rare disease that mainly affects newborns with greater severity. Knowing the anatomical changes and cardiac physiology is essential for the correct approach to this condition. Health professionals should be alert to detect symptoms suggestive of heart failure and request tests for confirmation. As it is characterized as an insidious disease, the early diagnosis of this condition corresponds to a point of great importance for the proper management of the patient. 112241 Modality: E-Poster Scientific Initiation – Case Report Category: HEART FAILURE/CARDIOMYOPATHY/TRANSPLANT ANA ROBERTA DE SOUSA ARAUJO1, Beatriz Nunes Deseyvan Rodrigues2, Rayanne Carvalho Vasconcelos de Azevedo2, Ana Roberta de Sousa Araujo1 (1) FIMCA; (2) FIMCA Opening: The peripartum cardiomyopathy (PPCM) is a cardiac insufficiency secondary to systolic dysfunction of the left ventricle that affects women at the end of their pregnancy or even 6 months after giving birth. It has left ventricle ejection fraction (LVEF) < 45%, with a condition of dyspnea, fatigue and lower extremity edema (LEE). The risk factors include being an afro-descendant, multiparity, maternal old age and preeclampsia. Furthermore, it presents high and variable mortality rate, because it can be avoided with an early diagnosis and specific treatment. So, documenting PPCM is important, given that it often gets a late diagnosis. Thus, this study objectifies reporting the case of a woman diagnosed with PPCM 6 months after giving birth. Case Report: 33-year-old woman, brown, married, home secretary, no prior diseases, multiparous, no cardiomyopathies in previous gestations, developed preeclampsia in the fifth gestation, which was treated. Her delivery occurred after a hypertensive peak via cesarean, ceasing the hypertensive disturbances. 6 months of gestation passed, reported tiredness, dyspnea and an edema on the lower limbs, that extended itself to the abdomen. There was a diagnosis of cardiomegaly, and with the condition getting worse, she was hospitalized in an intensive Therapy unit. It was identified a 120/94 mmHg blood pressure, pulmonary noises with sparse hisses, high troponin I in the laboratory tests and hepatomegaly in the total abdomen ultrasound. In the Echocardiogram, it was verified a LVEF of 30, left ventricle dilated, diffuse hypokinesia, sharp systolic dysfunction, right ventricle with moderate increase, significant aortic regurgitation and moderate mitral. In the cardiogram, it was observed sinus tachycardia (FC > 99 bpm), probable left ventricular hypertrophy and long QT interval. Hence, after clinical analyses the patient was diagnosed with PPCM, it was prescribed atorvastatin, carvedilol and enalapril. With a bad prognosis, the patient finds itself admitted to the transplant center. Conclusion: Therefore, PPCM being a hard to diagnose disease, probable to adversely evolve, might be necessary to perform a transplant, as in the discussed case. However, controlling the appearance of signs and specific symptoms, besides doing early diagnostic tests, can effectively avoid complications. So, it’s indispensable to increase the scientific knowledge to improve the prognosis.

PMC10000371

Kai Gai,Yu Ge,Dapeng Liu,He Zhang,Bailin Cong,Shihao Guo,Yizheng Liu,Kai Xing,Xiaolong Qi,Xiangguo Wang,Longfei Xiao,Cheng Long,Yong Guo,Xihui Sheng

Identification of Key Genes Affecting Flavor Formation in Beijing-You Chicken Meat by Transcriptome and Metabolome Analyses

28-02-2023

meat flavor,Beijing-You chicken,transcriptome,metabolome,regulatory network

The flavor of chicken meat is influenced by muscle metabolites and regulatory genes and varies with age. In this study, the metabolomic and transcriptomic data of breast muscle at four developmental stages (days 1, 56, 98, and 120) of Beijing-You chickens (BJYs) were integrated and 310 significantly changed metabolites (SCMs) and 7,225 differentially expressed genes (DEGs) were identified. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SCMs and DEGs were enriched in amino acid, lipid, and inosine monophosphate (IMP) metabolism pathways. Furthermore, genes highly associated with flavor amino acids, lipids, and IMP were identified by a weighted gene co-expression network analysis (WGCNA), including cystathionine β-synthase (CBS), glycine amidinotransferase (GATM), glutamate decarboxylase 2 (GAD2), patatin-like phospholipasedomain containing 6 (PNPLA6), low-specificity L-threonine aldolase (ItaE), and adenylate monophosphate deaminase 1 (AMPD1) genes. A regulatory network related to the accumulation of key flavor components was constructed. In conclusion, this study provides new perspectives regarding the regulatory mechanisms of flavor metabolites in chicken meat during development.

Identification of Key Genes Affecting Flavor Formation in Beijing-You Chicken Meat by Transcriptome and Metabolome Analyses The flavor of chicken meat is influenced by muscle metabolites and regulatory genes and varies with age. In this study, the metabolomic and transcriptomic data of breast muscle at four developmental stages (days 1, 56, 98, and 120) of Beijing-You chickens (BJYs) were integrated and 310 significantly changed metabolites (SCMs) and 7,225 differentially expressed genes (DEGs) were identified. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SCMs and DEGs were enriched in amino acid, lipid, and inosine monophosphate (IMP) metabolism pathways. Furthermore, genes highly associated with flavor amino acids, lipids, and IMP were identified by a weighted gene co-expression network analysis (WGCNA), including cystathionine β-synthase (CBS), glycine amidinotransferase (GATM), glutamate decarboxylase 2 (GAD2), patatin-like phospholipasedomain containing 6 (PNPLA6), low-specificity L-threonine aldolase (ItaE), and adenylate monophosphate deaminase 1 (AMPD1) genes. A regulatory network related to the accumulation of key flavor components was constructed. In conclusion, this study provides new perspectives regarding the regulatory mechanisms of flavor metabolites in chicken meat during development. The growth efficiency of chickens has increased rapidly as large-scale poultry production has increased, but the quality and flavor of chicken have decreased significantly. The meat color, odor, and taste of chicken are the decisive factors that affect consumption [1]. Among them, chicken flavor (taste and odor) is an important index to measure quality [2]. Therefore, improving meat flavor has become an important research topic in broiler breeding. Meat flavor is attributed to a variety of volatile compounds formed during cooking by reactions between low molecular weight water-soluble compounds and lipids [3]. However, flavor phenotypes are difficult to quantify and are strongly influenced by environmental factors [4], which makes it difficult to improve chicken flavor. Lipids and water-soluble components are the main flavor precursors of meat. Water-soluble components include free sugars, sugar phosphates, nucleotide-bound sugars, free amino acids, peptides, nucleotides, and sulfur-containing compounds [5]. The umami flavor of chicken meat is primarily derived from water-soluble precursors substances such as inosine monophosphate (IMP) and glutamate [6,7]. Lipids are the fat-soluble substances found in meat. The main components of intramuscular fat (IMF) and subcutaneous fat are triglycerides and phospholipids, which contain large amounts of unsaturated fatty acids such as linolenic acid, oleic acid, and arachidonic acid (ARA). It has been demonstrated that chickens with a high ARA content have better sensory quality [8,9], and oleic acid is also associated with the taste of meat [10]. According to research by Mottram et al., phospholipids are important precursors for meat flavor, whereas triglycerides have little impact on flavor [11]. Studies have shown that adenylosuccinate lyase (ADSL) is a key gene in regulating the IMP synthetic pathway, and fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor-γ (PPARγ) are important genes that regulate lipid transport and metabolism. However, the key metabolites and molecular mechanisms that influence chicken flavor have not been fully elucidated. The transcriptome may present different gene expression states under different conditions. Furthermore, metabolomic analysis reveals changes in metabolites caused by gene regulation. Many researchers have investigated the regulatory process of IMF formation in livestock and poultry meat through a joint analysis of the transcriptome and metabolome [12,13], which has improved our understanding of the regulatory mechanisms involved in meat flavor. The Beijing-You chicken (BJY) is a distinctive indigenous breed that is well known for its meat quality and flavor in China. The free amino acids in its muscles are significantly higher than those of other breeds [2], and it is also rich in essential fatty acids and phospholipids [14]. In this study, we performed joint analyses between the transcriptome and metabolome to investigate the metabolic dynamics of flavor precursors in breast muscle samples of BJYs obtained on days 1, 56, 98, and 120, and identified key metabolites and genes that can affect meat flavor. Our results provide a theoretical foundation for the molecular mechanisms underlying chicken flavor and utilization of the germplasm resources of BJYs. Animal welfare practices and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). All procedures were approved by the Animal Ethics Committee of the Beijing University of Agriculture. The BJYs used in this experiment were obtained from a farm in a suburb of Beijing. A total of 100 1-day-old chickens were randomly selected and raised under identical standard management conditions with free access to water. The ingredients and composition of the BJY diets at different feeding stages are shown in Table S1. The immune procedures at different developmental stages are shown in Table S2. Ten healthy chickens of similar body weight were chosen at 1 (birth stage), 56 (rapid growth stage), 98 (stage with high deposition of IMF), and 120 days of age (marketing stage), respectively, and were slaughtered by a conventional neck cut, bled, and plucked. Breast muscle without skin was then collected from similar sampling sites and stored in a refrigerator at −80 ℃. The body weight and breast muscle weight at the four developmental stages of BJYs are shown in Table S3. Total RNA was extracted from 40 frozen breast muscle samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. An ARNA 6000 Nano LabChip Kit for Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was used to determine the purity and concentration of RNA. Forty cDNA libraries were created by reverse transcription using an mRNA-Seq Sample Preparation Kit (Illumina, San Diego, CA, USA). A HiSeq 2500 instrument was used for paired-end sequencing (Illumina). Raw data (raw reads) in fastq format were filtered to ensure quality. Reads with adapter sequences and low quality were eliminated and all reads containing A bases and reads with N ratios >10 % were eliminated to ensure quality. Using Hisat2 with the default settings, clean paired-end reads were aligned to the chicken reference genome (version: GCF_016699485.2). Stringtie (v2.1.5, http://ccb.jhu.edu/software/stringtie/, accessed on 3 September 2021) was used to assemble transcripts. Gene expression levels were estimated using counts per million (CPM). The differentially expressed genes (DEGs) between the two stages were calculated using the edger R package, which was defined as genes with a false discovery rate (FDR) ≤ 0.05 and a |log2(fold change)| ≥ 1. The DEGs were analyzed, clustered, and visually represented using Short Time-series Expression Miner (STEM) software (Pittsburgh, PA, USA, v1.3.11). The minimum and maximum numbers of model profiles were 2 and 45, respectively. We standardized the data using Log2 (CPM), and the screening interval for a valid trend was p < 0.05. A hierarchical cluster analysis of the DEGs was performed using the ggplot2 package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R package clusterProfiler (Guangdong, China, v4.0.5). A total of 40 frozen samples from four developmental stages were defrosted. Following homogenization, the samples were centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatant was collected in a tube for the LC-MS/MS analysis. The sample extracts were analyzed using an LC-MS/MS system (UFLC, shimadzu UFLC SHIMADZU CBM30A, available at https://www.shimadzu.com) (accessed on 3 September 2021) and chromatographic separations were performed. In reverse phase separation, a Waters ACQUITY UPLC HSS T3 C18 (1.8 m, 2.1 mm × 100 mm) was employed. The HPLC conditions were as follows: solvent system, water (0.04 % acetic acid): acetonitrile (0.04% acetic acid); gradient program of 95:5 V/V at 0 min, 5:95 V/V at 11.0 min, 95:5 V/V at 12.1 min, and 95:5 V/V at 14.0 min; flow rate, 0.40 mL.min−1; temperature, 40 °C; injection volume, 2 μL. A QTRAP® 6500+ LC-MS/MS System (https://sciex.com/) (accessed on 3 September 2021), equipped with an electrospray ionization (ESI) Turbo Ion-Spray interface and operating in positive and negative ion mode was used. The operating parameters for the ESI source were as follows: the ion spray (IS) voltage was 5500 V (positive) and −4500 V (negative) and the turbo spray source temperature was 500 °C. The ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) pressures were 55, 60, and 25.0 psi, respectively. The level of the collision gas (CAD) was high. Instrument calibration and mass calibration were performed with solutions of 10 and 100 mol/L polypropylene glycol in the QQQ and LIT modes, respectively. A specific set of multiple reaction monitoring (MRM) transitions was observed based on the metabolites eluted each time. Metabolites were differentiated by contrasting the m/z values of precursor ions, retention times, and fragmentation patterns with the standards in a database created by MetWare Biotechnology Co., Ltd. (Wuhan, China). An MRM pattern was used for quantitative detection. The following screening thresholds were used to identify significantly changed metabolites (SCMs): |log2(fold change)| ≥ 1 and variable importance in projection (VIP) ≥ 1. Principal component analysis (PCA) was carried out using SIMCA14.1 software. MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) (accessed on 3 September 2021) was used to analyze metabolic pathways of the SCMs. Co-expression network modules of all DEGs were constructed using the WGCNA R package (v1.70-3) and analyzed in combination with metabolites. The automatic network creation function (blockwiseModules) with the default parameters was used to obtain co-expression modules. The parameters were minModuleSize = 30, mergeCutHeight = 0.25, and soft threshold power = 10. Screening was carried out utilizing a gene significance |GS| ≥ 0.7 and a module membership |MM| ≥ 0.7 in order to better investigate interactions between genes in the modules. The link between genes and important metabolites was determined using Pearson correlation analysis. Interaction networks were constructed using Cytoscape (https://cytoscape.org) (accessed on 3 September 2021). KEGG pathways that were jointly enriched by all SCMs and DEGs were analyzed. The ‘cor’ package in R software (www.r-project.org) (accessed on 3 September 2021) was used to calculate Pearson correlation coefficients between the SCMs and DEGs through pairwise comparisons. RNA-seq analyses were conducted to examine gene expression profiles of breast muscles in BJYs at different developmental stages. A total of 7225 DEGs were identified in this study: 6,521 at day 1 vs. 56, 819 at day 56 vs. 98, and 747 at day 98 vs. 120 (Figure 1A). The results of cluster analysis revealed differential expression of genes at different stages (Figure 1B). To identify genes that play key roles in breast muscle development, 82 critical genes were discovered at the intersection generated from a Venn diagram of DEGs (Figure 1C and Table S4). We investigated the functions of 7,225 DEGs using GO analysis. Biological processes contained 268 significant terms (p < 0.05), and the top five terms in this category were cellular developmental process, cell differentiation, tissue development, cell proliferation, and regulation of cell differentiation. Molecular functions involved 18 significant terms, including cytoskeletal protein binding, lipid binding, tubulin binding, and sulfur compound binding (Figure 2A and Table S5). The KEGG enrichment analysis identified 26 key KEGG pathway terms, including multiple pathways involved in muscle growth, including the Wnt, MAPK, and PPAR signaling pathways (Figure 2B and Table S6). The functions of 82 intersecting genes in a Venn diagram analysis were analyzed to determine their involvement in regulatory mechanisms surrounding meat flavor. KEGG pathway analysis showed that multiple genes were enriched in pathways associated with muscle development, including ECM–receptor interaction, PPAR signaling pathway, and fatty acid biosynthesis and degradation, in which secreted phosphoprotein 1 (SPP1), thrombospondin 1 (THBS1), thrombospondin 2 (THBS2), and acyl-CoA synthetase long chain family member 4 (ACSL4) were associated with the biological pathways of IMF deposition (Table 1). STEM analysis revealed that the expression patterns of 7225 DEGs in breast muscle during BJY development were enriched into forty-five profiles, of which nine profiles appeared significant, including six up-regulated and three down-regulated profiles. A total of 2931 DEGs exhibited an up-regulation trend and were significantly enriched in profiles 5, 10, 36, 38, 42, and 44, whereas 2830 DEGs showed a down-regulation trend and were significantly enriched in profiles 0, 2, and 4 (Figure 2C). The genes enriched in profiles 2 and 38 were the most abundant, and KEGG enrichment analysis revealed that genes in profile 2 were primarily enriched in the Wnt signaling and purine metabolism pathways, whereas the genes in profile 38 were primarily enriched in cytokine–cytokine receptor interaction and cell adhesion molecular pathways (Table S7). We further analyzed the metabolic alterations in the breast muscle of BJYs at four developmental stages using an LC-MS/MS approach. In total, 578 compounds were identified and classified into 33 classes; primarily organic acids, carbohydrates, lipids, nucleotides and their derivatives, vitamins, and amino acid derivatives. A PCA of the metabolic data of the four developmental stages indicated a good correlation between replicates, and SCMs from day 1 were clearly distinct from those of other stages (Figure 3A). We identified 310 SCMs using the criteria of a VIP ≥ 1 and a |log2(fold change)| ≥ 1. At day 1 vs. 56, we found 294 SCMs, of which 221 and 73 were down- and up-accumulated metabolites, respectively. At day 56 vs. 98, a total of 19 SCMs, including 13 down- and 6 up-accumulated metabolites were detected. A total of 21 SCMs were identified at day 98 vs. 120, including 12 down- and 9 up-accumulated metabolites (Figure 3B). KEGG enrichment analysis indicated that the majority of SCMs were involved in amino acid metabolism, such as arginine biosynthesis; arginine and proline metabolism; purine metabolism; glycine, serine, and threonine metabolism; and alanine, aspartate, and glutamate metabolism (Figure 3C and Table S8). To better understand the gene regulation mechanism of metabolites during BJY development, nine co-expression modules of DEGs were identified using WGCNA (Figure 4A). DEGs of BJYs from day 1 vs. 56 were mainly gathered in the blue module, whereas DEGs from day 56 vs. 98 and from day 98 vs. 120 were mostly located in the cyan module. According to the heatmap of module–trait relationships (Figure 4B), the accumulation of transcripts of the blue module correlated with amino acids and lipid metabolites, including serine, glycine, cysteine, threonine, glutamate, and lysophosphatidylcholine (LPC), which are the main flavor compounds of BJYs. The accumulation of transcripts in the cyan module was correlated with flavor-associated metabolites such as creatine and IMP. These results indicated that the DEGs in these modules were mainly associated with flavor formation during the development of BJY breast muscle. To further explore the relationship between DEGs and SCMs, DEGs in the blue and cyan modules with a gene significance, |GS|, of ≥0.7, and a module membership, |MM|, of ≥0.7 were used to analyze interactions with SCMs. We identified 11 genes in the blue module involved in the amino acid metabolic pathway, including cystathionine β-synthase (CBS), glutamate decarboxylases (GAD1 and GAD2), glycine amidinotransferase (GATM), glycine decarboxylase (GLDC), glycine N-methyltransferase (GNMT), low-specificity L-threonine aldolase 2 (ItaE), cysteine lyase (CYLY), sarcosine dehydrogenase (SARDH), cysteine dioxygenase (CDO1), and serine racemase (SSR) genes, the expressions of which were highly correlated with the accumulation of glutamate, serine, glycine, threonine, and cysteine (Figure 4C). LPC plays a vital role in the formation of meat flavor. We identified key regulatory genes involved in the LPC biosynthesis pathway in the blue module, including acylglycerol phosphate acyltransferases (AGPAT2 and AGPAT4), diacylglycerol kinases (DGKB, DGKH, and DGKI), glycerol-3-phosphate acyltransferases (GPAM and GPAT2), lecithin cholesterol acyltransferase (LCAT), phospholipid phosphatases (PPAP2B and PPAPDC1B), acetylcholinesterase (ACHE), ethanolamine kinase (ETNK2), patatin-like phospholipase (PNPLA6), and phospholipase (PLA2G4EL1 and PLA2G4A) genes (Figure 4D). IMP is an important flavor substance in chickens. Seventeen genes in the cyan module were identified as good candidates encoding key genes in the IMP biosynthesis pathway, including adenylate monophosphate deaminases (AMPD1 and AMPD3), adenylosuccinate synthetase (Adssl1), ectonucleoside triphosphate diphosphohydrolases (ENTPD5 and ENTPD6), phosphodiesterases (PDE1B, PDE4B, PDE8B, PDE10A, and PDE4D), adenylate kinases (AK1 and AK3), 5′,3′-nucleotidase (NT5C1A and NT5M), one ectonucleotide pyrophosphatase (ENPP3), nucleoside diphosphate kinase (NME3), and phosphoribosylglycinamide formyltransferase (GART) genes (Figure 4E). A KEGG enrichment analysis of DEGs and SCMs revealed 46 co-enriched pathways (Figure 5A). The pathways associated with amino acids, lipids, and IMP were highlighted in this analysis, and these processes were combined in the related network (Figure 5B). We observed that the network involved flavor metabolites including glycine, serine, cysteine, threonine, and glutamate. The content of these amino acids was higher during the early development of BJYs. In the glycerophospholipid metabolism pathway, the LPC content increased from day 1 to 56 and decreased gradually with continuing growth, and was the key compound that led to fat deposition in the late stage of BJY maturation. IMP content in the purine metabolism pathway significantly increased with the age of BJYs. There was a significant correlation between the regulatory genes and flavor metabolites in the network (Figure 5C). For example, the correlation of CBS with cysteine and serine was 0.93 and 0.9, respectively; glycine with GATM was 0.88; IMP with AMPD1 was 0.65; and LPC with PNPLA6 was 0.54 (p < 0.05). Elucidating the regulatory mechanisms involved in the synthesis and accumulation of flavor compounds is essential for improving chicken meat quality. As illustrated in this study, 310 SCMs and 7,225 DEGs were identified at four developmental stages of BJYs. The functions of DEGs and SCMs were analyzed by KEGG enrichment, and the results showed that they were co-enriched in glycerophospholipid metabolism; glycine, serine, and threonine metabolism; purine metabolism; and alanine, aspartate, and glutamate metabolism pathways. To further understand the molecular mechanisms involved in meat flavor formation during the development of BJYs, we identified genes significantly related to flavor metabolites using WGCNA and established a regulatory network related to the accumulation of key flavor components. CBS, GATM, GAD2, PNPA6, ItaE, AMPD1, glycine, serine, cysteine, and threonine have been identified as important genes and metabolites that affect flavor formation. This study not only helps to define the regulatory networks of specific flavor compounds in BJY meat but also provides a theoretical foundation for the improvement of meat flavor in broiler breeds. Amino acids are crucial components in meat flavor [15]. The degradation of peptides and amino acids in meat improves its sensory properties and taste [3]. Glutamate, succinic acid, and IMP are umami amino acids, whereas glycine, threonine, alanine, and serine are sweet amino acids [16]. Cysteine is the cause of sulfur-containing flavors in meat [17], as it can produce meat flavors during heating [18]. However, the precise regulatory pathways of amino acid-derived flavors in BJYs remain unknown. We identified genes related to amino acid metabolism by WGCNA, including CDO1, CYLY, CBS, GAD, GATM, and ItaE. CDO1, CYLY, and CBS are considered critical genes involved in cysteine metabolism. CDO1 regulates cysteine concentrations in mice by participating in cysteine degradation [19]. Cysteine is the only major substrate of CYLY [20]. CBS catalyzes the formation of cysteine by the condensation of serine and homocysteine with water [21]. The absence of CBS increases the risk of elevated plasma homocysteine levels and severe growth retardation [22,23]. In addition, we demonstrated a high correlation between CBS and cysteine (r = 0.93). The consistent expression trends of CBS and cysteine indicated that CBS may play an important role in the growth and flavor formation of BJYs. GAD transforms glutamate to γ-aminobutyric acid by decarboxylation, which is then converted to succinate [24]. This was also confirmed by the higher association between GAD2 and glutamate in this study (r = 0.72). GATM encodes a mitochondrial enzyme that belongs to the amidinotransferase family and is thought to be a key gene in the process of creatine metabolism [25]. In this study, the creatine and glycine expression trends were opposite. A significant association between glycine and GATM supported speculation that GATM expression controls creatine metabolism and leads to glycine accumulation. Under physiological conditions, ItaE performs threonine catabolism and glycine synthesis by catalyzing the cleavage of threonine into glycine and acetaldehyde [26]. Similarly, our results showed that decreased glycine levels were associated with a decreased ItaE expression. The coefficients of correlation of ItaE with threonine and glycine were 0.48 and 0.59, respectively. Finally, we speculated that CBS, GATM, GAD2, and ItaE were key genes involved in amino acid-derived flavor formation in breast muscle during BJY development. LPC is obtained by loss of a fatty acid group from lecithin. In vitro studies have confirmed that the ability of LPC to emulsify fat is 4–5 times higher than that of common oil. Our results showed that LPC expression levels decreased with the aging of BJYs, which may be related to fat deposition in late developmental stages of BJYs. We identified a total of 15 DEGs significantly associated with LPC in the blue module. The PNPLA6 enzyme can react with a variety of substrates, including retinol esters, triacylglycerols, and phospholipids [27]. However, it can preferentially hydrolyze phosphatidylcholine (PC) and LPC [28]. In both Drosophila and mice, PNPLA6 gene deletion leads to increased lipid deposition, motor impairments, and neurodegeneration [29]. In this study, we identified the transcription level of PNPLA6 was significantly positively correlated with LPC (r = 0.54). We speculated that the decrease in LPC content under the regulation of PNPLA6 led to the accumulation of large amounts of fat during the late development of BJYs. The AGPAT enzyme catalyzes the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA) [30]. PA is a substrate for the synthesis of other polar phospholipids (PL) such as PC, phosphatidylserine (PS), and phosphatidylethanolamine (PE) [31]. AGPAT2 encodes a member of the 1-acylglycerol-3-phosphate O-acyltransferase family, which is involved in the conversion of lysophosphatidic acid to phosphatidic acid during the second step of phospholipid biosynthesis. The GPAT enzyme converts glycerol-3-P to lysolecithin, which is subsequently acylated to PA [32]. In addition, PA contains two fatty acids in its glycerol backbone at the sn-1 and sn-2 positions. Although GPAT determines fatty acid specificity at the sn-1 (carbon 1) position, AGPAT enzyme esterification causes variability at the sn-2 (carbon 2) position [33,34]. As a result, these two enzymes can produce a wide range of PA species with varying fatty acids at the two carbon positions. Based on our results, we believe that these genes are involved in fat metabolism, and the regulatory effects need to be confirmed by further research The Maillard reaction refers to the polymerization, condensation, and other reactions involving compounds containing free amino groups and reducing sugars or carbonyl compounds at normal atmospheric temperatures or under heating. IMP degradation-forming ribose participates in the Maillard reaction, which is also an important reaction in flavor formation. Numerous studies have shown that IMP is one of the most important flavor components in meat [35,36]. The results of this study showed that IMP content was the highest at the age of 56 days, and then decreased gradually with the increase in age, which was consistent with Katemala et al.’s study on Korat hybrid chickens [37]. Previous studies involving the formation of IMP have confirmed that AMPD1 enzyme catalyzes the irreversible hydrolysis of adenosine 5′-monophosphate (AMP) to IMP and ammonia [38]. Our data verified a significantly high correlation between AMPD1 and IMP (r = 0.65). Therefore, this gene was selected as a key gene that controls IMP biosynthesis by regulating AMP metabolism. In addition, we also identified DEGs involved in the IMP metabolic pathway. GART and ADSSL1 genes were able to increase the synthesis of IMP by promoting the purine de novo synthesis. Purine de novo synthesis was divided into 10 steps, in which GART was involved in Steps 2, 3, and 5, and ADSLL1 was involved in the Step 8 reaction in this pathway [39]. AMPD3 and NT5C are involved in the degradation of purine to accelerate the de novo synthesis of purine [40]. We found that the expression levels of these genes were significantly correlated with IMP. These results showed that these genes were related to IMP metabolism in the breast muscle of BJYs. In conclusion, we identified four crucial metabolic pathways involved in flavor formation in BJY breast muscle by combining transcriptomic and metabolomic data, including glycerophospholipid metabolism; glycine, serine, and threonine metabolism; alanine, aspartate, and glutamate metabolism; and purine metabolism pathways. We also identified a number of important metabolites and regulatory genes affecting meat quality and flavor involving these pathways, including glycine, serine, glutamate, threonine, LPC, IMP, CBS, GATM, GAD2, PNPLA6, ItaE, and AMPD1. However, the genetic mechanisms of chicken quality and flavor and the molecular function of these key genes and metabolites need to be further verified. In brief, our research provides not only new perspectives regarding the regulatory metabolism of chicken meat flavor during development, but also a theoretical foundation for the utilization of BJYs and genetic improvement in broiler meat quality and flavor.

PMC10000373

Samrat Roy Choudhury,Billie Heflin,Erin Taylor,Brian Koss,Nathan L. Avaritt,Alan J. Tackett

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells

24-02-2023

melanoma,S100b,CRISPR,dCas9-KRAB,apoptosis,cell death

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma. Melanoma is one of the deadliest forms of skin cancer, is particularly prevalent (2.5%) among the Caucasian population, and has almost doubled in incidence over the past three decades in the United States alone [1]. Several serological biomarkers have been identified for early detection, staging, prognosis, and therapeutic determination of melanoma. Among them, a high serum level of S100B (S100 calcium-binding protein B) has emerged as the most reliable biomarker of progression and survival outcome of the disease [2,3]. S100B (10.7 kDa) is a member of the S100 protein family that binds to its molecular targets by undergoing conformational changes at the carboxy-terminal EF-hand motif. The affinity of S100B-mediated protein–protein interactions is strengthened with the increase in intracellular Ca2+ concentrations. Ectopic upregulation of the protein has been noted in metastatic melanomas, as well as proneuronal, neural, or classic types of gliomas [3,4,5]. In melanoma, intracellular S100B interacts with proteins of different signaling pathways [6], such that S100B activates the glycolytic enzyme fructose 1,6-biphosphate (aldolase) and increases metabolism of melanoma cells. The protein also interacts with cytoskeletal components such as tubulin, Rac1 (GTPase), or IQGAP1 (cdc42 effector), which alters motility of melanoma cells toward enhanced migration and invasion [7]. In contrast, when extracellularly secreted via the receptor for advanced glycation end products (RAGE) signal transduction pathway, S100B facilitates tumor development in a mouse model of melanoma [8]. Additionally, S100B triggers melanoma tumor growth by interacting with the C terminus of wild-type (WT)-p53, preventing protein tetramerization and covalent modifications (e.g., phosphorylation or ubiquitination) [9]. Therefore, S100B–p53 interaction lowers the intracellular availability of free WT-p53 and limits the tumor-suppressing function of TP53 [6,10,11]. Previously, siRNA-mediated cell-type-specific targeted inhibition of the S100B–p53 complex was shown to rescue protein levels of WT-p53, phosphorylated p53, and downstream p21 [10]. The targeted inhibition also evoked poly-ADP ribose polymerase (PARP)-mediated apoptosis involving activation of caspase-3 or caspase-8 or aggregation of Fas death receptor upon ultraviolet irradiation [10]. Several attempts have been made to develop small-molecule inhibitors (e.g., pentamidine) that inhibit molecular interactions between the Ca2+-S100B apoprotein and its binding proteins [12]; however, the small-molecule inhibitors, as well as siRNA-mediated knockdown, of S100B resulted in only transient restriction of melanoma cell growth. We aimed to develop a more stable and effective targeted approach that uses a CRISPR (clustered regularly interspaced short palindromic repeats) platform for stable suppression of S100B protein. Targeted epigenome editing previously was attempted by using a CRISPR and associated protein 9 (Cas9) endonuclease system that contained deactivated Cas9 (dCas9) fused to a transcriptional suppressor, Krüppel-associated box repressor (KRAB) [13]. The KRAB domain exerts widespread transcriptional suppression through enrichment of H3K9me3 and condensation of chromatin [14]. Herein, we report development of a dCas9-KRAB (DK) system for targeted perturbation of S100B expression to rescue WT-p53 and its associated tumor-suppression properties in melanoma cells. Data from 28 retrospective studies available through the Cancer Genome Atlas (TCGA) were used to examine S100B expression in samples from patients with cutaneous (n = 443) or ocular (n = 80) melanoma, as well as patients with different cancer types (n = 10,071; comparison group) (Table S1). From the TCGA Firehose Legacy study, we selected a cohort of patients with skin melanoma (n = 287) on the basis of available data for DNA methylation, copy number (CN) alterations, and gene expression from the matched patient samples (Table S2) [15]. Genetic and transcriptomic data from TCGA were extracted through the cBioPortal (www.cbioportal.org; accessed on 17 January 2022). GEP was analyzed from mRNA expression (RNA sequencing [RNA-seq]) data; we represent GEP data expressed in RNA-seq by expectation-maximization (RSEM), log2 scale. CN alterations are presented as the capped relative linear copy-number values, where we consider patients with diploid genomes for S100B, compared to those with gain (+1) or loss (–1) in CN. There were single cases for amplification (+2) or deep deletion (–2); these were excluded from the analysis. DNA methylation was assayed on an Illumina HumanMethylation450 (HM450) BeadChip platform (San Diego, CA, USA), and the average β-value from all probe sets against S100B was considered. Raw values for GEP, CN, and methylation from matched patients are summarized in Table S3. A proteomics data set was analyzed from the consented (study #204543) and deidentified tissue biopsies from a cohort of patients with melanoma (n = 23) that was diagnosed at stage IV and treated with first-line ICIs (i.e., anti-CTLA4, PD-1, or a combination of monotherapies) at the University of Arkansas for Medical Sciences. The proteomics sample was prepared, and data were analyzed as reported earlier [16]. Clinicopathological information of the patients, including response to ICIs, is summarized in Table S4. The chromatin state at the S100B promoter, including transcriptional start sites (TSSs) and intragenic regions, was examined by comparing DNase-sequencing data from SK-MEL-5 melanoma cell lines (accession ID: ENCFF627WEJ) to that from primary keratinocytes derived of newborn foreskin (accession ID: ENCFF910KFI); both data sets were acquired from the ENCODE database. The DHS signals were represented as read-depth normalized signal. S100B mRNA levels in the same cell line (accession ID: ENCFF249LKO) and primary keratinocytes (accession ID: ENCFF249BFY) were also analyzed. The bigwig (hg38) files for both DHSs and RNA-seq data were loaded on the integrative genomics viewer (v 2.14.0, Broad Institute, Cambridge, MA, USA) for visualization. The amino acid sequences of human (S100B) and murine (S100b) homologs of the protein were compared by using Clustal Omega (v 1.2.4) (EMBL-EBI, Hinxton, UK) function and were presented by using Jalview (v 2.11.2.0) [17]. The putative TFs that interact with the murine single-guide RNA (m-sgRNA)-binding sites on S100b were predicted by using the target regions as input for the PROMO database (http://alggen.lsi.upc.es/; accessed on 21 May 2020) that uses TRASFAC for the prediction analyses [18,19]. The output was specific to Mus musculus. A plasmid containing the KRAB domain fused to dCas9 and the programmable sgRNA vector were obtained from Addgene, Watertown, MA, USA (#99372, and #44248, respectively). The plasmid #44248 contains an EGFP (enhancer green fluorescent protein)-specific sgRNA and has been used as an off-target control for the present study. Three murine S100b-specific sgRNAs were selected for the present study, based on their predicted efficacy of inhibition at the target site. m-sgRNA-1 and m-SgRNA-2 were selected from the sgRNA library that was recommended for the mouse genome [20]; m-SgRNA-3 was custom designed by using the CHOPCHOP (https://chopchop.cbu.uib.no/; accessed on 2 July 2020) algorithm design tool (Table S5). All sgRNA-binding sites were mapped with Mouse Genome Informatics (http://www.informatics.jax.org/; accessed on 12 June 2020) and annotated against the GRCm38/mm10 genome. The selected sgRNAs in combination with the scaffolds were obtained as gene blocks (gblocks) from Integrated DNA Technologies, Coralville, IA, USA. The gblocks were PCR amplified (Table S6) with CloneAmpHiFi PCR Premix (Takara, Kusatsu, Shiga, Japan) and were integrated into the sgRNA plasmid, flanked by BstXI and XhoI (New England Biolabs, Ipswich, MA, USA) sites. Correct clones were confirmed with low-throughput sequencing (Table S6, sequencing primers). Murine B16-F1 (CRL-6323) melanoma and B16-F10 (CRL-6475) metastatic melanoma cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cell lines were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 1% penicillin–streptomycin (Thermo–Fisher, Waltham, MA, USA), and 1% glutamine and maintained at 37 °C and 5% CO2. Cells were transduced with plasmid encoding DK alone, or in combination with the plasmids encoding sgRNAs specific to S100b or EGFP (sgRNAEGFP). Transduction was accomplished with a one-step lentivirus packaging system (Takara, Kusatsu, Shiga, Japan) per the manufacturer’s instructions. Briefly, 4–5 × 106 LentiX-293T cells (Takara) were transfected with the plasmids in Opti-MEM reduced serum medium (Thermo–Fisher, Waltham, MA, USA) and were incubated for 4–6 h. Next, each culture was supplemented with 14 mL complete medium and then incubated for 48 h. Supernatants were collected, passed through 0.45 µM syringe filters, and added dropwise to polybrene (8 µg/mL) charged F1 or F10 cells. Cells co-transduced with DK and sgRNA constructs were selected with puromycin (1 µg/mL), followed by FACS sorting (FACSARIA III, BD, San Jose, CA, USA) for mCherry+ cells (>95%). Expression of target genes was determined in reference to endogenous GAPDH, out of total RNA extracted from the cells. From cells that had been treated for 24 h, total RNA was extracted (RNeasy Mini Kit, QIAGEN, Hilden, Germany) and converted to c-DNA (Iscript Advanced cDNA synthesis kit, Bio-Rad, Hercules, CA, USA). The fold change in S100b expression was then determined, in triplicate, with qPCR (StepOnePlus Real-Time PCR Systems; v 2.0 Applied Biosystems, Waltham, MA, USA), compatible with SYBR green master mix (Thermo–Fisher). Amplification reactions were carried out at 95 °C for 1 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Primers used to amplify murine S100b (qMmuCID0015305), TP53 (qMmuCIP0032520), and GAPDH (qMmuCED0027497) were purchased from commercial sources (PrimePCR SYBR Green Assay, Bio-Rad, Hercules, CA, USA). The primers for DIP2A, PRMT2, CDKN1A, AIFM1, CASP3, PARP-1, and GAPDH were custom synthesized with IDT (Integrated DNA Technologies, Coralville, IA, USA), and primer sequences are summarized in Table S7. We prepared whole-cell lysates by using RIPA buffer (Thermo–Fisher, Waltham, MA, USA) per the manufacturer’s instructions. To determine levels of S100b protein, an aliquot of lysate (40 µg total protein, determined with the BCA assay; Pierce) was loaded onto 4–20% Bis–Tris gels (Thermo–Fisher, Waltham, MA, USA); to determine levels of p21, p53, AIFM1, cleaved Caspase-3, or PARP-1 proteins, aliquots of lysate (approximately 20 µg total protein) were loaded onto 4–12% Bis-Tris gels (Thermo–Fisher, Waltham, MA, USA), and proteins were electrotransferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked in 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies in 1% milk. Blots were probed with primary antibodies (Table S8) per the manufacturer’s instructions. Blots were then washed 3 times with 1% milk and incubated with HRP-tagged secondary antibody for 1 hour. Finally, blots were washed 3 times in TBST and were developed with enhanced chemiluminescence reagents (Perkin Elmer, Waltham, MA). Densitometries of the bands from the blots (wherever applicable) were prepared with ImageJ (https://imagej.nih.gov/ij/; accessed on 23 February 2023), v 1.53t, National Institute of Health, Bethesda, MD, USA). F1 and F10 cells were seeded in 6-well plates (5 × 104 cells per well). After 24 h, they were treated with staurosporine (Seleckchem, Houston, TX, USA) at indicated doses for 24 h. Apoptosis and cellular mortality then were determined with flow cytometry (FACS Verse, BD Biosciences, San Jose, CA, USA); staining with annexin V-FITC (Biolegend, San Diego, CA, USA) and DAPI (Sigma, St. Louis, MO, USA) was used to determine the percent viable (i.e., Annexin V–, DAPI–) cells. Both B16 melanoma cell lines were seeded (5 × 104 cells per well) in 12-well plates and imaged (from at least 10 different fields) after 24 and 48 h to examine differences in cell proliferation between the treatment and control groups. Additionally, B16 melanoma cells were seeded in 96-well plates (5 × 103 cells per well), incubated for 24 h, and treated as indicated. Viability of co-transduced (DK + m-sgRNA-1) cells and untreated control cells was determined with a CellTiter-Glo Luminescent Cell Viability Assay kit (G7570, Promega, Madison, WI, USA) after 24 and 48 h in the presence or absence of two chemotherapeutic agents, cisplatin (13119, Cayman Chemicals, Ann Arbor, MI, USA) and tunicamycin (11445, Cayman Chemicals, Ann Arbor, MI, USA). Drugs were serially diluted from the stock solution (2 mM) and added to cell cultures in the range of 5 µM to 10 nM. At the end of 24 and 48 h of incubation, equal volumes (100 µL each) of assay solution and cell suspension were mixed and incubated at room temperature for 10 min. Relative luminescence was recorded with a spectrometer (Agilent BioTek, Winooski, VT, USA Gen5 Microplate reader), and the half-maximal inhibitory concentration (IC50) value for each drug was plotted with GraphPad Prism, v7.0, Boston, MA, USA. We used the non-parametric Mann–Whitney U test or Dunn’s multiple comparison test to determine the significance of differences between the groups being compared. Significance was defined as p < 0.05. Statistical analyses and associated graphs were generated with GraphPad Prism, Boston, MA, USA. We evaluated S100B expression (RNA-seq V2, RSEM, log2) in 10,071 patient-derived samples of 28 different cancers from the TCGA repository (Table S1). Expression of S100B was highest in glioblastoma, followed by skin cutaneous melanoma (SKCM). S100B expression in SKCM samples (n = 443) ranged (25–75% quartile) from 4287.6 to 16,048.4 with a median of 8959.4 ± 14,212.4 (standard deviation). In samples of ocular melanoma (UVM) (n = 80), the gene was expressed at a similar fashion to SKCM or was overexpressed (646.7 ± 1474.9), relative to other cancer types (Figure 1A). A separate in-house proteomics study of a cohort of patient-derived SKCM samples demonstrated that, in the subgroup (n = 12) that was non-responsive to ICIs anti-CTLA-4, anti-PD1, and anti-CTLA4 + anti-PD1 combined therapy, the S100B protein was upregulated (p < 0.05, >2-fold change), compared to the subgroup (n = 10) that was responsive (Figure 1B). These findings strongly suggest that ectopic upregulation of S100B might play a critical role in development of refractory SKCM [10,21] and could contribute to therapeutic resistance in the disease. Next, we combined the data on S100B expression with data on CN alterations and DNA-methylation profiles in matched SKCM samples (n = 285), available from TCGA. We observed that 17.5% (50/285) of samples had gains in S100B CN, and 21% (60/285) of samples had heterozygous deletions in CN of S100B, while 61.4% (175/285) of samples were diploid for S100B. A significant (p < 0.01) decrease in median expression of S100B (RSEM = 5624.3) was observed in samples with CN losses, compared to those with gains (RSEM = 14,119.5) (Figure 1C) but not to those with diploidy. However, weak correlations (p > 0.05; R2 < 0.2) were observed when the relative linear values of CN for individual subgroups (gain, loss, or diploid) and their expression profiles were compared (Figure 1D). We also investigated whether S100B expression correlates to DNA methylation (β-values, probe: cg12092309) of S100B. Methylation ranged (25–75% quartile) from 0.54 to 0.87, with a median value 0.74. We observed a moderate (R2 = 0.216; p < 0.01) negative correlation between S100B methylation level and expression in SKCM samples, such that samples with lower methylation levels have relatively higher expression and vice versa (Figure 1E). Additionally, we analyzed signal intensities based onDHS (DNAse hypersensitive sites) to assess the chromatin state of S100B in the SK-MEL-5 cell line, which is representative of human SKCM, and compared it to that in primary keratinocytes derived from foreskin of human newborns. We observed epigenetic priming in the melanoma cells, such that DHS enrichment was observed in SK-MEL-5 cells at the S100B TSS and upstream promoter, which spans 578 bp (chr21:46,604,961-46,605,537) (Figure 1F). The conformational change in chromatin state may be best linked to S100B upregulation in melanoma. This is supported by RNA-seq analysis of the same set of samples, which revealed no S100B transcript in primary keratinocytes but marked S100B transcript from exon (E)2 and E3 in SK-MEL-5. Next, we evaluated amino acid sequence homology between human S100B and its mouse ortholog S100b. The analysis showed that the proteins are almost identical. Each contains 92 amino acids with a single base substitution (asparagine to glutamic acid) at position 63 (Figure 1G), which is predicted not to significantly alter the protein’s secondary structure (Jalview 2.11.1.0). Mouse B16 melanoma cells have been used for expressing differential levels of S100b under different pathological conditions in conjunction with tumor development [22,23,24]. We used the F1 and metastatic F10 melanoma cell lines to determine whether suppression of the gene can evoke anticancer phenotypes in treated cells. S100b expression (signal intensity relative to endogenous GAPDH) in F10 cells (0.58) was significantly (p < 0.05) lower than in F1 cells (1.72) (Figure 1H). We aimed to suppress the S100b expression by using CRISPR-KRAB-mediated interference at selected loci of the gene. S100b-specific m-sgRNAs were selected from the previously reported CRISPR inhibition library specific for the mouse genome or were designed with an online algorithm tool [20]. Ref. [25] m-SgRNA-1 and m-SgRNA-2 were targeted at 10 bp and 55 bp, respectively, downstream from the S100b TSS (chr10:76253899-76253915); m-SgRNA-3 was targeted at 49 bp upstream of the TSS (chr10:76253817-76253822) (Figure 2A). The targeted S100b regions also contain putative binding sites (predicted by the TRANSFAC tool) for multiple TFs. In particular, we observed the prevalence of c-Fos, C/EBP-β, and NF-1 TFs within 20 bp of the target sites of the 3 m-sgRNAs, suggesting their putative regulatory roles in determining upregulation of S100b (Figure 2B) [26,27,28]. The DK repressor protein was stably expressed in F1 and F10 cells via lentiviral transduction and was maintained under puromycin selection (Figure 2C). Additionally, the m-sgRNA vectors harboring an mCherry fluorophore allowed the use of FACS sorting to enrich (>95%) co-transduced cells (Figure 2D). The cells were transduced with DK module with or without three individual S100B-specific sgRNAs. Cells transduced with DK in combination with sgRNAEGFP served as an off-target control for the current study. The inherent transcription-repressive nature of the KRAB protein resulted in reduced S100b mRNA levels in both F1 and F10 cell lines, whereas combinatorial treatment with DK plus selective m-sgRNAs resulted in different degrees of reduction in the gene expression, compared to the parental lines (except for m-sgRNA-3 in F10). Therefore, to control for the effects of KRAB, we used DK-transduced cells as the baseline to which we compared the cells that were also transduced with the m-sgRNAs, for determining their effects on S100b expression. S100b expression was significantly (p < 0.05) reduced in the B16-F1 cells treated with the combination of DK plus m-sg-RNA-1, compared to the cells treated with DK alone (Figure 2E). In contrast, S100b expression was increased in F1 cells when treated with the combinations of DK plus remaining S100b-specific sgRNAs (m-sgRNA-2/3) or sgRNAEGFP (Figure 2E). A similar pattern was observed in the change in S100b mRNA expression in the B16-F10 cells, such that the gene expression was further reduced (p > 0.05) in cells treated with the combination of DK and m-sg-RNA-1, compared to cells transduced with DK alone. The combination of DK with m-SgRNA-2/3 or with sgRNAEGFP in either type of B16 cells remained ineffective in suppressing S100b expression. Additionally, S100b expression remained unaltered or insignificantly changed in both cell lines when transduced with vectors containing individual m-sgRNAs specific to S100b or EGFP without the KRAB module, compared to non-transduced parental lines (Figure S1). We observed a significant decrease in S100b mRNA expression in both the B16 cells, transduced with DK fusion alone, whereas the protein expression completely disappeared in cells transduced with the combination of DK plus m-SgRNA-1. Therefore, the S100b protein-expression changes in the similar direction to the changes in mRNA level in both F1 and F10 cells. Western blot results also corroborated the fact that S100b is much less expressed, both at the level of mRNA (Figure 1H) and protein (Figure 2G) in the F10 cells compared to the F1 cells. However, the S100b protein-expression band was totally resolved in the F10 cells treated in combination with DK plus m-SgRNA-1. Next, to examine the specificity of the CRISPR-KRAB toolbox developed here, we looked into the possible changes in expression of PRMT2 and DIP2A, two adjacent genes to S100b on Chr.10 (mm10) (Figure 3A). We did not observe any significant (p > 0.05) reduction in the expression of PRMT2 and DIP2A, indicating that the action of CRISPR-KRAB interference was target-specific (Figure 3A). In summary, the combination of DK plus m-SgRNA-1 imparted the greatest repressive effects on S100b expression, and CRISPR-KRAB interference was more profound in F1 cells than in F10 cells. Therefore, we continued with this combination for the downstream biological analyses. Based on the previous literature [2,10], we hypothesized that DK-mediated suppression of S100b might increase the availability of intracellular free WT-p53 and reactivate the tumor suppressor function of TP53 (Figure 3B). DK plus m-SgRNA-1 resulted in no observed increases in mRNA levels of TP53 in F1 or F10 cells (Figure S2). However, in both cell types, co-transduction with DK plus m-SgRNA-1 resulted in significantly increased (p < 0.05) p53 protein levels (Figure 3C,D), while transduction with only DK resulted in no marked changes in p53 protein levels in both cell types. As previously mentioned, targeted inhibition of S100b may release intracellular S100b-bound p53, potentially increasing levels of WT-p53 and its downstream product p21 or apoptogenic proteins [10,29,30]. Therefore, to investigate the consequences of S100b suppression, we assessed the effects of DK on induction of apoptosis in the target cells. In both F1 and F10 cells, transduction with DK plus m-SgRNA-1 resulted in significantly increased (p < 0.05) expression of CDKN1A (p21) in F1 cells both at the level of mRNA (14% increase) (Figure S2) and protein (Figure 3C), compared to F1-control. In contrast, a marginal (p = 0.05) increase in CDKN1A (11%) expression was observed in F10 cells (Figure S2). Nonetheless, the p21 protein level was significantly increased (p < 0.05) in the F10 line, compared to the control (Figure 3D). To test the effects of elevated p53 and p21 levels on apoptosis, we treated the control (F1/F10) and DK-m-SgRNA-1 transduced cells with staurosporine (STS), a generic inducer of apoptosis. S100b-suppressed F1 cells, compared to the parental line, had increased cell death and susceptibility to serial concentrations (0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM) of STS. An untreated control and exceedingly high dose (10 µM) of STS were used as negative and positive controls for cell viability assays. For instance, at 0.1 µM STS, we observed 11% less cell viability in F1 cells transduced with DK plus m-SgRNA-1 (26.1%) than in the parental cells (37.5%) (Figure 4A); however, we did not observe similar significant changes in cell death in studies with the F10 cells (data not shown). Next, to further investigate the observed cell death in the S100b-suppressed cells, we examined expression of key caspase and non-caspase proteases that are involved in apoptotic signaling pathways. We started by determining expression of mitochondrial apoptosis-inducing factor (AIFM1) [31,32], which was significantly increased (p < 0.05) in both mRNA and protein expression in the DK+m-SgRNA-1-transduced F1 cells, compared to non-transduced and DK+/-sgRNAEGFP control cells (Figure 4B,C). The impact of differential upregulation of AIFM1 then was evaluated in conjunction with caspase-3 activation. We did not observe any significant changes in the CASP3 mRNA expression in DK+m-SgRNA-1 transduced cells (Figure 4B). In contrast, a capase-3 protein cleavage (~17 kDa) was evident in cells transduced with DK+m-SgRNA-1 (Figure 4C); however, we did not observe noticeable changes in caspase-8 mRNA or protein levels (data not shown). Finally, we examined whether DK-m-SgRNA-1 affects expression of PARP-1, the substrate of caspase-3 [33]. We detected a significant (p > 0.05) increase in PARP-1 mRNA level (Figure 4B), while the cleaved subunit (~95 kDa) of PARP-1 protein was observed in DK+m-sgRNA-1 transduced cells but not in control cells (Figure 4C). In contrast, F10 cells transduced with DK+/-m-sgRNA-1 or sgRNAEGFP failed to evoke any apoptogenic changes, compared to the control cells. In F10 cells, S100b suppression also did not result in altered protein levels of caspase-3/caspase-8 or in cleaved PARP-1 (Figure S3), indicating that an apoptotic response was not evoked. In summary, DK suppression of S100b efficiently evoked p53-mediated mitochondrial apoptotic machinery in F1 melanoma cells (Figure 4C) but not in F10 cells, despite elevation of WT-p53 and p21 proteins in these cells. This suggests a mechanistic difference in S100b regulation between the B16 lines, which needs further investigation. We observed reduced cell viability in both the F1 and F10 cells co-transduced with DK in combination with m-SgRNA-1. We determined the percentage of viability by determining the amount of ATP produced by live cells 24 h and 48 h after initial seeding of cells in a 96-well plate (1 × 104 cells per well). Compared to the control cells, co-transduced F1 cells had a 65% reduction and F10 cells had a 40% reduction (p < 0.05) in cell viability (Figure 5A,B) after 24 h. In contrast, after 48 h, F10 cells co-transduced with DK plus m-SgRNA-1 had only 13% less viability (p = 0.14) than control cells, but co-transduced F1 cells continued to have at least 32% less viability (p < 0.05) than control cells. Next, we treated S100b-suppressed F1 and F10 cells with chemotherapeutic agents against metastatic melanoma: cisplatin, a platinum analog, and tunicamycin, an inducer of endoplasmic reticulum stress [34,35]. We determined the IC50 after incubating cells with each drug for 24 h; as control comparison groups, we used F1 and F10 cells that were not CRISPR-transformed. For controlling melanoma cell growth, tunicamycin was found to be more efficacious than cisplatin. The IC50 against cisplatin was achieved at 77.5 nM (R2 = 0.83) in the S100b-suppressed F1 cells, compared to the F1 control cells (IC50 = 851.3 nM; R2 = 0.93); the IC50 against cisplatin was achieved at 138.4 nM (R2 = 0.97) in the S100b-suppressed F10 cells, compared to the F10 control cells (IC50 = 611.8 nM; R2 = 0.92) (Figure 5C). The IC50 against tunicamycin, however, was achieved at a concentration as low as 9 nM (R2 = 0.93) in the S100b-suppressed F1 cells, compared to 185.3 nM (R2 = 0.96) in the F1 control cells. In contrast, in the S100b-suppressed F10 cells, the IC50 of tunicamycin was attained at 50 nM (R2 = 0.91), compared to 165 nM (R2 = 0.96) in the F10 control cells (Figure 5D). Overall, our data suggest that S100B suppression cooperatively works with the chemotherapeutic agents that trigger cell death by evoking apoptotic responses, thus potentially sensitizing melanoma cells to these inhibitors. Overexpression of S100B and its clinicopathological relevance in melanoma has been well studied. The study reported here elucidated the role of genetic and epigenetic modifications underlying oncogenic overexpression of S100B. For instance, we demonstrated that S100B expression is significantly (p < 0.05) higher in patient-samples with gains in CN of the gene, compared to those with losses in CN of the gene (Figure 1C). However, this observation needs further validation and inclusion of more study cohorts, because we did not observe significant alterations in gene expression levels when comparing samples with losses in S100B CN and those diploid for S100B. At the epigenetic level, we investigated alterations in DNA methylation and chromatin state of the gene. We observed a moderate negative correlation between DNA methylation and expression, suggesting that the HM450 probe possibly is targeted to the promoter of the gene, where DNA methylation is inversely proportional to gene expression [36]. At the chromatin level, we observed an event of epigenetic priming at the S100B promoter, such that the TSS at the upstream promoter showed enrichment for DHS in SK-MEL-5 melanoma cells. This seems reliable because, in primary keratinocytes, the same region remains devoid of such DHS marks. The chromatin state alterations of S100B in melanoma cells could be logically ascribed to overexpression of the gene because it does not seem to be expressed in primary keratinocytes at levels comparable to those in melanoma cells. Enhanced molecular interactions between S100B and WT-p53 proteins in melanoma patient samples were demonstrated to impair WT-p53 function for tumor suppression through restricted cell cycle arrest [29], which leads to increased resistance to chemotherapeutics [11,12,37,38,39]. In the present study, we customized inactivation of S100b expression in two murine melanoma cell lines by using a CRISPR/dCas9 system that allowed for restoring intercellular levels of WT-p53 that might otherwise be bound to the S100b protein, thereby salvaging p53-mediated cell death and apoptosis. Our approach could be broadly applicable to most melanoma cases, irrespective of S100B CN gain or loss because the CRISPR/Cas9 system can intrinsically suppress multiple copies of the same gene [40]. We observed varying degrees of DK efficacy among the tested sgRNAs, specific to S100b or specific to EGFP and cell lines, which may be attributed to several factors. For instance, we observed little to no S100b-inhibition in either cell types transduced with a combination of DK plus m-SgRNA-2 or m-SgRNA-3. This could be partly due to the fact that the target region is already masked or occupied with endogenous TFs, leaving a relatively narrow window of DNA sequence available for binding sgRNAs or dCas9. We observed that the combination of m-SgRNA-1 and DK imparted the strongest suppression of S100b expression in both cell lines that were studied. However, combining DK with all three m-sgRNAs did not result in better suppression of S100b (data not shown), which could be partly related to the inefficacy of m-SgRNA-2 and m-SgRNA-3. For the current study, cells transduced with DK plus sgRNAEGFP were used as an off-target control. It was observed that induction of cells with DK alone or DK in combination with S100b-specific sgRNA-2/3 or gRNAEGFP reduced the gene expression, compared to the parental lines (Figure 2E,F), which, however, did not correlate to the changes in downstream apoptotic responses. Therefore, the generic reduction in S100b expression may be ascribed to the lentiviral effect on cells, which was not consistent with the downstream apoptotic signaling. In contrast, reduction of S100b expression was consistent with the expression of apoptosis-responsive proteins. This makes the combination of DK plus m-sgRNA-1 a unique and selective candidate for the intended purpose. S100b suppression significantly (p < 0.05) increased levels of p53 and its downstream p21 protein. However, CRISPR interference was not evident at the mRNA level for TP53 in either cell line (Figure S2). This could be explained by the fact that S100B does not act upstream of the TP53 signaling cascade and, therefore, may not have significant effects on expression of the gene. The disconnect between p53 mRNA and protein levels also aligned with results of a previous study in which siRNA-based perturbation of S100b did not alter TP53 mRNA levels but significantly increased levels of total p53 and of phosphorylated p53 [10]. Overall, our findings support the hypothesis that suppressed S100b possibly lost its affinity for intracellular p53, thereby facilitating elevated levels of WT-p53 protein. We also observed that the degree of S100b inhibition correlated with p53-mediated activation and apoptotic changes, such that S100b inactivation and apoptotic changes in the F1 cells were profound, compared to those in the F10 cells. The decreased efficacy of DK against the F10 cells may be due to intrinsically lower expression of S100b in F10, relative to F1 cells, or due to ineffective binding of m-SgRNA-1 to the target region, presumably due to occupancy by other endogenous TFs. Disparities in the degree of functional outcomes between the two cell lines also could be explained by findings from previous studies reporting differential cytogenetic properties [41]. In this case, optimization of the KRAB suppressor system by integrating additional repressors, such as MeCP2 or EZH2, may impart superior gene inactivation effects against the F10 cells [42,43]. Finally, S100b inhibition decreased cell viability (Figure 5A,B) and increased susceptibility (Figure 5C,D) of melanoma cells to the chemotherapeutics. A previous study found cisplatin to be moderately effective against melanoma patients [35]. Similar findings were observed for the tunicamycin treatment. Similar to the S100b inhibition and apoptotic responses, chemotherapeutic susceptibility was also achieved at relatively lower concentrations in F1, compared to the F10 cells. A rational follow-up of the present study is aimed at determining the efficacy and persistence of the DK/CRISPR toolbox in other human and murine melanoma lines having high levels of S100b. Another limitation of the current study is that the S100b-specific DK/CRISPR tool needs further functional validations in vivo. Accordingly, the toolbox might need further modifications such that we may need to introduce additional suppressor domains, such as MeCP2, to the KRAB domain for better suppression efficacy [43]. Nonetheless, based on the outcomes from the reported system, we can flexibly modify the current toolbox by introducing additional transcriptional repressors or epigenetic modulators for robust suppression of any gene or gene network. The proof of concept generated here strongly encourages studies of different signaling pathways involved in sustaining malignant growth in melanoma cells.

PMC10000377

Jung-Won Lee,You-Soub Lee,Min-Kyu Kim,Xin-Zi Chi,Dohun Kim,Suk-Chul Bae

Role of RUNX3 in Restriction Point Regulation

23-02-2023

RUNX3,K-RAS,R-point,cell cycle

A cell cycle is a series of events that takes place in a cell as it grows and divides. At the G1 phase of cell cycle, cells monitor their cumulative exposure to specific signals and make the critical decision to pass through the restriction (R)-point. The R-point decision-making machinery is fundamental to normal differentiation, apoptosis, and G1–S transition. Deregulation of this machinery is markedly associated with tumorigenesis. Therefore, identification of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in tumor biology. RUNX3 is one of the genes frequently inactivated in tumors by epigenetic alterations. In particular, RUNX3 is downregulated in most K-RAS-activated human and mouse lung adenocarcinomas (ADCs). Targeted inactivation of Runx3 in the mouse lung induces adenomas (ADs), and markedly shortens the latency of ADC formation induced by oncogenic K-Ras. RUNX3 participates in the transient formation of R-point-associated activator (RPA-RX3-AC) complexes, which measure the duration of RAS signals and thereby protect cells against oncogenic RAS. This review focuses on the molecular mechanism by which the R-point participates in oncogenic surveillance.

Role of RUNX3 in Restriction Point Regulation A cell cycle is a series of events that takes place in a cell as it grows and divides. At the G1 phase of cell cycle, cells monitor their cumulative exposure to specific signals and make the critical decision to pass through the restriction (R)-point. The R-point decision-making machinery is fundamental to normal differentiation, apoptosis, and G1–S transition. Deregulation of this machinery is markedly associated with tumorigenesis. Therefore, identification of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in tumor biology. RUNX3 is one of the genes frequently inactivated in tumors by epigenetic alterations. In particular, RUNX3 is downregulated in most K-RAS-activated human and mouse lung adenocarcinomas (ADCs). Targeted inactivation of Runx3 in the mouse lung induces adenomas (ADs), and markedly shortens the latency of ADC formation induced by oncogenic K-Ras. RUNX3 participates in the transient formation of R-point-associated activator (RPA-RX3-AC) complexes, which measure the duration of RAS signals and thereby protect cells against oncogenic RAS. This review focuses on the molecular mechanism by which the R-point participates in oncogenic surveillance. The major events that regulate cell proliferation occur during the G1 phase of the cell cycle. The growth of normal cells in culture is regulated by complex interactions involving growth factors, cell density, and cell attachments to substrates. Growth factors are necessary to initiate and maintain the transition through the G1 phase, leading to the S phase. Reductions in growth factor levels in cells, such as by the removal of serum, prevent the onset of the S phase [1]. However, once the cells have moved through a certain G1 decision-making period, the removal of serum no longer affects their progress through the cell cycle, with these cells proceeding through the remainder of the G1 phase and onward through S, G2, and M phases. The point in G1 at which commitment occurs and the cell no longer requires growth factors to complete the cell cycle has been termed the restriction (R)-point [1]. Once beyond the R-point, cells are committed to DNA synthesis and are independent of extracellular growth factors during the remainder of the cell cycle (Figure 1). R-point transition is regulated by R-point-associated proteins (R-proteins), including c-Myc, cyclins, CDKs, p21, p27, E2F, and pRB [2], with pRB serving as the primary molecular regulator [3]. One of the most important breakthroughs in the understanding of cell cycle regulation was the finding that mitogenic stimulation was connected to the cell cycle machinery. The expression of cyclin D and its assembly with CDK4 and CDK6 into active kinase complexes are regulated by growth factors [4,5], indicating that cyclin D is a growth factor sensor. In turn, the ability of cyclin D-dependent kinases to trigger the phosphorylation of pRB during the mid- to late-G1 phase of the cell cycle makes inactivation of the growth-suppressive function of pRB a mitogen-dependent step. pRB participates in controlling the G1/S-phase transition. Cyclin E complexes with CDK2 downstream of the cyclin D-CDK4/6 complex, with the cyclin E-CDK2 complex further phosphorylating pRB. This shift from cyclin D-CDK4/6 to cyclin E-CDK2 accounts for the loss of dependency on growth factors, indicating that the R-point lies between cyclin D-CDK4/6 and cyclin E-CDK2 (Figure 1). The mammalian G1/S cell cycle phase transition network is a highly nonlinear network that produces seemingly paradoxical results [6]. Numerous feedback loops lead to situations in which downstream events lie upstream of themselves. For example, pRB2/p130 and p27 are both involved in a negative feedback regulatory loop with cyclin E [7]. Moreover, although c-Myc expression is downstream from p21, CDKs, and E2F [8,9], c-Myc is also an upstream regulator of p21 and CDKs [10]. Similarly, the finding that cyclin E is transactivated by E2F-1 suggests that cyclin E is located downstream from pRB and E2F-1. However, cyclin E inactivates pRB and releases E2F. These positive loops ensure the irreversibility of commitments. Once expressed, cyclin E becomes independent of downstream growth-factor-dependent cyclin D1. The phosphorylation of pRB abrogates growth factor dependency, enabling the cells to pass through the R-point and commit to completing the remaining phases of the growth cycle [11]. The nonlinear networks include c-Myc, cyclins, p21, CDKs and E2F, which play key roles in R-point regulation. Therefore, the nonlinear networks producing seemingly paradoxical results appears to be associated with R-point, which regulates cell fate. The R-point lies between cyclin D–CDK4/6 and cyclin E–CDK2, suggesting that cyclin E–CDK2 is responsible for pushing the cells from the R-point through to the remainder of the G1 phase. Therefore, cyclin E–CDK2 complexes should form only after the cell is committed to proliferation at the R-point. Although cyclin D expression is induced earlier than cyclin E expression, the times of their expression were found to overlap considerably [3] (Figure 1), suggesting that the expression of cyclin E does not solely determine the time of exit from the R-point. Cyclin E–CDK2, however, is activated only after R-point commitment to proliferation. p21WAF1/CIP1/Kip (hereafter p21), originally identified as an inhibitor of CDKs [12], is encoded by an immediate-early gene, with p21 mRNA peaking approximately 2 h after stimulation with serum or growth factor [13]. However, the biological importance of the mitogen-stimulated immediate early induction of p21 was not understood at that time. Subsequent studies revealed that p21 is not a simple CDK inhibitor; rather, members of the p21 family were found to activate cyclin D–CDK4/6 by promoting the association of its component proteins, while inhibiting cyclin E–CDK2 [14,15,16]. Recently, it was shown that only the tyrosine-phosphorylated p21 family activates cyclin D–CDK4/6, and this tyrosine phosphorylation occurs in response to mitogenic signaling [17]. Therefore, p21, induced early after mitogenic stimulation, promotes cell entry into the R-point at the early-/mid-G1 phase by activating cyclin D–CDK4/6, but prevents further progression through the R-point by inhibiting cyclin E–CDK2. That is, cells remain at the R-point while p21 is expressed, but exit from the R-point when p21 expression is attenuated, indicating that the p21 gene is involved in R-point regulation. Understanding the molecular mechanisms underlying the mitogen-stimulated immediate early induction of p21 should enable greater understanding of the mechanisms underlying R-point regulation. RUNX3, which plays pivotal roles in lineage determination and functions as a tumor suppressor, is frequently inactivated in various types of human cancers, including stomach and lung cancers [18]. Conditional deletion of Runx3 from mouse lungs results in the development of lung adenomas (ADs), with these pre-invasive lesions progressing to adenocarcinomas (ADCs) following the additional introduction of heterozygous oncogenic K-Ras mutations [19]. Deletion of Runx3 from mouse lung epithelial cells results in the development of lung Ads, and Runx3−/− mouse embryonic fibroblasts (MEFs) develop into tumors without oncogenic mutations in nude mice [20]. Although these results indicated that cells acquire tumorigenic activity following the deletion of Runx3, the mechanism involved remained unclear. An important clue was provided by an analysis of the minimum serum exposure time required for Runx3−/− and Runx3+/+ MEFs to progress to S phase. Runx3−/− MEFs required only 1–2 hours, whereas Runx3+/+ MEFs required at least 4 hours [20]. The short exposure time required by Runx3−/− MEFs was similar to that required by Rb−/− MEFs [21]. Notably, Runx3 deletion abolished the immediate-early induction of p21 [20], suggesting that Runx3 is essential for R-point regulation in MEFs, and that p21, a key regulator of the R-point, is a target of Runx3. These findings were supported by results showing that the expression of Runx3 in Runx3−/− MEFs restored the R-point and the immediate-early induction of p21, while abolishing cell tumorigenicity [20]. In addition, the R122C mutation (substitution of arginine 122 to cysteine) in RUNX3, which was identified from human gastric cancer [22], disrupts the R-point [23]. Thus, the tumorigenicity acquired by Runx3 deletion was associated with a deregulation of the R-point. Cell commitment at the R-point involves the regulation of several hundred R-point-associated genes, which are induced by exposure to serum for 1–2 h and subsequently suppressed [20]. The p14-ARF (hereafter ARF) and p21 genes are included among the R-point-associated genes [19,20,24], but their mechanism of induction early after mitogenic stimulation was originally undetermined. The induction of expression of silent genes requires the target sites within their regulatory regions to be bound de novo by transcription factors. Transcription factors that bind to condensed chromatin independently of other factors, modulate chromatin accessibility, and regulate gene transcription are known as pioneer factors [25,26,27]. To mediate these activities, pioneer factors require a complex network of other proteins, including coactivators, corepressors, histone-modifying complexes, chromatin-remodeling complexes, mediator complexes, and the basal transcription machinery. For example, proteins of the Trithorax group (TrxG) modify histones to activate transcription. TrxG proteins can be classified into two categories: histone modifiers [28] and nucleosome remodelers [29]. TrxG histone modifiers include members of the mixed-lineage leukemia (MLL) family, which methylate H3 at lysine 4 (H3K4-me3, -me2, and -me1), enhancing transcriptional activation. In contrast, TrxG nucleosome remodelers include the SWI–SNF complex, which facilitates binding of transcription factors and the basal transcription machinery. Mediator complexes transduce signals from the transcription activators bound to enhancer regions in the transcription machinery, which is assembled at promoters, as the preinitiation complex, to control the initiation of transcription [30]. Members of the bromodomain (BRD) family of proteins (BRDs) are components of the mediator complex. BRDs are integral to transcription through their interactions with mediator coactivator complexes, which are required for the transcription of various genes [31,32]. BRDs possess two bromodomains, BD1 and BD2, which interact with acetylated histones and acetylated transcription factors. Runx3 plays a key role in recruiting these chromatin modulators to activate signal-dependent R-point-associated gene expression at the correct target loci at the right time. Immediately after mitogenic stimulation, Runx3 binds to its target chromatin loci and recruits the pRB–E2F complex and p300 acetyltransferase [20]. The interactions are promoted by RAS-activated MEK1 [24]. Runx3 and histones are acetylated by p300 acetyltransferase, with BRD2 subsequently interacting with acetylated Runx3 through its BD1 domain, and with acetylated histone H4 through its BD2 domain [24]. Therefore, RUNX3 guides the pRB–E2F complex and p300 to target loci, with BRD2 binding both acetylated RUNX3 and acetylated histones through its two bromodomains prior to the R-point (Figure 2A). Subsequently, the C-terminal region of BRD2 interacts with the SWI/SNF chromatin-remodeling complex, MLLs, which act as activated histone modifiers, and TFIID complexes, representing basal transcription machinery [24]. Thus, at the R-point, Runx3 forms a large complex, called the R-point-associated RUNX3-containing activator complex (Rpa-RX3-AC), at target chromatin loci (Figure 2B). This Rpa-RX3-AC complex subsequently opens the chromatin structures of the target loci by replacing the inhibitory histone H3K27-me3 with the activating histone H3K4-me3. Runx3 is an enhancer binding protein, and TFIID is a promoter-binding complex. Therefore, the enhancer interacts with the promoter through the Rpa-RX3-AC complex during the R-point at the target loci, inducing the expression of R-point-associated genes [24] (Figure 2B). This Rpa-RX3-AC complex is maintained, while the RAS-MEK pathway is activated [24]. The activities of RUNX3, including its association with condensed chromatin, its modulation of chromatin accessibility, and its activation of gene expression, fulfill the characteristics of a pioneer factor, making RUNX3 a pioneer factor of the R-point. Hypo-phosphorylated pRB is a component of the Rpa-RX3-AC complex that forms 1–2 h after mitogenic stimulation and contributes to R-point commitment [20]. Soon after, CDK4 is recruited to the target locus by interacting with RUNX3. p21, which is induced by the Rpa-RX3-AC complex, facilitates CDK4-–cyclin D1 interactions. Thus, pRB and E2F1, along with CDK4, cyclin D1, and p21, which play key roles in cell cycle regulation, are recruited to the R-point-associated target loci [24] (Figure 2C). At these loci, pRB is phosphorylated at Ser-795 by cyclin D1–CDK4/6. Subsequently, after the R-point, 4 h after mitogenic stimulation, the pRB–E2F1 complex is released from the Rpa-RX3-AC complex, and the expression of R-point-associated target genes is suppressed [24] (Figure 2C). Activation of CDK4 by the cyclin D1–CDK4 interaction triggers the suppression of previously activated R-point-associated target genes, including p21 and ARF. Proteins in the polycomb group (PcG) modify histones to suppress gene transcription. There are two kinds of PcG complexes, called polycomb repressor complexes 1 and 2 (PRC1 and PRC2). Both complexes consist of multiple proteins, with PRC1 containing BMI1 and ring finger protein 1 (RING1) or 2 (RNF2) [33], and PRC2 containing EED and an enhancer of zeste homologs (EZH1 and EZH2), which trimethylate H3 at lysine 27 (H3K27-me3), a characteristic of inactive chromatin [34]. Cyclin D1, induced soon after mitogenic stimulation, forms a complex with HDAC4 and PRC1 [24]. Therefore, when cyclin D1 binds to CDK4, HDAC4 and PRC1 are also recruited to the Rpa-RX3-AC complex. Because p300-mediated RUNX3 acetylation and histone acetylation are effectively abolished by HDAC4 [35], HDAC4 may play a key role in the deacetylation of RUNX3 and histones, causing the release of BRD2 and other BRD2-associated proteins. Inactivation of chromatin is associated with HDAC-mediated histone deacetylation and H2A ubiquitination at Lys-119, mediated by RNF2, a component of PRC1 [33]. Consistently, H4K12 acetylation was reduced, and H2A-K119-Ub was enriched at the p21 and ARF loci 4–8 h after stimulation [24]. These results demonstrate that cyclin D1, HDAC4, and PRC1 bind to the Rpa-RX3-AC complex through interactions with CDK4. These interactions are facilitated by Rpa-RX3-AC-induced p21, which contributes to the inactivation of chromatin at target loci by deacetylating H4K12 and ubiquitinating H2A (Figure 2C). At 4–8 h after mitogen stimulation, RUNX3 and BRD2 existed in separate complexes: RUNX3 formed a complex with Cyclin D1, HDAC4, and PRC2, which remained bound to target chromatin loci, whereas BRD2 formed the BRD2–PRC1 complex, which was released from the loci [24] (Figure 2C). Moreover, H3K27-me3 was enriched at these loci. EZH2 is a component of PRC2 that mediates the modification of the inhibitory histone H3K27-me3, suggesting that PRC2, associated with RUNX3, may play a key role in the inactivation of chromatin loci. Because the RUNX3–Cyclin D1–HDAC4–PRC2 complex inactivates chromatin, the complex was named as the R-point–associated RUNX3-containing repressor complex (Rpa-RX3-RE) (Figure 2C). Many studies have reported that K-RAS mutations, the genetic alterations most frequently detected in various cancers, are an early event responsible for the development of lung ADs [36,37,38]. By contrast, the ARF-p53 pathway has been found to effectively defend cells against aberrant oncogene activation [39,40,41], with p53 mutations being a hallmark of cancer and a prevalent feature of human cancers [42]. Therefore, the development of K-RAS-activated cancer might be accompanied by the inactivation of the ARF-p53 pathway. These findings, however, are contradicted by results in human cancers. Evaluation of the key genetic and epigenetic alterations that are responsible for clonal expansion following each step of colon tumorigenesis has shown that colon ADs are initiated by the inactivation of Adenomatous polyposis coli (APC) [43,44] (Figure 3). Moreover, K-Ras is activated after AD development, with the loss of p53 occurring at an even later stage. Although the p53 pathway can defend against colon ADCs, it remains unclear as to whether this pathway can defend against K-Ras-activated colon ADs, and, if so, whether the p53 pathway can defend against high-grade, but not low-grade, cancers. These questions have been partly answered in lung cancer. The progression of lung ADCs from adenomatous growth to carcinomas was found to be similar to the multistep tumorigenesis pathway in colon cancer (Figure 3). Although the activation of K-Ras and inactivation of p53 are frequently detected in lung ADCs, the order of these molecular events has not been clearly established in human lung cancer patients. Rather, the relationship between K-Ras activation and p53 inactivation was analyzed by restoring p53 expression in K-Ras-activated and p53-inactivated mouse lung tumors. p53 restoration eliminated only Kras-activated lung ADCs, leaving lung ADs intact in these mice models [45,46]. These results suggested that p53 is inactivated during late-stage AD or early-stage ADC, later than K-Ras activation; this is similar to findings in colon cancer (Figure 3). Previous studies have speculated that this was due to inherent limits in the capacity of the Arf-p53 pathway to respond to a persistent low level of oncogenic K-Ras activity [45,46,47]. However, another possibility remained, that the failure of eliminating lung AD by p53 restoration may be due to disruption of a hidden molecular mechanism responsible for sensing the aberrant persistence of oncogenic signals. The initial step of colon AD development is the inactivation of APC, not the activation of K-Ras. Similarly, RUNX3 is frequently inactivated in human atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC), which correspond to mouse lung ADs, and inactivation of Runx3 induces lung ADs in mice [19]. A lone, heterozygous oncogenic K-Ras mutation in a large number of cells can also lead to the development of lung ADs, although only a very small number of these cells in a specific cellular context are transformed by oncogenic K-Ras [48], indicating that certain spontaneously occurring rare molecular events are involved in the development of K-Ras-activated lung cancer. These rare molecular events may occur in only a small percentage of K-Ras-activated cells. Thus, the likelihood of these hidden molecular events can be reduced by reducing the number of K-Ras-activated cells. Indeed, these same K-Ras mutations, with or without p53 inactivation, in an extremely small number of cells, failed to induce any pathologic lesions for up to 1 year [49]. In contrast, when Runx3 was inactivated, and K-Ras was activated by the same targeting method, lung ADCs and other tumors were rapidly induced and caused lethality in all the targeted mice within 3 months [49]. Therefore, under physiological conditions, in which oncogenic mutations are very rare, K-Ras activation alone is not sufficient, whereas its combination with Runx3 inactivation is sufficient, for lung cancer development. In addition, evaluation of a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS-driven human lung tumors showed that Runx3 was inactivated in both ADs and ADCs, whereas K-Ras was activated only in ADCs [49]. Mutations in p53 were an even later event than K-Ras activation [49]. Therefore, the order of the molecular events for the development of mouse lung AD/ADC was likely Runx3 inactivation → activation of K-Ras → loss of p53 (Figure 3). The universal process of malignant transformation involves both genetic damage and oncogenic signaling. These two stresses are signaled to p53 through different pathways. Based on this, p53 plays two important roles in cells: “defense against genome instability”, which consists of sensing and reacting to DNA damage through ATM/ATR kinases, and “defense against oncogene activation”, which consists of responding to oncogenic signaling through the p53-stabilizing protein ARF [50] (Figure 4A). Recent genetic evidence in mice indicates that the ARF-dependent activation of p53 is critical for early-stage p53-mediated tumor suppression. In contrast, ATM/ATR-dependent activation of p53 protects late-stage tumors [50]. Therefore, p53 mutations at relatively late stages of colon and lung tumorigenesis may be associated with the disruption of ATM/ATR-dependent p53 activation (Figure 4B). Nevertheless, K-RAS-activated AD cells have been found to proliferate in the presence of wild-type ARF and p53. Because heterozygous oncogenic K-Ras mutations alone in a small number of cells did not induce lung AD [49], the process of AD development may require the inactivation of the ARF-p53 pathway. The mechanism underlying the inactivation of the ARF-p53 pathway in ADs was unclear. However, Runx3 is inactivated in most K-Ras-activated mouse and human lung ADs [19], with Runx3 inactivation abrogating the R-point program, which plays a key role in ARF induction in response to oncogenic K-RAS [24]. Thus, Runx3 inactivation may inactivate the ARF-p53 pathway in lung ADs, thus providing a mechanism underlying the proliferation of K-Ras-activated lung AD cells in the absence of mutated p53 (Figure 4B). Mitogenic signaling activates the GTPase activity of RAS, which decreases to the basal level soon after the signal is transduced to downstream kinase pathways. Oncogenic RAS is a constitutively active form, with GTPase activity not being downregulated. Therefore, heterozygous RAS mutations yield cells with 50% of the maximum level of RAS activity [19] (Figure 5A). The ability of cells to sense the duration of 50% rather than maximal RAS GTPase activity may confer protection against oncogenic RAS-induced abnormal proliferation. The ability of cells to recognize the aberrant persistence of RAS activity, however, was unclear. For example, oncogenic K-Ras expressed at the endogenous level did not activate the ARF-p53 pathway in mouse lungs [20,45,46]. Based on these results, it had been considered that the ARF-p53 pathway does not respond to the aberrant persistence of RAS activity, although the pathway responds to only abnormally high levels of RAS activity [45,46,47]. Mammals, however, were later found to have evolved an effective defense mechanism against a persistent low level of RAS activity [19,24]. When K-RAS is activated by normal mitogenic stimulation, RUNX3 forms Rpa-RX3-AC complexes in a MAPK activity-dependent manner; these complexes transiently induce ARF, which in turn transiently stabilizes p53. Soon after the mitogenic surge, MAPK activity is reduced, converting Rpa-RX3-AC complexes to Rpa-RX3-RE complexes and repressing ARF expression (Figure 5B). Mitogen-stimulated transient activation of the ARF–p53 pathway does not affect the cell cycle because it occurs only 1–3 hours after mitogenic simulation, and is then silenced before the G1/S checkpoint. In contrast, when K-RAS is constitutively activated, the Rpa-RX3-AC complex is maintained, and the expression of ARF and p53 is maintained until the G1/S checkpoint, leading to cell death (Figure 5B). These results indicate that cells can effectively defend against an endogenous level of RAS activity, and that the Rpa-RX3-AC complex functions as a sensor and as a decision maker regarding the abnormal persistence of RAS activity [24]. H460 human lung cancer cells were used to determine whether the Rpa-RX3-AC complex-driven activation of the ARF-p53 pathway was sufficient to defend against oncogenic K-RAS-induced lung tumorigenesis. In these cells, K-RAS was heterozyously mutated but not amplified, and RUNX3 was inactivated by hyper-methylation. Despite these cells having wild-type ARF and p53, Rpa-RX3-AC complexes were not formed, and the ARF-p53 pathway was not activated. In contrast, exogenous expression of RUNX3 led to the formation of Rpa-RX3-AC complexes, which activated ARF expression and stabilized p53, thereby inducing cell apoptosis [24]. Expression of mutant RUNX3, which is unable to form Rpa-RX3-AC complexes, failed to activate ARF expression [24]. Therefore, failure of ARF-p53 pathway activation in H460 cells was due, not to the absence of a mechanism for sensing low endogenous levels of oncogenic K-Ras activity, but to the disruption of the R-point by RUNX3 inactivation. These findings indicate that cells can recognize the aberrant persistence of RAS activity through the R-point program and kill these cells by activating the ARF-p53 pathway. Although several important regulators of cell differentiation govern lung development, deregulation of the differentiation program was generally insufficient to induce AD. Runx3 is inactivated in nearly all the human and mouse lung ADs and, to date, Runx3 is the only gene whose inactivation has been reported to induce lung AD [19]. Cancer development is considered to be a biological process that resembles Darwinian evolution: random mutations create genetic variability in a cell population, and the force of selection favors the outgrowth of individual mutant cells that happen to be endowed with advantageous traits. Based on a combination of Darwinian theory and the concept of multistep tumor progression, tumorigenesis is now understood as a succession of clonal expansions [43,44]. Great numbers of cells are required to select cells endowed with advantageous traits. The random inactivation of Runx3 in normal cells results in a deregulation of the differentiation program [51] and disruption of the R-point program [24]. Deregulation of the differentiation program is likely responsible for the development of AD, although it is not sufficient, whereas disruption of the R-point program likely results in the abrogation of the ARF-p53 pathway-mediated oncogene surveillance mechanism, enabling the subsequent selection of K-RAS-mutated cells. Although K-RAS-induced lung cancer development can proceed via multiple pathways, the high frequency of RUNX3 inactivation in K-RAS-induced mouse and human lung ADCs suggests that a major pathway involves R-point disruption by RUNX3 inactivation prior to K-RAS activation. To date, RUNX3 is the only gene whose inactivation has been shown to be sufficient for both the induction of AD and abrogation of the R-point. These steps may result from multiple molecular events (i.e., one involving each pathway) or a single molecular event, such as RUNX3 inactivation. Obviously, the probability of deregulation is much higher for events involving a single gene than multiple genes, explaining the importance of RUNX3 in lung tumorigenesis. Tumor suppressor genes are defined as genes that “help control cell growth,” indicating that tumor suppressors act broadly to inhibit diverse aspects of both normal and neoplastic physiology. By contrast, oncogenes are genes activated by mutations or overexpression of genes that act dominantly to induce tumorigenesis. These terms, however, can overlap, as some proteins with various functions affecting a spectrum of cellular outcomes can enhance and/or suppress tumor pathogenesis. Although RUNX3 generally acts as a tumor suppressor, RUNX3 expression can be enhanced during the course of progression of some cancers, with this gene playing a tumor-promoting or oncogenic role. For example, the acquired expression of RUNX3 in head and neck carcinoma correlates with poor histologic differentiation, invasion, and metastasis [52]. High RUNX3 expression has also been observed in ovarian cancer, basal cell carcinoma, and skin cancers [18]. Moreover, Runx3 has been shown to inhibit the early-stage growth of pancreatic cancers but facilitates their metastatic progression at early-stage [53]. The ability of RUNX3 to exhibit both tumor-suppressing and tumor-promoting activities has been associated with the R-point, a decision-making program for cell proliferation, differentiation, senescence, and apoptosis. The R-point could be deregulated by either the abnormally high expression or inactivation of RUNX3. For example, the tumor suppressors p21 and ARF are induced at the R-point and then subsequently suppressed, with RUNX3 playing key roles in both programs (Figure 2B,C). If RUNX3 is inactivated, p21 and ARF are not induced, even when an oncogene is activated due to the failure of Rpa-RX3-AC complex formation. In this context, RUNX3 functions as a tumor suppressor. If, however, RUNX3 is overexpressed, and Rpa-RX3-AC complex formation is not possible, then RUNX3 may preferentially form Rpa-RX3-RE complexes, suppressing the expression of p21 and ARF. Under these conditions, RUNX3 functions as an oncogene. Although RUNX3 is the only gene to date that has been shown to act as a pioneer factor of the R-point, many pioneer factors of the R-point are likely present in various types of cells. For example, RUNX1 and RUNX2, which are master regulators of hematopoiesis and osteogenesis, respectively [18,54], are involved in R-point regulation [55]. Other master regulators might also play roles in R-point regulation, since development is a sequential process with decisions made at the R-point. Many key R-point regulators may also exhibit ambipotent and context-dependent effects on tumorigenesis. Therefore, we propose designating RUNX3 and similar acting proteins as “decision makers,” with their activities as tumor suppressors or oncogenes being dependent on the intactness of the decision-making machinery in cells. A tumor is defined as an abnormal mass of tissue that forms when cells divide more than they should or do not die when they should. The determination of whether a cell divides or dies is made at the R-point. In theory, cells that make a correct decision at the R-point and correctly execute this decision cannot develop into tumors. Deregulation of the R-point decision-making machinery is involved in the formation of most, if not all, types of tumors [3], suggesting the importance of the R-point in tumor development. Understanding the molecular mechanisms that underlie R-point commitment should provide important insights into how normal cells become tumorigenic. This review summarizes the method by which the R-point distinguishes normal from oncogenic RAS and determines pathways for cell survival or death. Several fundamental questions underlying cancer development remain to be resolved, including the mechanisms underlying tumor initiation and its rapid recurrence after treatment with anticancer drugs. If oncogene activation is solely responsible for tumor development, then inhibition of the activated oncogene would be able to cure that cancer without the likelihood of recurrence. Although targeted agents that inhibit activated oncogenes have yielded clinical responses, almost all of these malignancies recur. For example, gefitinib was found to effectively eliminate EGFR-mutated lung ADCs at the beginning of therapy, but the cancers recurred in 90% of patients within 2 years [56]. Moreover, although oncogenic K-RAS-specific inhibitors have been developed [57], some cancer cells bypass the effects of these inhibitors and resume proliferation [58]. In addition, the knockdown of oncogenic K-Ras in a mouse lung cancer model was found to result in rapid tumor recurrence, not because the gene knockdown was unsuccessful, but because other oncogenes were activated [59]. Because tumor frequency is much lower in normal than in tumor-regressed mice, the rapid activation of secondary oncogenes in the latter suggests that a defense mechanism can be abrogated in established tumors. However, efforts to restore p53 expression in K-Ras-activated mouse lung cancers eliminated only malignant ADCs and failed to eliminate ADs [45,46]. Therefore, it is of great therapeutic importance to understand as to why cancers recur, despite the effective inhibition of the activated oncogene. Recurrence of lung cancer is due primarily to persistent early lesions that are resistant to oncogene inhibitors. These early lesions do not contain activated oncogenes. Therefore, to eradicate cancers, it is necessary to understand their mechanisms of initiation. Inactivation of RUNX3 is thought to be responsible for the initiation of lung ADs, as well as for abrogating the R-point-regulating ARF-p53 pathway. The proliferation of K-RAS-activated lung AD cells with wild-type ARF and p53 results from RUNX3 inactivation, which abrogates the ARF-p53 pathway in lung ADs. Normal cells recognize the aberrant persistence of oncogenic K-RAS signals through their RUNX3-containing R-point-associated activator (Rp-RX3-AC) complexes, which sense the duration of RAS signals and regulate the ARF-p53 pathway. p53 deletions may be required at later stages of AD for abrogation of the ATM/ATR-p53 pathway. K-Ras-activated mouse lung ADs acquire secondary oncogene activation rapidly, because R-point associated oncogene surveillance mechanisms are abrogated in the ADs. RUNX3 restoration has been shown to eliminate K-RAS-activated tumors in a human lung cancer cell line. It would be exciting indeed if Runx3 restoration eliminates K-Ras-activated lung cancer in an animal model. If that turns out to be the case, RUNX3 will be a promising target for curative cancer therapy.

PMC10000383

Amol Gupta,Razelle Kurzrock,Jacob J. Adashek

Evolution of the Targeted Therapy Landscape for Cholangiocarcinoma: Is Cholangiocarcinoma the ‘NSCLC’ of GI Oncology?

03-03-2023

cholangiocarcinoma,targeted therapy,molecular biomarkers

Simple Summary In the past 20 years, the development of targeted therapies that can be matched to a tumor’s molecular and immune abnormalities has resulted in the improvement of outcomes for patients suffering from advanced aggressive malignancies. Remarkably, non-small cell lung cancer (NSCLC) has become the poster child for a lethal malignancy in which numerous molecular aberrations have become druggable. Similar to NSCLC, there are limited responses in cholangiocarcinoma (CCA) to conventional chemotherapy. Next-generation sequencing has identified novel genomic alterations in CCA that vary between patients. Gene- and immune-targeted therapies are leading to a new era of precision/personalized medicine for patients with CCA. Herein, we review the current status of molecularly matched precision-targeted therapy for CCA. Abstract In the past two decades, molecular targeted therapy has revolutionized the treatment landscape of several malignancies. Lethal malignancies such as non-small cell lung cancer (NSCLC) have become a model for precision-matched immune- and gene-targeted therapies. Multiple small subgroups of NSCLC defined by their genomic aberrations are now recognized; remarkably, taken together, almost 70% of NSCLCs now have a druggable anomaly. Cholangiocarcinoma (CCA) is a rare tumor with a poor prognosis. Novel molecular alterations have been recently identified in patients with CCA, and the potential for targeted therapy is being realized. In 2019, a fibroblast growth factor receptor 2 (FGFR2) inhibitor, pemigatinib, was the first approved targeted therapy for patients with locally advanced or metastatic intrahepatic CCA who had FGFR2 gene fusions or rearrangement. More regulatory approvals for matched targeted therapies as second-line or subsequent treatments in advanced CCA followed, including additional drugs that target FGFR2 gene fusion/rearrangement. Recent tumor-agnostic approvals include (but are not limited to) drugs that target mutations/rearrangements in the following genes and are hence applicable to CCA: isocitrate dehydrogenase 1 (IDH1); neurotrophic tropomyosin-receptor kinase (NTRK); the V600E mutation of the BRAF gene (BRAFV600E); and high tumor mutational burden, high microsatellite instability, and gene mismatch repair-deficient (TMB-H/MSI-H/dMMR) tumors. Ongoing trials investigate HER2, RET, and non-BRAFV600E mutations in CCA and improvements in the efficacy and safety of new targeted treatments. This review aims to present the current status of molecularly matched targeted therapy for advanced CCA.

Evolution of the Targeted Therapy Landscape for Cholangiocarcinoma: Is Cholangiocarcinoma the ‘NSCLC’ of GI Oncology? In the past 20 years, the development of targeted therapies that can be matched to a tumor’s molecular and immune abnormalities has resulted in the improvement of outcomes for patients suffering from advanced aggressive malignancies. Remarkably, non-small cell lung cancer (NSCLC) has become the poster child for a lethal malignancy in which numerous molecular aberrations have become druggable. Similar to NSCLC, there are limited responses in cholangiocarcinoma (CCA) to conventional chemotherapy. Next-generation sequencing has identified novel genomic alterations in CCA that vary between patients. Gene- and immune-targeted therapies are leading to a new era of precision/personalized medicine for patients with CCA. Herein, we review the current status of molecularly matched precision-targeted therapy for CCA. In the past two decades, molecular targeted therapy has revolutionized the treatment landscape of several malignancies. Lethal malignancies such as non-small cell lung cancer (NSCLC) have become a model for precision-matched immune- and gene-targeted therapies. Multiple small subgroups of NSCLC defined by their genomic aberrations are now recognized; remarkably, taken together, almost 70% of NSCLCs now have a druggable anomaly. Cholangiocarcinoma (CCA) is a rare tumor with a poor prognosis. Novel molecular alterations have been recently identified in patients with CCA, and the potential for targeted therapy is being realized. In 2019, a fibroblast growth factor receptor 2 (FGFR2) inhibitor, pemigatinib, was the first approved targeted therapy for patients with locally advanced or metastatic intrahepatic CCA who had FGFR2 gene fusions or rearrangement. More regulatory approvals for matched targeted therapies as second-line or subsequent treatments in advanced CCA followed, including additional drugs that target FGFR2 gene fusion/rearrangement. Recent tumor-agnostic approvals include (but are not limited to) drugs that target mutations/rearrangements in the following genes and are hence applicable to CCA: isocitrate dehydrogenase 1 (IDH1); neurotrophic tropomyosin-receptor kinase (NTRK); the V600E mutation of the BRAF gene (BRAFV600E); and high tumor mutational burden, high microsatellite instability, and gene mismatch repair-deficient (TMB-H/MSI-H/dMMR) tumors. Ongoing trials investigate HER2, RET, and non-BRAFV600E mutations in CCA and improvements in the efficacy and safety of new targeted treatments. This review aims to present the current status of molecularly matched targeted therapy for advanced CCA. In the past two decades, molecular targeted and immune therapy has revolutionized the treatment landscape of several malignancies. Recent findings highlighted that matched targeted therapy improved the response rate and prolonged the survival of patients with advanced cancers [1,2]. Some of the most notable achievements are in non-small cell lung cancer (NSCLC), a disease for which the number of actionable biomarkers has increased rapidly. Indeed, NSCLC is now almost a poster child for the benefits of precision medicine, with almost 70% of NSCLCs having a biomarker-based therapy (including but not limited to EGFR and ERBB2 alterations, mismatch repair gene defects, high tumor mutational burden, BRAF V600E, NTRK fusions, ALK fusions, ROS1 fusions, and RET alterations) [3,4,5,6,7,8,9,10]. Moreover, molecular diagnostics have supported the development of drugs and therapeutic antibodies targeting specific receptors, antigens, or molecular pathways crucial for tumor cell proliferation and invasion, tumor growth, immunity, and metastases across various malignancies [11]. Cholangiocarcinoma (CCA) is a promising candidate for targeted therapy due to its diverse molecular features [12]. The incidence of CCA is low in the Western world, with between 0.35 and 2 cases per a 100,000 population per year [13]. The global incidence of CCA has steadily increased over the last 30 years, from 0.1 to 0.6 cases per a 100,000 population [13]. CCA is a highly aggressive malignancy, with a 5-year OS for locally advanced or metastatic disease of less than 10% [14]. CCA arises from the intrahepatic and extrahepatic biliary epithelium [15]. Anatomically, 60–70% of cases are classified as perihilar CCA (pCCA); 20–30% of cases are distal CCA (dCCA); and 5–10% of cases are intrahepatic CCA (iCAA) [15]. Current population statistics show an increased prevalence of CCA [15]. In early-stage CCA, the primary treatment includes surgical resection and adjuvant chemotherapy, while systemic chemotherapy is the standard treatment for advanced-stage CCA [16]. Patients with CCA often present with late-stage disease, and the prognosis is poor [16]. Molecular techniques, including next-generation sequencing (NGS), have identified novel mutations in tumors from patients with CCA (Figure 1) [17]. Patients with CCA show substantial variations in their molecular profiling and genetic aberrations according to their anatomic locations (Table 1). Promising therapeutic molecular targets for CCA have been identified and include isocitrate dehydrogenases 1 and 2 (IDH1 and 2) and products of fusions of the fibroblast growth factor receptor 2 (FGFR2) gene [17]. In 2019, the US Food and Drug Administration (FDA) granted accelerated approval for pemigatinib, an FGFR2 inhibitor, as the first targeted therapy for locally advanced or metastatic iCCA with FGFR2 fusions or rearrangement [18]. Another approval for a targeted therapy specifically for CCA was on 28 May 2021 [19]. The FDA granted accelerated approval to infigratinib, a kinase inhibitor approved for adults with previously treated, locally advanced, unresectable, or metastatic CCA with an FGFR2 gene fusion or rearrangement [19]. This specific approval for CCA required confirmation that an FDA-approved diagnostic test detected FGFR2 gene fusion or rearrangement in CCA [19]. More recently, the FDA granted accelerated approval to futibatinib, an irreversible FGFR1-4 Inhibitor, for previously treated, locally advanced, unresectable, or metastatic CCA with an FGFR2 gene fusion or rearrangement on 30 September 2022 [20]. In support of the recent developments in targeted therapies, in August 2021, the FDA approved ivosidenib as a targeted therapy for adult patients with unresectable locally advanced or metastatic CCA with a mutation in the IDH1 gene detected by an FDA-approved diagnostic test [21]. Finally, ALK and ROS1 mutations occur in between 3% and 9% of patients with CCA, and ALK-positive and ROS1-positive CCA may also be treated with ALK inhibitors [22]. There are also several tumor-agnostic approvals that encompass CCA. For example, in May 2017, the FDA granted accelerated approval for the therapeutic monoclonal antibody, pembrolizumab, for microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) unresectable or metastatic solid tumors that have progressed despite prior treatment [36]. In 2020, pembrolizumab received FDA approval for adults and children with high tumor mutational burden (TMB-H) solid tumors [37]. In August 2021, accelerated approval was granted for dostarlimab for adult patients with recurrent or advanced solid tumors identified as mismatch repair deficient (dMMR) as determined by an FDA-approved diagnostic test [38]. In 2018 and 2019, two therapies targeting neurotrophic tyrosine receptor kinase (NTRK) gene fusion, entrectinib and larotrectinib, were approved for locally advanced or metastatic solid tumors [39,40]. In June 2022, the FDA approved dabrafenib combined with trametinib to treat unresectable or metastatic solid tumors with a BRAFV600E mutation [41]. This review aims to present and discuss the advances in molecular targeted therapy for patients with advanced CCA. Molecular profiling of solid tumors has identified clinically actionable fusions of the NTRK1, NTRK2, and NTRK3 genes, which encode neurotrophic tropomyosin receptor kinase (NTRK) [42]. NTRK fusion products activate the TRK gene and, subsequently, the downstream signaling pathways, PI3K and MAPK, leading to tumor cell proliferation and invasion [43,44]. Therefore, NTRK inhibitors are promising targeted therapies for patients with NTRK fusion-positive cancers and have shown antitumor responses in NCSLC, melanoma, and other tumors [43,44]. NTRK gene fusions have been identified in 1–3% of patients with CCA [45]. Table 2 shows the results of pivotal clinical trials that assessed the outcomes of NTRK inhibitors in NTRK fusion-positive metastatic or unresectable locally advanced solid tumors. Larotrectinib is a first-generation, highly selective pan-NTRK competitive inhibitor that suppresses cancer cell proliferation [62]. It has shown immediate, robust, and long-lasting anticancer efficacy in pediatric and adult patients with solid tumors harboring TRK fusions [49]. Drilon and colleagues conducted a phase 1 trial in adults (NCT02122913), a phase 1/2 trial in children (NCT02637687), and a phase 2 trial in children and adults (NCT02576431) with locally advanced or metastatic solid tumors who had received previous standard systemic therapy and were then given larotrectinib (100 mg twice daily), see Table 1 [49]. A total of 55 patients with 17 TRK fusion-positive tumor types included two patients (4%) with CCA [49]. The objective response rate (ORR) was high, at 80% (95% CI, 67–90) [49]. At one year, 55% of patients were still progression-free [49]. The median duration of response (MDR) and the progression-free survival (PFS) remain unmet [49]. The most common toxicities ≥ grade 3 included a raised ALT or AST level (9%), anemia (3.6%), reduced neutrophil count (3.6%), and nausea (3.6%) [49]. Based on these findings, larotrectinib was granted accelerated approval by the FDA in November 2018 for adult and pediatric patients with NTRK-positive solid malignant tumors, either metastatic or where surgical resection is unfeasible due to severe morbidity, who have progressed on systematic therapy [39]. Larotrectinib is also approved for patients with no satisfactory alternative treatments [39]. Currently, the MD Anderson Cancer Center and the National Cancer Institute (NCI) are conducting a phase 2 trial to investigate the efficacy of larotrectinib in previously treated patients with locally advanced or metastatic solid tumors and NTRK gene amplification (NCT04879121). Another ongoing phase 2 trial aims to assess the efficacy of larotrectinib in pediatric patients with relapsed or refractory advanced solid tumors with NTRK gene fusion (NCT03213704), Table 3. Entrectinib is another selective pan-TRK inhibitor with activity against ROS1 and ALK [63,64]. In two phase I studies (ALKA-372–001 and STARTRK-1), entrectinib was administered to 119 patients with relapsed or refractory advanced/metastatic solid tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions [47]. Entrectinib was well-tolerated and only 15% of patients required a dose modification [47]. The most common grade ≥ 3 toxicities included fatigue/asthenia (4%), weight increase (2%), diarrhea (1%), and eosinophilic myocarditis (1%), Table 2 [47]. The phase 2 STARTRK-2 trial was an open-label, multicenter, global basket study that included patients with solid tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions (NCT02568267). A focused integrated analysis on NTRK fusion-positive tumors showed that at a median follow-up of 12.9 months (interquartile range (IQR), 8.77–18.76), the median duration of response was 10 months (95% confidence interval (CI), 7.1–not reached) and the objective response rate (ORR) was 57% (95% CI, 43.2–70.8) [48]. The median overall survival (OS) was 21 months (95% CI, 14.9–NE) and the median progression-free survival (PFS) was 11.2 months (95% CI, 8.0–14.9) [48]. Major adverse events (≥3 grade) were reported in 61.6% of patients and included anemia (12%), an increase in weight (10%), and fatigue (7%), with no patient mortality, Table 2 [48]. Based on these findings, entrectinib gained accelerated approval by the FDA in August 2019 at a dose of 600 mg once daily [40]. Approval was for use in patients with NTRK gene fusion and metastatic or unresectable locally advanced solid tumors, who have progressed on systemic therapy or have no satisfactory alternative treatment [40]. The approval of entrectinib provides an additional treatment option for patients with advanced cancer, potentially creating the opportunity for patients and their physicians to choose between different therapies [65]. Larotrectinib is available in a liquid formulation approved for children younger than 12 years old, whereas entrectinib is not. However, entrectinib may be effective for children with brain tumors, while the efficacy of larotrectinib for primary and metastatic brain tumors is currently being evaluated [65]. The two drugs also have different side effect profiles. The adverse events seen most frequently in the larotrectinib trials include increased ALT or AST, fatigue, and vomiting. Warnings and precautions for larotrectinib include neurotoxicity, hepatotoxicity, and embryo–fetal toxicity. Entrectinib has additional warnings and precautions, including congestive heart failure, CNS effects, and skeletal fractures [65]. The National Comprehensive Cancer Network (NCCN) guidelines recommend larotrectinib and entrectinib as first-line or subsequent-line (following disease progression) treatment options for unresectable or metastatic iCCA and eCCA with NTRK gene fusions. Both entrectinib and larotrectinib are approved in the United States and Europe for the treatment of unresectable or metastatic solid tumors with NTRK gene fusion and progression after previous therapy [66,67]. However, there are currently limited data for patients with CCA. Mitogen-activated protein kinase (MAPK) signaling is essential for cell growth and survival through the RAS/RAF/MEK/ERK pathway [68]. The BRAF gene is an oncogene whose protein product upregulates the RAS/RAR/MEK pathway [68]. BRAF mutations have been identified in several solid malignancies, including colorectal cancer, NSCLC, and melanoma [69,70]. More than 50 different BRAF mutations have been reported, with the V600E point mutation being the most common mutation (BRAFV600E) [71]. In BRAFV600E, valine (V) is substituted by glutamic acid (E) at amino acid 600, resulting in activating BRAF with subsequent tumor growth and spread [72]. In CCA, BRAF mutations are uncommon, occurring almost exclusively in iCCAs, with a prevalence ranging between 5% and 7%, and with the BRAFV600E mutation in 1.5% of patients with iCCA [26,73]. Dabrafenib is a competitive inhibitor of the RAF protein, which causes apoptosis by decreasing downstream phosphorylation of MEK and ERK, arresting the cell cycle in G1, and activating caspase-3/7 [74,75]. Even if a tumor initially responds to dabrafenib alone, it may eventually become resistant to treatment if another pathway activates the MEK protein [75]. Trametinib is a selective inhibitor of MEK1/MEK2 and is used with dabrafenib, which prevents tumors from using this escape mechanism [76]. Trametinib reduces cell proliferation, causes G1 cell-cycle arrest, and induces apoptosis [77]. The combination of these drugs in targeting MEK and BRAF has yielded promising results (Table 2). In the phase 2, single-arm, open-label trial ROAR, the BRAF cohort included 43 adult patients with BRAFV600E-mutated CCA with metastatic, locally advanced, unresectable, or recurrent disease that had progressed on prior therapy [50]. Patients received trametinib 2 mg once daily and dabrafenib 150 mg twice daily, with a mean follow-up of 10 months [50]. The ORR was 51% (95% CI, 36–67), the mean OS was 14 months (95% CI, 10–33), the mean PFS was 9 months (95% CI, 5–10), and the MDR was 9 months (95% CI, 6–14) [50]. Increased gamma-glutamyl transferase (GGT) (12%), low WBC count (7%), and pyrexia (7%) were the most common grade ≥ 3 adverse events, Table 2 [50]. Salama et al. reported results from the NCI-MATCH trial, a single-arm, open-label study that enrolled 29 patients with different solid tumors that progressed on standard lines of therapy, including four patients with iCCA [51]. Patients were given a continued dosing of dabrafenib (150 mg twice a day) and trametinib (2 mg once daily) [51]. The ORR was 38% (90% CI, 22.9 –54.9%), the median OS was 28.6 months, the median PFS was 11.4 months (90% CI, 8.4–16.3), and the median MDR was 25.1 months (90% CI, 12.8–NE) [51]. Three of the four patients with CCA had a partial response and grade ≥ 3 adverse events occurred in 65.7% of the enrolled patients [51]. Fatigue (11.4%), decreased neutrophil count (8.6%), and decreased WBC count (8.6%) were the most frequently reported grade ≥ 3 adverse events associated with the treatment, Table 2 [51]. The results of these trials supported the FDA approval of the combination of dabrafenib and trametinib for treating adults and children >6 years with BRAFV600E mutation-positive, unresectable, or metastatic solid tumors who have progressed on prior therapy [41]. In a phase 1 study, patients with BRAFV600-mutated solid tumors (including CCA) are currently being evaluated for a response to ABM-1310, a selective inhibitor of BRAFV600E mutation tumors (NCT04190628). Patients with advanced solid tumors, including biliary tract cancers, with BRAF mutations, are the focus of a phase 1 study of BGB-3245, a second-generation BRAF inhibitor (NCT04249843), Table 3. Combining the selective ERK1/2 inhibitor JSI-1187 with a BRAF inhibitor is another potential study strategy. Despite the significant advances in managing patients with BRAFV600 mutations, further studies are required. For example, in studying the efficacy of dabrafenib and trametinib and concurrent mutations of TP53 and BRAFV600E, early studies reported that this was associated with a more aggressive disease, resulting in less clinical benefits from dabrafenib and trametinib [78]. In addition, patients with BRAFV600E/TP53 mutations were associated with reduced PFS and OS [79]. Alterations in the FGFR gene and dysregulated FGFR signaling play a role in the development and progression of several types of cancer, including CCA. Four receptors belong to the FGFR family, including FGFR1, 2, 3, and 4, which share a cytoplasmic tyrosine kinase domain [80]. FGFR2 alterations include rearrangements, amplifications, and mutations, present in 10–16% of patients with iCCA [33]. These alterations activate mitogen-activated protein kinases (MAPKs), triggering constitutive signaling cascades that prompt tumor cell proliferation, survival, migration, and angiogenesis [81,82]. Therefore, FGFR inhibitors are promising targeted therapies that can potentially improve the survival of patients with CCA. Initially, non-selective tyrosine kinase inhibitors (TKIs) were investigated in phase 1/2 clinical trials and showed low antitumor activity and a limited survival benefit [83,84,85]. More recently, selective FGFR inhibitors were introduced for patients with FGFR2 fusion-positive iCCA and resulted in a significant clinical response, prompting phase 2/3 trials and accelerated FDA approvals [18]. Pemigatinib is a selective inhibitor of FGFR1, 2, and 3 that competitively inhibits the autophosphorylation and FGFR-mediated signaling cascades in tumor cells [86]. In the phase 2, multicenter, open-label FIGHT-202 trial, previously treated patients with metastatic CCA with FGFR2 fusions or FGFR2 rearrangements (n = 107), other FGFR mutations (n = 20), or wild-type FGFR (n = 18) received 13.5 mg of pemigatinib once daily on day 1–14 of a 21-day cycle [52]. In patients with FGFR2 fusions or FGFR2 rearrangements, the objective response rate (ORR) was 35.5% (95% CI, 26.5–45.4%) [52]. The median PFS and OS were 6.9 and 21.1 months, respectively [52]. Up to 64% of the patients had grade ≥ 3 toxicities that included hyper/hypophosphatemia (12%), arthralgia (6%), fatigue (5%), and retinal detachment (4%), Table 2 [52]. Based on these results, pemigatinib became the first FDA-approved targeted therapy for previously treated metastatic CCA with FGFR2 fusions or FGFR2 rearrangements [18]. Currently, FIGHT-302 is an ongoing phase 3 clinical study comparing pemigatinib with gemcitabine and cisplatin chemotherapy to determine the drug’s efficacy in the first-line treatment of CCA (NCT03656536), Table 3. Infigratinib is another highly selective ATP-competitive FGFR1–3 inhibitor that showed promising antitumor activity in patients with FGFR2 fusions in early-phase trials [87]. Patients with advanced iCCA resistant to chemotherapy were enrolled in a phase 2 study of infigratinib in a multicenter, open-label, phase 2 trial [53]. Of the 122 enrolled patients, 108 had positive FGFR2 fusions or FGFR2 rearrangements [53]. Patients received 25 mg once daily infigratinib for 21 days in a 28-day cycle [53]. In patients with FGFR2 fusions or FGFR2 rearrangements, the ORR was 23.1% (95% CI, 15.6–32.3%), indicating clinically significant activity after treatment [53]. In this trial, one patient had a complete response (CR) and 24 patients had partial responses [53]. The median duration of response (DOR) was five months (95% CI, 3.7–9.3), and eight patients had a maintained response for more than six months [53]. Two-thirds of the patients (66.2%) had grade ≥ 3 toxicities, with hypophosphatemia (14.1%) and hyperphosphatemia (12.7%) being the most common adverse events, Table 2 [53]. A phase 1 dose-escalation study showed that the risk of hyperphosphatemia in patients receiving infigratinib increased with higher drug exposure and was associated with higher antitumor activity [88]. On May 2021, the FDA granted accelerated approval to infigratinib for previously treated locally advanced CCA or for patients with metastatic CCA with FGFR2 fusions or FGFR2 rearrangements [19]. Infigratinib is also a potential targeted therapy for patients with untreated CCA with FGFR2 fusions or FGFR2 rearrangements. PROOF-301 is an ongoing phase 3 trial that recruited patients with untreated locally advanced CCA or patients with metastatic CCA with FGFR2 fusions or FGFR2 rearrangements [89]. In PROOF-301, patients received either infigratinib or a standard chemotherapy regimen of gemcitabine and cisplatin [89]. Infigratinib may potentiate the apoptotic activities of chemotherapies in multidrug-resistant tumor cells [90]. Therefore, there is a potential future role for infigratinib in combination therapy for patients with advanced CCA, and the results of further clinical trials are awaited with interest. Futibatinib is an irreversible FGFR1-4 inhibitor that binds to the conserved cysteine residues of the P-loop of the kinase domain [91]. Previous studies showed that futibatinib exhibited highly selective antitumor activities against tumor cells harboring FGFR mutations, particularly against mutations commonly associated with resistance to ATP-dependent FGFR inhibitors [91]. In addition, the oral futibatinib molecule was associated with a comparatively lower number of drug-resistant clones than ATP-dependent FGFR inhibitors [91,92]. Futibatinib was studied in a phase I trial that recruited 197 patients with previously-treated advanced solid tumors, 83 of which had CCA with a mutation, fusion, or amplification of FGFR2 (NCT02052778) [92]. In this trial, 64 patients were treated with futibatinib at 20 mg and 19 patients at 16 mg [92]. The results showed that futibatinib at 20 mg daily resulted in an ORR of 15.6%, a disease control rate (CDR) of 71.9%, a median DOR of 5.3 months, and a median PFS of 5.1 months [92]. The updated analysis of the single-arm FOENIX-CCA2 phase 2 trial included 103 patients with previously-treated advanced or metastatic iCCA with FGFR2 fusions or FGFR2 rearrangements (NCT02052778) [54]. Futibatinib 20 mg daily led to an ORR of 41.7% at a median follow-up period of 25.0 months [54]. The DC was 82.5%, the median PFS was 8.9 months, and the median OS was 20.0 months, Table 2 [54]. Based on these findings, on September 30, 2022, the FDA granted accelerated approval for futibatinib for adult patients with previously treated locally advanced or metastatic iCCA with FGFR2 gene fusion or rearrangement [20]. Derazantinib is another potent anti-FGFR1-3 that showed promising antitumor activity in iCCA harboring FGFR2 fusions or FGFR2 rearrangement. In the phase 2 FIDES, 143 patients with iCCA harboring FGFR2 fusions (n = 103) or FGFR2 mutations or amplifications (n = 40) received derazantinib 300 mg once daily [93]. In the cohort with FGFR2 fusions, the ORR was 21.4% (95% CI 13.9–30.5), with a median PFS and OS of 8.0 (95% CI 5.5–8.3) and 17.2 (95% CI 12.5–22.4) months, respectively [93]. In the FGFR2 mutations or amplifications cohort, the ORR and DCR were 6.5% (95% CI 0.8–21.4) and 58.1% (95% CI 39.1–75.5). The median PFS was 8.3 (95% CI 1.9–16.7) and the median OS was 15.9 (95% CI 8.4–not estimated) [93]. The most common grade ≥ 3 adverse events in the overall cohort were hyperphosphatemia (3%), asthenia/fatigue (5%), nausea (1%), and transaminase elevations (12%) [93]. The phase 1/2 ReFocus trial evaluated RLY-4008, a selective FGFR2 inhibitor that can target FGFR resistance mutations, in CCA patients with FGFR2 fusions or FGFR2 rearrangements who did not receive FGFR inhibitors before. The preliminary analysis of 38 patients showed an ORR of 63.2% (95% CI 46.0–78.2) and a DCR of 94.7%. There was no observation of grade 4/5 adverse events [94]. Despite the promising findings of selective FGFR inhibitors in patients with CCA and FGFR2 fusions or FGFR2 rearrangements, several issues remained unanswered. More than 150 fusion partners are associated with FGFR2 gene rearrangement, which results in significant molecular diversity in patients with FGFR2 fusions and rearrangements [95,96]. Furthermore, nearly 50% of the gene fusion and rearrangement partners are present within the same chromosome of the FGFR2 gene [95,96]. It is still unclear which patients might respond to FGFR2-targeted therapies and whether fusion partners can affect the response and survival with FGFR2-targeted therapies [96]. Therefore, future research should focus on the effect of combined genetic alterations on the responses to FGFR2 inhibitors and their role in the development of acquired resistance, as well as identifying reliable response biomarkers for FGFR2-targeted therapies [96,97]. TMB is a recently identified biomarker of the response to immune checkpoint inhibitors (ICIs) in several types of cancer [97]. The TMB is the number of somatic mutations per megabase (Mb) of the genomic sequence of a tumor [97]. A TMB score of ≥10 mutations/Mb has been proposed as a threshold with a high likelihood of neoantigen formation and represents TMB-H status [97]. In patients with several tumor types, including melanoma, NSCLC, and bladder cancer, patients with TMB-H had better outcomes when treated with programmed death protein-1 and programmed cell death ligand-1 (PD-1/PD-L1) checkpoint inhibitors, or a cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade [98,99,100]. TMB-H has been detected in 27.3% of patients with iCCA [101]. Pembrolizumab is a humanized antibody that inhibits the PD-1 receptors on lymphocytes by inhibiting the ligands that would block the receptor and inhibit an immune response [102,103,104]. In a subgroup analysis of 102 patients with TMB-H, who were enrolled in the phase 2 KEYNOTE-158 trial and received pembrolizumab, the ORR was 29% (95% CI, 21–39%), the median OS was 11.7 months (95% CI, 9.1–19.1), and the median PFS was 2.1 months (95% CI, 2.1–4.1) [105]. Notably, there were no patients with CCA in the TMB-H subgroup of this trial [105]. However, based on these findings, in 2017, the FDA approved pembrolizumab to treat adult and pediatric patients with unresectable or metastatic solid tumors (including CCA) [36]. The indications for this approval also required that the tumor tissue was TMB-H (≥10 mutations/megabase), and that the patients had progressed following prior therapy and for whom there were no satisfactory alternative treatment options [36]. However, it is unclear if the TMB levels for predicting the response to the PD-1 blockade are consistent throughout the spectrum of solid tumors. There are situations in which a high TMB does not indicate a response. Therefore, novel biomarkers are required that reflect the complexity of the tumor immune microenvironment and consider the effects of tumor mutations on the immune response. The production of neoantigens and CD8+ T cell infiltrations into the tumor microenvironment are both increased in tumors with dMMR or high levels of MSI [106]. MSI-H/dMMR makes the errors produced during DNA replication difficult to repair, which leads to mutations [106]. The prevalence of MSI-H/dMMR in patients with iCCA ranges from 4.7–18.2% [35]. As part of the phase 2 KEYNOTE-158 study, pembrolizumab 200 mg intravenously once every three weeks was administered to 233 patients with advanced solid tumors and confirmed MSI-H/dMMR [56]. In this study, 22 (9.4%) patients had CCA [56]. With a median follow-up of 13.4 months, the ORR was 34.3% (95% CI, 28.3–40.8%), the median OS was 23.5 months (95% CI, 13.5–NR), and the median PFS was 4.1 months (95% CI, 2.4–4.9 months [56]. The median duration of the response was not reached (range, 2.9–31.3 months) [56]. A subgroup analysis of CCA showed that the ORR was 40.9% (95% CI, 20.7–63.6), the median OS was 24.3 months (95% CI: 6.5–NE), and the median PFS was 4.2 months (95% CI, 2.1–NE) [56]. Adverse events ≥ grade 3 occurred in 34 patients (14.6%) [56]. Pneumonitis (1.3%), severe skin reactions (1.3%), and colitis (0.9%) were the most common adverse events, Table 2 [56]. In an open-label, single-arm, phase 2 clinical trial, 86 patients with 12 types of cancer, including four patients with CCA, with at least one prior cancer therapy and MSI-H/dMMR mutations, were included [55]. The patients received 10 mg/kg of pembrolizumab every 14 days [55]. The ORR was 53% (95% CI, 42–64%), the 2-year OS was 64% (95% CI, 53–78%), and the 2-year PFS was 53% (95% CI, 42–68%) [55]. A subgroup analysis of patients with CCA showed that the ORR was 50%, the CR was 25%, the SD was 75%, and the disease control rate was 100% [55]. Minor adverse events were reported in 20%, with the most common being diarrhea/colitis (6%), pancreatitis/hyperamylasemia (6%), fatigue (2%), and anemia (2%), Table 2 [55]. Based on these findings, in 2020, the FDA approved the use of pembrolizumab in adults and pediatric patients who have unresectable or metastatic MSI-H/dMMR solid tumors that have progressed despite prior treatment, without satisfactory alternative treatment options [37]. The results of other trials for MSI-H patients in solid tumors are awaited. For example, a phase 1/2 trial of pevonedistat, a selective NEDD8 inhibitor, in combination with pembrolizumab in patients with dMMR/MSI-H solid cancers is ongoing (NCT04800627), Table 3. Dostarlimab is another anti-PD-1 monoclonal antibody [107]. The phase 1 GARNET study was a non-randomized, multicenter, open-label trial to evaluate the safety and efficacy of dostarlimab (500 mg every 3 weeks for four cycles, then 1000 mg every 6 weeks) in 106 patients with advanced solid tumors (two of whom had CCA) with confirmed MSI-H/dMMR, one of whom had a biliary tract cancer [57]. The ORR was 38.7% (95% CI, 29.4–48.6) [57]. With a median duration of follow-up of 12.4 months, the median DOR was not reached [57]. At 12 months, the Kaplan–Meier estimate for the chance of response preservation was 91%, and at 18 months it was 80% [57]. Approximately 8.3% of patients reported at least one grade 3 adverse event, including anemia (3.9%), elevated lipase (2.3%), elevated ALT (1.6%), and diarrhea (1.6%), Table 2 [57]. Based on the findings from this study, the FDA approved dostarlimab in adult patients with MSI-H/dMMR recurrent or advanced solid tumors that progressed on or following prior treatment and were not candidates for satisfactory alternative treatment options [38]. Given the limited single-agent activity and the limited number of patients with CCA included in these clinical trials, further studies are required to investigate novel immunotherapy combinations to enhance the treatment efficacy in patients with CCA. IDH1 gene mutations commonly occur in CCA [108]. Missense mutations in the IDH1 R132 codon leads to the overproduction of the oncometabolite R-2-hydroxyglutarate (R-2HG) [109]. In tumor progenitor cells, an increase in the R-2HG levels inhibits cellular differentiation and drives oncogenesis by promoting histone methylation and DNA methylation [110]. The prevalence of an IDH1 mutation in iCCA has been estimated at 13% [34]. Ivosidenib is a small molecule inhibitor of IDH1. In a phase 1, multicenter, open-label study, 73 patients with IDH1-mutant CCA (89% iCCA and 11% eCCA), refractory to other systemic therapy, were enrolled and received ivosidenib (200–1200 mg daily in 28-day cycles) [58]. The ORR was 5% (95% CI, 1.5–13.4), the median OS was 13.8 months (95% CI, 11.1–29.3), and the median PFS was 3.8 months (95% CI, 3.6–7.3) [58]. Approximately 23% of the included patients had grade ≥ 3 adverse events, including ascites (5%), anemia (4%), and fatigue (3%), Table 2 [58]. This trial led to a multicenter, randomized, double-blind, placebo-controlled phase 3 study (ClarIDHy), which included 185 adult patients with advanced CCA with IDH1 mutations who had progressed on previous therapy [34]. Patients were randomly assigned to oral ivosidenib 500 mg or matched placebo once daily in continuous 28-day cycles [34]. In the intervention group, with a median follow-up of 6.9 months, the ORR was 2% (95% CI, 0.5–6.9), and the median PFS was significantly improved (median PFS 2.7 months; 95% CI, 1.6–4.2) [34]. The median OS after accounting for the cross-over was 10.3 months (95% CI, 7.8–12.4), which was significantly better than the placebo (median OS = 5.1 months) [34]. Grade ≥ 3 adverse events were reported in 30% of the patients [34]. The most frequently reported adverse event grades ≥ 3 were ascites (7%), increased AST (5%), anemia (3%), and fatigue (3%), Table 2 [34]. Based on this trial, the FDA approved ivosidenib for the treatment of adult patients with unresectable locally advanced or metastatic IDH1-mutated CCA who had been previously treated [21]. PARP inhibitors are also being studied in this subgroup of patients as they exhibit dysregulated homologous recombination repair. The National Cancer Institute (NCI) is conducting a phase 2 clinical trial that aims to investigate the safety and efficacy of olaparib as a subsequent line therapy for patients with advanced solid tumors (including CCA) with IDH1 or IDH2 mutations (NCT03212274). Another ongoing study combined ceralasertib and olaparib in patients with refractory CCA and advanced solid tumors with IDH1/2 (NCT03878095), Table 3. The use of IDH1 inhibitors as single treatments has shown promising results in targeted therapy of malignancies harboring IDH1 mutations in preclinical and clinical settings. However, these studies are preliminary and currently include small numbers of patients with CCA. The ERBB2 gene encodes HER2, a receptor tyrosine kinase found in the plasma membrane [111]. ERBB2 triggers several signaling pathways involved in tumor growth [112]. Tumorigenesis is associated with HER2 and MAPK pathway dysregulation [32,113]. The overexpression of HER2 has been reported in 5.8% of iCCA and 13–20% of eCCA [31,32]. Pertuzumab and trastuzumab are monoclonal antibodies that target HER2 and are used to treat HER2-positive malignancies [114]. The combination of pertuzumab and trastuzumab suppresses HER2-AKT signaling by inhibiting both the ligand-induced and ligand-independent HER2-HER3 complex formations [114]. A phase 2, single-arm, multicenter trial called MyPathway included 39 patients with previously treated HER2-positive metastatic CCA [59]. Patients were treated with pertuzumab (840 mg loading dose and 420 mg every three weeks) plus trastuzumab (8 mg/kg loading dose, 6 mg/kg every three weeks) [59]. The ORR was 23% (95% CI, 11–39), the median PFS was 4.0 months (95% CI, 1.8–5.7), and the MDR was 10.8 months (95% CI, 0.7–25.4) [59]. Grade ≥3 adverse events were reported in 46% of patients, with the most common being an increase in the ALT and AST (13%) and an increase in ALP (10%), Table 2 [59]. These were promising results for patients with HER2-mutated CCA. However, regulatory approval for patients with CCA is awaited. More recently, trastuzumab deruxtecan (T-DXd) showed promising antitumor activity in HER2-positive advanced solid tumors, including CCA. In a phase 1 trial of 60 patients with advanced, non-breast/non-gastric, HER2-mutant solid tumors (NCT02564900), the ORR was 28.3% and the median PFS was 7.2 (95% CI 4.8–11.1) months [115]. The phase 2 HERB trial recruited 32 patients (24 with HER2-positive and eight with HER2-low) BTCs who received T-DXd. The efficacy cohort included 22 patients (9 had CCA). In patients with HER2-positive BTCs, the ORR was 36.4%, the median PFS was 4.4 months, and the median OS was 7.1 months. In the HER2-low group, the ORR, median PFS, and median OS were 12.5%, 4.2 months, and 8.9 months, respectively. The rate of grade ≥ 3 adverse events was 81.3% [116]. Several ongoing studies aim to investigate targeted HER2 agents in treating patients with CCA with ERBB2 mutations (Table 3). In the front-line setting, gemcitabine combined with cisplatin and trastuzumab and the combination of gemcitabine, cisplatin, and varlitinib are currently being studied in two clinical trials (NCT03613168 and NCT02992340). In addition, HER2-targeting agents are currently being studied for subsequent lines of therapy, either as monotherapy or in combination with standard chemotherapy agents. Ongoing trials include: the oral HER2 covalent inhibitor, TAS0728, (NCT03410927); the HER2 antibody-drug conjugate, trastuzumab deruxtecan (NCT04482309 and JMA-IIA00423); the antibody-drug conjugate, RC48-ADC, (NCT04329429); capecitabine plus the dual HER2 and EGFR inhibitor, varlitinib (NCT03093870); chemotherapy (5-FU or IRI or Cape) plus trastuzumab (NCT03185988); and chemotherapy plus the HER2-targeted bispecific antibody, zanidatamab (NCT02892123). The results of these ongoing trials may provide multiple options for targeted treatments for patients with CCA in the molecular subgroup and improve patient survival. More than three decades have passed since the discovery of the gene encoding the receptor tyrosine kinase, RET [117]. RET rearrangements and mutations are recognized as treatable drivers of oncogenesis [117]. Certain RET fusion proteins and activating point mutations can drive oncogenesis and tumor progression by activating downstream signaling pathways, leading to uncontrolled cell proliferation [117]. RET gene fusions are present in between 1% and 2% of NSCLC and thyroid cancers and represent potential targets for therapeutic inhibition of RET kinase [118]. However, RET fusions seem rare in CCA, and data regarding their exact prevalence in CCA are limited [119]. Pralsetinib is a selective inhibitor of the RET receptor tyrosine kinase. In the phase 1/2, open-label ARROW trial, 29 patients with RET fusion-positive solid tumors were included, with three patients with CCA [60]. Patients received a starting dose of pralsetinib of 400 mg QD [60]. The ORR was 57% (95% CI, 35–77%), the median OS was 13.6 months (95% CI, 7.5–NE), the median PFS was 7.4 months (95% CI, 5.1–13.6), and the median duration of response was 11.7 months (95% CI, 5.5–19.0) [60]. Altogether, 69% of patients had grade ≥ 3 adverse events that included neutropenia (31%), anemia (14%), and increased AST (10%), Table 2 [60]. Based on the findings of this trial, the FDA approved pralsetinib for adult and pediatric patients with advanced or metastatic RET-fusion-positive lung and thyroid cancers for whom systemic therapy is indicated [120]. Selpercatinib is another highly selective inhibitor of the RET receptor tyrosine kinase, with CNS activity [121]. In the phase 1/2, open-label LIBRETTO-001 trial, 45 patients with RET fusion-positive solid tumors other than lung or thyroid tumors were included, with two patients with CCA [61]. Of the 45 patients, 43 received a starting recommended dose of 160 mg BID. In the 41 patients who were evaluated for efficacy, the ORR was 43.9% (95% CI, 28.5–60.3), the median OS was 18.0 months (95% CI, 10.7– NE), the median PFS was 13.2 months (95% CI, 7.4–26.2), and the median duration of response was 24.5 months (95% CI: 9.2–NE). One patient with CCA was evaluated for efficacy; the ORR was 100% and the duration of response was 5.6 months [61]. Altogether, 49% of patients had grade ≥ 3 adverse events that included hypertension (22%), increased ALT (16%), and increased AST (13%), Table 2 [61]. Selpercatinib is currently approved for locally advanced or metastatic RET-fusion-positive solid tumors [122]. Again, further studies are needed to focus on CCA to validate these findings in this group of patients. Liquid biopsies refer to blood sampling to detect circulating tumor DNA (ctDNA), circulating cell-free RNA (ccfRNA), and cell-free DNA (cfDNA) [123,124]. Liquid biopsy as an adjunctive diagnostic method has gained popularity over the last decade due to its potential benefits for cancer patients [123]. The most commonly interrogated element in liquid biopsies is ctDNA or DNA fragments produced from tumors [125]. Treatment resistance tracking, response monitoring, target selection, relapse detection, and early diagnosis are the potential benefits of identifying tumor-derived material in liquid biopsies [126]. Although liquid biopsy is a potentially useful diagnostic tool for patients with CCA, this modality remains underexplored in CCA. Advances in this diagnostic area for patients with CCA have been limited by several practical factors, including the small amounts of ctDNA shed into the bloodstream in patients with localized tumors [127]. On the other hand, CCA is an internal malignancy and it is often difficult to obtain a tissue biopsy. It is also challenging to acquire sufficient tumor cells for diagnosis on aspiration cytology, and these challenges can prevent adequate molecular tumor profiling for CCA [128]. Therefore, blood-derived ctDNA may play an important role in identifying molecular alterations in patients with CCA who may not have an adequate biopsy or cytology sample available for analysis [129]. Consistent mutation findings between tumor tissue and ctDNA were reported in 2015 by Zill et al. in a prospective study of 26 pancreaticobiliary cancers, including eight patients with CCA [130]. In this preliminary study, 93% of mutations found in tissue samples were also identified by cfDNA [130]. In 2019, Mody et al. analyzed ctDNA from 138 patients with biliary tract cancers and identified genetic alterations in 89% of the samples [131]. Recently, Kumari et al. evaluated the diagnostic role of cfDNA in gallbladder carcinoma [132]. Serum was obtained from 34 patients with gallbladder carcinoma and 39 matched controls without malignancy [132]. This study showed that the cfDNA levels were lower in healthy individuals than in patients with gallbladder cancer [132]. In addition, there was a significant correlation between the cfDNA and the presence of jaundice, lymph node metastases, and overall disease TNM stage [132]. Therefore, the quantitative analysis of cfDNA may have the potential to be a unique marker for the molecular detection of targeted therapies in CCA. Furthermore, the analysis of cfDNA may have a role in distinguishing between neoplastic and inflammatory conditions of the gallbladder and biliary tract identified by imaging but without available biopsy material. However, concerns remain about the overall sensitivity of ctDNA mutations for diagnosing early-stage CCA [133]. An additional feature of ctDNA/cfDNA is to track the development of resistance to chemotherapy and targeted treatments [134]. In 2019, Ettrich and colleagues sequenced 15 common gene mutations in ctDNA samples from patients with biliary tract cancer throughout their chemotherapy [135]. In the iCCA cohort (n = 13), there was a 92% agreement between tissue samples and blood-derived ctDNA [135]. The level of agreement for the overall cohort was 74% [135]. A change in the mutational profile was also seen in 63% of chemotherapy-naive individuals after treatment [135]. A study reported in 2017 showed that the integrative genomic analysis of cfDNA could identify acquired treatment resistance due to multiple recurrent point mutations in the FGFR2 kinase domain during tumor progression [136]. Until recently, clinical studies considered CCA a homogeneous entity, which may have led to the limited antitumor activity of conventional treatment regimens. It is now apparent that the behavior and responses of CCA vary substantially according to the underlying molecular profile. These relatively recent findings highlight the crucial role of precision medicine in guiding the treatment selection for patients with CCA. The number of investigational and approved targeted therapies for CCA has increased exponentially in the past decade. Several targeted agents are now approved as first-line and subsequent treatments for patients with locally advanced or metastatic CCA. The selective FGFR inhibitors pemigatinib and infigratinib and the IDH1 inhibitor ivosidenib are now approved for previously treated patients with FGFR2 fusions or FGFR2 rearrangements and IDH1 mutations, respectively. These gene fusions, rearrangements, and mutations are present in a subset of patients with iCCA. Pemigatinib and infigratinib are also currently being investigated in the first-line setting. Other targeted therapies are available across solid tumors, including CCA, and include: NTRK inhibitors (entrectinib and larotrectinib); a BRAF/MEK inhibitor combination (dabrafenib and trametinib); pembrolizumab (for high ≥ 10 mutations/mb tumor mutational burden or mismatch repair defect cancers); and RET inhibitors (selpercatinib). Targeted therapies for other genomic alterations in CCA and solid tumors in general continue to be developed and explored. Further therapeutic possibilities for patients with CCA may emerge as our knowledge of the tumor microenvironment and its impact on tumor growth increases. Combined treatments that target both actionable mutations and the tumor microenvironment is another approach that assists with patient selection for the most appropriate molecular targeted therapy. The value of blood-derived ctDNA in the clinic is currently being explored in CCA, especially since tissue biopsies can be difficult in this type of cancer. Despite multiple FDA approved targeted therapies for patients with CCA, the overall survival for these patients remains dismal. Utilizing combination approaches with targeted therapies may offer some patients more benefit than single agents [137,138,139]. Mechanisms of resistance to single agent therapies may include RNA silencing [140] as well as multiple gene driven pathogenesis, which could be overcome with ‘multiomic’ patient diagnostics using genomic, proteomic, transcriptomic, and immunomic data as well as n-of-1 customized combination therapeutic approaches [141,142,143,144]. Further, using patient-specific immunomic data may allow for a tailored immunotherapy-targeted treatment as well as being informed by specific genomic aberrations and the most logical immunotherapeutic target [145]. A next step in improving outcomes in these patients likely involves identifying more therapeutic targets as well as overcoming secondary resistance mechanisms via targeting as many genomic alterations present within a specific tumor as possible [144,146,147].

PMC10000388

Fei-Yuan Yu,Qian Xu,Xiao-Yun Zhao,Hai-Ying Mo,Qiu-Hua Zhong,Li Luo,Andy T. Y. Lau,Yan-Ming Xu

The Atypical MAP Kinase MAPK15 Is Required for Lung Adenocarcinoma Metastasis via Its Interaction with NF-κB p50 Subunit and Transcriptional Regulation of Prostaglandin E2 Receptor EP3 Subtype

22-02-2023

MAPK15,EP3,p50,LUAD,metastasis

Simple Summary Due to the lack of effective early diagnostic markers for lung cancer and the rich blood circulation in the lungs, it is very easy to cause lymph node metastasis and distant metastasis of lung cancer, making lung cancer as one of the top ten cancer types with the highest mortality rate in the world. This study found that MAPK15 is highly expressed in the tissues of patients with lung adenocarcinoma lymph node metastasis, and MAPK15 interacts with p50 to regulate the expression of EP3 at the transcriptional level, thereby promoting cancer cell migration. This suggests that MAPK15 plays a key role in the metastasis of lung cancer cells, and MAPK15 can be used as a molecular marker for the early diagnosis or prognosis assessment of lung cancer. Its molecular mechanism for regulating lung cancer metastasis can provide valuable information and insights on novel therapeutic options at molecular levels. Abstract Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD.

The Atypical MAP Kinase MAPK15 Is Required for Lung Adenocarcinoma Metastasis via Its Interaction with NF-κB p50 Subunit and Transcriptional Regulation of Prostaglandin E2 Receptor EP3 Subtype Due to the lack of effective early diagnostic markers for lung cancer and the rich blood circulation in the lungs, it is very easy to cause lymph node metastasis and distant metastasis of lung cancer, making lung cancer as one of the top ten cancer types with the highest mortality rate in the world. This study found that MAPK15 is highly expressed in the tissues of patients with lung adenocarcinoma lymph node metastasis, and MAPK15 interacts with p50 to regulate the expression of EP3 at the transcriptional level, thereby promoting cancer cell migration. This suggests that MAPK15 plays a key role in the metastasis of lung cancer cells, and MAPK15 can be used as a molecular marker for the early diagnosis or prognosis assessment of lung cancer. Its molecular mechanism for regulating lung cancer metastasis can provide valuable information and insights on novel therapeutic options at molecular levels. Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD. The incidence of lung cancer is high among malignant tumors, which seriously affects human health. The mortality rate of lung cancer patients is high because lung cancer is usually in an advanced stage when diagnosed, with lymph node metastasis or even distant metastasis. Radiotherapy and chemotherapy have very limited therapeutic effects on advanced lung cancer. Targeted therapies, such as the use of targeted drugs EGFR tyrosine kinase inhibitors [1,2], can improve the survival of lung cancer patients to a certain extent, but they still face the problem of chemotherapy resistance, recurrence of targeted therapy, etc. Therefore, it is still not possible to effectively control the malignant development of lung cancer [2]. The study of molecular markers related to lung cancer metastasis and their corresponding molecular mechanisms still needs to be further explored. The classical mitogen-activated protein kinases (MAPKs, e.g., ERK1/2, p38, and JNK/SAPK) play important roles in regulating gene expression, cell growth, proliferation, etc. Atypical MAPKs such as ERK3, ERK4, and NLK (nemo-like kinase) also play critical roles in many cellular responses [3,4]. MAPK15, alias extracellular signal-regulated kinase 7/8 (ERK7/8), is the most recently discovered atypical MAPK. Current research indicates that MAPK15 can promote the transformation of colon cancer by mediating the activation of the transcription factor c-Jun [5,6] or promoting the growth of gastric cancer cells [7]. MAPK15 has also been found to interact with autophagy-related proteins such as GABARAP and LC3 to control tumor development [8]. In addition, MAPK15 can be activated by carcinogenic factors such as RET/PTC3 [9] or involved in the regulation of telomerase activity [10] to participate in the development of tumors. Recently, our group has reported that MAPK15 can promote arsenic trioxide-induced apoptosis, as well as boosting the efficacy of combination therapy with cisplatin and TNF-α, in lung cancer cells [11,12]. At present, research about the function of MAPK15 is still limited, and its role in lung cancer metastasis remains unclear. EP3 is one of the four G protein-coupled receptors of prostaglandin E2 (PGE2), which plays an important role in cell proliferation, differentiation, apoptosis, cardiovascular system regulation, and inflammation. It has been reported that tumor angiogenesis and tumor cell growth were significantly inhibited in a mouse lung cancer model with EP3 knocked out [13]. Yamaki et al. found that PGE2 promotes the growth of lung adenocarcinoma (LUAD) cell line A549 via the EP3 receptor-activated Src signaling pathway [14]. However, the molecular mechanism of EP3 in regulating lung cancer progression is still not fully clarified. In this study, we detected the expression of MAPK15 in lung cancer tissues, and found that the expression of MAPK15 is positively correlated with lymph node metastasis in LUAD patients; remarkably, our results showed that the expression of EP3 was transcriptionally regulated by MAPK15, and the expression of EP3 was positively correlated with the expression of MAPK15 in LUAD tissues. Furthermore, we revealed the first time that MAPK15 promotes the expression of EP3 by interacting with p50, thereby enhancing the migration of lung cancer cells. Lung cancer tissue microarray (BC041115c, US Biomax, Rockville, MD, USA) was purchased and all human tissues were collected according to HIPPA-approved protocols as described by US Biomax (https://www.biomax.us/FAQs, accessed on 14 June 2022). Immunohistochemistry was performed to detect the expression of MAPK15 and EP3. Briefly, tissue microarray was deparaffinized thrice in xylene (10 min for each) and rehydrated in gradient series ethanol (100%, 95%, 90%, 90%, 5 min for each), respectively. After being rinsed with water, tissue slides were incubated with 3% hydrogen peroxide for 40 min to block endogenous peroxidase. Tissue slides were then rinsed with PBS and immersed in 0.01 M citrate acid antigen retrieval solution and heated at 98 °C for 20 min using a water bath. After natural cooling, tissue slides were washed with PBS and incubated with 5% BSA for 30 min. Tissue slides were then incubated with MAPK15 [15] or EP3 (Cat. 101760, Cayman Chemical, Ann Arbor, USA) antibody at 4 °C overnight. After being rinsed with PBS, tissue slides were incubated with secondary antibody for 45 min at RT. Subsequently, tissue slides were washed with PBS and reacted with 3,3′-diaminobenzidine (DAB, Zhongshan Golden Bridge Inc. Beijing, China) and counterstained with hematoxylin. Then, tissue slides were mounted with glycerogelatin and photographed with a light microscope. Immunostaining of tissue microarray were scored according to immunoreactive score (IRS) [16,17]. Each tissue in the microarray was semiquantitatively scored for intensity (0, absent; 1, weak; 2, moderate; 3, strong) and extent of staining (percentage of the positive tumor cells: 0, ≤5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, >75%). Intensity and extent of each tissue were multiplied to give a composite score: 0–3, deemed as low expression, “−”); 4–12, deemed as high expression (4–6, “+”; 7–9, “++”; 10–12, “+++”). All cells were grown at 37 °C in a 5% CO2 incubator. HEK293T, H1299, and A549 cells were purchased from ATCC Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in MEM, RPMI-1640, or F12-K medium supplemented with 10% FBS and 1% PS, respectively. MAPK15 stable knockdown LUAD H1299 cells (H1299-shMAPK15) and control cells (H1299-shCtrl) were established previously [12]. For transfection, cells were mixed with siRNA/plasmids-polyethylenimine mixture and cultured for the indicated time point. Negative control siRNA (siN05815122147) and siRNA duplexes against EP3 were purchased from IGE Biotechnology LTD (Guangzhou, China) and listed in Supplementary Table S1. RNA was extracted using RNAiso Plus (Takara, Dalian, China) from cells. Then, cDNA was synthesized using GoScript™ Reverse Transcription Mix (Promega, Madison, WI, USA) by following the manufacturer’s instructions. Specific primers were used, and real-time PCR was performed using GoTaq qPCR Master Mix (Promega, Madison, WI, USA) on Applied Biosystems 7500 Real-Time PCR System. The 2ΔΔCT method was used to calculate the relative expression of target genes compared to internal control (β-Actin) as described previously [18]. Primers were synthesized by IGE Biotechnology LTD (Guangzhou, China) and listed in supplementary Table S1. Equivalent amounts of extracted protein were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk in PBS containing 0.05% Tween 20 followed by incubation with primary antibody overnight at 4 °C. After reacting with primary antibody, membranes were incubated with secondary antibody and proteins were visualized with ECL reagent using Tanon 5200 system (Tanon, Shanghai, China). The optical density of each protein band was quantified by Gel-Pro Analyzer 4 (Toyobo, Osaka, Japan) software. Original blots and blot quantification are shown in Figures S3–S5, S7 and S8. Transwell assay was performed as described previously [16]. Briefly, 3.0 × 104 cells were seeded in the upper compartment of transwell inserts with 8 μm microporous membrane (cat no. 3422, Corning Inc., Corning, NY, USA). After being incubated for 24 h, unmigrated cells on the upper surface of the microporous membrane were wiped using a cotton swab. Cells on the lower surface of the microporous membrane were fixed with 4% PFA for 20 min and subsequently stained with 0.1% crystal violet for 15 min. The transwell chamber was rinsed with PBS to remove excess crystal violet, and images of migrated cells were captured using an Axiovert 40 CFL microscope (Carl Zeiss AG, Oberkochen, Germany) with CCD camera (magnified 100×). Finally, the crystal violet in the migrated cells was dissolved with 33% acetic acid, and absorbance was measured at OD595. Cells seeded on coverslip in 6-well plate were incubated for the indicated time point and fixed with 4% PFA for 15 min. After being rinsed with PBS, cells were permeabilized for 10 min with PBS containing 0.25% Triton X-100. Subsequently, cells were incubated with 5% BSA for 30 min to block unspecific binding of antibodies. Then, cells were incubated with primary antibody in a humidified chamber at 4 °C overnight. After decanting of primary antibody solution, cells were washed with PBS and incubated with secondary antibody for 1.5 h at room temperature in the dark. Coverslips were counterstained with 1 μg/mL Hoechst 33342 and mounted with mounting medium. Images were captured with Axiovert 40 CFL Microscope (Carl Zeiss AG, Germany) or Zeiss lsm 800 confocal microscope (Carl Zeiss AG, Germany). In vivo peritoneal metastasis assay was performed as described previously [19]. Briefly, 5 × 106 MAPK15 stable knockdown H1299 or control cells in 200 μL of phosphate-buffered saline were injected intraperitoneally into BALB/c nude mice (Beijing Vital River Animal Technology Co., Ltd., Beijing, China, licensed by Charles River). After 7 weeks, the mice were sacrificed, and tumor nodules were quantified. HEK293T cells cultured in 10 cm dish were transfected with pcDNA4/Xpress-MAPK15 plasmids [15] and incubated for 24 h. Prior to immunoprecipitation, 1 μg of Xpress antibody or normal IgG was pre-adsorbed with 20 μL Protein A/G Sepharose slurry for 2 h at 4 °C with rotation. After transfection, cells were harvested and lysed with NP-40 lysis buffer using repetitive freeze-thawing method. An amount of 300 μg of lysates to be used for immunoprecipitation was precleared with 20 μL Protein A/G Sepharose at 4 °C for 1 h with rotation. The supernatant was then incubated with the Xpress antibody–Protein A/G Sepharose complexes overnight at 4 °C with rotation (anti-mouse IgG was used as negative control). In total, 10% of the supernatant was used as input. The Sepharose beads were collected by centrifugation and washed extensively in 500 μL of lysis buffer, and eluted in 20 μL of SDS sample buffer by heating to 98 °C for 5 min. After centrifugation at 10,000× g, the supernatant was collected for immunoblot analysis. Chromatin immunoprecipitation assay was performed using SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA). Briefly, formaldehyde cross-linked H1299 cells were lysed, and chromatin was digested with micrococcal nuclease into DNA/protein fragments. Then, p50 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was added and the complex is captured by protein G magnetic beads. Seven p50 binding sites (site1–site7) in the EP3 promoter region (−2000 bp) were predicted by JASPAR databases and PCR was used to detect p50 binding. Five repeats of p50 binding sequence (site5, sequence: GGGGCTTCCC) and 12 bp linker sequences with AflII and NsiI sites were synthesized by IGE Biotechnology LTD (Guangzhou, China) and ligated to a modified pJC6-GL3 plasmid [11] to construct luciferase reporter plasmid (5 × p50-Luc). Then, the 5 × p50-Luc plasmid was co-transfected with/without pCMV-p50 plasmid into equal amount of H1299 cells in 12-well plate. Afterward, cells were lysed for luciferase assay following manufacturer’s instructions (dual-luciferase reporter assay system, Promega, Madison, WI, USA). Mean comparisons were performed using the GraphPad Prism 8 for unpaired t-test. Fisher’s exact test was used to study the correlation between MAPK15 expression and clinical parameters. Spearman rank correlation analysis was used to compare the correlation between the expression of MAPK15 and EP3 in lung cancer tissues using SPSS 19 software. The above statistical analysis was two-tailed; p < 0.05 suggested that the difference was statistically significant. To study the role of MAPK15 in lung cancer, we analyzed the relationship between MAPK15 and clinical–pathological parameters such as age, gender, depth of tumor invasion, lymph node metastasis, distant metastasis, tumor differentiation, clinical stage, etc. We found that there was a positive correlation between MAPK15 expression and lymph node metastasis (p = 0.012) as well as clinical stage (p = 0.033) (Supplementary Table S2). The expression of MAPK15 is higher in patients with lymph node metastasis (N1 + N2) as compared to patients without lymph node metastasis (N0) (Supplementary Table S2). Other clinical–pathological parameters such as age, gender, depth of tumor invasion, distant metastasis, and tumor differentiation were not significantly correlated with the expression of MAPK15 (Supplementary Table S2). Adenocarcinoma and squamous cell carcinoma are major types of non-small-cell lung cancer (NSCLC). As compared to squamous cell carcinoma, we revealed that the expression of MAPK15 is relatively higher in adenocarcinoma (Figure 1A, Supplementary Table S3) and is associated with lymph node metastasis (p = 0.013) (Table 1, Figure 1B). The above results indicate that MAPK15 is expressed more highly in lymphatic metastatic LUAD. In MAPK15 stable knockdown LUAD H1299 cells (Figure 1C,D), cell migration was significantly inhibited (Figure 1E,F). The expression of Snail1 was decreased in MAPK15 knockdown cells (Figure 1G), which can down-regulate the expression of E-cadherin by post-translational modifications such as deacetylation and methylation during EMT [20]. Consequently, the expression of epithelial marker E-cadherin was increased, while mesenchymal marker integrin β1 is decreased after MAPK15 knockdown (Figure 1G). Then, we performed an in vivo peritoneal metastasis assay using H1299-shMAPK15 cells and found that loss of MAPK15 significantly reduces metastasis to mesentery in vivo (Figure 1H,I). The above results indicate that H1299 cells undergo mesenchymal–epithelial transition after MAPK15 knockdown, thereby decreasing migration and metastasis. It has been reported that the expression of MMP2 was depressed in EP3 knock-out mice under hypoxic stress [21], which indicates a correlation between the expression of MMP2 and EP3. Our results showed that MMP2 was down-regulated in MAPK15 knockdown H1299 cells (Figure S1). To investigate whether EP3 is involved, we detect the expression of EP3 in H1299-shCtrl and H1299-shMAPK15 cells. We found that the mRNA and protein level of EP3 was significantly decreased in MAPK15-deficient cells (Figure 2A,B) and the protein level of EP3 was not affected by proteasome inhibitor MG132 (Figure 2B), suggesting that the decreased EP3 in H1299-shMAPK15 cells was transcriptionally regulated. Moreover, the migration of H1299 cells was inhibited (Figure 2F,G) after EP3 was knocked down (Figure 2C–E). The decreased EP3 in MAPK15 knockdown cells suggested that there might be a correlation between the expression patterns of these two molecules. Then, we detected the expression of EP3 in a serial section from the same tissue that we stained with MAPK15 antibody and found that the expression of EP3 is positively correlated with MAPK15 (r = 0.589, p < 0.001, Figure 2H and Table 2). Taken together, the above results show that MAPK15 affects cell migration through the regulation of EP3. The molecular mechanism of how EP3 is transcriptionally regulated by MAPK15 is unknown. It has been reported that the expression of MAPK15, NF-κB1 (p50), and NF-κB2 (p52) were obviously decreased in ovarian cancer cell lines [22], which indicate there are correlations between MAPK15 and the NF-κB family. To investigate the relationship between MAPK15 and NF-κB family members, we transfected the pcDNA4/Xpress-MAPK15 plasmid into 293T cells and the immunoprecipitation assay revealed that MAPK15 interacts with p50 but not p65 and c-rel (Figure 3A). We also detected the localization of MAPK15 and p50 in H1299 cells by confocal microscopy and found that MAPK15 is distributed both in the cytoplasm and nucleus, and colocalizes with p50 (Figure 3B), indicating that there is an interaction between these two proteins in LUAD cells which might contribute to the expression of EP3. To study the relationship between MAPK15/p50 and EP3, we overexpressed MAPK15 (Figure 3C) and p50 (Figure 3D) in H1299 cells and found that the expression of EP3 was increased (Figure 3E), which indicates that MAPK15 and p50 positively regulate the transcription of EP3. The chromatin immunoprecipitation assay found that p50 binds to two p50 binding motifs in the EP3 promoter (Figure 3F, site2 and site5). Subsequently, we chose site 5 (Figure 3F) to construct the luciferase reporter plasmid and co-transfect with/without pCMV-p50 in H1299 cells for the luciferase reporter assay. Our results indicate that the luciferase activity is significantly increased in cells overexpressed with p50 (Figure 3G), which revealed that p50 can transcriptionally regulate EP3 by binding to the EP3 promoter. MAPK15 interacts with p50 intracellularly, indicating potential gene regulation and cellular phenotypic change. Beinke et al. reported that the p105 pathway can positively regulate gene transcription under TNF-α stimulation [23]. We hypothesize that TNF-α might promote the expression of EP3 through the p50 pathway, thereby contributing to cell migration. In TNF-α-treated H1299 cells, we found that TNF-α promoted EP3 expression in a dose- (Figure 4A) and time-dependent manner (Figure 4B). Furthermore, TNF-α promoted the migration of H1299 cells but had no significant effect on the migration of MAPK15 knockdown cells (Figure 4C,D). This result suggests that TNF-α promotes cell migration through MAPK15. In TNF-α-treated A549 cells, we found that TNF-α promotes nuclear localization of MAPK15 and p50 (Figure S2). In H1299 cells, we found that p50 is distributed in both cytoplasm and nucleus, whereas in MAPK15 knockdown cells, p50 is mainly located in the cytoplasm (Figure 4E), indicating that nuclear localization of p50 is dependent on MAPK15. At the same time, we treated H1299 cells with TNF-α and found that p50 is mainly located in the nucleus, whereas in MAPK15 knockdown cells, p50 is distributed in both the cytoplasm (white arrows) and the nucleus (Figure 4E). The above results indicate that TNF-α-induced nuclear translocation of p50 is dependent on MAPK15. In addition, we found that the expression of EP3 in TNF-α-treated H1299 cells was increased, while the expression changes of EP3 in MAPK15 knockdown H1299 cells were not significant (Figure 4F), and TNF-α could not promote H1299 cell migration while EP3 was knocked down (Figure 4G,H). Taken together, these results reveal that TNF-α promotes H1299 cell migration through induction of MAPK15-p50 nuclear localization and EP3 expression in cells with MAPK15 expression. JSH-23 is an NF-κB inhibitor. When using JSH-23 to treat H1299 cells, we found that JSH-23 inhibited the expression of EP3 in a dose- (Figure 5A) and time-dependent manner (Figure 5B). Furthermore, JSH-23 inhibited the migration of H1299 cells but had no significant effect on the migration of knockdown MAPK15 cells (Figure 5C,D). This result suggests that JSH-23 inhibits cell migration through MAPK15. In addition, we found that the expression of EP3 in H1299 cells treated with JSH-23 was decreased, while the expression of EP3 in MAPK15 knockdown H1299 cells did not change significantly (Figure 5E), and JSH-23 could not inhibit H1299 cell migration when EP3 was knocked down (Figure 5F,G). The above results indicate that JSH-23 inhibits cell migration by inhibiting MAPK15-induced EP3 expression. Lung cancer is usually at an advanced stage with lymph node or distant metastasis when diagnosed, which leads to high mortality. Medical knowledge still lacks effective diagnostic molecular markers for metastatic lung cancer. In the present study, we revealed that MAPK15 is more highly expressed in the tissues of LUAD patients with lymph node metastasis (Figure 1B), and MAPK15 interacts with p50 to promote EP3 expression at the transcriptional level (Figure 6), thereby enhancing cancer cell migration and metastasis. MAPK15 is a member of the ERK subfamily, which is involved in the regulation of cell growth and differentiation like other well-known ERKs. Previous research indicates that MAPK15 is involved in the transformation of colon cancer [6], promotes gastric cancer cell proliferation [7], and is associated with autophagy [8]. However, its clinical pathological role has, until now, not been examined in lung cancer. The correlation between MAPK15 and lymph node metastasis in LUAD described here suggests that MAPK15 plays an important role in lung cancer development, which may lead to poor clinical outcomes. Since we used a commercialized lung cancer tissue array in this study, there is a lack of relevant information on disease progression, so it is impossible to conduct a longitudinal assessment of the relationship between MAPK15 expression and patients’ disease-free survival/overall survival, recurrence, metastasis, etc. However, with the in-depth study of MAPK15, we gradually realized its important role in LUAD. In future studies, multicenter, larger-sample-size studies should be conducted through longitudinal assessment of the patients’ critical long-term clinical outcome to further clarify MAPK15 expression and the significance of clinical parameters. Due to the significant correlation between MAPK15 and the clinical features of the LUAD patients we observed, MAPK15 and its signaling pathway in LUAD may be a potential therapeutic target for metastatic LUAD. As a kinase, MAPK15 carries out different functions in various cancers, indicating the deregulation of key pathways. Studies have indicated a pivotal role of MAPK15 in mediating the effect of gene transcription. We have previously shown that MAPK15 promotes the transformation of colon cancer by mediating the activation of c-Jun [6]. Here, the identification of MAPK15 as an upstream regulator for EP3 unveiled a previously unknown mechanism for the MAPK15 or EP3 signaling pathway and their roles in the regulation of cell migration in LUAD. The role of EP3 in tumor progression is still controversial. It has been reported that EP3 coupled with G proteins can effectively inhibit tumor growth. Shoji et al. found that EP3 can significantly inhibit the proliferation of tumor cells in advanced-stage colon cancer [24]. Sanchez et al. found that EP3 can promote the expression of p21 by reducing cAMP, thereby arresting the cell cycle in the S phase, and ultimately inhibiting the proliferation of 3T6 fibroblasts [25]. On the other hand, there are more and more studies showing that EP3 can promote the development of tumors. Finetti et al. found that EP3 is involved in regulating the formation of tumor blood vessels [26]. Amano et al. found that in an EP3-deficient mouse tumor model, tumor angiogenesis and tumor cell growth were effectively inhibited [13]. Yamaki et al. found that EP3 participates in the Src signaling pathway to promote the growth of LUAD A549 cells [14]. In this study, we reveal that knocking down EP3 can inhibit the migration of LUAD cells and that the expression of EP3 was positively regulated by MAPK15, which expands our understanding of EP3 and its regulation in lung cancer. NF-κB is a type of transcription factor that plays an important role in the occurrence and development of tumors. The ERK family was linked to the NF-κB pathway [27,28]. As the most recently discovered MAPK family member, the relation between MAPK15 and NF-κB is mainly uncharacterized. Previous studies on the NF-κB protein family mainly focused on the activity of IκB or p65 in the p50/p65 complex to promote gene transcription. However, more and more studies have shown that p50 can bind to the promoter of the gene and activate gene transcription. The study of Hong et al. showed that overexpression of p50 in BAR-T cells significantly enhanced the activity of the DNMT1 gene promoter [29]. Karst et al. showed that overexpression of NF-κB p50 in melanoma cells MMRU can promote angiogenesis and up-regulate IL6 expression. They confirmed by Chip assay that p50 can bind to the promoter region of IL6 gene and activate its transcription [30]. Similarly, Southern et al. found that the BAG-1 protein can interact with the p50-p50 homodimer and bind to the promoter region of downstream genes to play a positive role in regulating gene transcription [31]. Beinke et al. reviewed that TNF-α/IL-1/LPS can activate the classic p50/p65 dimer NF-κB signaling pathway and the p100/RelB non-canonical signaling pathway, as well as the p105/p50 signaling pathway [23]. In this study, we found that MAPK15 interacts with p50 in LUAD cells, and the nuclear translocation of p50 may require the assistance of MAPK15. In addition, we also found that the mRNA expression level of EP3 increased when p50 was overexpressed in H1299 cells, indicating that p50 can regulate the expression of EP3 at the transcriptional level, and CHIP assay and luciferase reporter assay confirmed that p50 can bind to the promoter region of EP3 and promote the transcription of EP3. Our results revealed that MAPK15 interacts with p50 to promote the transcription of EP3, thereby affecting biological functions such as the migration of LUAD cells. In conclusion, this study demonstrates the role of MAPK15 in the metastasis of LUAD. We revealed that MAPK15 promotes LUAD cell migration via p50 and EP3 signaling and is associated with lymph node metastasis in LUAD patients, which indicates that MAPK15 might be a potential prognostic biomarker for LUAD and a therapeutic target to inhibit metastasis in metastatic LUAD patients. The insights provided by this study could facilitate understanding the role of MAPK15 in lung cancer progression and its potential modulatory role in cancer metastasis.

PMC10000409

Eszter Emri,Oisin Cappa,Caoimhe Kelly,Elod Kortvely,John Paul SanGiovanni,Brian S. McKay,Arthur A. Bergen,David A. Simpson,Imre Lengyel

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration

28-02-2023

zinc,retinal pigment epithelium,single-cell RNA sequencing,age-related macular degeneration,maturation

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD. The retinal pigment epithelium (RPE) is a highly polarized monolayer of cells lining the back of the eye which provides critical support for the functioning of the adjacent photoreceptors. It is part of the outer blood–retina barrier that regulates the transport of metabolites between the bloodstream and the neural retina. The RPE undergoes structural and functional transitions during maturation, which are essential to fulfill its biological functions [1,2,3,4]. Because of its critical function, the RPE has been directly implicated in several retinal diseases, most notably age-related macular degeneration (AMD). A hallmark feature of AMD is the accumulation of protein-, lipid-, and mineral-rich deposits between the RPE and the choroidal microcapillary network [5,6,7]. The size and number of these sub-RPE deposits increase with disease progression [8]. Another hallmark is pigmentary changes associated with the RPE [9,10]. Both of these are linked to the progression to end-stage AMD [5] manifested as geographic atrophy (GA), characterized by progressive degeneration and loss of the RPE layer, or as neovascular (NV) AMD, which is characterized by abnormal leaky blood vessels that grow from the choroid into the sub-RPE space (Type 1), sub-retinal space (Type 2), or the retina (Type 3) [11], causing fluid accumulation and scarring [12]. Zinc is part of a nutritional supplement endorsed by the National Eye Institute (NEI) to slow the progression from mild/moderate to advanced AMD [13]. The biochemical pathways involved in these beneficial effects are not fully understood. Recent studies showed that human primary RPE cells in long-term culture model the hallmark features of AMD. RPE cell-based models develop as monolayers with tight junctions and high transepithelial resistance (TEER), extensive pigmentation, specific gene expression profiles, and also sub-RPE deposits [14,15,16,17], many of which can be affected by zinc supplementation directly [15,17]. This in vitro model system can be manipulated experimentally and interrogated longitudinally under conditions resembling health and disease. In this study, we identified dynamic changes in gene expression and the effects of acute (1 week) zinc supplementation using single-cell RNA sequencing (scRNA-Seq). Our results elucidate several specific pathways involved in the maturation of RPE to a stage that develops hallmark changes of AMD (sub-RPE deposition and pigmentary changes) and how these are modified by zinc supplementation. Primary human fetal RPE cells (ScienCell, Carlsbad, CA, USA) from one donor were purchased and used at passage three (P3) for the complete study in duplicates/triplicates with unknown clinical or genetic background. Cells were seeded onto Corning 6-well transwell inserts (10 µm thick polyester inserts with 0.4 µm pore size, 4 × 106/cm2 pore density, Corning, Wiesbaden, Germany) in 125.000/cm2 of epithelial cell medium (EpiCM, ScienCell, Carlsbad, CA, USA). After one week in culture, cell culture media were replaced with Miller medium with 1% FBS [18,19] and cells were cultured for two, nine, and nineteen weeks in duplicates. Two types of short-term zinc treatment were also conducted, where one–one extra replicates of untreated controls were taken for the two types of zinc treatment experimental setup. After one week or eighteen weeks in culture, cell culture media were replaced with Miller medium with 1% FBS for an additional one week in the absence or presence of 125 µM externally added zinc (as zinc sulphate; Thermo Fisher Scientific, Waltham, MA, USA) both in the apical and basal chambers, resulting in ~10 nM bio-available or free zinc [15,20]. The resulting replicates were the following: duplicates of zinc-treated samples, triplicates of untreated controls at the two- and nineteen-week time point, and duplicates of untreated controls at the nine-week time point. Cellular differentiation was monitored through the development of cobblestone cell morphology and increase in pigmentation using light microscopy. The increase in transepithelial resistance (TEER) was measured using the EVOM2 Epithelial Voltohmmeter and STX2 electrodes (World Precision Instruments, Sarasota, FL, USA). At the sample collection time, as detailed above, cells were washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA) two times for one minute. Cells were detached by incubation with 0.15 % Trypsin-EDTA for thirty minutes at 37 °C. The trypsinization was stopped using 100% FBS and trypsin neutralization solution (ScienCell, Carlsbad, CA, USA). The obtained single-cell suspensions were washed in PBS with 1% BSA (Thermo Fisher Scientific, Waltham, MA, USA) 2 times for 5 min at 1000 rpm. After automatic cell counting (EVE, Thermo Fisher Scientific, Waltham, MA, USA), 7 × 105 cells/mL were prepared, and the cells were kept on ice for a maximum of ten minutes before proceeding with single-cell RNA sequencing. In parallel to single-cell sequencing, adjacent samples were fixed for fifteen minutes in 4% PFA (Merck, Darmstadt, Germany) diluted in PBS (Thermo Fisher Scientific, Waltham, MA, USA) for immunofluorescence. Our previous study showed individual differences in assaying primary hfRPE from different donors [17]. To overcome the variations introduced by variability in donor samples and to generate a reproducible zinc effect, in this manuscript, experiments were performed on primary hfRPE cells from a single donor. In the initial scRNA-Seq run, samples were obtained from RPE cells cultured for two weeks (2W), nine weeks (9W), and nineteen weeks (19W) (Supplementary Figure S1A) in duplicates. Cells were collected from two wells at these time points. A total of 7000 cells from each sample were loaded on 10× Genomics Chromium v1.3 with a target recovery of 4000. Libraries made from each sample were pooled and sequenced. In the second run, samples originated from RPE cultures were treated with a zinc-supplemented medium for one week either after: (1) one week in culture or (2) eighteen weeks in culture in duplicates. We also included one–one sample from untreated RPE culture in this run and the transcriptomic profiles were generated in a pooled fashion as described above. The actual cell recovery of both runs ranged from 3000 to 4000 in each well, resulting in a total recovery of ~30,000 cells for the first run and ~15,000 for the second run. The raw scRNA-Seq data were processed using CellRanger v3.0.0. and then Seurat v3.1 to determine the heterogeneity of our specimens using unsupervised clustering, followed by annotation based on hierarchical clustering of a pre-defined set of canonical RPE marker genes [21,22,23,24] (Supplementary Table S3). For further analysis, we initially analyzed our samples of untreated control RPE cultures from the two runs (triplicates for 2W and 19W and duplicates for 9W cultures). We then separately analyzed the duplicate samples of our zinc-treated RPE cultures compared to the triplicate samples of untreated control RPE cultures of 2W and 19W. Approximately 7000 single cells per sample were processed with the Chromium system using the v3 single-cell reagent kit (10× Genomics, San Francisco, CA, USA). Barcoded libraries were pooled and sequenced on the NovaSeq platform (Illumina, San Diego, CA, USA), generating 150 bp paired-end reads as per 10× Genomics recommendations, with >30,000 reads per cell. The raw scRNA-Seq data were processed using CellRanger version 3.0.0 (10× Genomics). The resulting filtered expression matrices were then imported into R for analyses using scRNA-Seq packages, Seurat (Version 3.1) (Stuart et al. 2019) and Monocle (Version 3.0) (Trapnell et al. 2014; Cao et al. 2019). Cells were filtered to exclude those with <1000 or >8000 genes, or with >20% of counts aligned to mitochondrial genes, or >40% counts aligned to ribosomal genes. Cells passing QC were downsampled randomly to 1000 cells per sample to prevent over- or under-representation of any sample. Each sample was log-normalized using default Seurat parameters, with the top 3000 highly variable genes used for Seurat iterative pairwise integration. The integrated dataset was scaled to regress variance arising from read depth and mitochondrial and ribosomal expression. Principal Component Analysis was then performed on the integrated dataset, and Seurat’s JackStraw function was applied to determine the components used in UMAP and SNN clustering. Unsupervised clustering was run iteratively at resolutions ranging from 0.25 to 1, at increments of 0.25. At the highest resolution, a total of 13 clusters were detected. These clusters were observed in UMAP to form two overall, as-yet unannotated cell populations. Using untreated cells only, the average expression for the clusters was determined for a set of 213 canonical RPE marker genes [21,22,23,24] (Supplementary Table S3) to which hierarchical clustering was applied. The clusters were segregated into two distinct branches, exhibiting characteristics of more and less differentiated RPE, which matched the distinction observed in UMAP. As such, the 13 unsupervised clusters were annotated to reflect these two overall cellular populations for downstream differential expression analysis. Seurat’s Wilcoxon rank sum test was used for differential expression testing, using default FindMarkers parameters, with genes below 0.05 adjusted p-value considered significantly differentially expressed. Monocle 3 [25] was used for pseudotime analysis, for which downsampled count data were imported from Seurat and independently processed and batch-corrected in Monocle using default parameters. For continuity, a pseudotime trajectory graph was calculated and projected on the UMAP coordinates preserved from Seurat analysis. The data were filtered to focus on the main less differentiated to more differentiated pseudotemporal trajectory, by excluding small branches not contributing to the main trajectory. This was followed by graph autocorrelation analysis to detect gene expression changes correlating with progress along the trajectory, filtered for significance at p-value and Q-value <0.05. Genes with expression significantly correlated with the trajectory were grouped into ‘modules’ of co-regulated genes and the average expression of each gene module calculated across pseudotime. For pathway and network analysis, we used the GeneAnalytics (https://ga.genecards.org/#input; accessed on 14 March 2021) and STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) 11.0 (https://string-db.org/; accessed on 14 March 2021) [26] in combination with Cytoscape [27]. GeneAnalytics uses binomial distribution to test the null hypothesis that the queried genes are not over-represented within any superpath, GO term, or compound in the GeneAnalytics data sources. The presented score in each section is a transformation of the resulting p-value, corrected for multiple comparisons using the false discovery rate (FDR) method, with higher scores indicating a better match. The bar color, indicating the matching quality—high (dark green), medium (light green), low (beige)—is common for all sections. STRING in combination with Cytoscape implements classification systems such as Gene Ontology, KEGG, and systems based on high-throughput text mining and the used reference dataset was the human genome. The identified functional protein association network was validated via text mining, database information, co-expression, and experimental evidence. For immunofluorescence analysis, the cells on the transwell membrane were permeabilized in 0.5% Triton-X (Merck, Darmstadt, Germany) in PBS for ten minutes at 4 °C and then washed in 0.1% Tween20 in PBS (PBST) (Merck, Darmstadt, Germany) and blocked with 5% goat sera (Merck, Darmstadt, Germany) in PBST for one hour at room temperature. Samples were then incubated with primary antibodies overnight: COL1A1 (Abcam plc, Cambridge, UK, dilution 1:200) and RPE65 (Merck Millipore, Darmstadt, Germany, 1:50), diluted in PBST containing 1% goat sera. Following washing with PBST, the samples were incubated with secondary antibodies in 1:200 in PBST with 1% goat sera for one hour in the dark at room temperature. Samples were washed with PBST for five minutes, then with PBS. Cell nuclei were then labelled with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:1000 in PBS. Finally, samples were mounted onto Menzel–Glaser slides (Thermo Fisher Scientific, Waltham, MA, USA) in Vectashield (Vector Laboratories, Burlingame, CA, USA). For negative control, the primary antibody labelling was omitted. Cells were visualized using a Leica SP8 confocal microscope (Leica, Wetzlar, Germany). Images were obtained and analyzed with Leica Application Suite X Image software (Leica, Wetzlar, Germany). Primary human fetal RPE cells from a single donor were cultured for 2 weeks (2W = short term), 9 weeks (9W = medium term), and 19 weeks (19W = long term) (Supplementary Figure S1A). We used culture conditions that, in our hands, reproducibly recapitulated key aspects of RPE cells as described in previous studies [15,16,17]. As time in culture increased, RPE cells were observed to develop pigmentation, hexagonal morphology (Supplementary Figure S1B), and a progressively increasing epithelial barrier function (112.9 ± 3.9 Ohm × cm2 at 2W, 195.2 ± 16.6 Ohm × cm2 at 9W, and 201.36 ±49 Ohm × cm2 in 19W in culture). The cell cultures also began accumulating sub-RPE deposits (Supplementary Figure S1C) containing lipids and hydroxyapatite that we have shown earlier [15,16,17]. To identify the transcriptomic profiles of RPE cells at the three time points, we collected cells from three wells at 2W and 19W and two wells at 9W in culture (see Section 2 for detail). Approximately 3000–4000 cells were captured from each well and processed on the 10× Genomics Chromium v1.3 platform, with transcriptomes generated for a total of 30,000 cells. To ensure equal representation from all conditions, all samples were downsampled to include an equal number (1000) of randomly selected cells in Seurat 3.1 [28]. Based on 3417 differentially expressed transcripts (Supplementary Table S1), the cells were automatically allocated into thirteen clusters and visualized on a Uniform Manifold Approximation and Projection (UMAP) plot (Figure 1A). The lists of cluster-specific ‘marker’ genes were input into GeneAnalytics. The gene set analysis tool identified significant cluster-specific canonical pathways [29], labelled as superpathways for the functional analysis of the cell populations. Supplementary Table S2 contains information on the ‘marker’ genes, numbers of enriched pathways, and matched number of genes to the total number of genes in a pathway for each cluster. The software assigned the 312 genes in Cluster 0 to 18 superpathways, with respiratory electron transport and heat production of uncoupling proteins, metabolism, and visual cycle among the top five hits. The 205 genes in Cluster 1 were assigned to 44 superpathways, with degradation of extracellular matrix, ERK signalling, and phospholipase C pathway amongst the top five hits. The 270 genes in Cluster 2 were associated with 19 superpathways with metabolism, respiratory electron transport, heat production of uncoupling proteins, and visual cycle amongst the top five hits. In Cluster 3, the 210 differentially expressed genes were associated with 76 superpathways with cytoskeletal signalling, ERK signalling, and focal adhesion among the top five hits. The 87 differentially expressed genes in Cluster 4 were associated with 29 superpathways with cytoskeletal signalling, ERK signalling, and integrin signalling among the top five hits. The 30 differentially expressed genes in Cluster 5 were associated with two superpathways: melanin biosynthesis and tyrosine metabolism. The 270 differentially expressed genes in Cluster 6 were associated with 50 superpathways, degradation of extracellular matrix, metabolism of proteins, and cell adhesion and ECM remodelling amongst the top five hits. In Cluster 7, 313 differentially expressed genes were associated with 110 superpathways with cytoskeletal signalling, ERK signalling, and degradation of extracellular matrix among the top five hits. The 303 differentially expressed genes in Cluster 8 were associated with 61 superpathways, degradation of extracellular matrix, ERK signalling, and phospholipase C pathway among the top five hits. In Cluster 9, we identified 302 differentially expressed genes associated with 21 superpathways with metabolism, visual cycle, and copper homeostasis among the top five hits. In Cluster 10, 198 differentially expressed genes were associated with 24 superpathways with metabolism, visual cycle, and oxidative stress among the top five hits. The 807 differentially expressed genes in Cluster 11 were associated with 86 superpathways, degradation of extracellular matrix, protein processing in the endoplasmic reticulum, and cytoskeletal signalling amongst the top five hits. Finally, in Cluster 12, 164 differentially expressed genes were associated with 11 superpathways with organelle biogenesis and maintenance, intraflagellar transport, and mitotic cell cycle among the top five hits. The identification of thirteen clusters shows that cells in culture are not homogenous. We aimed to identify which of the unsupervised clusters most resemble mature RPE. We separated cells that were deemed to be more differentiated based on the expression of 213 RPE-specific genes we identified from several publications [21,22,23,24] (Supplementary Table S3). Hierarchical clustering based on the gene list divided the 13 clusters into two distinct groups (Figure 1B). We annotated clusters 0, 2, 5, 9, 10, and 12 as ‘more differentiated‘ RPE cells and the remaining clusters (1, 3, 4, 6, 7, 8, and 11) ‘less differentiated‘ cells (Figure 1B). We use the terms ‘more differentiated‘ and ‘less differentiated‘ from this point forward. The more and less differentiated cells are separated on the original UMAP (Figure 1(C1)). We calculated the proportion of more and less differentiated cells at the 2W, 9W, and 19W. Interestingly, nearly half of the cells were more differentiated even as early as 2W or 9W in culture (2W = 41%, 9W = 41%). By 19W, the proportion of the more differentiated cells increased to 73% (Figure 1(C2)), with 27% remaining less differentiated. Supplementary Table S4 lists the genes that define the more and less differentiated groups. The expression levels of three highly expressed representative genes from each group are presented as violin plots and UMAP plots in Supplementary Figure S2, highlighting the enrichment but not the exclusive presence of these genes in one or the other group. Next, we tested whether the protein products of the genes that distinguish more and less differentiated cells show differential expression. One of the highly expressed mRNAs in the less differentiated cells was Collagen Type I alpha 1 chain (COL1A1), fibril-forming collagen. The RPE secretes the protein encoded by this gene and it is found in the sub-RPE space [30]. We found that the expression of COL1A1 gradually increased in the less differentiated group and decreased in the more differentiated group (Figure 2A). In contrast, Retinoid Isomerohydrolase (RPE65), a visual cycle component marker for differentiated RPE, was mainly expressed in the more differentiated group (Figure 2A). Both genes were expressed in the other group but at a low level in the opposing groups (Figure 2B). Next, we determined the immunolocalization of the COL1A1 and RPE65 proteins in the 19W RPE monolayer. In line with the gene expression results, the cells with a strong RPE65 immunolabelling also had weak intracellular immunoreactivity for COL1A1 proteins, and cells with strong immunolabelling for COL1A1 showed weak labelling for RPE65 (Figure 2C; green: RPE65, red: COL1A1). Immunolabeling of COL1A1 is also present in the sub-RPE space. This extracellular immunoreactivity gradually increased with time in culture (Supplementary Figure S3), suggesting that the secreted COL1A1 accumulates as part of the developing extracellular sub-RPE material (Supplementary Figure S3). As we identified more and less differentiated RPE cells in our hfRPE, we investigated whether more and less differentiated cells are also present in RPE cells directly isolated from human eyes. We used two independent previously published datasets: the scRNA-Seq data obtained from human embryos [31] or adult human eyes [32] (Supplementary Figure S4). We applied our cell grouping strategy based on 213 RPE-specific signature genes (Supplementary Table S2). Indeed, our analysis showed that both the embryonic RPE (Supplementary Figure S4(A1,A2)) and adult RPE (Supplementary Figure S4(B1,B2)) could be classified into more and less differentiated cell populations. Of note, the number of cells analyzed in the publication using adult RPE [32] was relatively low. Hence, clusters were less well separated. To identify the genes associated with transitioning from the less to the more differentiated cells, we performed a pseudotemporal ordering of our scRNA-Seq transcriptome profile using Monocle3 (Figure 3). This unsupervised analysis identified a main trajectory with 11 nodes (Figure 3(A1)). Based on the original cluster analysis depicted in Figure 1, node 1 corresponded to the less and node 10 to the more differentiated cells (Figure 3(A2)). The main trajectory was correlated with 537 variably expressed genes. Based on their pseudotemporal expression profile, these clustered into seven modules (Figure 3(B1); Supplementary Table S5). Modules 2 and 5 contained 175 genes with high expression at the early stages of the trajectory that gradually declined towards the end of the trajectory. GeneAnalytics identified 62 potential significant superpathways associated with these genes (Supplementary Table S6). Degradation of extracellular matrix, focal adhesion, and cell adhesion–endothelial cell contacts were amongst the top five ranked pathways. Modules 3, 4, and 6 contained 172 genes. These gradually increased towards the late stages of the trajectory. GeneAnalytics identified nine potential superpathways defined by these genes, including the transport of glucose, metabolism, and visual cycle among the top five hits (Supplementary Table S6). Modules 1 and 7 contained 190 genes. The expression of these genes transiently increased to a maximum at the middle of the trajectory followed by a decrease over pseudotime. These identified sixteen superpathways with degradation of extracellular matrix, ERK signalling, and cytoskeleton remodelling among the top five hits (Supplementary Table S6). To identify potential transcriptional regulators of the pseudotemporal trajectory, GO term analysis was carried out in GeneAnalytics. This identified transcriptional regulator activity in two genes, ID1 and ID3, belonging to the combined Module 1 and 7, representing the transitional phase on the pseudotemporal trajectory. Next, we examined the potential relationships between the most highly significantly correlated in the main trajectory using a cut-off value of Moran I = 0.5 (see Section 2). We identify 44 genes strongly influencing the main trajectory. Using GeneAnalytics in combination with STRING database and Cytoscape, we found that these genes do not appear to be randomly distributed. A total of 31 out of 44 genes showed a significant biological connection, validated via text mining, database information, co-expression, or experimental evidence (p < 1.0 × 10−16; Figure 3(B2)). The 44 genes were associated with six potential superpathways, including visual cycle, extracellular matrix degradation, and cell adhesion–extracellular matrix remodelling as the top hits (Supplementary Table S6). Previous studies have shown that chronic zinc supplementation has clinical benefit associated with molecular and cellular changes [13,15,17,33,34], but the effects of acute or short-term zinc supplementation had not been studied in detail. To identify the effects of short-term zinc supplementation, we treated our RPE cultures for one week with a zinc-supplemented medium, using the same approach as we described earlier [15]. This acute zinc supplementation was carried out on less differentiated cells starting at the end of the first week in culture. Then, cells were harvested at the end of 2W or more differentiated cells at the end of the 18th week, and cells were harvested at the end of 19W. Gene expression changes with zinc supplementation were compared to cells in culture without zinc supplementation for either 2W or 19W. Cells with and without zinc supplementation were clustered using the process used in Figure 1(C1). While acute zinc supplementation did not noticeably change the proportion of the more and the less differentiated cells (Figure 4A), it significantly changed the expression of 472 genes in the more differentiated cells (Figure 4(B2)) and 149 genes in the less differentiated cells (Figure 4(B1)) at the two-week time point (Supplementary Table S7). At 19W, zinc altered the gene expression of 487 genes in the more differentiated cells (Figure 4(B4)) and 417 genes in the less differentiated cells (Figure 4(B3)) (Supplementary Table S7) (logFC > 0.25, adjusted p-value < 0.05). We displayed the four datasets in a four-way Venn diagram to further analyze specific temporal zinc-induced gene expression changes (Figure 4C). We found 81 overlapping genes differentially expressed under all four conditions. Two-thirds of these 81 genes were identified as housekeeping genes by GeneAnalytics, confirming previous studies showing that zinc plays a role in regulating cellular homeostatic processes [35]. Relevant proteins include metallothioneins (MT1E, MT1F, and MT1X) that act as essential stress proteins to regulate immune homeostasis. In the more differentiated cells, 222 uniquely affected genes were at 2W and 163 at 19W (Figure 4C). In the less differentiated cells, only four genes were specifically affected by zinc supplementation at 2W and 94 genes at 19W (Figure 4C). At 2W, we identified superpathways only in the more differentiated cells; these were cytoskeleton remodelling, focal adhesion, and degradation of extracellular matrix among the top five superpathways (Supplementary Table S8). At 19W, in the less differentiated cells, we identified presenilin signalling, SMAD signalling, and antigen-presenting cross-presentation amongst the top five superpathways (Supplementary Table S8). In contrast, in the more differentiated cells, we identified metabolism, ferroptosis, and protein processing in the endoplasmic reticulum amongst the top five superpathways (Supplementary Table S8). Information on the magnitude and direction of zinc-associated change in transcript abundance of these gene lists is provided in Supplementary Table S7. The analysis of these five gene lists by GeneAnalytics to identify superpathways is listed in Supplementary Table S8. We next determined the overlap between the 537 genes identified in the main trajectory in the pseudotemporal analysis (Figure 3(A2); Supplementary Table S5) and the list of the differentially expressed genes following the acute zinc supplementation (Figure 4(D1); Supplementary Table S7). This comparison identified 16 common genes (Supplementary Table S9). Using GeneAnalytics in combination with STRING database and Cytoscape, we found that these 16 genes show significantly (p-value < 1.0 × 10−16) more interactions than expected, validated by text mining, database information, co-expression, and experimental evidence (Figure 4(D2)) that relates to the respiratory electron transport and response to metal ions as biological function (Supplementary Table S9). Our hfRPE culture developed sub-RPE deposits even without photoreceptors and the supporting choriocapillaris (Supplementary Figure S1C). This allowed us to analyze the expression of genes potentially involved in the sub-RPE deposit formation process. We compiled lists of genes associated with various aspects of sub-RPE deposit formation and analyzed the changes in expression throughout cell maturation and zinc supplementation (Supplementary Table S11). Some genes belong to more than one gene list (Figure 5). This contains 55 genes previously genetically associated with AMD (Figure 5A; Supplementary Table S11 [36]). We found that 52 out of the 55 genes in AMD-risk-associated risk loci were expressed in our RPE model (Supplementary Table S11). Some genes were expressed higher at 2W, like CFHR3, LIPC, SYN3, and VTN, while others were expressed higher at 19W, like ARHGAP21, RDH5, SKIV2L, SRPK2, TGFBR1, and TRPM3. Among the genes expressed higher in less differentiated cells were CFHR3, LIPC, TGFBR1, and VTN. In more differentiated cells, we found higher expression of PRLR, RDH5, RORB, SLC16A8, SPEF2, and VEGFA. From these 52 genes, CFH, COL8A1, CD63, TSPAN10, APOE, TIMP3, and SLC16A8 were significantly upregulated, while CFHR1, VEGFA, TRPM3, and RDH5 were significantly downregulated in response to acute zinc supplementation (Supplementary Table S7). This contains 66 complement-regulation-related genes. Several complement proteins have been implicated in AMD and are found in sub-RPE deposits (Figure 5C, Supplementary Table S11 [37,38,39]). A total of 41 out of the 66 identified complement genes were expressed in our hfRPE cultures, most showing low expression levels (Supplementary Table S11). The genes that were expressed higher in 2W cultures were C4B, C4BPB, C8B, C8G, CFHR3, CFP, CSMD1, CSMD3, TPSG1, and VTN, while the genes expressed higher in 19W cultures were C1S, CD55, CD59, and PTX3. Among the genes expressed higher in less differentiated cells were C4BPA, C4BPB, `C8G, CFHR3, CFP, CR2, CSMD1, FHL-1, ITGB2, PTX3, and VTN. The expressions of C2, C4A, C5, CR1, and SERPING1 were higher in the more differentiated cells. CFH and C1R were significantly upregulated, while CFHR1 and CLU were significantly downregulated in response to zinc supplementation (Supplementary Table S7). This contains cholesterol-metabolism-related genes (Figure 5B, Supplementary Table S11 [40,41]). A total of 42 out of the 51 identified genes were expressed in hfRPE (Supplementary Table S11). The genes that were expressed higher at 2W were ABCG5, ANGPTL8, APOA1, CD36, LIPC, and STAR, while the genes expressed higher at 19W were APOC1, LDLRAP1, and NPC1. Among the genes expressed higher in less differentiated cells were CD36, LIPC, PCSK9, and STAR. In the more differentiated cells, the expressions of CYP27A1, LIPG, LPL, and PLTP were higher. PLTP, ANGPTL4, APOE, LRPAP1, VDAC2, and TSPO were significantly upregulated, and NPC2 was significantly downregulated in response to zinc supplementation. Interestingly, CYP27A1 showed significant upregulation at 2W and significant downregulation at 19W in response to zinc supplementation (Supplementary Table S7). This contains mineralization-related genes (Figure 5D, Supplementary Table S11 [42,43,44,45]) that could be associated with the inorganic hydroxyapatite component of sub-RPE deposits. A total of 80 out of the identified 99 calcification-related genes were expressed in the RPE (Supplementary Table S11). The genes that were expressed higher at 2W were COL10A1, NKX3-2, PHEX, SPP1, TNFRSF11B, and WNT7B, while others were expressed higher at 19W, including AP1S2, BMP2, CLCN3, LAMP1, POSTN, SMAD1, and SOX9. Among the genes expressed higher in less differentiated cells were AP1S1, COL10A1, COL1A1, DLX5, IBSP, JAM2, LAMP1, MGP, MYORG, NKX3-2, PDGFB, PHEX, POSTN, and RUNX2. The expressions of ABCC6, BMP2, BMP7, CNMD, SOX6, and WNT6 were higher in the more differentiated cells. COL1A1, POSTN, CD63, LAMP1, and BMP4 were significantly upregulated and SLC20A1, SOX9, and BMP7 were significantly downregulated in response to zinc supplementation (Supplementary Table S7). This contains genes that are related to pigmentation (Figure 5E, Supplementary Table S11 [9,46,47]). Pigmentary abnormalities show strong correlation with sub-RPE deposit formation and the development of AMD, and we found that 19 out of the identified 21 genes were expressed in hpRPE (Supplementary Table S11). At 2W, we found no differentially expressed genes. At 19W, however, we found that AP3B1, AP3D1, BLOC1S6, HPS5, HPS6, and SLC24A5 were expressed higher. There were no highly expressed genes in less differentiated cells. In the more differentiated cells, the expression of OCA2 and SLC24A5 was higher. TYR, TYRP1, and DCT were significantly downregulated in response to zinc supplementation in our acute treatment (Supplementary Table S7). The RPE plays a pivotal role in maintaining the health of the retina, and changes in RPE function have been linked to the development and progression of AMD [48,49]. Optimal zinc balance is key for RPE function [50], and zinc deficiency contributes to AMD pathogenesis [51]. Based on these findings, it has been suggested that zinc supplementation can slow the progression of AMD [51,52], although the mechanism of this beneficial effect is not fully understood [53]. In this study, we used primary human fetal RPE cells and scRNA-Seq analysis to identify the transcriptomic changes and biologically plausible molecular pathways involved in the maturation of the RPE and the changes associated with zinc supplementation. The specific transcriptional changes and molecular pathways identified provide an improved understanding of RPE cell maturation and insight into how the function of RPE might be affected by acute zinc supplementation, which has relevance for the progression of AMD. Maturation of RPE cells is key to developing appropriate morphology, pigmentation [54], and production of key signature proteins that determine the function of these cells [55]. Different studies use a variety of sources to study RPE maturation and function, ranging from the immortalized ARPE-19 cells [56] to induced pluripotent stem-cell-derived RPE [57] and primary porcine [16] or human RPE [58]. As with all model systems, cellular models for RPE must replicate the in vivo situation as closely as possible. Recently we have shown that primary human fetal RPE cells develop the most critical features of native RPE, including the formation of pigmentation, tight junctions with high TEER values, and the expression of RPE signature genes and proteins [15,16]. Most importantly, the cells in culture can lay down sub-RPE deposits, a hallmark feature of AMD [15,16]. Despite demonstrating these in vivo-like features, the molecular signature for RPE maturation has not yet been fully explored. Previous studies have reported a variety of approaches to map molecular maturation. Earlier studies used microarrays [23,59] or bulk RNA sequencing. Most recently, a powerful tool capable of sequencing individual cells has been introduced. Single-cell RNA sequencing provides an unparalleled opportunity to identify cell heterogeneity [60]. Lidgerwood et al. [57] used pluripotent stem-cell-derived RPE to analyze transcriptomic changes after 1 month or 12 months in culture and analyzed these separately, then combined the data. In a subsequent study, the same group combined scRNA-Seq and proteomics in iPSC cells obtained from individuals with or without AMD to identify regulations in geographic atrophy [61]. Exciting opportunities are presented by scRNA-Seq studies using freshly isolated RPE from human eyes. RPE cells from both fetal and adult human eyes were analyzed in previous studies [31] and [32], respectively). Although both studies used a limited number of cells, they provide invaluable insight for cell-culture-based observations. In our study, we used primary fetal RPE cells that recapitulated features of RPE cells in vivo (Supplementary Figure S1). Despite their fetal origin, these cells developed sub-RPE deposits and varied pigmentation, suggesting that they recapitulate the hallmarks of AMD (Supplementary Figure S1) despite the relatively short time in culture (19W). The generation of scRNA-Seq data from a large number of cells allowed us to confidently determine that there is a significant degree of heterogeneity between the cells. A key observation was that some RPE cells could develop into more differentiated cells even after 2 weeks in culture, but even after 19 weeks, we still observed less differentiated cells (Supplementary Figure S1). Heterogeneity of RPE had been reported after multiple passages and over the years in culture [62] (Supplementary Figure S1), reflecting what had been reported for RPE in vivo [63,64,65] and in situ [62]. Despite the long-lasting heterogeneity, the melanosome precursor PMEL17 was expressed in both less and more differentiated cells. In fact, from the 19 pigmentation-related genes expressed in our cells, the only transcripts that showed elevated expression in the more differentiated RPE cells were OCA2 and SLC24A2 (Supplementary Figure S2(B1) and Supplementary Table S11, Figure 5E), suggesting that all cells could become pigmented [46,66]. COL1A1 was amongst the top transcripts in the less differentiated cells, and immunoreactivity of COL1A1 protein was able to distinguish the less differentiated cells from the more differentiated cells that express the RPE65 gene highly and are immunopositive for the RPE65 protein (Figure 2C). Immunoreactivity to the COL1A1 protein gradually increased in the sub-RPE space with time in culture (Supplementary Figure S3), suggesting that the half-life of this extracellular matrix protein is long in our culture system. This increase in sub-RPE COL1A1 may correspond to the role this protein plays in forming the extracellular matrix of Bruch’s membrane [67]. Other collagens were also expressed highly in the less differentiated cell population (Supplementary Table S4), reflecting their reported involvement in increased attachment and spread of RPE cells [68]. The only highly expressed transcript for collagen in the more differentiated cells was COL8A1 (Supplementary Table S4). The COL8A1 protein is a component of basement membranes in the eye and contributes to the formation of the basement membrane of RPE [21,69] and a genetic risk variant of AMD [70]. The findings on COL1A1 and RPE65 might be mechanistically important: the mature RPE cells (RPE65 expressing) could enable the performance of the visual cycle, while the less differentiated cells (COL1A1 expressing) can support the formation of ECM throughout life. As more and less differentiated cells are present at all three time points, we combined the scRNA-Seq data from the three time points and analyzed these datasets together, an approach different from a previous study [57]. This integrated approach helped us to identify a pseudotemporal trajectory of gene expression from less to more differentiated cells (Figure 3(A1)). This approach identified a well-defined main trajectory (Figure 3(A2)). The top genes with the highest score in the main trajectory were associated with regulating the visual cycle (RPE65, LRAT, TTR, RDH5) (Supplementary Table S6). Transcriptomic analysis of the bulk RNA isolated from RPE cells from aging human donor eyes recently reported a positive feedback mechanism between the upregulation of visual cycle genes and the accumulation of retinoid by-products [71]. As visual cycle-related bisretinoids are constituents of the accumulating lipofuscin in RPE [72], this upregulation could eventually lead to AMD-like pathogenesis [73] in this cell culture model. Indeed, there are ongoing clinical trials for visual cycle modulators as therapeutic options for AMD [74], and our cell culture model has the potential to serve as a preclinical tool for testing novel compounds. The genes associated with the main trajectory could be clustered into seven modules based on their transcriptional change along the pseudotemporal trajectory (Supplementary Table S5). The transcripts whose expression is transiently upregulated on the pseudotemporal trajectory likely represent the genes mediating the transition from the less to the more differentiated cells (Supplementary Table S5). These genes were associated with cellular and extracellular remodelling and metabolic pathways (Supplementary Table S6). Therefore, our data support the hypothesis that extracellular matrix remodelling of the Bruch’s membrane could become a therapeutic target to combat RPE loss [75] due to topographic changes in the RPE–Bruch’s membrane interface [68]. Alterations of the extracellular matrix may impact immune response as well as the secretion of pro-inflammatory cytokines, such as MCP-1 and IL-8 [68], and promote sub-RPE deposit formation [76,77,78,79,80]. Our data highlights potential molecular targets to achieve a regulation of this process. Among the transiently expressed genes, we identified ID1 and ID3 (Supplementary Table S6). The corresponding helix–loop–helix (HLH) proteins form heterodimers with members of the basic HLH family of transcription factors, inhibiting DNA binding and preventing the formation of active transcriptional complexes [81]. ID proteins promote cell cycle progression and cell migration and restrict cellular senescence and the differentiation of a number of progenitor cell types [82,83]. Recent results indicate that the expression of ID family proteins may play an important role in regulating retinal progenitor cell proliferation and differentiation [84]. ID genes and proteins showed increased expression levels in the retina at embryonic and early postnatal stages and declined in the adult [84]. ID protein expression is silenced in many adult tissues but is re-activated in diverse disease processes [83,85,86]. ID proteins appear to play a crucial role in the angiogenic processes. It was proposed that inhibition of expression and/or function of ID1 and ID3 may be of therapeutic value for conditions associated with pathological angiogenesis [87]. In fact, the deletion of Id1/Id3 reduced ocular neovascularization in a mouse model of neovascular AMD [81]. In conclusion, drugs targeting ID1/ID3 could modulate RPE maturation and pathological changes in AMD. Treatment with zinc has been reported to prevent progression to advanced AMD (for review, see [88]), at least partly due to a direct effect of zinc on the RPE [15,89,90]. In previous in vitro studies, we investigated long-term supplementation with zinc and found altered selective gene expression, protein secretion, and increased pigmentation and barrier function [15,17]. We identified several molecular pathways, such as cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response, involved in the beneficial effects of chronic zinc supplementation on the RPE. However, these studies could not address the complexity associated with cell heterogeneity and detailed temporal changes. We were particularly interested in exploring how zinc supplementation could affect the less and more differentiated cells in the short term to understand the potential to develop a more targeted intervention through supplementation. To decipher the effects of acute zinc supplementation, RPE cells were treated with elevated zinc for 1 week following the protocols we used previously [17]. We found that acute zinc supplementation induced significant changes in gene expression in both short- and long-term cultures (Figure 4(B1–B4)) regardless of the temporal stage of the cells. We also identified 81 zinc-responsive transcripts (Figure 4C) that were common amongst all groups. These transcripts were enriched in housekeeping genes and contained transcripts for metallothioneins, ribosomal protein, and ATP synthases (Supplementary Table S8), indicating that zinc affects the cellular homeostasis of the RPE, similar to that of other systems [91]. Apart from the shared genes, specific changes were associated with the more or the less differentiated cell groups at both 2W and 19W in culture (Figure 4C). The four specific genes affected by short-term zinc supplementation in the less differentiated cell group (Supplementary Table S8) are genes linked to the integrity of Bruch’s membrane (COL8A1) [70], epithelial–mesenchymal transition (KRT17) [92], phagocytic activity and the rescue of the RPE (MFGE8) [93,94], and activity of heparan sulfate (SULF1) [22], suggesting that zinc might influence interaction with the local extracellular environment. In the more differentiated cell group in the 2W cultures, zinc affected biological processes including extracellular matrix organization, cellular polarity, and visual processes (Supplementary Table S8) that are critical for supporting the photoreceptors [95]. At 19W in culture, zinc affected the less differentiated cells via modulating proteolysis, DNA replication and RNA transcription, and amino acid metabolisms (Supplementary Table S8), probably to mitigate oxidative stress, one of the AMD-associated biological functions [96]. In the more differentiated cells at 19W in culture, zinc supplementation affected several metabolic pathways (Supplementary Table S8). Dysregulation of metabolic pathways is an important contributor to AMD pathophysiology [97]. This may directly explain the benefit of zinc supplementation in patients in the AREDS study [13,51,98]. Therefore, zinc supplementation has a multitude of effects on RPE, with some specific effects depending on cell differentiation and maturity. Identifying the specific molecular changes may help redefine treatment strategies based on zinc supplementation or nutritional interventions. Earlier we identified 537 genes (Supplementary Table S5) in the main pseudotemporal trajectory (Figure 3(A2)). Zinc supplementation did not affect 240 genes (Figure 4(D1)). Of the remaining 297 genes, 16 were housekeeping genes (Supplementary Table S8; Figure 4C) associated with the mitochondrion, the activation of cytochrome-c oxidase and ubiquinone, and response to metal ions (Supplementary Table S9). This is in line with a previous observation that zinc supplementation can protect the RPE from oxidative-stress-induced cell death by improving mitochondrial function [89], and this could be behind the positive effect of zinc supplementation in the AREDS studies [13,51,98] or increased zinc intake through diet [34,99]. Metallothioneins (MT1F and MT1E) that belong to this group (Figure 4(D2), Supplementary Tables S7 and S9) are well-recognized mediators of zinc supplementation in the RPE [100] via mediating oxidative-stress-induced RPE damage [90] and differentiation of RPE [57]. The remaining 281 genes in the main trajectory (Figure 4(D1)) were associated with various biological processes including extracellular matrix organization, angiogenesis, collagen fibril organization, and visual perception (Supplementary Table S10). The composition of extracellular matrix has a profound effect on how the RPE attaches to the Bruch’s membrane [101]. Thus, gene expression modification by zinc could directly affect sub-RPE deposit formation [76]. We also found that acute zinc supplementation upregulated the expression of transcriptional regulators ID1 and ID3, a finding that had not been reported before. In addition, in a previous study, we identified TGFB1 as a potential upstream regulator effect of chronic zinc supplementation [17]. In our current study, we found that TGFB1 expression was also upregulated by acute zinc supplementation. Therefore, we carried out an Upstream Analysis in Ingenuity Pathway Analysis (QIAGEN, Redwood City) for the 190 transiently expressed genes in the combined pseudotime-correlated groups 1 and 7 (Supplementary Table S5). We identified a strong relationship for TGFB1 (p < 6.98 × 10−19) and also for ID1 (p < 3.59 × 10−5) and ID3 (p < 1.23 × 10−3) as potential upstream regulators for a group of genes among the transiently expressed group. In fact, TGFB1 was an upstream regulatory element for ID1 and ID3 (Supplementary Table S12). A direct molecular link between ID1 and TGFB1 had already been suggested [102]. Therefore, the positive effects of zinc supplementation could be directly through TGFB1 signalling, which involves ID1 and ID3. The receptor of TGFB1, TGFBR1, is an AMD genetic risk variant [36], suggesting that these findings are directly relevant to further studies on AMD. Based on literature searches, we generated gene lists that have been shown to contribute to the pathological changes associated with AMD and we examined the effects of cell maturation and zinc supplementation on these genes (Figure 5, Supplementary Table S11). Specific attention was paid to the activation complement system and lipid-metabolism-related genes, as these were the genetically most significantly associated pathways with AMD [36,103]. We also scrutinized genes associated with pigmentary changes and mineralization-associated genes due to their potential link with RPE function and/or sub-RPE deposit formation in AMD [5,45]. Not all genes involved in complement regulation were expressed in RPE cells (Figure 5B, Supplementary Table S11). This is perhaps not surprising, as the local activity of the complement cascade is influenced by a complicated mix of local and systemic regulatory factors, which is altered in AMD retina [4,104,105]. However, some complement genes that were expressed in the RPE were affected by acute zinc supplementation, including CFH, C1R, CFHR1, and CLU (Figure 5B, Supplementary Table S7). These transcriptomic changes are in line with our previous reports that zinc supplementation has a functional effect on CFH secretion [17] as well as oligomerization and activity [106], and zinc levels can regulate interferon gamma systematically, which, in turn, regulates expression of complement genes [107,108]. Apart from CFH, several complement proteins can bind zinc, and this binding alters their activity [109,110]. In addition, network analysis has highlighted elements of the complement regulation as potential targets for nutrient-affected pathways [111]. Finally, there is also clinical evidence that zinc supplementation can directly inhibit complement activation in AMD patients [104], suggesting that modulation of the complement system could be one of the ways that zinc supplementation affects the progression to AMD. Of the 42 genes expressed in our RPE culture associated with cholesterol metabolism (Figure 5C, Supplementary Table S11), ANGPTL4, LRPAP1, VDAC2, APOE, PLTP, NPC2, TSPO, and CYP27A1 were altered in response to acute zinc supplementation (Figure 5C, Supplementary Table S7) [112,113,114]. These findings corroborate our previously reported effect of long-term zinc supplementation on lipid metabolism [17]. ANGPTL4 is a lipid-inducible feedback regulator of LPL-mediated lipid uptake. However it is also a multifunctional cytokine, regulating vascular permeability, angiogenesis, and inflammation [115]. The systemic level of ANGPTL4 is associated with NV AMD [116]. Reportedly, this protein indirectly induces RPE barrier breakdown [117]. LRPAP1 is a chaperon protein, generally controlling the folding and ligand–receptor interaction expression of the LRP receptors [118]. Its role in RPE and AMD remains elusive. VDAC2 is a ceramide sensor integrated into the mitochondrial membrane and its function relates to regulation of mitochondrial apoptosis [119,120]. Increased ceramide levels affect non-polarized RPE cells found in late stages of AMD [121]. APOE, a lipophilic glycoprotein with a major role in lipid transport, is one of the many constituents of the sub-RPE deposits and has been associated with increased AMD risk [122,123]. PLTP is a phospholipid transfer protein and is one of the main players in lipid homeostasis in ApoB-containing particles and high-density lipoprotein metabolism. PLTP plasma levels are associated with AMD [124], but their potential role in drusen formation remains elusive. NPC2 is a cholesterol transporter, effluxing cholesterol out of late endosomes in RPE. The lack of this protein is associated with age-related maculopathies [125]. TSPO is a translocator protein that transfers cholesterol from the mitochondrial outer membrane to the mitochondrial inner membrane and also plays role in oxidative stress and inflammation. It was recently implicated as a highly relevant drug target for immunomodulatory and antioxidant therapies of AMD [126,127]. CYP27A1 is involved in the elimination of 7-ketocholesterol from RPE, a toxic product of cholesterol auto-oxidation, which accumulates in drusen [128,129]. In summary, the aforementioned affected gene expressions in response to zinc suggest that zinc has an impact on sub-RPE cholesterol accumulation, oxidative stress, inflammation, and angiogenesis via the regulation of lipid–membrane interaction, lipid transport, and the elimination of toxic lipid byproducts. In our cultures, we found 80 RPE-expressed genes associated with mineralization (Figure 5D, Supplementary Table S11). Out of these, we found that COL1A1, POSTN, CD63, LAMP1, BMP4, SLC20A1, SOX9, and BMP7 were altered in response to acute zinc supplementation (Figure 5D, Supplementary Table S7). The POSTN gene encodes a secreted extracellular matrix protein that functions in tissue development and regeneration and a potential anti-fibrotic therapeutic target for NV AMD [130]. CD63 is involved in the regulation of cell development, activation, growth, and motility [131], and together with LAMP1, it plays a role in autophagy, exosome secretion, and drusen formation [132,133]. BMP4 has been implicated in the disruption of RPE cell migration and barrier disruption in NV AMD [134]. The protein encoded by SLC20A1 is a sodium–phosphate symporter involved in vascular calcification but not reported in association with RPE function or AMD [135]. SOX9 plays a key role in regulating visual cycle gene expression in RPE [136] but also plays a role in the prevention of calcification [137]. BMP7 is hypothesized to be critical for the differentiation of the retinal pigmented epithelium during development [138]. It also has been implicated in prevention of vascular calcification [139]. Zinc supplementation is reported to inhibit phosphate-induced vascular calcification [140], but, as our results indicate, it may also have a (indirect) role in the prevention of drusen calcification. In our cultures, most pigmentation-related genes were detected and their expression level either remained constant or increased throughout the culture time (Figure 5E, Supplementary Table S11). Only TYR, TYRP1, and DCT were altered in response to acute zinc supplementation (Figure 5D, Supplementary Table S7). TYR, TYRP1, and DCT are key to the production of melanin [46] and pigmentary abnormalities show a strong correlation with sub-RPE deposit formation and development of AMD [9]. TYR catalyzes the production of melanin from tyrosine, in which L-DOPA is produced as an intermediate [141,142]. The function of TYRP1 is in the biosynthesis of melanin from tyrosine, whilst TYRP1 catalyzes the oxidation of 5–6-dihydroxyindole-2-carboxylic acid to an indole, whilst DCT catalyzes the conversion of L-dopachrome into 5–6-dihydroxyindole-2-carboxylic acid [142]. These events lead to the activation of GPR143 signaling and may initiate several downstream effects, such PEDF, VEGF secretion, and/or exosome release [46]. Since we found an influence of zinc on the expression of these pigmentation-related genes, and given the data from literature above, zinc might also have an influence on GPR143 signaling. Surprisingly, acute zinc treatment resulted in downregulation of the aforementioned genes, despite long-term zinc supplementation enhancing RPE pigmentation [15]. At the transcriptional level, long-term zinc supplementation significantly altered the expression of 18 out of the 21 pigmentation-related genes (Supplementary Table S11, [17]), of which the majority were also downregulated, except for HPS5, HPS6, and LYST. These three upregulated genes are all related to intracellular trafficking, such as lysosomes and melanosomes [143,144,145].The negative effect of acute zinc supplementation on the gene expression of other pigmentation-related genes needs to be further investigated. Primary hfRPE cultures that recapitulate the main phenotypes of aged RPE in vivo can help to dissect the molecular changes associated with RPE maturation and experimental manipulation, such as zinc supplementation. This cellular model provides an excellent platform for further preclinical studies to identify new treatment strategies for AMD. As reported in vivo, these cells retain a high degree of heterogeneity even after extended time in culture, which may help to understand the role of this heterogeneity in the human eyes. Identifying the transcriptional machinery, including transcriptional regulators ID1 and ID3, may help us to target pathways previously not considered for AMD. The data also show that the differentiation of RPE into cells that resemble those in vivo requires an extended time in culture, and experimental manipulation will need to consider this. The wide-ranging effects of zinc supplementation, from the regulation of housekeeping genes to very specific AMD-associated transcripts, build confidence that this intervention could indeed be a suitable intervention strategy to slow the progression to advanced-stage AMD, as suggested by the AREDS studies.

PMC10000422

Danielle K. DePalo,Jonathan S. Zager

Advances in Intralesional Therapy for Locoregionally Advanced and Metastatic Melanoma: Five Years of Progress

23-02-2023

advanced melanoma,in transit metastases,intralesional therapy,intratumoral therapy,local therapy,locoregional therapy,melanoma,metastatic melanoma,regional therapy,regional chemotherapy

Simple Summary The prognosis for patients with locoregionally advanced and metastatic melanoma remains poor despite advances in systemic therapy. By delivering therapeutics directly to the site(s) of disease, intralesional therapies have the advantage of delivering the oncolytic agent directly into the metastatic melanoma while minimizing systemic side effects and resistance. Within the past 5 years, numerous potential intratumoral therapies have been investigated, though few have reached phase 2 clinical trials. We present a discussion of the scientific rationale for and status of intralesional therapies that have reached phase 2 or later clinical trials within the past 5 years in order to inform providers about current and upcoming intralesional therapeutic options for advanced melanoma. Abstract Locoregionally advanced and metastatic melanoma are complex diagnoses with a variety of available treatment options. Intralesional therapy for melanoma has been under investigation for decades; however, it has advanced precipitously in recent years. In 2015, the Food and Drug Administration (FDA) approved talimogene laherparepvec (T-VEC), the only FDA-approved intralesional therapy for advanced melanoma. There has been significant progress since that time with other oncolytic viruses, toll-like receptor agonists, cytokines, xanthene dyes, and immune checkpoint inhibitors all under investigation as intralesional agents. Further to this, there has been exploration of numerous combinations of intralesional therapies and systemic therapies as various lines of therapy. Several of these combinations have been abandoned due to their lack of efficacy or safety concerns. This manuscript presents the various types of intralesional therapies that have reached phase 2 or later clinical trials in the past 5 years, including their mechanism of action, therapeutic combinations under investigation, and published results. The intention is to provide an overview of the progress that has been made, discuss ongoing trials worth following, and share our opinions on opportunities for further advancement.

Advances in Intralesional Therapy for Locoregionally Advanced and Metastatic Melanoma: Five Years of Progress The prognosis for patients with locoregionally advanced and metastatic melanoma remains poor despite advances in systemic therapy. By delivering therapeutics directly to the site(s) of disease, intralesional therapies have the advantage of delivering the oncolytic agent directly into the metastatic melanoma while minimizing systemic side effects and resistance. Within the past 5 years, numerous potential intratumoral therapies have been investigated, though few have reached phase 2 clinical trials. We present a discussion of the scientific rationale for and status of intralesional therapies that have reached phase 2 or later clinical trials within the past 5 years in order to inform providers about current and upcoming intralesional therapeutic options for advanced melanoma. Locoregionally advanced and metastatic melanoma are complex diagnoses with a variety of available treatment options. Intralesional therapy for melanoma has been under investigation for decades; however, it has advanced precipitously in recent years. In 2015, the Food and Drug Administration (FDA) approved talimogene laherparepvec (T-VEC), the only FDA-approved intralesional therapy for advanced melanoma. There has been significant progress since that time with other oncolytic viruses, toll-like receptor agonists, cytokines, xanthene dyes, and immune checkpoint inhibitors all under investigation as intralesional agents. Further to this, there has been exploration of numerous combinations of intralesional therapies and systemic therapies as various lines of therapy. Several of these combinations have been abandoned due to their lack of efficacy or safety concerns. This manuscript presents the various types of intralesional therapies that have reached phase 2 or later clinical trials in the past 5 years, including their mechanism of action, therapeutic combinations under investigation, and published results. The intention is to provide an overview of the progress that has been made, discuss ongoing trials worth following, and share our opinions on opportunities for further advancement. While early-stage localized melanoma disease has a 5-year survival of greater than 90%, regional and distant disease have 5-year survival rates of 66% and 27%, respectively [1]. Therefore, there remains significant room for improvement in the treatment of advanced melanoma. Many of the major advancements in the treatment of advanced disease have been with systemic immune checkpoint inhibitors (ICIs) and targeted therapies [2,3,4]. These therapies have revolutionized the treatment of advanced disease, which previously had an even poorer prognosis; however, they can have permanent, life-altering side effects in some and responses are limited [2,3,4,5]. In patients with locoregionally advanced and metastatic melanoma that is unresectable but amenable to injection, intralesional therapy provides a promising alternative or adjunct to systemic therapy. Intratumoral therapy has been explored for the treatment of melanoma since the 1970s. Early investigations included the injection of bacillus Calmette–Guerin (BCG), a live, attenuated strain of Mycobacterium bovis developed as a tuberculosis vaccine, as an intralesional therapy for melanoma in 1974 [6,7]. More recent randomized trials have not supported the efficacy of BCG [8], and it has fallen further out of favor due to significant systemic toxicity [7,9]. However, since that time, innumerable other intralesional therapies have been and continue to be investigated for the treatment of advanced melanoma. To limit the scope of this discussion to the most current and clinically relevant therapies, the focus of this review is on intralesional therapies that have reached phase 2 or later clinical trials within the past 5 years. To identify clinical trials, the clinicaltrials.gov Advanced Search feature was used. The condition or disease was specified as melanoma and phase 2, phase 3, and phase 4 were selected. Key terms searched were: intralesional, intratumoral, inject, xanthene, PV-10, Rose Bengal, intratumoral interleukin, intratumoral interferon, Aldesleukin, tavokinogene telseplasmid, tavo, electroporation, Darleukin, L19IL2, Daromun, L19TNF, stimulator of interferon genes, STING, DMXAA, MIW815, MK1454,, oncolytic virus, talimogene laherparepvec, T-VEC, GM-CSF, vusolimogene oderparepvec, RP1, OrienX010, Oncos-102, BT-001, canerpaturev, C-REV, TBI-1401, HF-10, coxsackievirus A21, CA21, CAVATAK, V937, gebasaxturev, lerapolturev, PVSRIPO, Telomelysin, OBP-301, Voyager-V1, VV1, VSV-IFNβ-NIS, VSV-IFNβTYRP1, Ad-p53, RheoSwitch, Ad-RTS-hIL-12, IXN-2001, veledimex, IXN-1001, oca511, Toca FC toll-like receptor, TLR, tilsotolimod, IMO-2125, SD-101, vidutolimod, CMP-001, cavrotolimod, AST-008, Lefitolimod, MGN1703, NKTR-262, G100, CV8102, LHC165, intratumoral ipilimumab, LL37, MAGE-A3, Hiltonol, poly-ICLC, APX005M, ABBV-927, dendritic cell, INT230-6, and TTI-621 polidocanol. Trials were included if they had published results in 2018 or later or were ongoing as of December 2022. Trials were excluded if at least one agent was not delivered intralesionally. PV-10 is a formulation of 10% Rose Bengal xanthene dye in saline. The dye was first found to have antitumor effects when it was observed to have a dose-dependent survival benefit in a strain of mice with a propensity for spontaneous tumor development that were used to test its safety as a food dye [10]. PV-10 is theorized to cause photolytic release of lysosomal enzymes resulting in tumor cell lysis and has been found in vitro to cause cell lysis in melanoma cell lines, but not in normal fibroblasts [11,12]. Bystander lesion response has also been observed with intralesional PV-10 injection and is theorized to result from High Mobility Group Box 1 activation and the maturation of dendritic cells and tumor-specific CD-8+ T cells [13,14,15,16]. In 2015, a phase 2 clinical trial of single-agent intratumoral PV-10 in 80 patients with treatment-refractory American Joint Committee of Cancer 8th Edition [17] stage III-IV melanoma found an overall response rate (ORR) of 51% with an ORR of 33% in uninjected lesions [18]. AEs were observed in all patients, mostly grade 1 to 2, while 15% experienced possibly related grade 3 adverse event (AE); the most common AEs were injection site pain (80%), edema (41%), and vesicles (39%). This was continued in two single-institution studies that reported ORRs of 53–87% with a 50% response in non-injected lesions and consistent AE profiles [19,20]. The dosing schedule examined, intralesional injections on Day 0 then repeated on Weeks 8, 12, and 16 for additional or incomplete responding lesions, makes this therapy particularly appealing. The addition of external beam radiotherapy (XRT) to intralesional PV-10 was examined in stage IIB-IV melanoma in a phase 2 trial at a single institution [21]. A total of 98 target lesions in 15 patients were treated, the ORR was 87%, and all patients experienced at least one grade 1 or 2 AE, including injection site pain (87%), swelling (60%), and blistering (20%) with one patient experiencing a grade 3 AE of injection site pain. A phase 3, randomized controlled trial (NCT02288897) sought to compare PV-10 to investigator choice chemotherapy (dacarbazine or temozolomide) or T-VEC, but was terminated early due to an inadequate rate of enrollment and the changing landscape of therapy for advanced melanoma. Since PV-10 exerts its antitumor effects through T cell activation [13], the addition of immune checkpoint inhibitors provides a potential pathway for overcoming resistance or potentiating PV-10′s effects. The efficacy of intralesional PV-10 with systemic pembrolizumab, anti-programmed cell death protein 1 (PD-1) antibody, in stage IIIB-IVM1c unresectable melanoma is undergoing evaluation in a phase 1b/2 trial (NCT02557321) in which intralesional PV-10 is dosed every 3 weeks with intravenous pembrolizumab for up to 5 weeks followed by intravenous pembrolizumab alone (Table 1). In phase 1b, 21 ICI-naive patients experienced a 67% ORR with primarily grade 1 and 2 injection-site-related AEs and one grade 3 AE related to PV-10 of injection site pain as well as AEs consistent with pembrolizumab’s known toxicity profile [22]. An expansion phase 1b cohort in 14 evaluable ICI-refractory patients had an ORR of 29% and AEs were consistent with known drug profiles, primarily grade 1 or 2 injection site events related to PV-10 and grade 1–3 immune-related events related to pembrolizumab [23]. Phase 2 with arm 1, PV-10 with pembrolizumab, and arm 2, pembrolizumab alone, is ongoing. IL-2 acts to activate numerous pathways modulating lymphocyte proliferation, activity, and survival by binding the IL-2 receptor [31]. It received Food and Drug Administration (FDA) approval in 1998 for the treatment of advanced melanoma; however, its systemic application has been limited by significant side effects at efficacious doses [32]. Intralesional injection of IL-2 is an appealing way to minimize systemic effects while locally delivering high doses of the drug. This premise was first investigated in 1994 [33], and in 2010 a phase 2 trial in 48 evaluable patients reported a complete response (CR) of 69%, though the response was not observed in any uninjected lesions [34]. Only grade 1/2 AEs were reported, including injection site reaction and pain in most patients as well as fever (58%), fatigue (36%), and nausea (34%). The addition of ICIs to cytokine therapy should theoretically potentiate cytokine effects by antagonizing immune regulatory pathways [31]. In 2017, a phase 2 trial (NCT01480323) sought to investigate the combination of intratumoral IL-2 plus systemic ipilimumab, an anti-CTLA-4 antibody, in 15 patients with treatment-refractory stage IV melanoma, but found a disappointing ORR of 0%, a disease control rate (DCR) of 20%, and grade 3/4 AEs in 40%, most commonly fatigue and pain (excluding injection pain) [35] The combination of systemic ICI with intratumoral IL-2 is under further investigation in a phase 1/2 trial (NCT03474497) evaluating intralesional IL-2 in combination with systemic pembrolizumab and hypofractionated radiotherapy in patients with metastatic melanoma and other solid tumors who failed to respond or progressed on previous anti-PD-1/PD-L1 therapy (Table 1). One limitation to the use of IL-2 in the above-mentioned trials is the frequent dosing schedule of injections, two times per week. Every-2-week dosing is under investigation in a phase 2/3, randomized controlled trial (NCT03928275), a 2-stage study of intralesional IL-2 versus combination intralesional IL-2 and BCG in stage IIIC-IVM1a melanoma. Interleukin-12 (IL-12) bridges the innate and adaptive immune systems, stimulates Interferon-gamma (IFN-γ), regulates natural killer (NK) and T cell production, and promotes T helper type 1 (Th-1) differentiation [36,37]. Systemic administration of IL-12 has been found to have high toxicity with limited efficacy [37,38]. Tavokinogene telseplasmid (tavo) is a plasmid that encodes IL-12 that is delivered intratumorally and is typically combined with electroporation (EP) to improve plasmid cell uptake by increasing cell permeability [39,40]. Simply, this results in increased local IL-12 and IFN-γ expression, which activates the innate and adaptive immune systems [39,41,42]. Intratumoral tavo-EP alone in stage III-IV melanoma was investigated in a phase 2 trial (NCT01502293) (Figure 1). The results published in 2020 included a CR rate of 18% and ORR of 36% in the 28 patients in the standard dosing cohort, with a median PFS of 3.72 months [42]. The most common AEs reported were procedural pain in 80% and various injection site reactions. Since cytokines increase tumor-specific T cells, the addition of ICIs should potentiate their effects by inhibiting immune regulatory pathways [31]. This combination is under investigation with intratumoral tavo-EP and intravenous pembrolizumab in a phase 2 trial (NCT03132675) in 54 ICI-refractory patients with stage III/IV melanoma (Table 1). On interim analysis, ORR was 30% with a reduction in non-injected tumor burden seen in all 12 patients with non-injected disease and the most common AEs were low-grade fatigue, procedural pain, and diarrhea [24]. Though tavo-EP injection occurs on Days 1, 5, and 8 of each cycle, cycles span for 6 weeks, making overall dosing frequency more manageable. As previously discussed, IL-2 acts to increase tumor immunogenicity but is not well tolerated when delivered systemically at efficacious doses [31,32]. A fusion protein of IL-2 to L19, a monoclonal antibody targeted to an angiogenesis marker, L19IL2, enables preferential delivery and activation of the cytokine within the tumor cells [43]. It has been investigated as a single agent in a phase 2 trial (NCT01253096) in 24 evaluable patients with unresectable stage IIIB/C melanoma and found to generate a CR in 25% at lower doses than non-targeted IL-2 with the most common toxicity being injection site reaction, seen in 76%, with few grade 3 cases [44]. Other AEs were seen in 25% or less and included fatigue, edema, and fever. The addition of intratumoral L19IL2 to systemic dacarbazine vs. systemic dacarbazine alone is now under investigation for the treatment of stage IVM1a-b melanoma in a phase 1/2 trial (NCT02076646) (Table 1). Daromun is the combination of the previously mentioned, L19IL2 and L19TNF, a fusion protein of L19 and human recombinant tumor necrosis factor-alpha (TNFα). The addition of L19TNF acts synergistically in murine models to enhance the antiangiogenic and, therefore, antitumor effects [45]. A phase 2 trial (NCT02076633) of intratumoral daromun injection weekly for 4 weeks in stage III-IVM1a melanoma in 2015 found that of 20 evaluable patients, 50% had PR, 25% had SD, and 20% had PD for an ORR of 55% and DCR of 80%. Additionally, in 13 lesions not amenable to injection, 54% demonstrated CR [46]. Injection site reaction was the most common AE, seen in 73%, and was the only grade 3 AE reported. Other common grade 1 and 2 AEs were fever, headache, edema, and erythema. The efficacy of intralesional daromun in the neoadjuvant setting with systemic investigator’s discretion ICI compared to neoadjuvant systemic ICI alone is now under investigation in a phase 3 trial (NCT03567889) in resectable stage IIIB/C melanoma in the United States and Europe (Table 1) [47]. A similar trial (NCT02938299) is ongoing at European centers with neoadjuvant intratumoral daromun versus surgery alone. Talimogene laherparepvec (T-VEC) is a live-attenuated herpes simplex virus type-1 (HSV1) that has been engineered to express the gene for granulocyte-macrophage colony-stimulating factor (GM-CSF) [48]. Two mutations in the engineered HSV1, γ34.5 and α47 gene deletions, enable the virus to selectively replicate in tumor cells and enhance the immune response, respectively [49]. T-VEC enters tumor cells through herpes virus glycoproteins and ultimately results in cell lysis. Lysis of injected cells releases viral particles, tumor-derived antigens, and GM-CSF, promoting both cell-mediated and humoral immune responses and linking innate and acquired immune systems through T cell proliferation and activation and DC growth and maturation [49,50]. While the results of intralesional injection of GM-CSF have been underwhelming [51,52], intralesional T-VEC has received FDA approval. This is based on the results of the OPTiM phase III trial in stage IIB-IV melanoma, in which 47% of injected lesions, 22% of uninjected non-visceral lesions, and 9% of uninjected visceral lesions had CR [53]. The most common adverse events seen in those treated with T-VEC were fatigue (50%), chills (49%), and pyrexia (43%), with an 11% incidence of grade 3 or 4 treatment-related AEs including cellulitis (2%), fatigue (2%), and vomiting (2%) [54]. Single-agent T-VEC neoadjuvant therapy with surgery (arm 1, n = 76) versus surgery alone (arm 2, n = 74) in resectable Stage IIIB-IVM1a melanoma was compared in a phase 2 randomized controlled trial, NCT02211131, which demonstrated that 2-year recurrence-free survival (RFS), the primary endpoint, was improved with the addition of neoadjuvant T-VEC, 29.5% vs. 16.5% (overall hazard ratio 0.75, 80% confidence interval 0.58–0.96) for a 25% recurrence risk reduction with the addition of neoadjuvant T-VEC (Figure 1) [55]. Side effects included flu-like illness and pyrexia and were consistent with the known T-VEC safety profile established by the OPTiM trial [54,55]. The application of T-VEC monotherapy as a neoadjuvant therapy in surgically resectable, high-risk melanoma is also being evaluated in a phase 2 trial, NCT04427306 (Table 1). Theoretically, oncolytic viral therapies and ICIs should have synergistic effects since the viral infection increases susceptibility to immune surveillance and facilitates antigen-presenting cell (APC) processing [49,56]. A phase 2 trial in which CD8+ T cell levels were monitored in response to intralesional T-VEC demonstrated that, while CD8+ T cell levels were not associated with response, a 2.4-fold increase in CD8+ T cells was observed in non-injected lesions and the CD8+ T cells identified in non-injected lesions had increased expression of PD-1, programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoints [57]. In a 2018 phase 2 trial (NCT01740297) of 98 patients who received intratumoral T-VEC with intravenous ipilimumab versus 100 who received systemic ipilimumab alone, ORR was significantly improved with the combination, 39% vs. 18%, respectively, without a significant increase in toxicity, the most common AEs in the combination arm being fatigue (59%), chills (53%), and diarrhea (42%) (Figure 1) [58]. However, in 2022, a phase 3 (NCT02263508) randomized controlled, double-blinded trial evaluating intralesional T-VEC with systemic pembrolizumab versus an intralesional placebo with systemic pembrolizumab in 692 patients with Stage IIIB-IVM1c unresectable, anti-PD-1-naïve melanoma found no significant difference in overall survival (OS) or progression-free survival (PFS) and similar rates of grade 3 or greater treatment-related AEs between the two groups [59]. However, it is important to note that the lack of statistical significance in survival may in part be due to the inclusion of stage IVM1b and IVM1c patients in this trial when the most benefit from T-VEC has been demonstrated in stage IIIB-IVM1a in the past [60]. An ongoing phase 2 trial (NCT04068181) is evaluating the combination of intralesional T-VEC with systemic pembrolizumab in patients who have previously progressed on anti-PD-1 therapy (Table 1). Preliminary results have shown limited efficacy in those who previously progressed on anti-PD-1 in a locally recurrent or metastatic setting, 0% ORR in Cohort 1 (primary resistance, n = 26) and 7% ORR in Cohort 2 (acquired resistance, n = 15), but more promising results in those who progressed after receiving anti-PD-1 only in the adjuvant setting, 40% ORR in Cohort 3 (disease free < 6 months, n = 15) and 47% ORR in Cohort 4 (disease free ≥ 6 months, n = 15) [25]. The most common treatment-related AEs in all cohorts combined were pyrexia (30%), fatigue (16%), and flu-like illness (16%) with 13% experiencing grade 3 or higher treatment-related AEs. The combination of intratumoral T-VEC with systemic ICI is also being examined in the neoadjuvant setting. A phase 2 trial, NCT04330430, is evaluating neoadjuvant T-VEC with nivolumab for resectable Stage IIIB-IVM1a melanoma (Table 1). In patients with clinically node-positive disease, neoadjuvant intranodal T-VEC injection with pembrolizumab is being evaluated in a phase 2 trial, NCT03842943. The addition of T-VEC to other treatment modalities also provides an avenue for therapeutic benefit. Intralesional T-VEC with and without radiotherapy is under investigation in a phase 2 trial in Stage IIIB or higher melanoma and other solid malignancies (NCT02819843) (Table 1). Additionally, a phase 1/2 trial (NCT03555032) examining the efficacy of intralesional T-VEC with isolated limb perfusion in stage IIIB-IVM1b melanoma as well as sarcoma is ongoing. RP1 is a genetically modified HSV1 encoding GM-CSF and the gibbon ape leukemia virus fusogenic membrane glycoprotein with R sequence deletion (GALV-GP R-) that has been demonstrated to have potent antitumor activity in vitro, abscopal effects in murine models, as well as potentiated effects when administered with anti-mouse PD-1 antibody in murine models [61]. In humans, a phase 1/2 trial (NCT03767348) of intratumoral RP1 alone and in combination with intravenous nivolumab in advanced solid tumors, including Stage IIIB-IVM1c melanoma, is underway (Table 1). Interim results in cutaneous melanoma reported an ORR of 63% in eight anti-PD-1-naïve patients who received combination therapy and 38% in 16 patients who previously failed anti-PD-1 therapy [26]. More recent interim results of 75 patients who failed anti-PD-1/L1 therapy reported an ORR of 36% and DCR of 53% at a median follow-up of 10 months [27]. An additional phase 1b/2 trial of RP1 (NCT04349436) is ongoing for cutaneous malignancies, including locally advanced or metastatic melanoma, in solid organ transplant patients. OrienX010 is an oncolytic HSV engineered to express GM-CSF like T-VEC does; however, it uses a strain isolated in China, HSV1 CL1 [62]. It is undergoing phase 2 investigation (NCT04200040) as a single, intratumoral agent compared to systemic dacarbazine in treatment-naïve, unresectable Stage IIIB-IVM1b melanoma in China (Table 1). Oncos-102 is an adenovirus that is genetically modified to express GM-CSF that has preferential tumor cell binding through desmoglein 2 receptors [63]. As with T-VEC, the intratumoral GM-CSF increases the antitumor response by increasing NK and CD8+ T cells. Furthermore, combination with pembrolizumab in murine models has increased antitumor activity, thought to be related to increased immune surveillance against tumor-antigens exposed by viral cell lysis. A phase 2 trial (NCT05561491) of intratumoral ONCOS-102 alone or in combination with intravenous anti-PD-1 inhibitor, balstilimab, in anti-PD-1-refractory unresectable or metastatic melanoma is not yet accruing (Table 1). BT-001 is an oncolytic vaccinia virus containing genes encoding human GM-CSF and the human recombinant anti-CTLA-4 antibody [64]. This allows for T-VEC-like effects of local GM-CSF delivery as well as local immune checkpoint blockade from delivery of the anti-CTLA-4 antibody. In murine models, it has also been demonstrated to have abscopal effects despite low systemic levels of the anti-CTLA4 antibody. An ongoing, phase 1/2a, multipart clinical trial, NCT04725331, is evaluating intratumoral BT-001 alone and in combination with systemic pembrolizumab in multiple solid tumors, including locally advanced or metastatic melanoma (Table 1). C-REV is a strain of HSV1, HF10, with naturally occurring genomic alterations that result in preferential infection of and replication in tumor cells [65]. This causes cytolysis and intratumoral penetration of CD4+, CD8+, and NK cells. As previously discussed, oncolytic viruses are theorized to synergize with ICIs by increasing immune surveillance [49,56]. Therefore, intratumoral C-REV in combination with intravenous ipilimumab was investigated in a phase 2 trial (NCT02272855) in 44 evaluable patients with ipilimumab-naïve, stage IIIB-IV melanoma (Figure 1). The results presented in 2018 included an ORR of 41% and DCR of 68% [66]. Grade 3 or higher AEs were reported in 37%, though only 7% were attributed to C-REV and were classified as gastrointestinal, musculoskeletal, metabolism/nutrition, and vascular disorders. In 2019, the results of a similar phase 2 trial (NCT03153085) in 27 evaluable Japanese patients who had failed prior therapies were presented and included an 11% ORR and 56% DCR [67]. Unspecified severe AEs were seen in 36% of patients. Further investigation of the combination of intralesional C-REV with systemic ICI was undertaken in a phase 2 trial (NCT03259425) examining C-REV in combination with nivolumab in the neoadjuvant setting for resectable stage IIIB-IVM1a melanoma, but it was terminated in 2022 at the recommendation of the Data Safety Monitoring Committee. CVA21 is an enterovirus that preferentially infects melanoma cells since it binds to receptors that are overexpressed on melanoma cells, decay-accelerating factor (DAF) and intracellular adhesion molecule-1 (ICAM-1) [68]. A phase 2 trial (NCT01227551) of intratumoral CVA21 in 57 patients with stage IIIC-IV melanoma published in 2021 reported an ORR of 39% (unconfirmed per irRECIST) and 28% (confirmed) as well as 12-month PFS and OS of 33% and 75%, respectively (Figure 1) [69]. All treatment-related AEs reported were grade 1 or 2 with the most common being injection site pain, fatigue, and chills. Patients with stable or responding disease could continue on NCT01636882, the extension study. The synergistic potential of CVA21 delivered both intratumorally and intravenously in combination with systemic pembrolizumab versus pembrolizumab alone in anti-PD-1-naïve stage III-IV melanoma is under phase 2 investigation (NCT04152863) (Table 1). The same combination is under investigation in the neoadjuvant setting in one arm of a multi-arm, phase 1/2 study (NCT04303169) comparing systemic pembrolizumab in combination with five different investigational agents in stage III melanoma. PVSRIPO is a recombinant, live-attenuated poliovirus Sabin type 1 with human reovirus type 2 in the internal ribosomal entry site [70]. It preferentially enters melanoma cells, which have a high rate of CD155 poliovirus receptor expression, and stimulates the host antiviral immune response against the tumor cells. It is currently under investigation in a phase 2 randomized clinical trial (NCT04577807) as a single, intralesional agent or in combination with systemic anti-PD-1 therapy in unresectable, anti-PD-1/L1 refractory stage III-IVM1b (Table 1). OBP-301 is a telomerase-specific replication-competent oncolytic adenovirus 5 in which human telomerase reverse transcriptase enables selective replication in tumor cells [71]. Preclinically, it has been shown to increase local infiltration of CD8+ T cells and APCs and decrease regulatory T cells in both injected and uninjected tumor sites. A phase 2a trial, NCT03190824, examining this drug as an intralesional therapy in unresectable Stage III-IV melanoma is ongoing (Table 1). VV1 is a vesicular stomatitis virus engineered to express interferon-beta (IFN-β), potentiating the immune response to viral cell lysis, as well as a sodium/iodide symporter (NIS) gene for tracking through single-photon emission computerized tomography (SPECT) or positron emission tomography (PET) imaging [72,73]. It has been shown to increase inflammation and T cell infiltration in injected and non-injected lesions in phase 1 trials, and clinical benefit was observed as a monotherapy and in combination with systemic ICI in ICI-refractory disease. A phase 2 trial (NCT04291105) is underway in anti-PD-1/L1-refractory solid tumors, including advanced or metastatic melanoma; one study arm includes intratumoral and intravenous VV1 with intravenous cemiplimab in melanoma (Table 1). Ad-p53 is a recombinant human adenovirus with wild-type p53. It functions as a gene therapy, delivering the wild-type p53 gene to tumor cells, which otherwise often suppress p53 function as a part of regulatory evasion [74]. It has been applied to other malignancies without any reported serious adverse events, the most common AEs being transient fever, flu-like symptoms, muscle aches, and injection site pain, and is approved in China for the treatment of head and neck squamous cell carcinoma. It is now under phase 2 investigation (NCT03544723) in combination with ICI in recurrent or metastatic solid tumors including melanoma (Table 1). RheoSwitch is comprised of a replication-incompetent adenoviral vector (Ad-RTS-hIL-12, IXN-2001) administered via intratumoral injection in combination with an oral activator ligand (veledimex, IXN-1001) [75]. Preclinical models demonstrated increased intratumoral IL-12 and CD8+ T cells with a corresponding dose-dependent decrease in tumor volume. Preliminary results of a phase 1/2 trial (NCT01397708) of this therapeutic combination in unresectable Stage III–IV melanoma reported a similar increase in tumor IL-12 mRNA and tumor-infiltrating lymphocytes and well-tolerated dose escalation (Table 1) [28]. TLR 9 agonists act to induce DC maturation; cytokine secretion; APC uptake, processing and presentation; NK cell activation; and T cell response, thereby improving tumor immunogenicity [76,77,78]. Additionally, this improves tumor cell susceptibility to ICIs as the antitumor response is dependent upon tumor immunogenicity [65,79,80,81,82]. TLR9 agonist, tilsotolimod, activates the Th-1-type immune response, which increases expression of ICIs by promoting the maturation of local APCs [83]. The results from 49 evaluable patients with Stage IIIC-IV, anti-PD-1 refractory melanoma treated with tilsotolimod and ipilimumab in a phase 2 trial (NCT02644967) published in 2020 included an ORR of 22% and median OS of 21 months (Figure 1) [84]. The most common AEs seen related to this combination were fatigue, nausea, and anemia with 48% experiencing grade 3 or 4 treatment-emergent AEs and 32% experiencing at least one serious AE. This combination was compared to systemic ipilimumab alone in anti-PD-1-refractory, stage III-IVM1c melanoma in a phase 3, randomized trial (NCT03445533); however, it was terminated in 2022 for a lack of improvement in the ORR and OS. SD-101, a TLR9 agonist, is a synthetic cytosine–phosphate–guanine (CpG) oligonucleotide that exerts its effects by inducing the production of CD8+ T cells and T cell infiltration [85]. A phase 1/2 trial (NCT02521870) of SD-101 in combination with pembrolizumab in metastatic melanoma or head and neck squamous cell carcinoma was terminated by the sponsor in 2021 with no plans for additional investment in SD-101 development. Vidutolimod is comprised of CpG-A DNA, a TLR9 agonist, packaged in a virus-like particle, which ultimately induces IFN-α production by DCs and creates a Th-1-type immune response [86,87]. The combination of intralesional vidutolimod with systemic nivolumab is under phase 2 investigation (NCT04698187) in anti-PD-1-refractory and phase 2/3 investigation (NCT04695977) compared to systemic nivolumab alone in treatment-naïve, unresectable or metastatic melanoma (Table 1). The role of vidutolimod in the neoadjuvant setting is also being explored. Neoadjuvant intralesional vidutolimod in combination with intravenous nivolumab was evaluated in a phase 2 clinical trial (NCT03618641) in 30 patients with Stage IIIB-D, clinically node-positive melanoma (Figure 1) [88]. On surgical pathology, CR was seen in 50% with 70% having a >50% reduction in tumor volume. There were no dose-limiting toxicities, three patients experienced an unspecified grade 3 or 4 immune-related AE and two discontinued vidutolimod as a result. A similar trial of neoadjuvant intratumoral vidutolimod with systemic nivolumab versus nivolumab alone is being evaluated in an ongoing phase 2 study, NCT04401995, in stage IIIB-D melanoma, but with palpable nodal disease or nodal recurrence (Table 1). Neoadjuvant intralesional vidutolimod is also being investigated in combination with systemic pembrolizumab versus pembrolizumab alone in stage III, N1b-N3c, resectable melanoma in a phase 2 trial (NCT04708418). Cavrotolimod (AST-008) is a spherical nucleic acid configuration, with densely and radially arranged oligonucleotides on liposomal nanoparticles, of a TLR9 agonist that has been shown, preclinically, to elicit a Th-1-type immune response similar to other TLR9 agonists [89]. Clinical investigation of intratumoral cavrotolimod in combination with systemic pembrolizumab is ongoing in a phase 1b/2 trial (NCT03684785) in solid tumors including anti-PD-1/L1-refractory, locally advanced or metastatic melanoma (Table 1). Interim phase 1b data reported that in 19 evaluable patients, the ORR was 21% and two of the four responders had anti-PD-1 refractory melanoma [29]. There were no dose-limiting toxicities and the most common treatment-related AEs were grade 1 or 2 injection site reactions. NKTR-262, a TLR7/8 agonist, has similar effects to TLR9 agonists; it increases CD8+ T cell and NK cells, resulting in amplified tumor antigen release and presentation [90,91]. In combination with bempegaldesleukin, it has been shown to increase innate immune signaling and antigen presentation [92]. Therefore, the combination of intralesional NKTR-262 with systemic bempegaldesleukin with or without systemic nivolumab was under investigation in unresectable or metastatic solid tumors including melanoma in a phase 1/2 trial (NCT03435640); however, it was recently terminated by the sponsor based on phase 1 findings. Ipilimumab is an anti-CTLA-4 antibody that acts to disinhibit the antitumor T cell response and is FDA-approved for metastatic melanoma [93]. However, its efficacy as an intravenous therapy is often limited by significant systemic adverse effects [93,94]. Intratumoral delivery offers the potential to avoid systemic dosing and, in early clinical investigation, was well tolerated and elicited responses in combination with intratumoral IL-2 [95]. Specifically, of the 12 patients, none experienced dose-limiting toxicities, there were no grade 4 or 5 treatment-related AEs, and the most common were injection site reaction, pain, and ulceration as well as fatigue and chills. Intratumoral ipilimumab is now under investigation in combination with systemic nivolumab compared to intravenous administration of both drugs in a phase 1/2 trial (NCT02857569) in stage III-IV melanoma (Table 1). This concept is being expanded to include localized delivery of various ICIs in a phase 2/3 trial (NCT03755739) assessing the efficacy of intralesional and trans-arterial delivery of investigator’s choice ICIs compared to standard venous administration in advanced sold tumors, including melanoma. LL37 is an endogenous antimicrobial peptide that can increase DC and B cell recognition and binding of CpG oligonucleotides [96]. A phase 1/2 trial (NCT02225366) investigating intratumoral LL37 in Stage IIIB-IVA melanoma is awaiting publication (Table 1). Hiltonol is a toll-like receptor 3 and melanoma differentiation-associated protein 5 ligand, a synthetic double-stranded RNA mimic of a pathogen-associated molecular pattern [97]. In early clinical investigation with intratumoral and intramuscular injection, it was shown to increase intratumoral levels of CD4+ and CD8+ T cells, PD-1, and PD-L1. A phase 2 trial (NCT02423863) of intratumoral and intramuscular hiltonol alone or in combination with anti-PD-1/L1 in advanced, unresectable solid tumors, including melanoma, is now underway (Table 1). APX005M is a monoclonal antibody agonist of CD40. CD40 is present on APCs and macrophages and the CD40 ligand is on T cells; therefore, APX005M has the potential to activate CD8+ T cells and increase major histocompatibility complex (MHC) class I expression on tumor cells [98]. It is now under investigation as an intralesional agent in a phase 1/2 trial (NCT02706353) in combination with systemic pembrolizumab in ICI-naïve, unresectable stage III-IVM1c melanoma (Table 1). DCs are APCs, which, in their mature state, express MHC-antigen complexes and costimulatory molecules, and elicit a T cell response [99]. Since DC activation is an important part of the antitumor immune response, autologous DCs have been a therapeutic avenue under investigation for many malignancies. A phase 1b/2 trial (NCT03325101) examining the efficacy of intratumoral autologous mature DCs in combination with cryosurgery and systemic pembrolizumab for anti-PD-1/L1-refractory unresectable stage III-IV melanoma is ongoing (Table 1). INT230-6 is a combination of cisplatin and vinblastine with an amphiphilic penetration enhancer for targeted tumor cell delivery [30]. It has been shown to result in CD4+ and CD8+ T cell activation and DC recruitment with abscopal effects in preclinical models as well as synergistic effects when combined with ICIs. It is under phase 1/2 investigation (NCT03058289) in solid tumors, including treatment-refractory metastatic melanoma, alone and in combination with ICIs, and interim results show it is well-tolerated without dose-limiting toxicity or grade 4 or 5 treatment-emergent adverse events (Table 1) [30]. The most common treatment-related AEs reported were localized pain, nausea, fatigue, and vomiting. Polidocanol, an intralesional sclerosant that has long been used in the treatment of varicose veins [100], was under investigation as an intralesional therapy in Stage IIIB-IV melanoma (NCT03754140); however, the phase 2 study was recently withdrawn due to poor accrual. There remains significant room for advancement in the treatment of advanced melanoma. In patients with lesions amenable to injection, intralesional therapy provides a promising option. Though intralesional therapies for melanoma were under investigation nearly 50 years ago, the field has seen the most growth within the past decade. Following the FDA approval of T-VEC in 2015, the field has continued to grow and evolve. In the past 5 years, we have seen numerous therapies under investigation, with most modulating or activating the host immune response in some way. This has followed the trend of systemic immunotherapy for advanced melanoma. While systemic ICIs have revolutionized the treatment of advanced disease, response and durability is limited and there can be significant treatment-limiting immune-related adverse events. Local delivery of therapeutics that have the potential to modulate the immune response at the site(s) of disease provide promise as a way to enhance efficacy, circumvent resistance, and limit systemic adverse effects, as we have seen in some of the trials discussed above. Unfortunately, not every intralesional regimen investigated has been successful. Perhaps the most notable and surprising were the recent results of NCT02288897, the phase 3, randomized controlled trial of intralesional T-VEC with systemic pembrolizumab versus an intralesional placebo with systemic pembrolizumab in which no survival benefit was seen [59]. Since this study was conducted in anti-PD-1-naïve advanced melanoma, it highlights the importance of identifying the appropriate timing of therapeutic regimens. Additionally the inclusion of stage IVM1b-IVM1c melanoma despite past trials demonstrating greater efficacy in less advanced (stage III-IVM1a) disease [60], emphasizes the importance of patient selection for future trials. Further to this, the trial outcome suggests that optimal benefit of T-VEC in combination with ICI may either be in the neoadjuvant, or second-line or later setting, as is currently under investigation. Additionally, multiple trials evaluating intralesional toll-like receptor agonists in combination with systemic immunotherapies in metastatic melanoma (NCT03445533, NCT02521870, NCT03435640) have recently been terminated due to their lack of efficacy or concern from the sponsor. Other TLR-9 agonists, vidutolimod and cavrotolimod, remain in phase 2 trials with systemic ICIs and bear watching. Ultimately, the major limitation of intralesional therapy is administration. Injection should be performed by an experienced provider and drug delivery is dependent on provider technique. T-VEC and other intralesional therapies have attempted to account for variability by providing specific dosing instructions based on lesion size and number; however, the nature of intralesional therapy makes it impossible to completely standardize dosing. For many patients, a significant barrier to receiving intralesional therapies is access to healthcare facilities in which they are offered. This is further exacerbated by frequent dosing schedules. In general, it seems that the cytokine-based therapies (IL-2) and TLR-9 agonists have the most intensive dosing regimens, while other therapies have less frequent dosing, but even the less intensive schedules are every 3–4 weeks. Lastly, the intratumoral delivery of these therapies is advantageous in generating a response to specific disease sites and limiting systemic side effects; however, it also appears to limit efficacy in uninjected sites of disease. The abscopal effects of intralesional therapies are of great interest since in many cases of advanced disease, it is not possible to inject every tumor and microscopic disease may not be identified. While intralesional therapies have demonstrated a response in some sites of uninjected disease, to our knowledge, no studies of intratumoral agents alone have demonstrated an equivocal response in injected and uninjected lesions. In discussing the limitations and shortcomings of current intralesional therapies, we highlight areas for improvement and future directions. One of the greatest areas for advancement is sorting out the appropriate drug regimens and timing. There are numerous possible combinations of intratumoral and systemic therapies, even if only using the drugs that have been discussed here. We have already seen instances of regimens that appear promising in other settings or earlier phase trials that have not delivered expected results. Therefore, we should expect more clarity regarding effective timing and combinations in the coming years. Accessibility of intratumoral therapy is another avenue for future improvement. As more intratumoral therapies and combination regimens become FDA-approved or widely available, we can expect to see more providers administering intralesional therapies. Additionally, once more efficacious intratumoral therapies and combination regimens have been identified, accessibility can be further improved with optimization of the dosing schedules to make them more patient friendly where possible. Finally, the application of precision medicine to intratumoral therapy is largely unexplored. The use of autologous mature DCs as intralesional therapy is one of the few personalized therapies currently under investigation. With more widespread tumor genome sequencing, identification of targetable mutations, and development of targeted therapies, we can hope for a more individualized approach to intralesional therapy in the future.

PMC10000424

Atharva Kale,Natasha M. Rogers

No Time to Die—How Islets Meet Their Demise in Transplantation

03-03-2023

islet transplantation,beta cells,rejection,IBMIR,ER stress

Islet transplantation represents an effective treatment for patients with type 1 diabetes mellitus (T1DM) and severe hypoglycaemia unawareness, capable of circumventing impaired counterregulatory pathways that no longer provide protection against low blood glucose levels. The additional beneficial effect of normalizing metabolic glycaemic control is the minimisation of further complications related to T1DM and insulin administration. However, patients require allogeneic islets from up to three donors, and the long-term insulin independence is inferior to that achieved with solid organ (whole pancreas) transplantation. This is likely due to the fragility of islets caused by the isolation process, innate immune responses following portal infusion, auto- and allo-immune-mediated destruction and β-cell exhaustion following transplantation. This review covers the specific challenges related to islet vulnerability and dysfunction that affect long-term cell survival following transplantation.

No Time to Die—How Islets Meet Their Demise in Transplantation Islet transplantation represents an effective treatment for patients with type 1 diabetes mellitus (T1DM) and severe hypoglycaemia unawareness, capable of circumventing impaired counterregulatory pathways that no longer provide protection against low blood glucose levels. The additional beneficial effect of normalizing metabolic glycaemic control is the minimisation of further complications related to T1DM and insulin administration. However, patients require allogeneic islets from up to three donors, and the long-term insulin independence is inferior to that achieved with solid organ (whole pancreas) transplantation. This is likely due to the fragility of islets caused by the isolation process, innate immune responses following portal infusion, auto- and allo-immune-mediated destruction and β-cell exhaustion following transplantation. This review covers the specific challenges related to islet vulnerability and dysfunction that affect long-term cell survival following transplantation. Diabetes mellitus (DM) is a developing global health emergency. Current estimates suggest that >500 million people worldwide are affected, with an increasing prevalence in low- and middle-income countries. DM, regardless of aetiology, has a unique capacity to affect multiple organ systems that predispose to a substantially increased risk of cardiovascular disease, chronic kidney and liver diseases, malignancy and neurological impairment. The long-term economic burden that accompanies a diagnosis of DM and the development of complications create concerns about the cost and utilisation of healthcare resources over the duration of the disease. Type 1 DM (T1DM) is characterised by the autoimmune destruction of β-cells, although the notion of β-cell complicity in their own destruction [1] due to limited responses to survive an inflammatory insult is touted as a competing hypothesis for disease development. Type 2 DM (T2DM) is caused by a combination of β-cell dysfunction and insulin resistance [2]. The failure of insulin-sensitive tissues to respond appropriately to insulin leads to compensatory hyperinsulinemia, which facilitates β-cell dysfunction and death through exhaustion. Islet transplantation is an accepted treatment option for patients with T1DM [3]—and not yet for patients with T2DM primarily due to a lack of sufficient donors—but despite the initial success in reducing hypoglycaemia unawareness, long-term allograft survival and insulin independence are limited [4] due to an inexorable decline in β-cell number and function. The loss of critical islet mass is common in both type 1 and type 2 DM, as well as in islet transplantation. Our understanding of islet biology, particularly how these cells die in response to exogenous stressors, remains key to developing novel treatments that protect the endocrine pancreas and facilitate the survival of transplanted β-cells. Insulin replacement has remained the standard management for patients with T1DM and for patients with late-stage T2DM. However, it is not able to provide complete physiological metabolic control. Allogeneic islet cell transplantation remains an effective treatment for patients with T1DM who have concurrent hypoglycaemia unawareness and metabolic instability [3]. Hypoglycaemia can pass as unrecognised in a subset of patients with diabetes—this condition is known as hypoglycaemic unawareness—where the development of neuroglycopenia is not preceded by autonomic warning symptoms (e.g., tremors and sweating) due to a loss of sympathetic and adreno-medullary counterregulatory mechanisms, as well as a lack of α-cell responsiveness [5,6]. The development of hypoglycaemia unawareness is associated with the duration of diabetes in the context of tight metabolic control. It is most commonly observed in patients with T1DM, affecting 30–40% of patients, compared to patients with insulin-dependent T2DM [7]. Hypoglycaemic unawareness is associated with the development of severe hypoglycaemia leading to increased morbidity and mortality [8,9]. Islet transplantation provides the replacement of β-cell (and potentially α-cell) function, and it has been shown to effectively reduce hypoglycaemia unawareness, improve quality of life and provide durable insulin independence [3,10]. Islet transplantation also slows the micro- and macro-vascular complications that impact morbidity and mortality [11], as well as healthcare resource use, although the procedure is not necessarily cost-effective [12]. The ability to apply this therapy to a broader range of patients with diabetes is also limited by donor pancreas availability. Patients typically require three separate transplantation procedures to maximise the efficacy and stability of islet cell mass. The majority of islets are thought to be lost in the immediate peri-transplant period following infusion and engraftment. This is typically followed by a second phase of cellular loss, where an inexorable decline in β-cell function can be mediated by allo- and auto-immune destruction, as well as by non-immunological functional impairment and destruction that resembles the failure of islets in T2DM (inflammatory, ER amyloid and metabolic stressors, Figure 1) [13]. This results in long-term insulin independence rates of <30% [14]. Inflammation during the early stages of islet transplantation has been identified as one of the major reasons for poor long-term graft survival. Approximately 25% of transplanted islets are lost immediately [15] as they come into contact with intraportal ABO-compatible whole blood, triggered by exposed tissue factor on the surface of the islets [16]. Tissue factor interacts with factor VIIa to activate the extrinsic coagulation pathway. IBMIR is therefore characterised by coagulation, complement activation, the recruitment and infiltration of leukocytes and the production of proinflammatory cytokines/chemokines, all of which lead to the regulated necrosis [17] of β-cells. Concurrent increases in thrombin–antithrombin levels and c-peptide are indicative of islet damage and cell lysis, although this can be mitigated by the use of heparin [18]. Extra-hepatic transplantation sites have been considered alternative locations for islet deposition, as they eliminate the potential for IBMIR, and while they have been effective in small animal models, these have typically produced poorer clinical outcomes [19]. The use of encapsulation technology as a cytoprotective mechanism has been extensively explored (well-reviewed in [20]) but has not been robustly translated to clinical practice. The study of IBMIR in vivo is difficult to recapitulate due to multiple interacting components; however, whole blood models of human allo-islet transplantation have been developed, and the early (<6 h) innate immune response has been well characterised. A recent study investigated IBMIR for up to 48 h [21], demonstrating ongoing thrombin–antithrombin complexes and platelet activation at 12 h post-transplantation. This was accompanied by increased expressions of chemokines, including interferon-inducible T-cell chemoattractant (the CXCR3 ligand), soluble CD40 ligand and monocyte chemoattractant protein-1 (the CCR2 ligand), and a massive infiltration of neutrophils and monocytes. Natural killer (NK) cells and macrophages also contribute to IBMIR. The liver—a typical site of β-cell deposition following intraportal infusion—and its substantial mononuclear cell population contain a large proportion of NK cells, which are highly cytotoxic compared to peripheral blood NK cells. NK cells are part of the innate immunity and mediate their cytotoxicity by secreting cytokines and via direct cell-to-cell contact. One of the dominant apoptotic pathways utilised by NK cells is the TNF-α-related apoptosis-inducing ligand (TRAIL) to TRAIL receptor pathway. It has been discovered that pancreatic β-cells express the TRAIL receptor. TRAIL-mediated islet destruction by NK cells was partially but significantly inhibited following the administration of anti-TRAIL mAb in mice. The inhibitory effect was more profound following the co-administration of anti-TRAIL mAb and concanamycin A, an inhibitor of perforin-mediated cytotoxicity [22]. NK cells are traditionally believed to not differentiate into memory cells (cells that have the ability to remember a past encounter with a foreign antigen and stimulate an efficient and enhanced response when re-encountering the same antigen). This is a pathognomonic feature of both T cells and B cells in the adaptive immune system. There is now accumulating evidence that NK cells may possess memory-cell-like properties. NK cells specific to the liver in particular exhibit memory-like properties and responses and lack a classical NK marker DX5, the a2 integral chain CD49b. TNF-α induces the activation of DX5− NK cells through interactions with both TNF-α 1 and 2 receptors on DX5− NK cells. The blocking of TNF-α 1 and 2 receptors on DX5− NK cells with anti-TNF-α antibody treatment prior to transplantation protects islets from NK cell attack during IBMIR. The memory-like liver-resident DX5− NK cells significantly expand in number after primary syngeneic islet transplantation in mice and may target both the originally engrafted primary and secondary transplanted islets. Anti-TNF-α antibody treatment also significantly inhibited the expansion of DX5− NK cells and the prolonged CD69/TRAIL expression on liver NK cells after sequential islet transplantations [23]. NF-κB inhibitors can mitigate the effects of IBMIR on human islets in vitro [24], partly due to their regulation of tissue factor expression. The overexpression of the integral membrane ectonucleotidase CD39 on islets enhances ATP degradation, and it has been shown to limit platelet activation and coagulation without effecting glucose metabolism [25]. The use of complement inhibitors—a C5a inhibitory peptide in a rodent model [26] and a complement inhibitor in a xenotransplant model [27]—showed improved graft survival. Islets are particularly susceptible to damage mediated by the pro-inflammatory cytokines interleukin (IL)-1, interferon-γ and tumour necrosis factor (TNF)α [28] through NK-κB signalling, the activation of mitogen-activated protein kinases (MAPKs) and Fas-triggered apoptosis. Macrophage-mediated IL-1β secretion may represent the final common pathway for functional islet impairment and destruction through pro-inflammatory cytokine stimulation [29] by enhancing inducible nitric oxide synthase activity and impairing glucose-stimulated insulin secretion [30,31]. IL-1β gene expression was significantly upregulated in in vitro cultures of islets [32], which was exacerbated by serum deprivation and mitigated following incubation with an IL-1 receptor antagonist [33]. Post-transplantation, IL-1β is detectable even in syngeneic grafts [34]. The TNF decoy receptor etanercept has been shown to reduce inflammation and oxidative stress in islets [35]. However, its incorporation into clinical practice proceeded with limited clinical data after being trialled in eight patients [36]. The attractiveness of anti-inflammatory therapeutics to limit IBMIR has persisted, and drug repurposing studies using the IL-1 receptor antagonist anakinra demonstrated improved marginal mass islet engraftment by limiting apoptosis [37]. The combined use of etanercept and anakinra (an IL-1 inhibitor) in allogeneic and autologous islet transplantation demonstrated both safety and tolerability [38,39,40], although larger clinical trials are required to show definitively improved clinical outcomes. CD47 is a universally expressed cell membrane receptor that ligates signal regulatory protein (SIRP), particularly the alpha domain, to monitor self (versus non-self) and oversee the don’t-eat-me signal. The interaction of parenchymal cell CD47 with myeloid-based SIRPα places it as a checkpoint of innate, allogeneic and xenogeneic immunity. Enhanced CD47 expression correlates with improved solid organ engraftment. The generation of a chimeric CD47-SIRPα protein (containing binding and signal transduction domains) linked to streptavidin was able to limit the macrophage-based phagocytosis of biotinylated cells [41]. Mouse islets engineered to express this protein were protected from the development of an IBMIR-type reaction in vitro and in vivo, facilitating engraftment and long-term syngeneic graft function. These findings were associated with decreased inflammatory cell infiltrates, particularly CD11b+Ly6Chi/CD11b+/Ly6Cint inflammatory monocytes and CD11bhiGr1hi neutrophils. Allogeneic islet transplantation requires immunosuppression to limit the rejection of islets, with the potential added benefit of suppressing autoimmunity. Patients with T1DM and a concurrent burden of autoimmune antibodies have a lower rate of islet transplant success due to the presence of memory CD4+ and CD8+ T cells that are rapidly reactivated to target islet antigens (IA-2, GAD-54 and ZnT8) and destroy β-cells [42,43]. Despite their clear prognostic role in the development of T1DM [44], the association between autoantibodies and long-term islet allograft function has been difficult to demonstrate robustly due to small patient numbers. Indeed, patients with T1DM receiving adequate (calcineurin inhibitor and mammalian target of rapamycin inhibitor-based) immunosuppression following islet transplantation demonstrated lymphopenia and a concurrent chronic elevation of IL-7 and IL-15 that promoted T-cell turnover and the expansion of auto-antigen-specific T cells [45]. Human islets bind complement proteins, particularly IgG, IgM, C1q and C3b/iC3b (18431241), leading to lysis and the release of c-peptide. This may be crucial in bridging innate (IBMIR-based) and allogeneic (HLA-based) immune responses in islet transplantation, as C3 can trigger rejection in pre-clinical models of solid organ transplantation [46] and humans [47]. The presence or the development of alloimmunity (to human leukocyte antigens, HLAs) and its effect on allograft survival is well-defined in the solid organ transplantation literature. Donor-specific antibodies (DSAs), pre-formed or de novo, are a leading cause of graft failure due to the development of antibody-mediated rejection in the kidney [48], heart [49] and lung [50], which is associated with graft dysfunction and poorer long-term graft survival. The introduction of molecular typing and Luminex technology has facilitated our ability to precisely define HLAs, the presence (or absence) of antibodies and their (relative) abundance. The risk factors for developing de novo DSAs encompass inadequate immunosuppression (including nonadherence) and inflammation within the graft (rejection) or systemically (infection), which can incite graft immunogenicity and/or heterologous immunity [51]. As many islet transplant patients receive grafts from two–three HLA mismatched donors, the presumed risk of allosensitisation is higher. DSAs binding to endothelial cells or islets (that constitutively express class I and aberrantly upregulate class II HLAs [52]) can activate the classic complement pathway. Even in the absence of complement, some DSAs can promote antibody-dependent cytotoxicity, and innate immune cells bind Fc fragments that trigger degranulation and the release of lytic enzymes from neutrophils and NK cells. C4d, the degradation product of the classical complement pathway, binds covalently to the endothelium and can be used as an immunological marker of antibody-mediated rejection. However, the breadth of islet dispersal throughout the liver parenchyma increases the risk–benefit ratio of a liver biopsy to provide histological assistance for the diagnosis of rejection. The association of DSAs with islet transplant failure is not as well characterised. It is not known whether the hepatic location of islets is relatively protective given the tolerogenic environment provided by the liver [53]. The prevalence of pre-formed DSAs in islet transplant recipients has been shown to be similar to that of other solid organ transplant cohorts [54], with the suggestion that pre-existing IgM antibodies against HLA class II are associated with improved outcomes. De novo DSAs have been shown to be predictive of islet graft failure [54,55], particularly the development of class I HLAs [56], although this has been disputed [57,58]. Indeed, allogeneic islets may be resistant to DSA-mediated rejection [58] despite the susceptibility with direct binding in vitro, and this is directly due to the endothelial sequestration of DSAs in neo-vascularised islets. The chronic attrition of islet allograft function over time, despite initial engraftment success, is multifactorial and contributed to by rejection, chronic fibrosis within a non-physiological environment and the drug-induced toxicity of immunosuppression. Both tacrolimus and sirolimus, which are part of standard immunosuppression protocols [3,59], have diabetogenic properties. Calcineurin inhibitors (tacrolimus and ciclosporin) act by limiting the dephosphorylation and translocation of the nuclear factor of activated T cells (NFATs). Calcineurin signalling is required for insulin secretion and β-cell proliferation [60], and the specific inactivation of calcineurin in β-cells is associated with hyperglycaemia with increasing age [61]. Tacrolimus has been shown to increase blood glucose and reduce the homeostasis model assessment of β-cell function (HOMA-β) and the insulin sensitivity index in animals with intact native endocrine pancreatic function [62], following transplantation with human islets [60] and following solid organ transplantation [63]. The effects of short-term tacrolimus exposure promoting hyperglycaemia and compensatory hyperinsulinemia transition to the pseudo-normalisation of insulin, indicative of the loss of insulin secretory capacity, with evidence of β-cell death [64]. Islet apoptosis associated with calcineurin inhibition is also thought to occur by limiting the cAMP response element binding protein (CREB) [65], which decreases IRS-2 expression, limits the phosphorylation of Akt and impacts insulin secretion [66]. CNIs also reduce the cell surface expression of GLUT4 and decrease insulin-stimulated glucose uptake in adipocytes [67], which potentially contributes to peripheral insulin resistance. Tacrolimus promotes a decrease in mitochondrial Ca2+ uptake, which has been shown to impair respiration and ATP production, leading to compromised glucose-stimulated insulin secretion (GSIS) [68]. CNIs, tacrolimus in particular, potentiate the deleterious effect of glucolipotoxicity on β-cells, inducing nuclear FoxO1 expression (which, in turn, limits proliferation [69]) and reducing insulin content and secretion [70]. The incidence of post-transplant diabetes mellitus in solid organ transplantation is the highest in tacrolimus-treated recipients [71,72]. However, mTOR inhibitors are not innocuous in terms of diabetogenic capacity, although much of the literature derives from clinical studies in solid organ (kidney transplant) recipients. An analysis of >20,000 patients in USRDS revealed that kidney transplant recipients without DM receiving sirolimus as part of their immunosuppression regimen were most likely to have Medicare billing for post-transplant DM [73], and the highest HR associated with post-transplant DM was sirolimus and calcineurin inhibitors combined. Tacrolimus and sirolimus both induce reversible graft dysfunction, characterised by amyloid deposition and macrophage infiltration in transplanted islets [60], but without evidence of frank β-cell death. An ultrastructural examination of grafts demonstrated decreased insulin granules, and an accompanying genomic analysis revealed transcripts associated with extracellular matrix deposition and inflammation. Insulin signalling and β-cell proliferation/survival require intact mammalian target of rapamycin (mTOR), particularly mTORC1 function, which occurs via the insulin receptor substrate 1-Akt-mTOR pathway, where the final step is the phosphorylation of p70 ribosomal protein S6 kinase. In rodent studies, the administration of sirolimus worsened hyperglycaemia, abolished the hyperinsulinemic response and decreased muscle insulin sensitivity in diabetic animals [74]. The latter effect is mediated by glycogen synthase 3β activity [75], and further work has demonstrated that the sirolimus-based dephosphorylation of Yin Yang 1 in skeletal muscle limits insulin signalling [76,77]. Sirolimus has been shown to cause islet death [78] and impair proliferative cell recovery [79]. mTORC2 is required for the insulin-mediated suppression of hepatic gluconeogenesis [80], which is disrupted by sirolimus at higher doses. Not all studies concur with sirolimus impacting insulin action and glucose homeostasis, and this may be related to overall drug exposure at concentrations that affect both mTORC1 and 2. Immune cell infiltration [81] and amyloid deposition [82] have both been described in liver biopsy results following intraportal transplantation, but these correlate poorly with clinical phenotype. The transplantation of islets from islet amyloid polypeptide (IAPP)-expressing transgenic mice demonstrated early amyloid deposition post-transplantation, reduced β-cell volume and graft failure. This was thought to be due to apoptosis and a concurrent reduction in β-cell replication [83], and both phenomena have been observed in vitro in response to amyloid fibrils. Although IAPP is secreted by β-cells, the human form can aggregate to form cytotoxic fibrils [84]. Interestingly, heparin, which is used to reduce the cyto-destructive effect of IBMIR on islets, promotes the fibrillogenesis of human IAPP, and it has been shown to simultaneously promote amyloid deposition and decrease β-cell apoptosis. Heparinase treatment significantly reduced amyloid deposition and subsequent β-cell cytotoxicity [85]. Islets are three-dimensional structures, occupying only 2% of the total pancreatic volume but intrinsically linked to abundant vasculature. The islet vascular network comprises 7–8% of the total islet volume and, overall, receives approximately 10× more blood than the surrounding exocrine pancreas [86]. Intact, this capillary system is critical for islet survival and function, and it is central to the secretion of insulin into the circulation for subsequent systemic distribution. Intra-islet endothelial cells (EC) are highly fenestrated. Islet-EC crosstalk provides a number of paracrine effects that promote angiogenesis and β-cell survival, including vascular endothelial growth factor [87], angiopoietins [88], ephrins [89,90] and insulin [91]. The basement membrane also provides a barrier function, structural support and signalling moieties for cellular integrity. The process of islet isolation for transplantation strips β-cells of their vascular infrastructure, and islets are therefore devoid of endothelial cells to support neoangiogenesis, an absolute requirement for engraftment. The basement membrane is also lost during isolation, particularly laminin and collagen [92], and matrix detachment promotes apoptosis [93]. The recovery of structure and function is seen in syngeneic mouse models of islet transplantation, and this is incomplete in allotransplantation [94]. Islets make little matrix and are dependent on endothelial cell recruitment (donor and recipient) for basement membrane repair. Islets are clusters of approximately 2000 β-cells. Due to the loss of blood flow, the availability of oxygen and nutrients becomes diffusion-dependent, and isolated islets are hypothesised to become hypoxic typically at the central core; however, formal quantitation is difficult. Previous studies have suggested that smaller islets produce superior outcomes [95,96] as a surrogate marker of islet survival. Studies have shown that approximately 70% of transplanted islets remain hypoxic 1 month post-transplantation [97]. However, the exposure of isolated islets to hyperoxic conditions paradoxically worsens the damage of cells residing on the periphery, despite a reduction in central necrosis [98]. The liver is currently considered to be the most suitable site for islet transplantation because, compared to other vascularised beds, such as the spleen and kidney, it is easily accessible with minimal invasion and is (potentially) the least immunogenic. The liver provides a significantly lower oxygen tension (5–10 mmHg) than native pancreatic tissue (30–40 mmHg); however, it allows for a better dispersal of oxygen and nutrients from hepatic sinusoids to transplanted islets. The root of the cause of low revascularisation and oxygen deficiencies in transplanted islets may be intrinsic, as transplanted islets have been reported to have low nitric oxide production, which is essential for regulating vascular tone and blood flow, as well as mitochondrial oxygen consumption. Furthermore, the physiological route of secreted insulin from the pancreas is first directed to the liver via the hepatic portal vein, where it is first consumed before being dispersed to adipose tissue and skeletal muscles [99,100]. β-cells require large amounts of oxygen to meet mitochondrial respiration demands and to facilitate efficient insulin secretion. Hypoxic stress is a contributing factor to cellular dysfunction, islet death during isolation and low islet survival post-transplantation. High numbers of islets are therefore required at transplantation to compensate for cell death and loss. Hypoxia activates the HIF-1α cascade in islets following initial procurement and isolation. HIF-1α accumulates in islet grafts [101], which presumably drives an adaptive response to hypoxia, inducing the expression of pro-angiogenic factors, such as VEGF, AngptI4, Pgf and Anxa2, as well as downstream effectors that initiate angiogenesis [102]. The protective effect of HIF-1α was shown by a poorer survival of transplanted null islets; iron chelation using desferrioxamine increased HIF-1α expression (as well as concurrent ATP content and glucose oxidation) leading to improved islet transplant outcomes [103]. HIF-1α also drives impaired glucose-stimulated insulin secretion [104] and apoptosis [101], an effect mediated by metabolic switching to glycolysis and reduced ATP generation [105]. This has been confirmed in microarray analyses of human isolated islets, where a range of upregulated hypoxia-response genes were identified [106]. The transplantation of hypoxic islets demonstrated a defect in (but not the absence of) β-cell function, which was a consequence of stabilised HIF-1α. HIF-1α activation also initiates apoptosis and mitochondrial caspase-mediated cell death pathways [106], a loss of glucose-stimulated insulin secretion [101] and pro-inflammatory cytokine activation and release, followed by the structural defragmentation of islets. This is exacerbated by significant delays in revascularisation post-transplantation. The mechanisms of hypoxia-induced β-cell death and dysfunction have been widely investigated, and they are key to facilitating post-transplantation survival. Pancreatic β-cells exposed to acute hypoxia induce caspase-3-mediated apoptosis and endoplasmic reticulum (ER) stress [107]. Recent studies have alluded to ER stress as a causal factor in β-cell dysfunction and islet transplant failure [108]. β-cells are highly metabolic and have a highly developed ER involved in post-translational modification and the assembly and folding of newly synthesised proteins (e.g., insulin in β-cells). An overload of protein production in cells can lead to ER stress. A clinical feature of islet allograft failure (as well as T2DM) is the overproduction of insulin to meet high demands in the context of significant β-cell loss. Insulin overproduction results in protein misfolding and the induction of ER stress through the activation of the C/EBP homologous protein (CHOP), X-box binding protein 1 (XBP-1) and immunoglobulin heavy chain (BIP). To re-establish cellular homeostasis, the adaptive unfolded protein response (UPR) is triggered via three main sensors: activating transcription factor (ATF) 6, double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and inositol requiring kinase 1 (IRE1). The UPR responds to ER stress by inducing the upregulation of genes encoding ER chaperone proteins to mitigate protein aggregation and accumulation and to initiate the proteasomal degradation of misfolded proteins. However, in the context of chronic ER stress, the cytoprotective role of the UPR fails, and apoptotic pathways are initiated to induce cell death. The ER stress marker CHOP is also a pro-apoptotic transcription factor. In response to acute hypoxia, CHOP is upregulated in β-cells. Upon the silencing of CHOP, hypoxia-induced apoptosis is prevented [106]. In addition to environmental hypoxia, intracellular hypoxia is also present in isolated islets. Despite culturing islets in normoxic conditions following isolation, intracellular hypoxia remains. Studies have found that HIF-1α remains overexpressed in isolated islets and that CHOP is upregulated within 4 h of isolation, suggesting early apoptosis. Hypoxia-mediated apoptosis is also implicated in the pathogenesis of T2DM, as pancreatic islets obtained from various murine models (including db/db, ob/ob and kky mice) were discovered to be significantly hypoxic and overexpressed ER stress markers compared to non-diabetic counterparts [106,109]. Chronic hyperglycaemia, which is a clinical feature of both DM and islet allograft failure, damages islets further by triggering β-cell de-differentiation, ER-stress and a loss of function. This phenomenon is known as glucotoxicity. The hyperglycaemic environment in diabetic islet transplant recipients has been implicated in the early loss of transplanted islets. The elimination of glucocorticoids from the immunosuppressive regimen was a significant contributing factor to the success of the Edmonton protocol [59]. One study recently investigated the effects of in vivo chronic hyperglycaemia on ER stress and UPR gene expression in transplanted mouse islets [110]. Diabetic recipients receiving a suboptimal islet transplant that failed to restore euglycemia demonstrated a significant reduction in UPR gene expression, including PERK and IRE1/ATF6, in transplanted islets compared to non-diabetic mice receiving the same β-cell mass. The recovery of adaptive UPR gene expression was observed in diabetic mice transplanted with a sufficient islet mass that established normoglycaemia. Glucotoxicity is also known to exacerbate other stressors within the graft environment, including inflammation, oxidative stress, hypoxia and impaired vascularisation. Hypoxia decreases ER-to-Golgi protein trafficking and induces cell death by inhibiting the adaptive UPR [111]. Hypoxia mediates these responses independent of HIF-1α activation via several effectors of ER stress, including CHOP, DNA-damage inducible transcript 3 (DDIT3) and c-Jun N-terminal kinase (JNK). Islets isolated from diabetic mice have decreased expressions of adaptive UPR genes, such as Hspa5, spliced Xbp1 and Fkbp11. The overexpression of Hspa5 was found to protect islet β-cells from hypoxia-induced cell death. The silencing of CHOP or JNK also restored UPR gene expression and protein trafficking, providing protection against apoptosis induced by hypoxia [111]. Emerging strategies to overcome the current limitations of islet transplantation and to improve their success include the encapsulation of islets, the co-transplantation of islets with endothelial cells, stem cell sources for islet transplantation, genetic manipulation and immunosuppression strategies. To mitigate the effects of IBMIR, extrahepatic locations, including the kidney capsule, the spleen, the omental pouch and subcutaneous sites, have been explored in pre-clinical [112,113,114] and human [19] studies. The subcutaneous site is minimally invasive and easily accessible; however, the main limitation is poor vascularisation [115]. Multiple strategies have been developed to counteract this problem and improve the outcomes of transplanted islet survival, including the bioengineering of functional cell sheets using bone-marrow-derived mesenchymal stem cells (MSCs) [116]. MSCs are a source of pro-angiogenic and anti-apoptotic cytokines, including VEGF, HGF, IL-6 and TGFβ1. TGFβ1 triggers the production of heat shock protein 32 (HSP32) and X-linked inhibitor of apoptosis protein (XIAP), which protect islets by supressing oxidative stress [117], inflammation and β-cell apoptosis [118,119]. MSCs can also differentiate into endothelial cells to facilitate peri-islet vascularisation. When engineered into cell sheets, they provided an ideal substrate for human islets and the extracellular matrix that improved islet function and survival [116]. The co-transplantation of islets with adipose-derived mesenchymal stem cell sheets into the subcutaneous site normalised blood glucose levels in diabetic pig recipients [120]. Human dermal fibroblasts have also been studied in vitro as an alternative source to bone-marrow-derived MSCs, as they can be easily harvested from the skin and are highly proliferative. Fibroblasts are also a source of pro-angiogenic factors, such as VEGF and fibroblast growth factor, both of which can improve vascularisation and islet viability post-transplantation. Furthermore, when engineered into cell sheets, fibroblasts maintain the natural structural integrity of islets while also improving their function and survival in vitro [121]. Overcoming insufficient revascularisation to facilitate islet engraftment has been a substantial challenge clinically. The increasing evidence of molecular crosstalk between intra-islet endothelial cells and β-cells [122] suggests that the co-transplantation of these cells may be advantageous. In addition to their contribution to angiogenesis, intra-islet endothelial cells produce various endocrine factors, including thrombospondins, hepatocyte growth factors, collagen and laminins, which promote β-cell survival and improve insulin secretion. Similarly, intra-islet endothelial cells benefit from proliferative factors secreted by β-cells, including ephrins, VEGF, angiopoietins and insulin. Bone-marrow-derived endothelial progenitor cells co-transplanted with islets in diabetic mice have shown promising results in restoring euglycemia through rapid revascularisation [123,124,125]. This was also associated with a strong downregulation of PECAM-1, which is involved in mediating inflammatory responses by promoting the trans-endothelial migration of monocytes, NK cells and neutrophils [123]. Alternative vascularisation units, such as adipose-tissue-derived microvascular fragments (MVFs), may be more efficient at reassembling into microvessels in the initial post-islet-transplantation phase [126,127]. MVFs are also composed of substantial numbers of mesenchymal stem cells, which are known to secrete angiogenic factors and reduce inflammation, thereby protecting islets from hypoxia-induced death. In pre-clinical studies, MVFs co-transplanted with islets under the kidney capsule, as well as the subcutaneous space, were found to improve islet engraftment and restore normoglycaemia in recipients with diabetes [127]. Plasma insulin levels post-transplantation were similar to non-diabetic healthy controls, which was accompanied by enhanced angiogenesis in the grafts. Engineered vascularised organoids have also been shown to re-assemble into larger interconnected channels for perfusion [128], which has been applied to islet transplantation for integration with recipient vasculature [129]. To bypass immune attack following islet transplantation, transplanted islets can be encapsulated in biomaterials (well-reviewed in [130]), including alginate, a polysaccharide derived from seaweed with hydrophilic and biocompatible properties. However, foreign-body responses and fibrotic overgrowth have limited long-term islet viability and graft function [131,132]. Chemical modifications [133] or the addition of anti-TNFα to capsules [134] can improve viability. Cell subsets of myeloid, mesenchymal and T cell lineage have the capacity to regulate immune responses and show po-tential as adjuvant immunosuppressive agents in pre-clinical and clinical studies [135]. The intrahepatic infusion of autologous bone-marrow-derived mesenchymal stem cells co-transplanted with islets improved glycaemic control, islet engraftment and quality of life in patients with total pancreatectomy [136]. However, there are no current clinical trials investigating cell therapy in allogeneic islet transplantation. Islet transplantation is an effective treatment that reduces severe hypoglycaemia, re-establishes hypoglycaemia awareness, stabilises glycaemic control and can provide insulin independence. Its long-term benefits also include reduced morbidity from microvascular complications, particularly retinopathy and nephrotoxicity [137]. The benefits of islet transplantation are balanced by the need for immunosuppression—and the concordant risks of malignancy, infection and cardiovascular disease that manifest with long-term exposure to immunomodulatory agents—and the relatively ephemeral duration of successful islet transplantation (only 40% of patients achieve long-term insulin independence). The failure to meet graft survival outcomes similar to those of solid organ transplantation may be due to the susceptibility of β-cells to demise. This effect is multifactorial, but the development of strategies to improve β-cell durability or to reverse the adverse changes that are initiated by the islet isolation process will improve the success and duration of islet transplantation. The use of alternative sources of islets—xenotransplantation or stem-cell-derived sources—may help to reverse the current imbalance between supply and demand, but much remains unknown regarding their immunogenicity and physiological function. Further refinements in immunosuppressive drug regimens are also required to limit the harmful effects on both β-cells and remote organs.

PMC10000425

Hye-Jung Moon,Youn-Soo Cha,Kyung-Ah Kim

Blackcurrant Alleviates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice

02-03-2023

blackcurrant,whole foods,ulcerative colitis,anti-inflammation,tight junction proteins,gut microbiota

Previous studies have reported that anthocyanin (ACN)-rich materials have beneficial effects on ulcerative colitis (UC). Blackcurrant (BC) has been known as one of the foods rich in ACN, while studies demonstrating its effect on UC are rare. This study attempted to investigate the protective effects of whole BC in mice with colitis using dextran sulfate sodium (DSS). Mice were orally given whole BC powder at a dose of 150 mg daily for four weeks, and colitis was induced by drinking 3% DSS for six days. Whole BC relieved symptoms of colitis and pathological changes in the colon. The overproduction of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 in serum and colon tissues was also reduced by whole BC. In addition, whole BC significantly lowered the levels of mRNA and protein of downstream targets in the NF-κB signaling pathway. Furthermore, BC administration increased the expression of genes related to barrier function: ZO-1, occludin, and mucin. Moreover, the whole BC modulated the relative abundance of gut microbiota altered with DSS. Therefore, the whole BC has demonstrated the potential to prevent colitis through attenuation of the inflammatory response and regulation of the gut microbial composition.

Blackcurrant Alleviates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice Previous studies have reported that anthocyanin (ACN)-rich materials have beneficial effects on ulcerative colitis (UC). Blackcurrant (BC) has been known as one of the foods rich in ACN, while studies demonstrating its effect on UC are rare. This study attempted to investigate the protective effects of whole BC in mice with colitis using dextran sulfate sodium (DSS). Mice were orally given whole BC powder at a dose of 150 mg daily for four weeks, and colitis was induced by drinking 3% DSS for six days. Whole BC relieved symptoms of colitis and pathological changes in the colon. The overproduction of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 in serum and colon tissues was also reduced by whole BC. In addition, whole BC significantly lowered the levels of mRNA and protein of downstream targets in the NF-κB signaling pathway. Furthermore, BC administration increased the expression of genes related to barrier function: ZO-1, occludin, and mucin. Moreover, the whole BC modulated the relative abundance of gut microbiota altered with DSS. Therefore, the whole BC has demonstrated the potential to prevent colitis through attenuation of the inflammatory response and regulation of the gut microbial composition. Inflammatory bowel disease (IBD) refers to a chronic inflammatory condition of the intestinal tract that increases health and economic burdens due to an increase in global prevalence and lowers the quality of life [1]. Ulcerative colitis (UC), one of the typical IBDs, appears only in the colon and is marked by supercritical mucosal inflammation [2]. A cross-sectional study of 15 countries in Asia and the Middle East reported that UC is twice as prevalent as Crohn’s disease and occurs more frequently in men in their 30s [3]. In addition, it is essential to treat UC because it can develop into colorectal cancer if it persists for a long time [4]. UC is characterized by diarrhea, bloody stools, urgency, increased frequency of defecation, and, in severe cases, fever and weight loss [5]. It is estimated that UC is caused by the disruption of intestinal homeostasis due to genetic, microbiological, immunological, and environmental factors including diet, smoking, and stress [5,6]. Drugs such as 5-aminosalicylic acid (5-ASA), biological drugs (anti-tumor necrosis factor-α (anti-TNF-α) and anti-adhesion molecule inhibitors), immunosuppressants, and corticosteroids have been used to treat UC [5]. However, it has been reported that the remission rate of UC is only 15% to 44.9%, and adverse events such as infection, UC flare, nasopharyngitis, myelosuppression, liver toxicity, and malignancy occur [5,6,7,8]. Therefore, to develop other safe and effective treatments, natural products using polyphenols such as apigenin and curcumin, and polysaccharides such as Scutellaria baicalensis Georgi, are being studied [9,10]. Anthocyanins (ACN), belonging to the flavonoid subgroup of polyphenols, are found in flowers, vegetables, and fruits and are water-soluble pigments in red, blue, and purple [11]. Various health benefits of ACNs have been discovered, in particular, ACN supplements have been shown to improve gut health by modifying the gut microflora and enhancing the intestinal barrier, thereby reducing the potential risk of inflammation [11,12,13]. ACN-rich foods include berries (blackcurrants, blueberries, and raspberries) and dark red vegetables (red cabbage, eggplant, and purple wheat), among which blackcurrants have been reported to have a higher total ACN content than blueberries [11,14]. Blackcurrant (BC) has been suggested to possess various health effects, including prevention of obesity, improvement of cognitive impairment due to aging, and reduction of diabetes-related cardiovascular dysfunction [15,16,17]. Recently, ACN dietary supplements consisting of BC and bilberry extracts have shown anti-inflammatory effects in intestinal epithelial cells [18]. Additionally, silver nanoparticles based on BC extracts were observed to restore inflammation of induced colitis in mice [19]. However, these studies are insufficient to confirm the effect of BC on improving intestinal inflammation. Furthermore, most of these studies have verified the physiological activity of BC extracts, and studies on BC in its whole form are rare. Therefore, the aim of this study was to investigate whether the intake of whole BC in mice alleviates dextran sulfate sodium (DSS)-induced colitis. The commercial freeze-dried powder of whole BC was obtained from Sujon Berries (Nelson, New Zealand). According to Willems et al. (2017), 1 g of Sujon’s BC powder contained 23.1 mg of anthocyanin, 0.9 g of carbohydrates, and 8.2 mg of vitamin C [20]. DSS was bought from MP Biochemical (MW: 36–50 kDa; Solon, OH, USA). A TNF-α enzyme-linked immunosorbent assay (ELISA) kit was bought from Invitrogen (Vienna, Austria), and interleukin (IL)-1β and IL-6 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). The RNAiso Plus kit, PrimeScript RT Master Mix, and bicinchoninic acid (BCA) protein assay kits were purchased from Takara Bio, Inc. (Shiga, Japan). RIPA buffer was procured from Thermo Scientific Inc. (Rockford, IL, USA). Primary antibodies including phosphorylated-p65 (p-p65), p65, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). The animal experiment was approved by the Animal Ethics Committee of Chungnam National University (IACUC approval number: 202112-CNU-214). Five-week-old male C57BL/6J mice were acquired from Central Lab Animal, Inc. (Seoul, Republic of Korea). The experimental design is illustrated in Figure S1. The mice were housed under the same conditions (temperature of 22 ± 2 °C, relative humidity of 50 ± 5%, and 12 h/12 h light/dark cycles) and acclimatized for six days. After the adaptation period, 24 mice were separated into three groups (n = 8 per group): Vehicle group, normal control group not treated with DSS; DSS group, DSS-treated control group; DSS + BC group, DSS and blackcurrant treatment group. In the DSS + BC group, BC powder diluted in phosphate-buffered saline (PBS) was orally administered at a dose of 150 mg/mice per day throughout the experimental period. The PBS dosage given to the Vehicle and DSS groups was the same as that given to the DSS + BC group. To induce colitis in the DSS group and the DSS + BC groups, 3% DSS (w/v) in drinking water was given for six days from the 21st day of the experiment. One day before the experiment’s termination, DSS was replaced with normal water. Symptoms of colitis were monitored daily using the DAI (disease activity index) while DSS was administered. The DAI, which was slightly modified from what Peng et al. (2019) described, was measured as scores for body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, >10%), stool consistency (0, normal; 1, slightly loose feces; 2, loose feces; 3, watery diarrhea), and bloody stools (0, none; 1, slightly bloody; 2, bloody; 3, gross bleeding) [21]. Feces were collected the day before the sacrifice. Mice were euthanized after fasting for 12 h. The blood and colon tissues were obtained after the experiment was completed. Blood was centrifuged at 1100 g for 15 min to obtain the serum. After measuring the length and weight of colonic tissue samples, some were fixed in 4% formalin for histological assessment. The remaining colon tissues were immediately frozen in liquid nitrogen and kept at −80 °C until the experiment. Hematoxylin and eosin (H&E) staining was accomplished on 4 μm thick sections of colon tissues fixed in 4% formalin. Colon slides were examined using a light microscope (DM2500, Leica Microsystems, Wetzlar, Germany) installed at the Center for University-wide Research Facilities (CURF) at Jeonbuk National University (Jeonju, Republic of Korea). Histological damage to the colon tissue was evaluated by the scores of epithelium loss (0–3), crypt damage (0–3), depletion of goblet cells (0–3), and infiltration of inflammatory cells (0–3) [22]. Colon tissue was homogenized with lysis buffer, and the supernatant was separated. ELISA kits were used to quantify inflammatory cytokines (TNF-α, IL-1β, and IL-6) contained in the separated supernatant and serum, according to the manufacturer’s procedure. qRT-PCR analysis was performed with reference to Song et al. (2021) and the instructions of the manufacturer of the reagent [15]. Following the manufacturer’s directions for the RNAiso Plus kit (Takara Bio, Inc.), total RNA was extracted from the colon tissue. cDNA was synthesized from total RNA using PrimeScript RT Master Mix (Takara Bio, Inc.). The TOPrealTM SYBR green qPCR Premix (Enzynomics, Daejeon, Republic of Korea) and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) were used to carry out the qRT-PCR. The relative expression of the target gene was determined using the 2 −ΔΔCt method and normalized to that of the internal reference GAPDH. Western blotting was carried out by referring to the experimental method of Jang et al. (2019) [22]. Total protein lysates were extracted by homogenizing the colon tissue in a radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The protein content of the supernatant obtained by centrifugation of the extract was quantified using a BCA assay kit. Loading buffer was added to the supernatant and inactivated at 95 °C for 10 min. Protein samples were electrophoresed on SDS–polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking the membrane with 5% skim milk, the antibody diluted to an appropriate concentration was applied for 24 h at 4 °C. After washing the membrane with tris-buffered saline with 0.1% tween 20 (TBST), the secondary antibody was added, and the protein was identified using enhanced chemiluminescence (ECL) solution and the ChemiDoc system (ATTO LuminoGraph II, ATTO, Tokyo, Japan). The bands of the target proteins were quantified using Image J software (US National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin. Song et al. (2021) and Jang et al. (2019) were referred to for fecal collection and gut microbiota analysis [15,21]. The day after the DSS drinking was completed, feces were collected and stored at −80 °C in order to analyze the gut microbial community. The microbial community of the collected feces was analyzed by Macrogen Inc. (Seoul, Republic of Korea). In summary, a library for 16S metagenomic sequencing was prepared by amplifying the V3–V4 region of 16S rRNA using the Hercules kit on the Illumina platform to construct a library of DNA extracted from fecal samples. The sequencing results were analyzed using the QIIME2 program, and taxonomic information classification was confirmed using the BLAST program of the NCBI 16S database. Data were shown as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). The significance of differences among groups was assessed using a one-way analysis of variance (ANOVA) by Duncan’s post hoc tests at p < 0.05. UC symptoms of colitis were identified as changes in body weight, disease activity index (DAI), colon length, and weight per length of the colon (Figure 1A–C). There was no significant difference in the change in body weight before DSS administration, but from the 6th day after DSS administration, both the DSS and DSS + BC groups were significantly reduced compared with the Vehicle control group (Figure 1A). Changes in DAI were checked daily during the DSS drinking period (Figure 1B). The DSS group showed a significantly higher DAI than the Vehicle group from the 22nd day. In contrast, the DSS + BC group showed significantly lower values than the DSS group until the 25th day. The DSS + BC group also showed an improved DAI on the final day of the experiment. The colon length was 4.73 ± 0.66 cm in the UC-induced DSS group, which was significantly shorter by about 29.9% compared with 6.75 ± 0.33 cm in the Vehicle group (Figure 1C). In the DSS + BC group, colon length was 5.70 ± 0.42 cm, and a DSS-related decrease in colon length was significantly restored. In addition, the DSS + BC group showed a significantly reduced colon weight-to-length ratio. Sections of the colonic tissue were stained with H&E and histopathological scores were given to confirm the extent of damage (Figure 2A,B). The Vehicle group had no damage or inflammatory response to the mucosa, submucosa, crypt structure, or goblet cells in the colon. However, severe epithelial erosion, deficiency of goblet cells, destruction of the crypt structure, and infiltration of many inflammatory cells into the mucosa and submucosa were observed in DSS-treated mice. Supplementation with blackcurrant alleviated damage to the mucosal layer of colonic tissue and infiltration of inflammatory cells caused by DSS, and significantly reduced the histological damage score. The levels of proinflammatory cytokines in the serum and colon are shown in Table 1. The DSS group showed significantly higher levels of serum TNF-α and interleukin (IL)-6 than the Vehicle group. The DSS + BC group showed significantly attenuated levels of serum TNF-α, which were elevated by DSS. In colon tissue, the levels of TNF-α and IL-1β in the DSS group were increased significantly compared with the Vehicle group. However, the levels of TNF-α and IL-1β increased by DSS treatment were significantly reduced in the DSS + BC. We investigated whether BC affects the expression of genes and proteins related to the NF-κB signaling pathway, mucin, and TJ proteins (Figure 3A–D). The DSS group upregulated the genes of toll-like receptor-4 (TLR-4) and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) related to the NF-kB signaling pathway compared with the Vehicle group (Figure 3A). Furthermore, an increase in the expression of iNOS, COX-2, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and monocyte chemoattractant protein-1 (MCP-1), which are downstream genes of NF-κB, was observed in the DSS group. However, the expression levels of these excessive mRNAs were inhibited in the DSS + BC group, with a value similar to those of the Vehicle group. Next, the effects of BC on the expression of genes encoding TJ proteins and mucin involved in barrier function were evaluated (Figure 3B). The DSS group significantly downregulated expression of all genes associated with TJ proteins and mucin compared with the Vehicle group. In contrast, the DSS + BC group showed higher expression of all such genes than the DSS group. The expression of proteins related to the NF-κB signaling pathway, an inflammatory response pathway, was also examined (Figure 3C,D). As a result, it was found that the phosphorylation of NF-κB p65 (p-p65) and the protein expression of its downstream enzymes, iNOS and COX-2, were significantly increased in the DSS group compared with the Vehicle group. However, the DSS + BC group was revealed to inhibit the overexpression of p-p65, iNOS, and COX-2 increased by DSS. That is, it was shown that the administration of BC decreased the inflammatory response by inhibiting the NF-κB signaling pathway activated by DSS in the colon. The influence of BC on the diversity and relative abundance of the gut microbiome was analyzed (Figure 4). To confirm the α-diversity of the gut microbiota, the observed amplicon sequence variant (ASV), an index of evenness, and Chao1, an index of richness, were evaluated. There was no significant difference between all groups, but the α-diversity of the DSS + BC group tended to increase slightly compared with the DSS group (ASV; Vehicle, 116.00 ± 32.33; DSS, 106.80 ± 9.36; DSS + BC, 125.20 ± 36.53, Chao1; Vehicle, 117.61 ± 32.30; DSS, 108.66 ± 11.33; DSS + BC, 127.79 ± 37.95). Regarding the composition of gut microbiota, the DSS group showed a distinct alteration from that of the Vehicle group (Figure 4A–D). In taxonomic community analysis at the phylum level, Firmicutes and Actinobacteria were reduced in the DSS group compared with the Vehicle group, whereas Bacteroidetes and Verrucomicrobia were increased (Figure 4A). Meanwhile, the DSS + BC group was found to modulate the changes in the phylum caused by DSS. The abundance of Ligilactobacillus, Enterococcus, and Bifidobacterium at the genus level was high in the Vehicle group (Figure 4B). However, DSS treatment diminished these genera and elevated the levels of Bacteroides, Escherichia, and Akkermansia. BC decreased Bacteroides levels and increased Ligilactobacillus compared with the DSS group. Moreover, at the species level, the administration of BC was shown to regulate the change in microbial composition due to DSS (Figure 4C). As a result of analyzing β-diversity with a principal coordinate analysis (PCoA) plot to confirm the relative similarity in the gut microflora between each group, it was distinguished by the first principal component (PC1) between the Vehicle and DSS-treated groups (Figure 4D). Moreover, the DSS and DSS + BC groups were distinguished by the second principal component (PC2), and supplementation with BC tended to modulate the gut microbial community. The cause of colitis is considered to be an imbalance in intestinal homeostasis due to the influence of genetic, microbiological, immunological, and environmental factors [5,6]. Natural products are being developed to treat UC, and ACNs are known to have positive effects on gut health [9,10,12,13,18,19]. Thus, the current study aimed to analyze how the beneficial effects of ACN-rich BC caused immunological and microbiological changes in the colon in mice with DSS-induced colitis. Indeed, a previous study reported that nonalcoholic steatohepatitis was prevented in mice fed a high-fat/high-sucrose diet containing 6% whole BC powder, which was equivalent to consuming two cups of fresh BC per day in humans, for 24 weeks [23]. Based on a previous study, we explored the effect of oral administration of 150 mg/day (7.5 g/kg body weight (BW), total ACN content; 165 mg/kg BW) of whole BC powder to mice, which was less than the dose administered in the previous study. In addition, the anti-inflammatory effects in colitis mice induced by DSS when administered BC at this dose were confirmed as a result of this study. Chemical induction of colitis using DSS in mice is the most widely used method because it reflects clinical symptoms and histological changes observed in humans [6,24,25]. DSS, which has a highly negative charge, acts directly on colonic epithelial cells as a chemical toxin and damages them, resulting in the depletion of mucin and goblet cells, epithelial erosion, and ulcers [24,25]. Destruction of the intestinal epithelial layer also increases colonic epithelial permeability, allowing commensal bacteria and related antigens to infiltrate the mucosa, followed by infiltration of immune cells such as neutrophils [22,24,25]. Immune cells infiltrating the lamina propria and submucosa reportedly secrete pro-inflammatory cytokines and disseminate inflammatory responses to underlying tissues [24,25]. The results of this work revealed that, when colitis was induced with DSS, clinical symptoms such as a decrease in body weight and colon length, as well as an increase in DAI and colon weight, were observed. Furthermore, histological changes were observed after inducing colitis with DSS, including epithelial loss, crypt damage, depletion of goblet cells, and infiltration of inflammatory cells. In contrast, the administration of BC had no effect on weight loss but showed beneficial effects on other clinical symptoms and histological changes following colitis induction. In another study, the intake of 200 mg/kg BW of crude ACN isolated from the fruits of Lycium ruthenicum Murray had no effect on weight loss induced by DSS, similar to our results [21]. Previous studies also demonstrated that giving mice ACN-containing materials such as the water extract of maqui berry, ACN extracted from mulberry fruit and black rice relieved the pathological changes in the colon caused by DSS, like inflammatory cell infiltration and mucosal damage [26,27,28]. Additionally, when silver nanoparticles with a diameter of 213 nm based on blackcurrant extract were supplied to the DSS colitis mice model at a concentration of 2 mg/kg, only the macroscopic score and colon shortening were significantly improved [19]. Similar to the previous study, our study in which whole BC powder was administered also showed an improvement effect in these indicators, as well as a relieving effect in the colonic weight-to-length ratio. This difference is likely due to the difference in dose concentration. Damage to intestinal epithelial cells caused by DSS was reported to worsen the inflammatory response by increasing the generation of pro-inflammatory cytokines [9,25,29]. It was also reported that the levels of TNF-α and IL-6 were altered in the serum of mice with early-stage colitis induced by one week of DSS administration [29]. Elevated levels of pro-inflammatory cytokines due to colitis can be reduced by various polyphenols, including ACNs [9,13,22]. In this study, except for IL-1β in the serum and IL-6 in the colon tissue, DSS treatment increased the levels of other pro-inflammatory cytokines, whereas BC administration decreased these levels. It was reported that treatment with petunidin 3-O-[rhamnopyranosyl]-(trans-p-coumaroyl)-5-O-[β-D-glucopyranoside] (P3G), isolated from the fruits of Lycium ruthenicum Murray, reduced all pro-inflammatory cytokines in the serum, but there was no difference in IL-1β levels in the crude ACN-administered group compared with the DSS-treated group, as in our study [21]. When mulberry ACN was administered, the inhibitory effect on pro-inflammatory cytokines in the colon decreased all indicators at a high concentration (200 mg/kg BW), but there was no change, except for IL-1β, at a low concentration (100 mg/kg BW) [26]. The major ACNs in BC are delphinidin-3-rutinoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and cyanidin-3-glucoside, and each food item contains different types of ACNs [11]. Therefore, the difference in effects on weight loss and pro-inflammatory cytokines was presumed to be due to differences in the types and intake of different ACNs in food, and differences in UC mouse models and disease stages. Moreover, previous studies have shown that BC extract decreases inflammation-related cytokines in bone-marrow-derived macrophages and vascular tissue in mice with type 2 diabetes mellitus [17,30]. Similarly, in the present study, BC was observed to reduce the production of pro-inflammatory cytokines, even when consumed in the form of whole BC powder. Intestinal homeostasis is maintained by a barrier consisting of mucus, epithelial, and immune cells that prevent the penetration of bacteria and other antigens into the colon tissue [2,31]. DSS-induced loss of TJ proteins (ZO-1 and occludin) in mucus and mucin in the intestinal epithelial layers [21,26,31,32]. NF-κB is an inducible transcription factor that regulates the expression of genes encoding cytokines associated with immune and inflammatory responses and is involved in maintaining intestinal homeostasis [33]. When cells are stimulated externally through gut microbes, pro-inflammatory cytokines and toll-like receptors activate NF-κB (p-p65), which is known to be involved in the onset of inflammatory diseases by upregulating the expression of inflammation-related cytokines (TNF-α, IL-1β, and IL-6), chemokines (MCP-1), and inducible enzymes (COX-2, iNOS) [21,28,32,33]. In previous studies, the administration of ACN in mice with DSS-induced colitis and mice fed a high-fat diet increased the expression of factors related to mucin and TJ proteins in the colon, while downregulating the expression of target genes in the NF-κB signaling pathway [21,34]. In vitro, ACN-rich bilberry and BC extracts, as well as the 3-O-glucosides of cyanidin and delphinidin, have been shown to inhibit the activity of TNF-α-induced NF-κB in intestinal epithelial cells [18,35]. The results of this study demonstrated that BC intake enhanced the expression of genes related to mucin and TJ proteins in colitis-induced mice. Additionally, BC decreased the phosphorylation of the NF-κB subunit and downregulated the expression of NF-κB target genes and proteins, such as COX-2 and iNOS, which were shown to improve DSS-induced colitis. Many studies have reported that changes in the community structure of gut microflora are associated with the development of colitis [10,21,22,24,25,26,28]. In the DSS-induced colitis model, maqui berry extract and ACNs of mulberry and Lycium ruthenicum Murray changed the α-diversity of gut microflora [21,28], but BC did not change it significantly. However, it was confirmed that the treatment with BC had an effect on the β-diversity and gut microbial composition, which was distinct from that of the DSS group. Several studies using DSS-induced colitis mouse models revealed a reduction in Firmicutes and an increase in Bacteroidetes at the phylum level, and the intake of ACNs and flavonoids modulated their composition [36,37,38]. The genera Lactobacillus (some of the reclassified genera, Ligilactobacillus [39]) and Bifidobacterium in the colon, known to have beneficial effects on health in several studies, are reduced by DSS [22,28,40], and our results were similar. Similar to another chronic DSS animal study, this study observed that treatment with DSS increased the genus Akkermansia, and this increase was a positive correlation with IL-1β, a pro-inflammatory cytokine [40]. Although the genus Akkermansia is known to have anti-inflammatory effects, it is still controversial and more studies are required because its exact role in IBD is not known [41]. As a change in relative abundance at the species level, BC decreased Bacteroides acidifaciens, known colitis-associated bacteria, after DSS treatment, and increased Bacteroides caecimuris, which rose in the recovery phase after stopping DSS treatment [42]. In addition, BC administration tended to increase the abundance of Mucispirillum schaedleri, which has been reported to have a preventive effect against colitis caused by Salmonella and Alistipes putredinis, which decreases in IBD [43,44]. As such, BC modulated the composition of gut microbiota that was altered by DSS. However, further studies are required to investigate the precise mechanism for the role of gut microbiota in each in the alleviation of colitis by BC. The intake of whole BC powder has been shown to prevent clinical symptoms and histological destruction caused by colitis. BC was observed to attenuate the levels of pro-inflammatory cytokines in serum and colon tissues and enhance the gene expression of mucin and tight junction proteins. Additionally, it downregulated the expression of target proteins and genes involved in the NF-κB signaling pathway. Furthermore, BC showed the potential to alleviate the intestinal inflammatory response by modulating the composition of gut microbiota altered by DSS. Therefore, in this study, whole BC powder showed a protective effect against DSS-induced colitis by regulating the inflammation-related NF-κB signaling pathway and gut microflora, confirming its potential as a natural dietary material to improve UC.

PMC10000430

Marta Santiago,Alessandro Liquori,Esperanza Such,Ángel Zúñiga,José Cervera

The Clinical Spectrum, Diagnosis, and Management of GATA2 Deficiency

03-03-2023

GATA2 deficiency,GATA2 haploinsufficiency,germline mutation,predisposition to myeloid neoplasms

Simple Summary A predisposition to myeloid neoplasms has recently been recognized as a defined clinical entity by the World Health Organization. One of the most well-known syndromes within this group is GATA2 deficiency, which is a highly heterogeneous disorder that can include pulmonary and vascular involvement, immunodeficiency, and myeloid neoplasms. The only curative treatment for this syndrome is allogeneic hematopoietic stem cell transplantation (HSCT), which should be performed in patients with GATA2 deficiency before irreversible organ damage. These patients should be referred to a multidisciplinary team to assess all potential and specific organ-system manifestations that could impact the patient’s treatment, and consultations with appropriate subspecialists should be facilitated. Additionally, genetic testing should be offered to first-degree relatives, particularly those considered for donation when an HSCT with a sibling donor is feasible. Abstract Hereditary myeloid malignancy syndromes (HMMSs) are rare but are becoming increasingly significant in clinical practice. One of the most well-known syndromes within this group is GATA2 deficiency. The GATA2 gene encodes a zinc finger transcription factor essential for normal hematopoiesis. Insufficient expression and function of this gene as a result of germinal mutations underlie distinct clinical presentations, including childhood myelodysplastic syndrome and acute myeloid leukemia, in which the acquisition of additional molecular somatic abnormalities can lead to variable outcomes. The only curative treatment for this syndrome is allogeneic hematopoietic stem cell transplantation, which should be performed before irreversible organ damage happens. In this review, we will examine the structural characteristics of the GATA2 gene, its physiological and pathological functions, how GATA2 genetic mutations contribute to myeloid neoplasms, and other potential clinical manifestations. Finally, we will provide an overview of current therapeutic options, including recent transplantation strategies.

The Clinical Spectrum, Diagnosis, and Management of GATA2 Deficiency A predisposition to myeloid neoplasms has recently been recognized as a defined clinical entity by the World Health Organization. One of the most well-known syndromes within this group is GATA2 deficiency, which is a highly heterogeneous disorder that can include pulmonary and vascular involvement, immunodeficiency, and myeloid neoplasms. The only curative treatment for this syndrome is allogeneic hematopoietic stem cell transplantation (HSCT), which should be performed in patients with GATA2 deficiency before irreversible organ damage. These patients should be referred to a multidisciplinary team to assess all potential and specific organ-system manifestations that could impact the patient’s treatment, and consultations with appropriate subspecialists should be facilitated. Additionally, genetic testing should be offered to first-degree relatives, particularly those considered for donation when an HSCT with a sibling donor is feasible. Hereditary myeloid malignancy syndromes (HMMSs) are rare but are becoming increasingly significant in clinical practice. One of the most well-known syndromes within this group is GATA2 deficiency. The GATA2 gene encodes a zinc finger transcription factor essential for normal hematopoiesis. Insufficient expression and function of this gene as a result of germinal mutations underlie distinct clinical presentations, including childhood myelodysplastic syndrome and acute myeloid leukemia, in which the acquisition of additional molecular somatic abnormalities can lead to variable outcomes. The only curative treatment for this syndrome is allogeneic hematopoietic stem cell transplantation, which should be performed before irreversible organ damage happens. In this review, we will examine the structural characteristics of the GATA2 gene, its physiological and pathological functions, how GATA2 genetic mutations contribute to myeloid neoplasms, and other potential clinical manifestations. Finally, we will provide an overview of current therapeutic options, including recent transplantation strategies. Familial myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML), also known as hereditary myeloid malignancy syndromes (HMMSs), have been recognized phenotypically for more than a century, with the first molecular basis discovered in 1999 through the identification of germline RUNX1 mutations [1]. Since then, and recently accelerated by the advent of next-generation sequencing (NGS), a growing number of genes have been associated with germline predisposition to myeloid malignancies, including the ANKRD26 [2,3,4], ETV6 [5,6,7], CEBPA [8], DDX41 [9], GATA2 [10], RBBP6 [11], TERT, TERC [12], and, most recently the SAMD9 [13] and SAMD9L genes [14,15]. Although they are traditionally considered very rare entities, it is now known that 4–13% of pediatric and 5–15% of adult MDS/AML cases are caused by germline predisposition [16,17,18,19,20,21]. Although most of these entities have only recently been described, the World Health Organization (WHO) incorporated some of them as provisional categories in its fourth revised classification [22]. In recognition of the robustness of data, HMMSs have also been integrated into other guidelines and expert recommendations, such as the Nordic Guidelines and the European Leukemia Network [23,24], highlighting the need to identify, diagnose, and correctly manage patients with hereditary syndromes. Finally, the growing recognition and molecular identification of this subset of myeloid malignancies have led to their being formalized in the most recent revisions by the WHO and the International Consensus Classification (ICC) of myeloid neoplasms [25,26]. The WHO 2022 update reinforces this category and includes it within the group of secondary myeloid neoplasms [25]. On the other hand, the 2022 ICC proposes to place these entities within the category of pediatric and/or germline mutation-associated disorders due to their overlap with other childhood disorders [26]. This review focuses on one of these entities, specifically the phenotypic spectrum of patients diagnosed with GATA2 deficiency, recognized as a major myeloid neoplasia predisposition syndrome with pleiotropic manifestations. We discuss the structural characteristics of the GATA2 gene and describe how its genetic alterations might contribute to the onset of myeloid neoplasms as a result of aberrant induced hematopoiesis [27]. In addition, we will summarize diagnostic clues for proper identification and management of this syndrome. The GATA binding protein 2 (GATA2) gene is located on the long arm of human chromosome 3 at cytoband 21.3 (i.e., 3q21.3) and encodes two main isoforms (NM_032638 and NM_001145661) identical in their coding regions, but differing in the 5′ untranslated region [28,29]. The GATA2 protein belongs to the GATA binding factors family, which modulates the expression of several genes by binding to the DNA motif GATA and other transcription factors [30]. This is managed by two highly conserved zinc finger domains (ZF1 and ZF2), which are responsible for the DNA-binding ability of GATA2. In addition, the GATA2 protein contains two transactivation domains, a nuclear localization signal, and a negative regulatory domain [29]. The precise role of GATA2 in hematopoiesis is still not entirely understood. Hematopoietic stem cells (HSCs) found in the bone marrow of GATA2+/− mice were found to be impaired in terms of both number and functionality, as evidenced by serial transplantation assays [31]. GATA2 heterozygosity is associated with decreased proliferation ability and increased quiescence and apoptosis in HSCs [31]. Moreover, GATA2 haploinsufficiency impairs the function of granulocyte-macrophage progenitors but not that of other committed myeloid progenitors [32]. Despite this, GATA2+/− mice do not develop MDS/AML, which makes it challenging to study the impact of GATA2 haploinsufficiency on leukemic progression in these models. On the other hand, the overexpression of GATA2 results in the self-renewal of myeloid progenitors and hampers lymphoid differentiation in mouse bone marrow [33]. Additionally, the overexpression of GATA2 promotes proliferation in human embryonic stem cells (hESCs) but quiescence in hESC-derived HSCs [34]. Elevated levels of GATA2 have been observed in AML patients, both adults and children, who have poor prognoses [35]. These findings indicate that, in addition to its function as a tumor suppressor, GATA2 may also act as an oncogene when overexpressed. In line with these data, and focusing on adult hematopoiesis, the GATA2 protein, together with several transcription factors (e.g., FLI1, LMO2, and RUNX1), is involved in HSC survival and self-renewal, thus participating in early lineage commitment. Meanwhile, during hematopoietic differentiation, GATA2 modulates downstream fate decisions by interacting with CEBPA, GATA1, and SPI1 [36,37]. To date, roughly 500 GATA2-deficient patients have been reported, and the syndrome was confirmed to be inherited according to an autosomal dominant pattern in 50% of cases, de novo in 5% of cases, and uncertain in the rest of the cases [38]. This is unexpectedly different from previous studies, in which de novo occurrence was estimated in two thirds of all cases [39,40]. However, there is a lack of a well-characterized series in which segregation studies have been carried out systematically or in which penetrance or expressivity were considered. Therefore, these data should be viewed with caution. In addition, almost 200 unique (likely) pathogenic variants have been described that can be classified into four groups: truncating mutations (splice site, nonsense, frameshift, and whole-gene deletions) proximal to or within the ZF2 domain; missense mutations within the ZF2 domain; mutations resulting in aberrant mRNA splicing (e.g., synonymous changes) (Figure 1) [38,41,42]; and other regulatory variants, such as those located in the GATA2 intronic +9.5 kb enhancer site (e.g., c.1017+572C>T, the c.1017+532A>T, and the c.1017+513_1017+540del [c.1017+512del28]), which is essential for hematopoiesis [42,43,44,45,46]. Overall, germline GATA2 (likely) pathogenic variants are hypothesized to result in haploinsufficiency because truncated alleles lead to clinical phenotypes similar to missense variants [31,45]. Strikingly, some variants have been associated with only partial loss-of-function (p.T354M) or even gain-of-function (p.L359V) mechanisms, suggesting more complex pathways [47,48]. Although most deleterious changes are private, it is possible to recognize some mutational hotspots. Recurrent variants in the extended ZF2 domain have been identified, including p.T354M and p.R396W/Q/W, found in roughly one fifth of the reported cases, as well as the c.1017+572C>T intronic variant, found in 20 patients [38]. Germline GATA2 mutations are usually necessary but not sufficient for myeloid disease development. It has been proposed that different environmental stressors may modify the expression of these germline variants during embryogenesis or after birth, inducing disorder in tissues where limited GATA2 expression is inadequate for their normal cellular function [38]. Particularly in bone marrow (BM), such stressors can lead to certain cytogenetic and molecular alterations that accumulate over time, selecting clonality and triggering myeloid transformation. Indeed, the germline variant can also modify the BM microenvironment, contributing to clonal selection [38]. In patients with progression to a malignant neoplasm, certain cytogenetic and molecular alterations appear recurrently. The most frequent cytogenetic alterations in patients with germline GATA2-mutated myeloid neoplasms involve chromosome 7, including its monosomy, partial deletion of 7q and der(1;7)(q10;p10), and trisomy of chromosome 8 [27,40,49]. These neoplasms tend to show fewer somatic mutations and a different molecular landscape compared to non-GATA2 MDS/AML. The most frequent recurrent somatic mutations identified in GATA2-MDS/AML patients are in the SETBP1, ASXL1, and STAG2 genes, and the RAS pathway. By contrast, deleterious SF3B1, U2AF1, NPM1, and FLT3 changes are infrequent in GATA2-mutated myeloid neoplasms [21,50,51,52,53,54,55,56,57,58,59,60]. Interestingly, GATA2 can also be mutated in somatic cells of sporadic MDS/AML. Different from germline GATA2 mutations, which mainly include truncated and ZF2 missense changes, somatic GATA2 mutations are usually missense variants located in the ZF1 domain (e.g., p.N317-L321 hotspot) or in-frame indels in the C-terminus (Figure 1) [38]. This suggests a likely difference in GATA2 function during the leukemogenic process between germline and somatic cases [61]. Of note, somatic GATA2 mutations are often associated with both monoallelic and biallelic CEBPA somatic mutations [62,63,64]. Additionally, somatic mutations in GATA2, although rare, have also been linked to milder forms of the immunodeficiency phenotype observed in patients with germline mutant GATA2 [65,66]. Heterozygous pathogenic variants in the GATA2 gene cause a highly heterogeneous disorder with incomplete penetrance [67]. This may present with immunodeficiency (including monocytopenia with Mycobacterium avium complex (MonoMAC) infection and dendritic cell (DC), monocyte, B, and natural killer (NK) lymphoid (DCML) deficiency syndromes); syndromic features, such as congenital deafness and lymphedema (originally defining Emberger syndrome), or pulmonary and vascular involvement [49], and there is a high probability of evolving to MDS and/or AML. In 2011, these diverse clinical syndromes were linked to define a common genetic diagnosis of the GATA2 deficiency syndrome [10,45,68,69]. Except for a few cases, the relationship between genotype and phenotype in these patients is poorly understood due to significant variations in clinical presentation, even among individuals within the same family [41,49]. Therefore, determining the true clinical penetrance of this disorder would require a comprehensive examination of the genotypes of a large number of first-degree relatives of patients. It is worth noting some of the reported phenotype/genotype correlations: (1) patients with noncoding variants (which can account for up to 10% of cases) have been observed to exhibit reduced disease penetrance [41,49,70,71]; (2) the p.T354M variant seemed to be associated with a predominance of myeloid malignancies (83% of cases; 44/53), while p.R398W/Q variants were more commonly associated with immunodeficiency (88% of cases; 23/26) in a relatively large series [38]; (3) there have been indications that complete haploinsufficiency or loss of GATA2 function, rather than missense mutations, may be required for the development of lymphedema [72]. These complex and variable presentations pose a significant challenge for clinicians when diagnosing and managing patients with GATA2 mutations. The first hematoimmunologic manifestation typically occurs between the second and third decade of life, with a median age that varies in different studies (ranging from 12 to 19 years) [38,39,41,49,71]. While some patients present with cytopenias, immunodeficiency, or BM failure during childhood, others can develop MDS without preexisting clinical features during young adulthood (Figure 2) [27]. Unlike other germline alterations predisposing to HMMSs that preferentially lead to thrombocytopenia (e.g., ANKRD26, RUNX1, ETV6) [73,74,75], neutropenia may be the first and leading form of cytopenia in these patients. Although a decreased white blood cells (WBC) count can lead to a complex differential diagnosis, neutropenia with profound monocytopenia should prompt consideration of GATA2 deficiency [67]. Paradoxically, monocytosis can be the initial presenting sign in patients who develop GATA2-related MDS [27]. Bone marrow morphology can reveal altered cellularity (hypo- and normal or hypercellular marrow in patients with cytopenia or MDS, respectively), pronounced erythropoiesis, multilineage dysplasia, and fibrosis [40,67]. GATA2 haploinsufficiency is a major contributor to MDS/AML in adolescents and young adults. While some patients who develop MDS have a high risk of progressing to AML or chronic myelomonocytic leukemia (CMML), a small subset presents directly with AML [27]. Other reported hematological disorders include acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML), and myelofibrosis [71,76,77]. The prevalence of GATA2 deficiency is currently unknown, but given the significant disease penetrance and low tolerance to pathogenic mutations in the GATA2 gene, it is likely that most carriers of the mutation will develop hematologic or immunologic complications over the course of their lifetime. In one study that reviewed 18 published series (>350 individuals), the penetrance of myeloid neoplasms was estimated to reach 75% in GATA2-mutated carriers [27], with an increased risk of developing MDS/AML as they aged. The risk of developing MDS/AML was calculated to be 6% at 10 years, 39% at 20 years, and 81% at 40 years in a series of 79 patients [39,71,76]. While MDS/AML is the most common neoplasm in GATA2 deficiency, the EWOG-MDS study [49], which included 426 patients, found that GATA2 germline mutations were present in up to 7% of all pediatric cases with primary MDS and 15% of advanced MDS in examined series [49,78,79]. Monosomy 7 is the most frequent cytogenetic alteration, being present in 37–57% of all patients with GATA2 MDS and 48–72% of adolescents (>12 years old) with GATA2 MDS [22,49,76]. Since MDS is very uncommon during childhood, it would seem mandatory to screen all children with this diagnosis for GATA2 germline mutations [22,49,76]. GATA2 deficiency is a unique primary immune deficiency that is also known as immunodeficiency 21, DCML, or MonoMAC (OMIM #614172). The immune defect may appear in adult life, as the number of hematopoietic stem and progenitor cells (HSPCs) decreases with age, which makes GATA2 deficiency a unique primary immune deficiency [80]. It is characterized by immunophenotype features resembling those seen in chronic infection or age-related immunosenescence. The spectrum of alterations can include dendritic cell deficiency, monocytopenia, loss of transitional B cells, the absence of CD56 bright NK cells (which presents an altered CXCL12/CXCR4-dependent chemotaxis [76,81,82,83,84]), reversed CD4:CD8 ratio, an excess of CD45RA+ CD8+ T cells, and poor-quality humoral response [27,85] despite normal levels of immunoglobulins and an adequate presence of bone marrow plasma cells in most patients [40,86,87]. As a result of immune deficiency, GATA2 carriers have an increased frequency of infections, with significant differences in the severity between patients [80]. Due to the deficit and dysfunction of dendritic cells, NK cells, and monocytes/macrophages, the identification of viruses and intracellular pathogens is compromised, leading to the severe spread of viral infections and mycobacterial susceptibility [40,41]. Donadieu and colleagues described severe bacterial infections as the most frequent pathogenic occurrences in GATA2 carriers, with a cumulative rate of 33% at 20 years and 64% at 40 years [71]. On the other hand, Spinner et al. reported that severe viral infections were the most common ones in their series (70%), in particular those related to the human papilloma virus (HPV), which occurred in about two thirds of carriers [41]. The most important complication derived from underlying HPV infection is the development of recurrent warts or condyloma that can lead to dysplasia and/or squamous carcinoma [88]. Infections with other disseminated pathogens are frequently observed in GATA2-deficient patients, including non-tuberculous mycobacteria, herpes virus (varicella zoster virus, Epstein–Barr virus, and cytomegalovirus), and fungi (invasive aspergillosis, disseminated histoplasmosis, and candidiasis) [41]. Therefore, various immunological factors are highly suggestive of GATA2 deficiency and should make the clinician think of this disorder. These include prior immunodeficiency in a patient with MDS, atypical mycobacterial infections in patients with monocytopenia, persistent warts or severe herpes virus infections in cytopenic patients, and loss of B cells and their precursors, especially in patients who develop MDS [27,41,84,85]. Eventually, as in other immunodeficiencies, these patients can also present with autoimmune manifestations, described in 11–30% of cases [41,71,89], which may overshadow typical features of GATA2 deficiency and delay the diagnosis. Amarnani et al. reported rheumatological findings in 18% of their GATA2 deficiency cohort, with notable manifestations, including early onset osteoarthritis, piezogenic pedal papules, ankylosing spondylitis, and seronegative erosive rheumatoid arthritis [89]. Pulmonary dysfunction is a common finding in up to 50% of patients with GATA2 deficiency [90], even in the absence of hematopoietic disease, leading to progressive compromised pulmonary function with diffusion defects, ventilatory defects, or a mixed pattern, along with significant clinical and radiographic disease [41,71,76,91]. In addition to recurrent infections, pulmonary alveolar proteinosis (PAP) is one of the most distinctive lung features. This rare disorder is characterized by the lack of anti-GM-CSF autoantibodies and the accumulation of surfactant proteins and subsequent impaired gas exchange [40]. It results from impaired function of the alveolar macrophages in GATA2-deficiency patients, which are responsible for inadequate clearance, and is associated with increased restrictive ventilatory defects and pulmonary arterial hypertension (PAH) [40,90]. Depending on the studied cohort, PAP and PAH may be present in 4–20% of patients [41,71,90]. Therefore, it is recommended to screen patients with PAP and/or immunodeficiency and/or myeloid malignancies without anti-GM-CSF antibodies for GATA2 alterations. It is important to note that clinical variability within families, including asymptomatic relatives identified through family screening, has also been reported in the case of pulmonary dysfunction [41,90]. Radiographic findings might be unspecific and will depend on the underlying disorder. Several structural abnormalities have been identified on chest computed tomography, including nodular and reticular opacities, ground-glass opacities, consolidations, a “crazy-paving” pattern, subpleural blebbing, and paraseptal emphysema [41,76,90]. Although some of the lung manifestations, including PAP, PAH, and underlying infections, can be reversed as a result of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) [41,92,93], it should be noted that HSCT toxicity related to the conditioning regimen and pulmonary graft-versus-host disease (GvHD) can also harm lung function [41,90]. Therefore, individuals with GATA2 deficiency should undergo regular, ongoing monitoring of their lung function throughout their lifetime. Although there are no guidelines for the pulmonary follow-up of these patients, it should be individualized and tailored to each patient’s needs. This may involve regular visits to a pulmonologist for symptom monitoring and pulmonary function testing to assess respiratory capacity. Imaging tests, such as chest X-rays or computerized tomography (CT) scans, may also be used to evaluate lung changes. Additionally, if there is suspicion of alveolar proteinosis, a diagnosis confirmation can be made through bronchoscopy with bronchoalveolar lavage (BAL) and/or parenchymal biopsy. Emberger syndrome (OMIM #614038) is characterized by the association of primary lymphedema (a common feature found in 11–20% of GATA2 carriers, typically affecting one or both lower limbs, frequently involving the genitals in the form of a hydrocele), with AML (often preceded by pancytopenia or MDS), with or without congenital sensorineural hearing loss [38,40,41,68,71,76,94,95,96,97]. Additional dysmorphic features that have been described, include hypothyroidism, bilateral syndactyly of the toes, hypotelorism, and epicanthal folds, behavioral disorder, and urogenital malformations, among others [27,41]. Although allo-HSCT is the only curative therapy for the impaired hematopoietic and lymphoid systems of patients with GATA2 deficiency [93,98,99,100], it represents a therapeutic challenge due to disease-associated comorbidities and clinical heterogeneity. Meanwhile, determining who should be candidates for allo-HSCT and when it should be performed (so that the benefits outweigh the risks) are questions that remain under debate [93,100]. Moreover, due to the low prevalence and relatively recent description of GATA2 deficiency syndrome, most outcomes and complications following allo-HSCT have been described in case reports or small series [93,98,100,101,102,103,104]. While some studies have reported an overall survival (OS) rate in 5-year posttransplant patients with clonal events at a rate of 55–60% [41,71,101], other reports have shown superior outcomes after the procedure [98,99,100,103]. Notably, Nichol-Vinueza et al. showed a 4-year posttransplant OS rate of 85.1% [100]. However, these cohorts are not necessarily comparable due to the heterogeneity of conditioning regimens and GvHD prophylaxis, donor type source, HSCT-related risk factors, duration of follow-up, and the clinical status or comorbidities of the GATA2 patient population [98,99,101]. While hematologic malignancy development may be the most dangerous complication and a primary indication for transplant, it is not the only one. Restoring normal immunity and lung function is also important in the decision to proceed with SCT [93]. The lack of a genotype-phenotype correlation makes the natural history of GATA2 deficiency unpredictable, to the point that there are patients who become symptomatic after many decades. However, once symptoms appear, survival declines, with a probability of survival by 40 years of 60–80% according to different series [41,71]. In this regard, the ideal time for allo-HSCT should be after the onset of symptoms but before irreversible organ damage occurs [93,98,99], although more specific criteria for the timing need to be defined [105]. While some authors report better outcomes when HSCT is performed earlier after diagnosis and when there are fewer comorbidities [71,100,101], the EWOG-MDS 2017 guidelines on childhood MDS recommend watchful waiting if blood cells are stable and high-risk genetic aberrations are absent [49]. By contrast, other authors go as far as to propose that preemptive allo-HSCT could improve overall outcomes before malignancy develops [106,107,108]. More specific treatment strategies have yet to be fully elucidated. There are three major indications for HSCT. Firstly, diagnosis of MDS/AML, however, it is not clear if better timing for HSCT is during the hypocellular MDS phase or when the patients develop cytogenetics abnormalities/excess of blasts [40,41,49,71,98,99,102,103,105]. Secondly, history of severe, recurrent, or treatment-refractory infections, particularly aggressive HPV infection. Relapsed/refractory precancerous or malignant disease due to HPV should be an indication for allo-HSCT. In this sense, considering the iatrogenic immunosuppression after HSCT, rigorous evaluation for HPV must take place before and after transplantation so that surgical and other therapeutic measures can be undertaken in cases with new or persistent disease [93,99,102,103,104,105]. Thirdly, progressive lung injury from infection and PAP, which leads to deteriorated lung function [93,99,102,103,105]. Transplanting GATA2-deficient patients is a controversial topic due to the variable disease progression and the timing of HSCT [100,109]. Although nonmyeloablative HSCT can reverse clinical manifestations and was the strategy used in the earlier years, relapse rates, engraftment failure, and late graft rejections led to the consideration of more intensive conditioning regimens [93,102]. In this regard, several reports have demonstrated similar outcomes when using myeloablative regimens in patients with mutated and wild-type (wt) GATA2 [49,98,101]. However, in patients with low-stage and hypocellular MDS, myeloablation may not be necessary due to low rates of relapse [98], and the intensity can be reduced by using a controlled approach [110]. Therefore, some authors propose that the choice of conditioning scheme choice for GATA2-deficient patients should be based on the patient’s MDS phenotype and cytogenetics [101,103,105,110]. The donor source constitutes a critical variable in the outcome of HSCT. Although it is still unclear which donor source will yield better outcomes for GATA2-deficient patients [102], it has been suggested that bone marrow should be preferred over peripheral blood, while umbilical cord blood should be avoided [102,103]. Matched related donors remain the best choice, although haploidentical HSCT could be an appropriate alternative [103,110]. Bortnick et al. conducted a study of 65 cases and found that pediatric patients with GATA2 deficiency had a similar risk of transplant-related toxicity (TRT) or transplant-related mortality (TRM) as compared to those with wt GATA2 [98]. However, they also reported that three patients developed transplant-associated microangiopathy, which might indicate a distinct endothelial vulnerability in GATA2 patients, consistent with the known role of GATA2 in the perturbation of normal vascular development [41,111,112]. Simonis et al. conducted a systematic review of 183 patients (median age 23 years) from January 2010 until March 2018 and reported that the risk of TRT was not higher in patients with GATA2 deficiency compared to those without it [93]. Similarly, Hofmann et al. reported no differences in TRM and overall organ toxicity between a pediatric cohort with GATA2 deficiency and controls [101]. However, they did observe a small number of serious and unusual infectious/immunologic complications and neurologic toxicities in the GATA2 population, as well as a higher rate of thrombotic events in GATA2 patients, with complete resolution after transplantation [101]. Although information about GvHD is often not available in these series, it seems that the proportion of patients with acute or chronic GvHD is similar to that of other transplant cohorts [93,100,101]. Reducing the severity of both acute and chronic GvHD is being evaluated in GATA2 deficiency patients with promising outcomes by administering post-cyclophosphamide (PTCy) after HSCT, as seen in wt GATA2 patients [100,103,105]. However, when HSCT is indicated but no preexisting malignancies are present, strategies to prevent GvHD are of the greatest importance, as there is no advantage to this complication [110]. In summary, considering that there are no formal recommendations on the indications for allo-HSCT, conditioning regimen, GvHD prophylaxis, donor source, and antibiotic prophylaxis in GATA2-deficiency patients, the decision to perform an allo-HSCT requires careful and individualized management [99]. Although treatment-related morbidity is manageable in these patients, an individualized approach should be taken into consideration for optimal outcomes. Prior to performing allo-HSCT, it is crucial to effectively treat any severe infections to create a favorable environment for the transplanted donor stem cells to thrive [105]. Although opportunistic infections that manifest before transplantation do not seem to pose a major issue in terms of overall outcomes, patients are typically kept on antibiotic prophylaxis to prevent such infections. While most case reports of allo-HSCT do not provide details on the antibiotic prophylaxis regimen, in a study by Simonis et al., patients treated for non-tuberculous mycobacterium before HSCT took prophylactic azithromycin until the time of transplant and for about one year afterward [93]. For patients still receiving treatment for active infection at the time of HSCT, antimycobacterial drugs were administered for 6–12 months after the transplant [93]. Spinner et al. also recommend azithromycin for all patients with GATA2 deficiency even before HSCT is indicated [41]. Although rare, GATA2 patients may experience humoral deficiency [87,113]. In such cases, immunoglobulin replacement may be necessary [40]. Due to the high susceptibility of patients to HPV and the potential for recurrent and life-threatening oncogenic HPV lesions, early vaccination is likely to be beneficial [40,41,71]. Given the complexity of information available on GATA2 deficiency syndrome and other HMMSs, patients should be referred to multidisciplinary teams that include physicians who are well-versed in these conditions. This would facilitate the assessment of potential organ-system manifestations that could impact the patient’s treatment, and promote consultation with appropriate subspecialists. Since most patients with symptomatic GATA2 deficiency will eventually require an allo-HSCT, close monitoring is crucial in order to perform the procedure before organ damage occurs [93,98]. Therefore, a donor search should be conducted as soon as the deficiency is diagnosed, with systematic testing of potential relatives considered for donation [71,99]. Allo-HSCT can eradicate clonal malignancy, restore normal hematopoiesis, clear underlying infections, and improve pulmonary symptoms and function in patients with PAP [93,98,103]. However, it cannot reverse extra-hematopoietic manifestations of GATA2 deficiency, so patients remain at risk for non-hematopoietic issues and will require lifelong follow-up [101,105]. It is worth mentioning that HPV can persist after allo-HSCT, so gynecologists play an important role in guiding the management and surveillance of these patients [104,114], especially during the period of immunosuppression following the procedure. Genetic testing should be offered to first-degree relatives, particularly to potential donors of HSC progenitors, to identify asymptomatic carriers with GATA2 deficiency. According to some authors, hematological surveillance of carriers should include annual bone marrow analysis with morphological, cytogenetic, and molecular evaluation to prevent the appearance of new driver acquisitions [99]. Moreover, some researchers recommend avoiding exposure to corticosteroids and immunosuppressive drugs and monitoring pulmonary function regularly to prevent complications [27]. It is important to note that genetic counseling should be offered to family members who test positive for GATA2 mutations to help them understand the implications of the diagnosis and the potential risks of passing the condition on to their own children, and they should receive proper information about the different reproductive or prenatal diagnostic options. Recognizing GATA2 deficiency in clinical care is crucial for several reasons [115]. Firstly, an accurate diagnosis can help patients understand their specific disorder and avoid inappropriate treatments. Secondly, a genetic diagnosis can aid in selecting the most suitable HSCs donor for an allo-HSCT. TShirdly, identifying GATA2 syndrome can impact treatment recommendations and disease management for patients and their families. As patients with this condition face various complications affecting many systems, HSCT is often an attractive therapeutic option. The choice of therapy largely depends on the patient´s age, the availability of a compatible donor, and any co-existing medical conditions. Thus, early and accurate diagnosis of these patients allows for tailored therapy. As awareness of GATA2 deficiency grows within the scientific community, early diagnosis will help in avoiding unnecessary diagnostic procedures and enable tailored strategies, for both treatment and surveillance [49]. Moreover, we may be able to identify patients who are at high risk of transforming to myeloid malignancies based on factors such as molecular alterations, cytogenetic evolution, or severity of cytopenias. By managing these patients early, we can aim for better outcomes before organ dysfunction occurs.

PMC10000431

Carlo Antonio Beretta,Sheng Liu,Alina Stegemann,Zheng Gan,Lirong Wang,Linette Liqi Tan,Rohini Kuner

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections

23-02-2023

quantitative analysis,immunohistochemistry,in situ hybridization,Fos protein,c-fos mRNA,2D automated cell counts,open-source ImageJ/Fiji tool

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes’ expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called ‘Quanty-cFOS’, with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes’ expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called ‘Quanty-cFOS’, with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells. Analysis of neural circuits frequently relies on the use of immunohistochemistry assays to identify specific cell types using neurochemical marker proteins or mRNAs target genes. Similarly, quantitative analyses of cell counts expressing plasticity markers, such as the activity-induced immediate early gene, c-fos, represent a cornerstone of studying neural plasticity processes over large cellular networks in histological specimens. However, reliably counting cells immunohistochemically positive for protein markers or for mRNAs visualized in in situ hybridization experiments in large histological specimens, such as brain sections, remains a major challenge. Manual counting is extremely time-consuming, cumbersome and prone to subjective variations. Recently, several automated digital image analysis tools have been developed. A major part of this development has focused on an automated analysis of the expression levels of proteins over regions of interest in histological specimens, such as tumor or immune markers, as clinical diagnostic or prognostic tools [1,2,3]. Despite this progress, availability of automated tools that enable easy-to-use, reproducible and reliable identification and quantitative counting of positive cells in immunohistochemical or mRNA in situ hybridization experiments on thick slices of tissue remains limited [4,5]. Often, user is required to have image analysis skills and experience, in-depth coding knowledge or access to expensive commercial software. Furthermore, automated tools for the identification of positive cells need high signal-to-noise levels, thus favoring highly expressed proteins. Signals representing nuanced differences in expression levels, high background and difficult antibodies, in contrast, are not suitable for conventional automated tools. This is particularly relevant to the protooncogene c-fos, the immediate early gene that is directly induced in expression upon neuronal activation, leading to a rapid and transient build-up of c-fos mRNA and consequently of the Fos protein, which decays shortly after cessation of neuronal activity. Over the recent years, mapping Fos expression has emerged as a technically simple and reliable global marker for analyzing neurons that are activated by diverse external inputs, such as sensory stimuli [6]. Moreover, because Fos expression is well-correlated with behavioral readouts in animals, Fos-based mapping enables spatial analysis of regions and cells recruited during particular behaviors [6,7,8,9]. Finally, Fos expression is frequently used to characterize the effects of diverse therapeutic regimens on the central nervous system [10,11]. The recent development of Fos-based transgenic tools for labelling ensembles, i.e., cells that are co-active during particular functional tasks, as well as approaches that enable the consecutive labelling of two distinct cell populations with c-fos mRNA and the Fos protein, provide tremendous scope for studying functional encoding in the nervous system [7]. Thus, although they lack the spatial resolution of direct electrophysiological measurements, Fos-based mapping approaches represent attractive, highly useful and popular tools that are delivering unprecedented insights into neural function. Quantifying Fos-expressing neurons, however, still represents a major problem owing to high background levels and non-linear expression with different levels of signal-to-noise within and across samples preparations. Batch-to-batch variability of both antibody-based signals as well as c-fos mRNA in situ probes leads to several confounding effects. This has led to the necessary use of experimenter-based manual counting in studies employing Fos-based mapping in a quantitative manner, which is highly laborious, time-consuming, not entirely objective and highly subject to experimenter bias. Here, we report the development of an open-source tool for ImageJ/Fiji [12], called ‘Quanty-cFOS’, with an easy-to-use, streamlined pipeline for automated or semi-automated quantitative analysis of cells positive for the Fos protein and/or c-fos mRNA on two-dimensional images (2D) or confocal maximum intensity projections (MIP) derived from tissue sections. Using example data sets of brain tissue from mice subjected to somatosensory stimuli, we demonstrate the entire process in a step-by-step manner and with the use of video tutorials, making it easy for novice users to apply on their images. Using manual counting to establish ground truth, we demonstrate both the fidelity of Quanty-cFOS and its ability to overcome user-to-user subjective variability. The tool also takes into account day-to-day and sample-to-sample variations in staining efficiency and enables for the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. Thus, by delivering reliable and fast automated cell quantification across complex, technically non-optimal data sets, Quanty-cFOS accelerates the use of Fos mapping for analyses of neural circuits and thus provides an impetus to a wide range of research fields, including memory, chronic pain, addiction and psychiatric disorders. Importantly, although this tool was optimized and validated for quantitating Fos-expressing cells, it is just as readily applicable to any antibody, and is particularly suitable for proteins that show a variable baseline and induced expression within and across samples. All experiments were conducted in C57BL/6J male mice (20–30 g) at 8 weeks of age that were obtained from Janvier Labs. In total 12 animals were used. Mice were housed individually in separated cages and kept under a 12 h light/dark cycle at a controlled temperature (22 ± 2 °C), humidity (40–50%), with food and water provided ad libitum according to ARRIVE guidelines. All experimental procedures were approved by the local governing body (Regierungspräsidium Karlsruhe, Germany, Ref. 35-9185.81/G-184/18), and abided to German Law (TierSchG, TierSchVersV) that regulates animal welfare and the protection of animals used for scientific purpose. A heat stimulus was presented to mice on a hot plate at 50 °C for 30 s (Ugo Basile Inc., Gemonio, Italy). Mice were exposed only once. To allow sufficient expression of Fos and to validate the Quanty-cFOS ImageJ/Fiji tool, C57BL/6J mice were kept in a home cage after stimuli for 20 min, 1 h or 3 h after the application of the stimulus prior to perfusion. Mice were sacrificed with an overdose of carbon dioxide, transcardially perfused with pre chilled phosphate-buffered saline (PBS) followed by 10% formalin fixative solution (Merck, Darmstadt, Germany). The brains were extracted and fixed in 10% formalin for 24 h at 4 °C. Coronal brain sections were collected at 50 μm with a vibratome (Leica VT100S, Wetzlar, Germany). Free-floating sections were incubated in antigen retrieval solution (2.94% Tri-sodium citrate in dH2O, pH 8.5) for 25 min at 85 °C, and after cooling down, washed at room temperature with 50 mM Glycine (AppliChem, Darmstadt, Germany) for 10 min, followed by PBS for 5 min and 0.2% Triton X-100 (Carl Roth, Karlsruhe, Germany) in PBS (PBST) for 15 min. Lastly, sections were treated with 5% horse serum in PBST for 1 h, before incubating with the rabbit anti-Fos primary antibody (Ab190289, 1:1000 in 5% horse serum in PBST, Abcam, Cambridge, UK) at 4 °C overnight. The next day, sections were washed with 5% horse serum in PBST 3 times for 10 min and incubated with the donkey anti-rabbit Alexa 594 secondary antibody (Ab21206, dilution 1:700 in 10% NHS in PBS, Thermo Fischer Scientific, Waltham, MA, USA) for 2 h. After washing again with 5% horse serum in PBST 3 times for 10 min and in PBS for 10 min, Hoechst (#H367, 1:10,000 in PBS, Thermo Fischer Scientific, Waltham, MA, USA) was added for 10 min, followed by washing 3 times in PBST for 10 min each and in 10 mM TRIS-HCl for 10 min before mounting on glass slides with Mowiol. For brain tissue preparation, mice were killed with CO2 at a defined time interval after the application of the external sensory stimulus and perfused with chilled PBS, followed by chilled 4% PFA. Brains were removed and held in 4% PFA for 3 h and transferred to 30% sucrose-PBS at 4 °C for 18–24 h. Brains were coronally sectioned with a cryotome (Leica CM1950, Wetzlar, Germany) at 50 µm and slices collected into 24-well plates with chilled PBS. All equipment was precleaned with RNaseZAP (Sigma RNaseZAP, Darmstadt, Germany), and all reagents were prepared using diethyl pyrocarbonate (DEPC)-treated PBS to avoid RNase contamination. For mRNA in situ hybridization, the c-fos mRNA in situ probe was constructed according to the information on the Allen Brain Atlas website (http://www.brain-map.org, accessed on 15 February 2023). The RNA probe was generated via an in vitro transcription and labeled using the DIG-RNA-Labeling Mix kit and T7 RNA polymerase (Merck, Darmstadt, Germany), dissolved as a 1 µg/mL concentration in the hybridization solution (50% formamide (v/v), 5× SSC, 0.3 mg/mL Yeast tRNA and 0.5 mg/mL Salmon Sperm DNA). For c-fos tyramide-amplified in situ hybridization, slides were firstly washed 3-times with ice-cold PBS for 3 min and treated with acetylation buffer (0.25% acetic anhydride (v/v) in 0.1 M triethanolamine) for 10 min at room temperature. After rinsing once with cold PBS, cells were permeabilized with 0.3% TX100-PBS for 20 min at 4 °C. For in situ hybridization, tissues were pre-hybridized in hybridization buffer for 30 min. For hybridization, a c-fos anti-sense probe (diluted 1:200 in hybridization buffer) was applied and incubated overnight at 65 °C. The sense probe was applied to control slides. Post-hybridization, the tissue was washed twice with 2 × SSC/0.1% N-Lauroylsarcosine/50% formamide at 60 °C, rinsed in RNAse buffer (10 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA) and then digested with 20 μg/mL RNaseA in the RNase buffer for 30 min at 37 °C. This was followed by washing with 2 × SSC/0.1% N-Lauroylsarcosine and 0.2 × SSC/0.1% N-Lauroylsarcosine twice for 20 min at 37 °C and then rinsed once again with MABT (Maleic acid buffer with 1% of Tween 20). Tissue was then blocked with MABT++ (MABT with 10% heat-inactivated goat serum and 1% Blocking reagent) for 1 h at room temperature. Next, the tissue was incubated in MABT++ solution with the anti-digoxygenin antibody (anti-DIG-POD, 1:1000, Roche, Basel, Switzerland) at 4 °C for 16 h. For signal amplification, the slides were washed with MABT for 30 min at least 6 times, then rinsed with TSA buffer (10 mM imidazole) and incubated with TSA staining solution (Dilute Rhodamine tyramide 1:75 in TSA buffer, add in 0.001% H2O2) for 20 min at room temperature in the dark, followed by washing with PBST (PBS with 0.1% Tween 20) for 10 min, 5 times at room temperature in the dark. Tissues were mounted on slides with Mowiol after washing with PBS for single mRNA staining, or further used for immunofluorescence co-staining. For immunofluorescence co-staining, the tissue was first washed with T-BST (0.05% Tween 20 and 0.05 M Tris-HCl in PBS) for 10 min, 5 times at room temperature in the dark and afterwards incubated with the anti-Fos primary antibody (1:1000, abcam, ab190289) in T-BST at 4 °C overnight. On the second day, the tissue was washed 3 times for 5 min in T-BST and then incubated with species-specific fluorescent secondary antibodies in T-BST for 1 h at room temperature. Finally, slides were washed 3 times for 15 min with 0.3% T-BST, then again with T-BST 3 times for 10 min and finally rinsed with 10 mM Tris-HCl for 10 min before mounting the coverslips with Mowiol. As examples of the region showing robust Fos expression upon somatosensory (cold) stimulation as well as reasonable baseline activity, coronal sections from the prelimbic and insula cortex (from 2.4 mm to 0.37 mm anterior to the bregma) were used for analysis. A confocal microscope (Leica TCS SP8, Wetzlar, Germany) was used to acquire immunofluorescence image stacks with 2 μm-thick planes using the 20× objective (N.A.: 0.75, oil immersion). Laser diode wavelengths of 405, 488, 552 and 638 nm in combination, respectively, with filters sets for DAPI (ex BP360/403 em LP425), FITC (ex 470/40, em LP515) and TRITC (ex 540/45, em LP590) were used. This resulted in an average z-optical section of 20 µm. The Fos protein signal showed nuclear staining pattern, whereas the c-fos mRNA appeared mostly in the cytoplasm. For manual counting, the experimenter was blind to the different test groups, and images from groups were assigned a random number prior to analysis that was decoded after analysis. All confocal images were overlaid with the corresponding atlas section to anatomically define the regions of interest. All labeled cells within the boundaries of the defined sites were marked using a self-developed tool (https://github.com/cberri/cFOS_ManualAnnotations_ImageJ-Fiji, accessed on 15 February 2023) for ImageJ/Fiji and manually counted on MIP obtained from confocal stacks [12]. Brightness and contrast were optimized for each image. Background subtraction was performed by subtracting the mean intensity value estimated from a single background ROI placed within an unlabeled region in the same image. The Fos protein and c-fos mRNA signals were analyzed as separate images taken from the same slice using their respective excitation wavelengths. Positive cells on the XY boundary were excluded, and Fos protein signals were typically 6–8 µm in diameter and located in or near the nucleus. Nuclei were identified via DAPI staining. The c-fos mRNA signals were located in the cytoplasm as regions of 8–16 µm diameter surrounding the nucleus. In order to be counted positive, a cell had to display an intensity value above the intensity threshold of the background. Image intensity features were extracted from 60 images acquired by 4 experimenters for the anti-Fos antibody staining and from 63 images acquired by 2 experimenters for the c-fos mRNA in situ using a customized ImageJ/Fiji script (https://github.com/cberri/Quanty-cFOS/blob/main/scripts/Extract-ImageProperties_V0.ijm, accessed on 15 February 2023). The script extracts intensity features to measure Fos staining variability between different preparations and image acquisition settings. The following features were computed for each image: mean intensity, standard deviation intensity, minimum intensity, maximum intensity, mode intensity and mean background intensity. Here, we developed Quanty-cFOS.ijm as an ImageJ/Fiji tool to semi-automatically or automatically count in an unbiased manner cells expressing the Fos protein or c-fos mRNA in fixed stained brain slices. It can be extended to generally count cell markers in 2D fluorescent images or on MIP. For flexibility reasons, this tool is developed as a macro-set (IJ1) for ImageJ/Fiji (tested on ImageJ version 1.53s and later) [12]. The proposed workflow consists of two major steps: - Automated cell segmentation, - Cell counting using the automated or the manual optimization method. The Quanty-cFOS tool can be downloaded from https://github.com/cberri/Quanty-cFOS (accessed on 15 February 2023) and we provide a detailed step-by-step documentation of how to use it, including supplementary videos and several scripts to validate the cell counting (the GitHub validation folder). Quanty-cFOS cell detection is implemented using two different state-of-the-art segmentation strategies, based on deep learning and machine learning. The first uses the StarDist 2D Versatile (Fluorescent-Nuclei) inference available in the StarDist ImageJ/Fiji plugin and applies it on the raw images to segment convex shape structures [13]. The second uses the ilastik software pixel classification machine learning workflow to generate a probability map image using manual user annotations for different classes of pixels in an image [14]. In this case, the corresponding probability map image is loaded in addition to the raw input image in the Quanty-cFOS and intensity-thresholded to segment the cells. The user can decide which method is more suitable to process the images depending on the signal-to-noise ratio and on the shape of the cells that need to be segmented. The ilastik pixel classification workflow needs to be trained in ilastik software before to run the Quanty-cFOS tool (https://www.ilastik.org/documentation/pixelclassification/pixelclassification, accessed on 15 February 2023). A key feature defining the novelty of the Quanty-cFOS counting method is the z-score intensity cutoff used for the Automated Optimization. The proposed algorithm computes the mean intensity value and the intensity standard deviation of each single segmented cell in the images, averages these two values and computes the z-score (Zi): single cell intensity mean cell intensity mean cell intensity standard deviation The intensity values in the significant z-score range (sigma) are averaged and used to set the intensity thresholds cutoff to count Fos/c-fos-positive (above) or negative (below) cells. The user can specify the range of standard deviations (sigma) to optimize the cutoff value for the Fos/c-fos cell counts. The larger the sigma value, the less restricted is the intensity cutoff value, and vice-versa. intensity cutoff computed on an image z-score significant range of standard deviations (sigma) single cell intensity number of positive cells in an image The cutoff optimization is critical to gain an accurate and robust estimation of cell numbers. To consistently calculate the mean and standard deviation intensity, an arbitrary number of images can be used as input to compute the intensity cutoff (see Batch Analysis with Optimization Steps). In this case, the average intensity values of the images used for the optimization are accounted to calculate the intensity cutoff. intensity cutoff with the optimization steps intensity cutoff x image optimization steps The results of the automated intensity method can be validated by manually counting cells in fewer images and by running the MATLAB correlation analysis provided together with the Quanty-cFOS (CorrelationAnalysis.mlx, see also the ValidationTable.xlsx file as an example). Manual counting can be performed using any favorite tool or by using the following ImageJ/Fiji IJ1 script that we developed for this purpose (https://github.com/cberri/cFOS_ManualAnnotations_ImageJ-Fiji, accessed on 15 February 2023). The intensity threshold value used for Fos/c-fos cell counting is the key parameter to decide the cutoff for positive or negative counts. This is rather important if images have been acquired with different settings or high staining variability occurs between samples. To optimize this process and to help test different threshold values in a semiautomated unbiased way, we implemented the Manual Optimization function. By using this method, images can be previewed, and different intensity values can be tested for Fos/c-fos-positive cell counting. The Manual Optimization default intensity value displayed in the user setting dialog box is computed via the Automated Optimization function to help in choosing the appropriate intensity cutoff value. Moreover, different size filters for the cell area can be applied to remove small and large detected objects in the images. The number of images previewed is specified using the Optimization Steps. Indeed, only these images are used for testing different thresholds and the average intensity value of these thresholds is applied as an intensity cutoff on all the subsequently listed raw images. Counting all cells without intensity optimization is also possible as an option and can be achieved by unchecking the Automated Optimization and the Manual Optimization. In this way, all the cells in the image are counted without an intensity cutoff. Only a size cutoff filter (based on the cell area) is applied to exclude cells below the cutoff value and 5 times above the specified cutoff value. This option is supported only with Batch Analysis and the number of Optimization Steps is ignored. Select Multiple Sub-Brain Region was added to select specific regions of interest in the input images and to count positive cells only inside the selected regions. This option works only without batch analysis. Select Allow Preview User Setting was added to preview the intensity threshold and area cutoff used for the ilastik probability map segmentation (simple method). The intensity threshold method and cell size filter (area) can be modified to gain the best segmentation results. Currently, we support simple ImageJ/Fiji thresholding methods to segment cells in the ilastik probability map. Matlab (R2019a) was used for the correlation analysis and statistics. Figures were prepared using Adobe Photoshop CS6 (Adobe) and Adobe Illustrator CS6 (Adobe). DaVinci Resolve was used to edit the supplementary movies. All statistical analyses were performed in Matlab (R2019a). Box plots were created using the Matlab box plot function and show the mean intensity value +/− and the standard deviation (S.D.) of each plotted feature. The black horizontal line in each box represents the median value z-score. Analysis was computed in Matlab, and a positive correlation was considered in the range of two standard deviations. Box plots were generated for each time point for c-fos mRNA and Fos protein counts. Each box shows the mean intensity counts, the vertical lines show the S.D. (+/−). Our past experience has shown that quantifying Fos-expressing cells is challenging, not only because major differences exist in expression levels across cells as well as across samples, but also owing to technical aspects of sample preparation and imaging parameters. This was again observed when we acquired Confocal Laser Scanning Microscopy (CLSM) z-stacks after Fos protein immunostaining and c-fos mRNA in situ hybridization, as described under methods. To address differences in the image acquisition, four different experimenters prepared the samples, optimized the confocal settings and acquired the images (Figure 1A–D,A’–D’). We quantified Fos protein expression by extracting intensity features along the different staining and acquisition settings (ImageJ/Fiji Set Measurement plugin) [12]. Our analysis revealed major differences between the different extracted features across samples and experimenters. Indeed, we observed a high variability for the mean image intensity, mode intensity and mean background intensity features (Figure 1E). Intriguingly, the maximum intensity feature also showed a large dynamic range, suggesting fluctuation in the signal-to-noise ratio between acquired images. Moreover, for the minimum and the mode intensity, several data points were detected outside the whiskers in box-and-whisker plots, highlighting the differences between stained images (Figure 1E, + symbol). The c-fos mRNA was evaluated in the same way on two different sets of samples prepared by two experimenters. The analysis revealed an even larger variability in terms of the mean image intensity, mode intensity and mean background intensity features (Figure 1E’). Differently from the Fos protein, the maximum intensity value is set to 255 for an 8-bit dynamic range (0–255), indicating that all the images have been saturated while being acquired. This is indicative of a low signal-to-noise ratio for the c-fos mRNA that required a high confocal gain or/and laser power during image acquisition. Considering the extracted intensity variability between the tested images, fewer data points were detected outside the whiskers (Figure 1E, plus symbol). Intensity fluctuations between different staining rounds, acquisition settings and in between images acquired to investigate a specific physiological problem can lead to bias, especially if cell counting is the main readout. These experiments thus demonstrate the need to reduce bias in manual counts, which served as the starting point of our efforts toward developing the Quanty-cFOS ImageJ/Fiji tool for the automated/semiautomated counting of cells positive for the Fos protein and c-fos mRNA. The Quanty-cFOS was developed to be a user-friendly, unbiased ImageJ/Fiji tool for Fos protein and c-fos mRNA counts. The workflow consists of four major steps: input, detection, quantification and results (Figure 2, Fos protein and c-fos mRNA workflow). An input directory containing all the Fos protein images to process can be chosen. For the Fos protein detection, we used the StarDist 2D versatile fluorescent nuclei model in the Quanty-cFOS (Figure 2A and Figure S1A, Movies S2–S5) [13]. This method generated labeled images (Figure 2B) and it was optimized to segment convex objects in two dimensions (2D). The cell detection can be easily improved in the Quanty-cFOS tool by training a custom StarDist 2D model [15]. For the c-fos mRNA counts, we used two input directories. The detection method uses pre-processed images obtained from ilastik pixel classification workflow [14] in combination with the raw images (Figure 2A1′,A2′; Movie S4). We implemented this method to detect any cell shape, from convex to more elongated shapes. Moreover, this option allows us to choose any pre-processed method in case cell detection is inefficient and upload the pre-processed images in the tool. MIP are automatically created using the Quanty-cFOS tool or can be generated by the user prior to the cell counting. The Quanty-cFOS tool supports three methods for cell counting: ‘automated optimization’, ‘manual optimization’ and ‘all cells counts’–‘with batch analysis’ and ‘without batch analysis’ (Figures S1B and S2; Movies S1–S4). The option ‘with batch analysis’ has been implemented to help the user in choosing the detection parameters and the intensity cutoff to batch process all the images in the input source directory by applying the same settings. This modality allows us to apply the ‘automated optimization’, the ‘manual optimization’ and ‘all cells counting’ methods (Figure S1B left; Movies S1–S4). Choosing the detection parameters can be difficult, in particular when the images are different from each other, or the counting method settings needs to be changed during the processing. Therefore, to simplify cell counting, we developed the option ‘without batch analysis’ (Figure S1B right; Movie S5). Optimization methods and parameters can be changed for each image to achieve the best counts. This method is recommended if the images to process are very different from each other, or different parameters need to be tested for the cell counting. Chosen parameters are thereby saved in the root output directory to document the analysis (output Log.txt file). This modality can be used only for the ‘automated’ and the ‘manual optimization’ methods. Moreover, the option ‘without batch analysis’ supports the multiple ‘sub-brain regions selection’ function to count Fos-positive cells in selected subregions of an image (Figure S3, Movie S5). The ‘automated optimization’ method is used to compute the intensity threshold cutoff on a predefined number of images specified via the ‘optimization steps’ and applies this cutoff to all the following images listed in the input directory (Figure S2, Movie S2). The cutoff intensity threshold is computed on the optimization images by calculating the z-score. Only cell intensity values in the range of the specified z-score sigma (number of standard deviations) are averaged and contribute to the final intensity cutoff. The ‘manual optimization’ method allows the user to choose the intensity cutoff by previewing a selected number of images specified via the ‘optimization steps’. These values are averaged and used as an intensity cutoff for cell counting (Figure S2; Movie S3). For both methods, an area filter is applied. For the ‘automated optimization option’, the area is set two times above and below the area standard deviation. In the ‘manual optimization’, the area can be measured on the previewed images and the cutoff value can be specified. The option ‘All cells counts’ can be used to count all the positive cells in the images without any ‘optimization steps’ (Figure S2, Movie S1). This has been included to count in 2D all the cells in an image independently of an intensity cutoff. To simplify further analysis and statistics, the Quanty-cFOS output consists of an output root directory created outside the input path with subdirectories named as the input processed images. Each subdirectory saves the labeled image for positive and negative cells (Figure 2C,C’), the ImageJ/Fiji ROI Manager ROIs and a comma separator values (csv) file with the coordinates of the center of mass of each detected cell. Moreover, the output root directory contains the summary of the counts as an csv file and the Log.txt file with the analysis steps and the chosen parameters (Figure 2 and Figure S1). An additional subdirectory with all the labeled images for positive and negative cells is created in the main output path for further analysis. After establishing the methodology, cell counting results generated using Quanty-cFOS on cells expressing the Fos protein were compared to manual counts of Fos-expressing cells using a test data set of randomly selected images from mouse brain sections (Figure 3). Fos-expressing cells were manually annotated in the validation images by four different experimenters (Figure 3A–D) and the results compared with the Quanty-cFOS output (Figure 3E). For the manual counting, we developed an ImageJ/Fiji tool that allows the user to select positive and negative cells by clicking the left and right mouse button. Positive cells can be counted by clicking the left mouse button, negative cells with the right mouse button (https://github.com/cberri/cFOS_ManualAnnotations_ImageJ-Fiji, accessed on 15 February 2023). By comparing the manual counts from the four experimenters, we observed consistent results between the single human counts in certain ROIs but also miscounted cells in other parts of the images (Figure 3A’–D’). Indeed, the single counts analysis of Fos protein-expressing cells over 30 images showed a discrepancy in the absolute number of positive cells counted manually (Figure 3F, Manual Count: experimenter one, experimenter two, experimenter three, experimenter four). The discrepancy in counts was also seen when the absolute single counts from manual counting were compared with the Quanty-cFOS output (Figure 3E’,F, automated Quanty-cFOS counts). To evaluate the relation between the two methods, we compared the manual counts average slope with the Quanty-cFOS slope. The slope for both, the manual average and Quanty-cFOS, showed a similar distribution in the number of positive cells counted, suggesting a consistent relation between the manual and the Quanty-cFOS counts (Figure 3G). To further verify this observation, we computed the correlation analysis between the manual and Quanty-cFOS counts using the z-score method. The analysis showed a significant correlation in two standard deviations range (sigma) between the manual and the Quanty-cFOS counts (Figure 3H). These results support the accuracy of the automated Quanty-cFOS method. Furthermore, the large differences in the absolute manual counts between the four experimenters further revealed the need of an unbiased algorithm for cell counting. In Quanty-cFOS, we developed a similar approach for counting cells positive for c-fos mRNA in in situ hybridization experiments, as was used for the Fos protein. First, we compared the manual counting results performed by four experimenters on 30 images from the mouse brain with the output of the automated Quanty-cFOS algorithm (Figure 4A–E). The manual counts were obtained as described for the Fos protein. The automated counts were achieved by using the combination of raw and ilastik pixel classification probability map as pre-processing step. Ilastik pixel classification workflow was trained using 15 raw c-fos mRNA images and all the images were batch processed in ilastik [14]. A larger amount of training data or different pre-processing methods can be used to gain higher accuracy in the segmentation results, e.g., ilastik Autocontex [16], image denoising with noise2void [17], image restoration with CARE [18] and a suitable deep learning model from BioImage Model Zoo [19]. The manual counts comparison showed consistent results between the experimenters’ counts in certain ROIs but also revealed miscounted cells in other parts of the images (Figure 4A’–D’). Similar results are observed for the automated c-fos mRNA counts (Figure 4A’–E’). Manual cell counts showed differences in the absolute number of c-fos-positive cell counts (Figure 4F: manual counts: H1, H2, H3, H4). This was also seen when comparing the c-fos manual counts with the automated counting results (Figure 4F: automated Quanty-cFOS counts). However, the discrepancy within the absolute counts, within the manual counts and in between the manual and the automated counts was smaller in comparison to what we observed for Fos protein counts (Figure 3F). We evaluated the counts relation by comparing the manual counts average slope with the mRNA Quanty-cFOS slope. Both slopes, manual and automated, showed the same distribution with many overlapping data points (Figure 4G). We further tested the counts for significance by computing the z-scores in two S.D. ranges. Manual and automated counts showed a strong correlation. As with the Fos protein counts, these analyses demonstrate the validity and accuracy of the Quanty-cFOS automated method for counting c-fos mRNA-positive cells in complex tissues (Figure 4H). Having established the Quanty-cFOS automated method, we then demonstrate its utility for studying activation in neuronal networks in the rodent brain by applying it to follow changes in both c-fos mRNA and Fos protein expression after sensory stimulation. In mice subjected to a heat stimulus of 50 °C applied to the hindpaw, c-fos mRNA levels started to increase in the prefrontal cortex within 20 min, peaked at 1 h and the mRNA was degraded by 3 h after stimulation (Figure 5A–C). In contrast, Fos protein increased only after 1 h and strong expression was evident 3 h after stimulation (Figure 5D–F). The automated counts were compared with manual counts performed by four experimenters, as described above. Both automated and manual counts for mRNA and protein show the expected c-fos expression. Indeed, c-fos mRNA could be seen at 20 min, reached a peak after 1 h and was degraded after 3 h, while protein expression was evident at 1 h and not seen at 20 min post-stimulation (Figure 5G, blue and red dash lines). Thus, we validated the Quanty-cFOS method, showing that it can be reliably used to automate the Fos protein and c-fos mRNA cell counts. This is an important prerequisite for using Quanty-cFOS for the automated quantification of positive cell counts in methods involving dual counting of mRNA and protein within the same specimen. While this can apply to any biological marker mRNAs or proteins, the ability to reliably count cells expressing c-fos mRNA and protein within the same specimen in the context of the different time course of their expression using Quanty-cFOS will allow for the implementation of this tool in dual-epoch labelling methods, such as TAI-FISH, that involve an analysis of cells responding in an activity-dependent manner to different stimuli (e.g., two distinct sensory stimuli, such as heat and cold) applied with a temporal gap [7]. The variability of manual cell counts can affect the final experiment outcome depending on how an experimenter visually counts cells (Figure 5G, orange and green arrowheads). Instead, the Quantity-cFOS tool can be used to obtain unbiased cell counts making analysis reproducible and objective, as shown in our results. This study introduces an open-source, novel and fully validated ImageJ/Fiji tool for the unbiased counting of cells expressing Fos protein or c-fos mRNA. The main advantages of this tool are its objectivity and lack of human bias in cell counting, consistence of methodology and analyses across different experiments and its ability to set thresholds objectively in experiments with inter- and intra-experiment variability. Further, Quanty-cFOS allows for higher speed and efficiency in analyzing a large number of images and applicability to antibodies or RNA probes that yield high experimental variability as well as graded nuances in expression levels which can lead to large errors when viewed subjectively. Importantly, the study provides an easy-to-use tool, including in-depth step-by-step videos for different cell counting applications that can be quickly learned and efficiently applied by non-experts in image analysis. Analysis of the activity-induced expression of the immediate early gene c-fos has rapidly established itself into a major surrogate for addressing activity in neurons [11]. While levels of expression of the gene can be measured quantitatively in terms of quantitative PCR analyses for the mRNA transcript or Western blot analyses for the protein product, they lack spatial and cellular resolution. Therefore, immunohistochemistry and in situ hybridization of the c-fos gene expression is critical in yielding information on activity-induced changes in distinct regions, pathways and individual cells of the brain. Although it lacks the fine temporal resolution of electrophysiology, analysis of spatial profiling of Fos expression is technically much easier, faster and can be carried out simultaneously across the whole brain, making it a broadly applicable method. Cells that induce and express Fos simultaneously following a particular external stimulus, such as painful sensory stimuli, or in association with a particular internal function, such as establishment or recall of particular memories, have been considered to be part of assemblies or ensembles that subserve particular functions. A very recent study has employed simultaneous Fos monitoring and in vivo calcium imaging of the hippocampus in mice to demonstrate that neurons with high Fos induction form cellular ensembles which show highly correlated activity and play an important functional role in spatial memory, thus leading further credence to the use of Fos mapping for identifying functionally relevant cells [20]. Thus, spatial profiling of the Fos protein is a valuable and widely employed method in the neurosciences. Unfortunately, quantitative analysis of Fos-expressing neurons has not evolved at the same rate, and most studies have relied on manual counting, which is not only highly cumbersome and time-consuming, but also prone to subjectivity, bias and variability within and across groups and experiments. While an automated algorithm was successfully developed for counting Fos-expressing cells in light sheet microscopy on cleared whole brains [21], analysis of thick brain slices or sections has proven to be more complex, owing to the high background from the brain parenchyma as well as difficulties in estimating cells that are cut out of the section in the z dimension. Fewer, automated solutions for cell counting on tissue sections are available as open-source standing alone software or as ImageJ/Fiji plugins. For instance, Cellpose and CellProfiler can be used to automatically count the number of cells in microscopy sections; however, these tools lacked specific thresholding optimization methods essential for mapping immunoreactive cells [4,5]. Basal expression of Fos/c-fos in neurons requires the development of ad hoc optimization algorithms necessary to count only reactive cells. Therefore, to cover this gap, we developed the Quanty-cFOS tool, optimized to count cells above an automated computed intensity or manual intensity cutoff. The option to count only cells above a specific cutoff, in a step-by-step designed workflow, is the major novelty of the Quanty-cFOS tool in comparison to the tools publicly available. To the best of our knowledge, there is no other open-source tool that is easily accessible and usable by non-experts, can run in a standard image analysis program such as ImageJ/Fiji, is accompanied by detailed video tutorials and which has been validated and optimized for both the Fos protein and c-fos mRNA. Currently, Quanty-cFOS can be used to count cells only on two-dimensional images and this could limit its application if cells need to be counted in three-dimensional stacks. Indeed, extending the Quanty-cFOS to count cells in three-dimensional stacks will be part of the future development of this open-source tool. Here, we took care to include human experimenters and manual counting in an iterative process while developing and optimizing the Quanty-cFOS tool. Moreover, emphasis was placed on collecting data sets showing a high level of inter-experimenter variability to challenge and optimize Quanty-cFOS. Furthermore, an important challenge in counting Fos-positive cells in response to a given stimulus or function is given by the fact that natural, spontaneous activity yields background Fos expression and stimulus-derived Fos expression can vary in strength across cells within the sample and across samples, rendering it difficult to set a threshold. This aspect is dealt efficiently in Quanty-cFOS using the automated and manual optimization methods. Nevertheless, the experimenter can choose to either employ the automated optimization method, which represents the most unbiased option, or to use the manual optimization method, or to count all stained cells without any intensity cutoff, as required per experimental conditions, thus providing maximum flexibility. Although in situ mRNA hybridization represents a more cumbersome method of spatially testing activity-induced expression of c-fos, requiring stringent control of RNA degradation, methods such as RNAscope have led to widespread use in recent times. Counting c-fos-positive cells harbors the same difficulties as discussed above for the Fos protein while carrying the additional hindrance that mRNA is diffusely distributed in a spotty, dotted appearance across the cytoplasm in contrast to nuclear localization of the Fos protein, rendering it harder to distinguish between neighboring cells. In Quanty-cFOS, using image preprocessing with ilastik pixel classification enabled us to circumvent this problem and facilitate the segmentation and the counting of mRNA-positive cells. This will not only foster the use of the c-fos mRNA as a spatial marker of activity-induced changes in networks, but also support methodologies in which analyses of the c-fos mRNA and Fos protein are combined with the same samples to identify differentially-activated cohorts, such as TaiFISH [7]. This method applies sequentially given stimuli and it takes advantage of the early expression and short lifespan of the c-fos mRNA in comparison with the later induction and longer expression of the Fos protein. Methods such as these are becoming increasingly prominent in yielding insights into the differential cellular encoding of distinct functions within the same region in the nervous system, e.g., aversion vs. reward processing in the prefrontal networks. Furthermore, it deserves to be noted that emphasis was placed on making Quanty-cFOS user-friendly by developing several workflow options and leaving control in the hands of the user. Additionally, with the tool we provide in-depth video tutorials to make it easily useable by scientists without expertise in image processing and image analysis. This is of critical importance since the lack of bioimage analysis and computing skills often limits stringent standards fundamental for reproducibility in image quantification. Finally, the efficiency of using Quanty-cFOS to quantitate activity-dependent changes in cellular responses deserves to be discussed. Having conducted and published a number of in-depth studies with c-fos-based activity mapping using manual counting that required several weeks to months of work [8,9], frequently leading to experimenter fatigue, we are confident that automated counting via Quanty-cFOS can achieve the same goals in the fraction of time required for manual analyses. In conclusion, making this fully validated tool freely available to the scientific community will help overcome human bias in spatial activity mapping and foster unbiased, efficient and rapid analyses. Moreover, although the tool was designed and optimized for quantitating cells expressing the Fos protein or c-fos mRNA in the nervous system, it is in principle applicable to any antibody or mRNA being investigated and to any type of tissue, thus rendering its multiple applications.

PMC10000432

Enrique Pérez-Riesgo,Elena Hernando-Pérez,Verónica Feijóo,Sendoa Tajada,Lucía Núñez,Carlos Villalobos

Transcriptional Basis of Ca2+ Remodeling Reversal Induced by Polyamine Synthesis Inhibition in Colorectal Cancer Cells

04-03-2023

colorectal cancer,polyamines,intracellular calcium,transcriptomic analysis,DFMO,store-operated calcium entry,mitochondria,TRP channels

Simple Summary The common activation of the c-Myc oncogene in colorectal cancer (CRC) induces the overexpression of ornithine decarboxylase (ODC), the limiting step in polyamine synthesis, which is a process blocked by α-Difluoromethylornithine (DFMO), an ODC suicide inhibitor and potential CRC treatment. We showed previously that intracellular Ca2+ homeostasis is remodeled in CRC and contributes to cancer hallmarks. We investigated whether polyamine synthesis inhibition induced by DFMO may reverse this remodeling in CRC and, if so, the molecular basis for this phenotypic reversal. To this end we used calcium imaging and transcriptomic analysis in both normal and CRC cells. We found that CRC cells showed enhanced resting Ca2+ and store-operated Ca2+ entry (SOCE) but decreased Ca2+ store content relative to normal cells. Polyamine synthesis inhibition reversed not only this Ca2+ remodeling but also reversed the changes in the transcription of a dozen genes involved in Ca2+ transport in CRC cells, including genes modulating Ca2+ entry into the cells as store-operated channels and TRP channels as well as Ca2+ extrusion systems in the plasma membrane and mitochondria. These results provide a molecular basis for the role of polyamine synthesis in Ca2+ remodeling in cancer. Abstract Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer.

Transcriptional Basis of Ca2+ Remodeling Reversal Induced by Polyamine Synthesis Inhibition in Colorectal Cancer Cells The common activation of the c-Myc oncogene in colorectal cancer (CRC) induces the overexpression of ornithine decarboxylase (ODC), the limiting step in polyamine synthesis, which is a process blocked by α-Difluoromethylornithine (DFMO), an ODC suicide inhibitor and potential CRC treatment. We showed previously that intracellular Ca2+ homeostasis is remodeled in CRC and contributes to cancer hallmarks. We investigated whether polyamine synthesis inhibition induced by DFMO may reverse this remodeling in CRC and, if so, the molecular basis for this phenotypic reversal. To this end we used calcium imaging and transcriptomic analysis in both normal and CRC cells. We found that CRC cells showed enhanced resting Ca2+ and store-operated Ca2+ entry (SOCE) but decreased Ca2+ store content relative to normal cells. Polyamine synthesis inhibition reversed not only this Ca2+ remodeling but also reversed the changes in the transcription of a dozen genes involved in Ca2+ transport in CRC cells, including genes modulating Ca2+ entry into the cells as store-operated channels and TRP channels as well as Ca2+ extrusion systems in the plasma membrane and mitochondria. These results provide a molecular basis for the role of polyamine synthesis in Ca2+ remodeling in cancer. Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer. Colorectal cancer (CRC) is one of the most common types of cancer and causes of cancer deaths worldwide, with nearly 1,250,000 new CRC cases every year and a mortality rate as high as 50% of all cases [1]. The molecular basis of CRC involves in most cases the activation of the Wnt–β-catenin signaling pathway by the mutation of intracellular components such as APC, AXIN, and CTNNB1/β-catenin genes or the epigenetic alteration of Wnt inhibitors such as DKK-1, SFRPs, and WIF, considered the initial steps in colorectal tumorigenesis [2]. These changes result in the activation of c-myc and K-ras, which lead to adenoma, adenocarcinoma, and colon carcinoma [2]. Myc activation induces the overexpression of multiple genes, including ornithine decarboyxlase (ODC), the limiting step in the synthesis of the polyamines putrescine, spermine, and spermidine. ODC activation can be efficiently prevented by the suicide inhibitor Difluoromethylornithine (DFMO, also named eflornithine) [3]. Substantial evidence links ODC overexpression, excess polyamines synthesis, and CRC. For instance, ODC is overexpressed in most CRCs, and different tumor promoters induce ODC1 and tumor formation [4,5]. ODC polymorphisms have also been reported in CRC. In addition, targeting ODC and polyamines using cell lines, animal models, and even clinical trials may efficiently prevent CRC [6,7,8]. For instance, DFMO inhibits colon carcinogenesis in ApcMin/+ mice with increased levels of ODC and polyamines in intestinal tissues and suppresses carcinogenesis in the small intestines of these mice [3,4]. Interestingly, the grade of intestinal polyps is polyamine-dependent, and the anti-carcinogenic effects can be rescued by putrescine. DFMO may work in humans as well. An ongoing trial is presently evaluating the effectiveness of the combination of DFMO and sulindac in preventing colon adenomas [9]. However, the mechanisms by which polyamines promote carcinogenesis remain to be fully established. Polyamines are physiological molecules that are produced transiently during epithelial restitution for epithelial tissue repair. This process involves the transient activation of cell migration and/or proliferation after wounding. Evidence suggests that this process could be mediated by Ca2+ signaling induced by changes in the expression of TRPC1 channels as well as an increased ratio of STIM1 to STIM2 [10]; molecular players involved in SOCE in epithelial cells, mediated by caveolin [11]; and the small guanosine-5′-triphosphate-binding protein RhoA [12]. Interestingly, SOCE and its molecular players have recently been found to be involved in carcinogenesis in CRC and other forms of cancer [13,14,15]. For instance, we recently reported that intracellular Ca2+ homeostasis is remodeled in CRC cells [14,16,17]. Specifically, CRC cells display enhanced SOCE and decreased Ca2+ store content relative to normal colonic cells, and these changes contribute to cancer hallmarks, such as increased cell proliferation, cell invasion, and resistance to apoptosis [16,17]. At the molecular level, enhanced SOCE was linked to the increased expression of TRPC1 and an increased ratio of STIM1 to STIM2 in CRC cells [16,17], thus mimicking changes previously reported to be induced by transient polyamine exposure during epithelial restitution [10]. In addition, we also reported that store-operated currents (SOCs) are quite different in normal and colon cancer cells. Specifically, normal colonic cells display typical CRAC-like currents driven by Orai1 channels, which are very small, Ca2+-selective, inward-rectifying currents. In contrast, CRC cells display larger, non-selective currents with both inward and outward components that are mediated by both Orai1 and TRPC1 channels [16]. We recently conducted a transcriptomic analysis of 77 selected gene transcripts involved in intracellular Ca2+ transport that provided the first insights into the transcriptional basis of this remodeling [18]. In short, we found the differential expression of selected voltage-operated Ca2+ channels and SOCE players, transient receptor potential (TRP) channels, Ca2+ release channels, Ca2+ pumps, Na+/Ca2+ exchanger isoforms, and genes involved in mitochondrial Ca2+ transport [18]. Therefore, the evidence suggests that intracellular Ca2+ homeostasis is largely remodeled in CRC, and these changes could be mediated by excess polyamine synthesis linked to CRC. To address this issue, we recently tested the effects of polyamine synthesis inhibition on Ca2+ remodeling in CRC cells [19]. In accordance with this hypothesis, we reported that CRC cells overexpressed ODC1 relative to normal cells. In addition, polyamine synthesis inhibition in CRC cells that were resistant to cell death reversed this phenotype and sensitized CRC cells to apoptosis. Importantly, polyamine synthesis inhibition promoted changes in intracellular Ca2+ homeostasis consistent with phenotype reversal, including changes in store-operated currents and SOCE, Ca2+ store content, and the expression of a few proteins involved in SOCE [19]. However, whether polyamine synthesis inhibition reverses the whole calcium signature linked to carcinogenesis remains to be addressed. Here, we combined calcium imaging and transcriptomic analysis using next-generation sequencing and microarray technology to determine the molecular basis of Ca2+ remodeling in CRC and the effects of polyamine synthesis inhibition on transcriptomic remodeling and changes in intracellular Ca2+ homeostasis. Our results indicated that polyamine synthesis inhibition partially reversed the remodeling of intracellular Ca2+ in CRC cells. In addition, polyamine synthesis inhibition induced expression changes in 25% of the whole transcriptome of CRC cells but had nearly negligible effects on normal cells. Finally, we found that the reversal of Ca2+ remodeling depended on the changes in a dozen genes, including SOCE modulators, several TRP channels, two Ca2+ pumps, and two channels involved in mitochondrial Ca2+ transport. The HT29 cell line was obtained from LONZA (Basel, Switzerland). NCM460 cells were from INCELL Corporation (San Antonio, TX, USA). SW480 cells were a kind gift from Prof. Alberto Muñoz (CSIC, Madrid, Spain). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were sourced from Lonza (Basel, Switzerland). L-glutamine was from Gibco (Barcelona, Spain). Trypsin-EDTA was from LONZA (Verbiers, Belgium). Poly-L-Lysin was from Marlenfeld GmbH (Lauda-Könlgshofen, Germany). Six-well plates were from NUNC (Thermo Scientific, Waltham, MA, USA). Dishes 10 cm2 in diameter were from Corning (NY, USA). DFMO was from TOCRIS (Bristol, UK). Fura2/AM and qPCR primers are from Invitrogen (Eugene, OR, USA). Cyclopiazonic acid (CPA) was from Sigma-Aldrich (Steinheim, Alemania). Antibodies against MCU and β actin were from Sigma (Madrid, Spain). The RNA extraction kit was a GeneMATRIX Universal RNA Purification Kit from EURx (Gdansk, Poland). Clariom D human microarrays (Affymetrix) were supplied by CABIMER (Andalucía, Spain). RNA-seq (Illumina) was provided by Sistemas Genómicos S.L (Valencia, Spain). PolyamineRED was from Funakoshi Co., Ltd., Tokyo, Japan). All other reagents were obtained from Sigma and Merck. As human colon cancer models, we used the HT29 and SW480 colon cancer cell lines, and the NCM460 cell line was employed as the normal control; all of these have been widely used and validated in the colon cancer research field. Cells were cultured in a 25 cm2 flask with DMEM plus 1 g/L of glucose, 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, which were placed into an incubator at 37 °C under a 10% CO2 atmosphere. All cells were used in passage 2. From each cell culture seeded in one flask, cells were detached with trypsin-EDTA and broken into two parts. One of these parts, which was used for the calcium imaging experiment, was seeded on four glass coverslips pretreated with poly-L-Lysin; each of them was then placed into a well in a 6-well plate with a cell density of 3000 cells per coverslip. The other part, which was used for the transcriptomic experiment (microarrays), was seeded on two dishes 10 cm2 in diameter with a cell density of 10.5 × 105 cells per dish. Notably, two flasks, one flask of HT29 and the other of NCM460 cells, were processed on the same day. Then, two flasks (one from HT29 cell cultures and the other from NCM460) were processed every day on four different days. After an entire day at 37 °C under a 10% CO2 atmosphere, of the eight coverslips and four dishes obtained from the two flasks (with one flask per cell line), two coverslips and one dish from each flask were treated with DFMO (DFMO 5 mM in DMEM 1 g/L glucose plus 10% dialyzed FBS), whereas the other two coverslips and dish from each flask were used to represent the non-treated conditions, i.e., treated without DFMO (DMEM 1 g/L glucose plus 10% dialyzed FBS). Then, the eight coverslips and four dishes were kept in an incubator at 37 °C under a 10% CO2 atmosphere for 96 h. Next, the coverslips were used for calcium imaging experiments, and the dishes were used to extract and isolate their mRNA for transcriptomic analysis (see procedure schematic in Figure 1a–c). Notice that a transcriptome analysis was previously carried out for both the NCM460 and HT29 cell lines without DFMO treatment using Illumina RNA-seq for comparison. Intracellular polyamines were estimated using fluorescence imaging and PolyamineRED (Funakoshi Co., Ltd., Japan), an intracellular polyamine detection reagent. Treated and control cells were cultured with 30 µM of the reagent in free-serum media for 30 min according to manufacturer’s procedure. Then, cells were fixed, and nuclei were stained with DAPI. Fluorescence images were obtained using a Nikon Eclipse 90i fluorescence microscope and analyzed with ImageJ software. Figure 2 shows that PolyamineRED fluorescence in cells treated with DFMO was largely reduced relative to untreated cells, in accordance with polyamine depletion. Similar results were obtained in SW480 and NCM460 cells. The effect of the inhibition of the polyamine synthesis pathway through DFMO on both HT29 and NCM460 cells was assessed at both the transcriptomic and functional levels (Figure 1). On the one hand, the transcriptomic experiment was conducted using Clarion D human microarrays from Affymetrix and, on the other hand, the functional test was carried out using calcium imaging. Then, four experimental conditions were evaluated: NCM460 cells, both treated and non-treated with DFMO, and HT29 cells, again both treated and non-treated with DFMO. Furthermore, since we were interested in obtaining the transcriptomic expression profile through calcium imaging assays, we acquired from the same flask, at the same time, a replicate for every experimental condition (for the same cell line) and for both transcriptomic and calcium imaging assays, as shown in Figure 1c. Specifically, on the same day, a replicate for both treated and non-treated experimental conditions was extracted from the same flask, and two replicates for calcium imaging for the experimental condition “g” were obtained and processed from one replicate for the same experimental conditions in the transcriptomic experiment. Furthermore, prior to the experiments described above, both non-treated HT29 and NCM460 cells were assessed at the transcriptomic level through Illumina RNA-seq technology. Regarding the factors that needed to be considered for later data analysis, on the one hand, in the calcium imaging experiment, there were two random factors (day and coverslip) and another two fixed factors (cell line and treatment). Then, we had to use linear mixed models to control the effects of random factors. On the other hand, concerning the transcriptomic experiment, there were two fixed factors (cell line and treatment) and just one random factor. We took into account this random factor, since it was necessary to control it. The data analysis for each type of experiment is explained more in detail in the corresponding paragraph. Fluorescence calcium measurements were conducted in colon cancer HT29 and SW480 cells as well as normal NCM460 cells, all treated and non-treated with DFMO. First of all, cells were washed with a standard external medium (SEM) containing (in mM) NaCl 145, KCl 5, CaCl2 1, MgCl2 1, glucose 10, and Hepes/NaOH 10 (pH 7.4). Then, cells were loaded with 4 μM of Fura2/AM, the acetoxymethyl ester form of the calcium-sensitive dye fura2, in SEM for 45 min at room temperature and in the dark. After loading, coverslips with attached cells were mounted in the perfusion chamber of a Zeiss Axiovert 100 TV inverted microscope and perfused with prewarmed (37 °C) SEM. Then, the cells in the chamber were excited alternately by light at 340 and 380 nm using a xenon lamp and a filter wheel, and the light emitted by the cells at 520 nm was filtered through a dichroic mirror and recorded every 5 s by a Hamamatsu ER camera (Hamamatsu Photonics France). Finally, the 340 nm/380 nm ratios between each pair of pixels were calculated, and all those belonging to the same region of interest (ROI), corresponding to a single cell, were averaged and interpreted as an intracellular [Ca2+] measurement from that ROI, as detailed before [20,21]. For resting [Ca2+] measurements, we used the median 340 nm/380 nm ratio of the first 30 s from each cell. After that, to obtain a measure of the Ca2+ store content from each cell, cells were treated with the reversible sarcoplasmic and endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) at 10 μM in a calcium-free SEM. Accordingly, the Ca2+ store content from each cell was measured as both the increment in and area under the curve (AUC) of the 340 nm/380 nm ratio for the signal corresponding to the rise in intracellular [Ca2+] induced by CPA in Ca2+-free medium. Finally, after store depletion, cells were perfused with SEM containing CPA and 1 mM Ca2+ to induce SOCE. Then, the SOCE corresponding to every single cell was measured as both the rise in and the area under the curve (AUC) of the F340 nm/F380 nm ratio for the signal corresponding to the rise in intracellular [Ca2+]. After RNA isolation (using a GeneMATRIX Universal RNA Purification Kit from EURx), microarrays were carried out in Centro Andaluz de Medicina Regenerativa (CABIMER, Seville, Spain), and RNA-seq assays were conducted by Sistemas Genómicos S.L (Valencia, Spain). Then, data analysis based on the microarrays was performed by our group using .CEL files, whereas data analysis for RNA-seq was performed using count-matrix, provided by Sistemas Genómicos S.L. (Valencia, Spain). HT29 cells were treated with vehicle or DFMO (5 mM, 96 h) and used for western blotting. Total protein was extracted from cells and used to quantify the expression of MCU. Whole-cell lysate was obtained using RIPA buffer (20 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, 1 mM EDTA, 0.05% SDS) supplemented with the Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) from ThermoFisher Scientific (ref #1861281) (Waltham, MC, USA). Protein concentrations were determined by a Bradford protein assay. Proteins were fractionated by SDS-PAGE; electroblotted onto PVDF membranes; and probed with the antibodies at a dilution of 1/200, except for the anti-β-actin, which was used at a dilution of 1/5000. The antibody against MCU (SC-246071) had been previously characterized and was visualized by the addition of goat anti-rabbit IgG or rabbit anti-mouse IgG. Detection was performed using Pierce ECL Western Blotting substrate (Thermo Scientific) and a VersaDoc Imaging System (BioRad, Munich, Germany). The quantification of protein expression was carried out using Quantity One software (BioRad, Munich, Germany). The datasets were analyzed by adjusting a linear model to fit the data, the model assumptions were evaluated, the response variables were transformed with Box–Cox family transformations because the normality assumption was violated, and outlier detection was carried out by an analysis of the Cook’s distance and the studentized residual. After transformation, the model assumptions were fulfilled as follows: residual normality with the Shapiro–Wilks test (p value = 0.1961) and homoscedasticity with the Bartlett test (p value = 0.8771). The sample size was equal to 6:3, corresponding to HT29 cells without treatment and HT29 cells treated with DFMO, respectively; p < 0.01. Total RNA from HT29 and NCM460 cells was isolated with a GeneMatrix Universal RNA purification kit (Eurx®, Molecular biology products) following the manufacturer’s instructions. A confluent 60 mm Petri dish per condition was employed for each assay. The quality of the RNA was determined by optical density measurements at 260 and 280 nm and by electrophoresis on agarose gels. After DNAse I treatment with a RapidOut DNA removal kit (Thermo Scientific), 1000 ng of RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) at 37 °C for 120 min to obtain cDNA. Quantitative PCR was carried out on equal amounts of cDNA in triplicate for each sample using a Kapa Sybr® Fast qPCR kit (Kapa Biosystems, Wilmington, MA, USA) in a LightCyler® 480 (Roche, Basel, Switzerland) thermocycler. The primers used are shown in Table 1 and were designed using OligoPerfect Primer Designer (ThermoFisher Scientific). The cycling conditions were 5 min at 95 °C, 40 cycles of 95 °C for 15 s, and 60 °C for 20 s. These amplifications were used to compare the different samples and melting curves to determine the specificity of the PCR products. The qPCR data were analyzed using the threshold cycle (Ct) relative quantification method (ΔΔCt). The gene expression levels were normalized by the housekeeping gene RP18S. The relative abundance of the genes was calculated as 2ΔCt, where ΔCt = Ctgene–Ct18S. Differences between cancer (HT29) and control (NCM460) cells were calculated from 2ΔΔCt, where ΔΔCt = ΔCt(HT29)–ΔCt(NCM460), using ΔCt(NCM460) as the calibrator. In this analysis, a value of 0 indicated no change, negative values indicated decreased expression, and positive values indicated increased expression relative to the calibrator. The statistical analysis method used was a Student’s t test with Bonferroni’s correction for two independent samples (ΔCt(HT29) and ΔCt(NCM460)) obtained with RP18S as endogenous control. * p < 0.05, ** p < 0.01, *** p < 0.001 For the calcium imaging experiments, we were interested in testing which experimental conditions were different from each other in terms of resting intracellular [Ca2+], Ca2+ store content, and SOCE. Thus, as response variables, at the single-cell level, we considered the median from the absolute value of the signal across the first 30 s for resting intracellular [Ca2+], and the corresponding increase in [Ca2+] levels and the area under the curve (AUC) for both Ca2+ store content and SOCE. We evaluated the effect of two factors, treatment and cellular line, on the response variable, each of them with just two levels (control and DFMO for the treatment factor, and NCM460 and HT29 for the cellular line factor), so there was a total of 4 experimental conditions: control-NCM460, DFMO-NCM460, control-HT29, and DFMO-HT29. Due to the experimental design, the dataset showed a hierarchical structure, since observations were clustered into, or organized at, different levels: the experiment was carried out on 4 different days; each day we performed 2 replicates corresponding to each of the 4 experimental conditions; and within each replicate, we measured several cells from the same experimental conditions. Furthermore, both replicates and day were random factors. Since there were random factors and there could be a correlation between observations belonging to the same cluster (i.e., they were non-independent), it became necessary to use linear mixed models, also known as multilevel models. Regarding the most adequate random structure, we followed the top-down strategy suggested by Brady and West [22]: several models with the same full structure (i.e., with all possible variables and interactions) but with a different random structure were estimated using restricted maximum likelihood estimation (REML) and compared using the conditional Akaike criterion information (cAIC), such that the model whose cAIC was lower was selected. We fitted the linear mixed models for each variable response: both Ca2+ rise and the AUC of the calcium signal for SOCE and Ca2+ store content or the median of the first 30 s of the recording. Specifically, we evaluated the assumptions of the models, such as the normality and homoscedasticity, through both graphical and test methods, since the non-independence of the observations from the same cluster was controlled, precisely, by the linear mixed models. On the one hand, for the normality assumption of both the Pearson residuals and random effects, we used QQ plots, histograms, Pearson residuals against fitted values plots, Shapiro–Wilks tests, and Jarke Bera tests. On the other hand, we employed Pearson residuals versus fitted values plots and Breush–Pagan and Barlett tests for the homoscedasticity assumption. If any model assumption was violated, different approaches were followed depending on the assumption violated: normality violations were addressed by removing outliers and transforming the response with Box–Cox transformation; heteroscedasticity violations were solved using generalized linear mixed models and different variance–covariance matrix structures and weighting observations. Furthermore, re-sampling methods, such as parametric bootstrapping for mixed models, were performed to evaluate the models and to estimate both the coefficients and their confidence intervals. Notably, the results from the bootstrapping procedures agreed with those from the classic linear mixed models that slightly violated the assumptions. That is why, in the real world, some deviations from assumptions are expected, since, if these deviations are not too large, the inferences extracted from the analysis will be acceptable, which is known as the robustness of validity [23]. For example, the non-normality of the residual distribution results in neither bias nor inefficiency from models; indeed, several authors claim that the violation of normality is not a serious problem as a consequence of the central limit theorem and can even be disregarded when the sample size is large (our experiment consisted of nearly 1000 observations), since the residual distribution will approximate to normality [24,25]. Regarding which experimental conditions were different from each other, all possible pairwise comparisons were made based on combinations between levels for the two explanatory variables considered: treatment and cellular line. At first, the expected value for each experimental condition was estimated from a linear mixed model. Secondly, the differences between each pair of expected values for each experimental condition were estimated; p-values were corrected using the Tukey method with a significance level of 0.05; and experimental conditions were grouped according to a pairwise comparison, i.e., if there were no differences between two experimental conditions, they belonged to the same group. In the graphs, this is shown by letters, which indicate the group to which each experimental condition belonged. The last step was based on graph theory and was carried out using the Bron–Kerbosch algorithm to find all maximal cliques. All data analyses and data collection processes were performed in the R environment [26]; the lmer [27] and nlme [28] packages were used for creating linear mixed models, lmer [27] and boot [29,30] packages for performing bootstrap methods, and multcomp [31] and igraph [32] for pairwise comparison and experimental condition grouping. First at all, the .CEL files, one per sample, were read with R software (oligo and affy packages). Then, samples were subjected to quality control through different methods, such that those samples which did not pass the quality control were removed from the posterior analysis. At first, the distribution of each sample and the possible presence of any batch effect or the need to normalize was tested through box plots and kernel estimators from non-normalized data. Then, pattern matching was conducted. To achieve this, it was necessary to normalize (quantile) and transform (log2) the datasets to make the samples comparable between them. Next, a principal component analysis (PCA) across all samples was conducted, and outlier detection was carried out by determining the robust Mahalanobis distance within each class (group of interest, e.g., observations from NCM460 cells treated with DFMO), where the input variables were the first principal components such that they explained at least 70% (PC70%) of the variability [33]. Another pattern-matching method employed was hierarchical cluster analysis with the Ward method, PC70%, and the Euclidean distance. Finally, probe-level models were employed from which both the normalized unscaled standard error (NUSE) and the relative log expression (RLE) were extracted and evaluated [34]. After quality control, the dataset was preprocessed, i.e., the dataset background was corrected, normalized, and finally summarized, all through the RMA method [35]. Subsequently, data were filtered by employing a non-specific filter implemented by the gene filter R function, and we kept those genes (probes) whose interquartile ranges (across all samples) were larger than the median of all interquartile ranges. Furthermore, we removed all those genes whose ALIAS annotation was unknown. Nevertheless, since we were concerned about a near 80 calcium gene set, all of them were retained in the dataset after the filtering process. In this way, the dataset passed from 138,745 to 12,933 probes. Afterward, differential expression analysis was carried out through linear models, specifically, through linear models for the microarray data (limma) method [36]. Regarding the variables in the model, the fixed factors were cellular line (levels: NCM460 and HT29) and treatment (levels: control and DFMO), whereas the random factor was day, i.e., the day on which the experimental units were processed, taking into account that each day a replicate corresponding to each experimental condition was processed. Then, the linear model selected was: where Day is a random factor. Finally, to control I-type error rates, we had to employ the false discovery rate (FDR) [37]. Differences were considered significant at FDR < 0.05. To assess changes in intracellular Ca2+ homeostasis during tumoral transformation (i.e., HT29 vs. NCM460) and the effect of DFMO treatment on each cell line, we employed the calcium imaging technique and Fura2/AM as a fluorescence ratiometric probe. Specifically, we assessed the free resting , the Ca2+ store content, and the SOCE (Figure 3 and Figure 4) in vehicle and DFMO-treated NCM460 and HT29 cells. Firstly, the free basal was measured as the median of the absolute F340/F380 signal during the first 30 s of the recordings, and the perfusion medium contained 1 mM Ca2+. Secondly, the Ca2+ store content was measured as the AUC and Δmax of the signal due to calcium release induced by CPA. Finally, SOCE was assessed as the increase in the AUC and Δmax of the signal due to perfusion with medium containing 1 mM Ca2+ after calcium store depletion. In accordance with our previous results [16], resting Ca2+ and SOCE levels appeared higher in colon cancer HT29 cells than in normal NCM460 cells, whereas the Ca2+ store content was larger in the normal NCM460 cells than in the colon cancer HT29 cells. Furthermore, DFMO treatment decreased resting [Ca2+] and SOCE in colon cancer HT29 cells and increased the Ca2+ store content in the same cells. In contrast, DFMO treatment had little or no effect on normal NCM460 cells. These results were similar to our previous results [19]. Figure 4 shows the statistical analysis of these data. Notably, due to the hierarchic structure of the data generated by this kind of experiment, we employed linear mixed models and Box–Cox transformation when needed. Furthermore, through pairwise comparison, the four experimental conditions (i.e., NCM460 control, NCM460 + DFMO, HT29 control, and HT29 + DFMO) were grouped into several clusters, such that the number of conditions in the each group was equal (clusters are indicated by the letters at the top of the bars plotted in Figure 4a–e). As result, we found that basal was significantly higher in non-treated HT29 than in non-treated NCM460 cells, and DFMO treatment decreased it in both cell lines (Figure 4a). Ca2+ store content was significantly lower in HT29 control cells than in NCM460 control cells (Figure 4b,c). Furthermore, DFMO treatment affected each cell line differently, since this treatment slightly reduced the calcium store content in NCM460 cells (just in terms of Δmax) but increased it in HT29 cells (in terms of both the AUC and Δmax). Notably, HT29 cells treated with DFMO showed a Ca2+ store content equal to that of the NCM460 control in terms of the AUC (Figure 4b,c). Finally, the data (Figure 4d,e) indicated that the SOCE was higher in HT29 cells than in NCM460 cells, and DFMO treatment reduced the SOCE in HT29 cells but not in NCM460 cells. Collectively, these data indicated that DFMO treatment was able to reverse, at least partially, the Ca2+ remodeling observed in HT29 cells relative to normal NCM460 cells. Furthermore, our previous results showed that DFMO treatment also reduced proliferation and death resistance in HT29 cells in accordance with the reversal of Ca2+ remodeling [19]. We also investigated the effects of DFMO treatment on another colon cancer cell line, SW480. Experiments were carried out again in parallel to the Ca2+ imaging experiments in normal NCM460 cells. As expected, the Ca2+ store content in colon cancer SW480 cells was much lower than in normal NCM460 cells, whereas the SOCE was higher in SW480 cancer cells than in normal cells. Treatment with DFMO significantly increased the Ca2+ stores in SW480 cancer cells but had no effect on normal NCM460 cells. In contrast to HT29 cells, DFMO treatment had no significant effect on the SOCE in SW480 cells (Supplementary Figures S1 and S2). The transcriptomic differential expression assays that we carried out could be separated into two parts. The first was conducted through two different technologies (RNA-seq and microarrays) to ascertain the differential expression between the HT29 and NCM460 cell lines. Thus, we established the following criteria to consider the information obtained by the two technologies and infer if a gene was differentially expressed or not: a gene was differentially expressed if it was indicated as such by at least one of the two technologies at a 0.05 significance level or by both technologies at a 0.1 significance level, as long as the logFC provided by both technologies fell in the same direction. The second part, i.e., the differential expression analysis between DFMO-treated and non-treated cells, both for HT29 and NCM460 cells, was conducted only by microarrays. Furthermore, the transcriptomic microarray-based experiments were conducted in parallel to the calcium imaging experiment, i.e., each sample assessed through microarrays was extracted from the same culture as a sample assessed by calcium imaging. In addition, both treated and non-treated samples processed on the same day were also extracted from the same culture. Finally, differential expression analyses were carried out for all genes (65,219 genes with ENSEMBL annotation for RNA-seq and more than 57,500 for Clariom D Human microarray). On the one hand, we identified 16,375 differentially expressed genes (DEGs) between HT29 and NCM460 through Illumina and 12,973 through microarrays. On the other hand, we found out that DFMO treatment was very much selective for HT29 cells, since treatment affected the expression of 3385 genes in HT29 cells against only 61 genes in NCM460 cells. Among all these genes, we focused only on 89 genes related to intracellular Ca2+ homeostasis, which we split into six clusters: voltage-operated calcium channels (VOCCs), SOCE modulators, TRP channels, Ca2+ release channels, Ca2+ pumps and exchangers, and mitochondrial Ca2+ transporters. Interestingly, none of these genes were affected in NCM460 cells, as opposed to 17 in HT29 cells. Furthermore, among these 17 genes, the change in the expression of 11 reversed the changes associated with cancer, i.e., the expression of these 11 genes was partially reverted by DFMO treatment. Regarding the effect at the transcriptomic level, DFMO treatment in NCM460 cells affected the expression of only 61 genes, whereas up to 3385 genes were differentially expressed in HT29 cells after treatment, indicating that DFMO is a treatment highly selective for colon cancer cells that is able to reverse, at least partially, the colon cancer phenotype. Regarding VOCCs, our results showed that CAV1.2 was downregulated in colon cancer HT29 cells, whereas CAV1.3 was overexpressed in colon cancer HT29 cells (Figure 5a,b). Furthermore, CAV2.3 and CAV3.2 were downregulated, and CAV3.1 and CAV3.3 were overexpressed according to RNA-seq technology. Regarding the effect of DFMO, no gene belonging to the VOCCs was affected by this treatment (Figure 5c,d). Regarding the 23 genes involved in SOCE, as many as 18 of them were identified as differentially expressed in cancer cells relative to normal cells by RNA-seq technology, whereas only 13 of them were differentially expressed according to microarrays (Figure 6a,b). Specifically, RNA-seq showed the overexpression of ORAI1, STIM1, STIM2, MBP, and SEPTIN2,4,7-11 and the downregulation of CRACR2A, STIMATE, ORMDL3, SARAF, SEPTIN1,3, and SEPTIN6 in HT29 cells relative to NCM460 cells (Figure 6a). Microarrays showed the overexpression of STIM1, MBP, SEPTIN9, and SEPTIN10 and the downregulation of ORAI1, CRACR2A, ORMDL3, SARAF, SEPTIN3,6-8, and SEPTIN11 (Figure 6b). Accordingly, both technologies reported the overexpression of STIM1, MBP, SEPTIN9, and SEPTIN10 and the downregulation of CRACR2A, ORMDL3, SARAF, SEPTIN3, and SEPTIN6 in colon cancer cells. Thus, some discrepancies occurred for SEPTIN7,8 and SEPTIN11, so these genes were not considered as differentially expressed. Consequently, the data suggested that HT29 cells overexpressed ORAI2, STIM1, STIM2, MBP, SEPTIN2, SEPTIN4, SEPTIN9, and SEPTIN10 relative to NCM460 cells, and showed a lower expression of ORAI1, CRACR2A, STIMATE, ORMD3, SARAF, SEPTIN1, SEPTIN3, and SEPTIN6 relative to the normal cells. Notably, treatment with DFMO did not affect NCM460 but did affect HT29 cells (Figure 6c,d). Specifically, DFMO treatment induced the overexpression of STIM1, STIM2, CRACR2A, ORMDL3, SEPTIN6-9, and SEPTIN11 in HT29 cells only (Figure 6d). Thus, it was clear that DFMO treatment was able to partially reverse the differential expression found for CRACR2A, ORMDL3, and SEPTIN6. Furthermore, if we consider the differential expression between HT29 and NCM460 cells identified by microarrays, DFMO treatment was also able to partially reverse the differential expression of SETPIN7, SEPTIN8, and SEPTIN11. Regarding the 27 genes that coded for TRPs, the most remarkable result was that over half of them were downregulated in HT29 cells relative to NCM460 cells (Figure 7a,b). Specifically, by combining the outcomes from both transcriptomic technologies, we could infer, on the one hand, that the following 13 genes were less expressed in HT29 cells: TRPC7, TRPM2, TRPM3, TRPM5, TRPM6, TRPM8, TRPML1-3, TRPV1, TRPA1, TRPP3, and TRPP5 (Figure 7c,d). On the other hand, it was clear that TRPC4, TRPC5, and TRPV6 were upregulated in HT29 cells. It is important to note that three genes showed an opposite differential expression pattern according to both technologies: TRPV5, TRPP1, and TRPP2. Regarding the effects of DFMO treatment, again, the treatment affected HT29 but not NCM460 cells (Figure 7c,d). Specifically, DFMO treatment decreased the expression of TRPC1, TRPC5, TRPV6, and TRPP1, whereas it increased the expression of TRPP2 (Figure 7d). Thus, DFMO treatment was able to partially reverse the tumoral phenotype in terms of the expression of TRPC5 and TRPV6, and, by taking into account just the results from the microarrays, DFMO treatment also partially reversed the differential expression of both TRPP1 and TRPP2. Regarding the expression of genes coding for Ca2+ release channels, our results indicated that Illumina identified five out of six genes belonging to the CRC set as differentially expressed between HT29 and NCM460 cells, against only three identified by microarrays (Figure 8a,b). Combining the results from both technologies, our findings suggested that IP3R1 and IP3R3 were upregulated in HT29 cells, whereas IP3R2 and RYR2 were downregulated. Nevertheless, RYR3 may also be overexpressed in HT29 cells. However, DFMO treatment had no effect on gene expression either in NCM460 or HT29 cells, suggesting that the possible differences could be mediated by excess polyamines. The calcium extrusion systems were also differentially expressed between HT29 and NCM460 cells (Figure 9a,b). Indeed, among the 12 genes considered, seven were identified as DEGs by Illumina (PMCA1, SERCA2,3, and NCX2 overexpressed, and PMCA3,4 and SPCA1 less expressed in HT29) and six by microarrays (PMCA1, NCX2,3, and SPCA2 overexpressed, and PMCA4 and SPCA1 less expressed in HT29). Again, DFMO treatment only affected HT29 cells and not NCM460 cells. Interestingly, tumor cells showed an increased expression of PMCA4 and decreased expression of SPCA2, and DFMO treatment reversed both changes in HT29 cells (Figure 9c,d). We also studied the differential expression of genes involved in mitochondrial Ca2+ transport. These genes showed clear differential expression between both cell lines (Figure 10a,b). On the one hand, Illumina identified five overexpressed genes (MCU, MICU1, MCUR1, EMRE, and VDAC2) and five downregulated genes (MICU2, MCUb, VDAC1, VDAC3, and NCLX) in HT29 cells. On the other hand, microarrays identified just one overexpressed gene, MCU (also identified by Illumina), and the same five downregulated genes identified by Illumina. Again, DFMO treatment affected only HT29 cells (Figure 10c,d). Specifically, DFMO treatment upregulated the expression of MCU and VDAC3, indeed reversing the effects of cancer in the case of VDAC3. Western blotting confirmed that MCU was upregulated by DFMO treatment in HT29 cells at the protein level (Supplementary Figure S3). In order to validate the most important results, the differential expression of selected genes was confirmed by qPCR analysis. For this analysis, we selected those genes whose differential expression in cancer cells vs. normal cells was reversed by DFMO treatment. The selected genes included those coding for channels TRPV6, TRPP1, and TRPC5; the SOCE modulators SEPTIN6 and ORMDL3; the pumps SPCA2 and PMCA4; and the mitochondrial transporters VDAC3 and MCU. Figure 11a confirms that all selected gene transcripts were differentially expressed in HT29 colon cancer cells relative to normal NCM460 cells, except for TRPC5 and ORMDL3, which showed no significant difference. Interestingly, when we compared the expression of the same selected genes in DFMO-treated normal and tumor cells, most differential expression was largely dampened (Figure 11b), thus also confirming the microarray data shown above. Finally, when we compared qPCR data on the expression of the selected genes before and after DFMO treatment in HT29 cells, the results confirmed that, in accordance with the microarray data shown above, DFMO treatment significantly increased the expression of the Ca2+ extrusion systems PMCA4 and VDAC3 in HT29 colon cancer cells and decreased the expression of the TRPV6 and TRPP1 channels and the Orai1 positive modulator SPCA2 (Figure 11c). However, in contrast to the microarray data, the qPCR data did not confirm the effects of DFMO treatment on TRPC5, SEPTIN6, ORMDL3, or MCU (Figure 11c). We investigated whether polyamine synthesis inhibition using DFMO as an ODC suicide inhibitor was able to reverse Ca2+ remodeling in cancer cells. To this end, HT29 and NCM460 cells were used as well-established cell models representing CRC and normal colonic cells, respectively [16]. CRC cells displayed significantly enhanced levels of resting intracellular [Ca2+] and SOCE relative to normal cells. Likewise, CRC cells showed decreased Ca2+ store content relative to normal cells. These findings are similar to previous results reported in [16,17]. We also showed here that SW480 colon cancer cells displayed enhanced SOCE and decreased Ca2+ stores compared to NCM460 cells, thus resembling the same intracellular Ca2+ remodeling as HT29 colon cancer cells. Although these changes were observed in cell lines representative of normal and tumor cells and should be confirmed in primary normal and tumor cells, changes in intracellular [Ca2+] homeostasis, collectively referred to as Ca2+ remodeling, could be part of the phenotypic changes associated with carcinogenesis; they may contribute to the hallmarks of cancer displayed by CRC cells, including enhanced cell proliferation and resistance to cell death [18]. We found that the incubation of cells with DFMO, a treatment that abolishes polyamine biosynthesis and decreases levels of intracellular polyamines, also significantly decreased both resting Ca2+ and SOCE, whereas it increased the Ca2+ store content in CRC cells, thus reversing the hypothetical Ca2+ remodeling associated with CRC. These results are similar to our recently reported results [19]. In contrast, in normal cells, the effects of DFMO were less important. These data suggested that polyamine synthesis inhibition may reverse, at least partially, the hypothetical Ca2+ remodeling associated with CRC, having minor effects on normal cells, where the ODC expression is low. Conversely, our results suggested that the excess polyamine synthesis observed in c-myc-related cancers may contribute to the hypothetical Ca2+ remodeling associated with CRC. The transcriptomic analysis of normal and CRC cells treated with DFMO showed that polyamine synthesis inhibition was able to significantly modify a large fraction (nearly 25%) of all gene transcripts studied in CRC cells. In striking contrast, in normal colonic cells, the same treatment affected less than 0.5% of the transcripts. Therefore, DFMO treatment was highly specific for cancer cells and had a nearly negligible effect on normal colonic cells. Again, we could explain these results by the fact that normal cells do not express ODC or express very low ODC levels unless this gene is induced during epithelial restitution, for instance. In this scenario, DFMO treatment probably induces only off-target effects or nearly negligible effects due to polyamine synthesis inhibition. These data also suggested that polyamines may induce rather dramatic effects on the transcriptome and the differential expression of nearly 25% of all the transcripts studied. In this work, we focused on the transcriptional effects on genes directly involved in intracellular Ca2+ homeostasis. We analyzed the effects of DFMO treatment (polyamine synthesis inhibition) on the transcription of the 10 voltage-gated Ca2+ channels, the 23 genes known to be involved in SOCE, the 28 TRP channels, the 6 Ca2+ release channels of the ER, the 12 Ca2+ pumps and exchangers, and the 11 genes known to be involved in mitochondrial Ca2+ transport. Figure 12 summarizes the changes in the Ca2+ transport systems in cancer cells relative to normal cells and the effects of DFMO. We used two independent approaches for transcriptomic analysis, i.e., Illumina next-generation RNA sequencing (RNAseq) and microarrays. Illumina data include sequence data that are critical in cases where polymorphisms or mutations are the main target. Microarrays only provide data on the relative transcription level of selected sequences. We selected microarrays instead of RNAseq for our analysis of the effects of DFMO treatment for several reasons. First, because microarray sensitivity is lower than that of RNAseq, we expected that the differentially expressed genes would be those more significantly influenced by DFMO treatment, leading to less artefactual DEGs. Second, the computational and preprocessing methods of transcriptomic outcomes are simpler with microarrays, i.e., RNAseq outcomes warrant more sophisticated and more computationally powerful methods than microarray outcomes. Finally, transcriptomic microarrays are generally more affordable. Regarding voltage-gated Ca2+ channels, the data obtained using Illumina were quite similar but not identical to our previous results obtained using Ion Torrent technology [18]. Microarray analysis showed that only two transcripts were differentially expressed in cancer cells, whereas changes in three other transcripts were only significant according to Illumina and not microarray. Specifically, we found that Cav1.2 was downregulated, whereas Cav1.3 was upregulated in CRC cells. Polyamine synthesis inhibition had no effect on the expression of any of the voltage-gated Ca2+ channels, either in the CRC cells or in the normal cells. Accordingly, these data indicated that, although the previous results suggested the differential expression of certain voltage-gated Ca2+ channels in CRC, they could not be attributed to excess polyamine synthesis in CRC. In marked contrast, a number of molecular players involved in SOCE (ORAI1,2,3 and STIM1,2) and its modulatory proteins were differentially expressed in CRC cells relative to normal cells. Moreover, the transcription levels of SOCE molecular players were deeply influenced by polyamine synthesis inhibition. However, these changes were only observed in CRC and not in normal colon cells. Specifically, we found that DFMO treatment induced the increased expression of Stim1 and Stim2 transcripts in HT29 cells. These data are similar to our previous results using qRT-PCR [19]. However, at the protein level, DFMO treatment decreased Stim1 expression in HT29 cells without changing Stim2 expression [19]. In addition, DFMO treatment increased the expression of a number of SOCE modulators, including CRACR2A, ORMDL3, and several septins. In nearly all these cases, except for SEPTIN9, the change induced by DFMO reversed the change associated with cancer. These data suggested that a number of SOCE modulators are downregulated in CRC, leading to a loss in SOCE modulation that might contribute to the enhanced SOCE in cancer cells. However, in contrast to the microarray data, the qPCR data did not confirm that DFMO increased either SEPTIN6 or ORMDL3. Although qPCR is considered the gold-standard for testing differential expression, the larger number of primers used for the microarrays (20 instead of just one for qPCR) may explain this difference. Further research is required, therefore, to confirm the effects of the polyamine depletion of SOCE modulators. SPCA2, a secretory pathway Ca2+ ATPase that has recently been reported to interact and modulate Orai1 channels to activate them independently of store depletion [38], was overexpressed in CRC cells. Interestingly, we found that SPCA2 was downregulated after DFMO treatment in HT29 colon cancer cells but not in normal cells. These data were also confirmed by qPCR analysis. Therefore, these data suggested that overexpressed SPCA2 in CRC cells might contribute to enhance resting [Ca2+] and Orai1 activation independently of store depletion in CRC, a process that is limited by polyamine synthesis inhibition. In addition, it has recently been reported that the epigenetic modulation of SPCA2 reverses the epithelial-to-mesenchymal transition in breast cancer cells [39]. It is tempting to speculate that overexpressed SPCLA2 in CRC cells due to polyamine excess may play a similar, previously unrecognized role in CRC as well. TRP channels were also differentially expressed in CRC cells relative to normal cells. In general, both the Illumina and microarray technologies showed the decreased expression of a number of different TRP channels in CRC cells, particularly TRPML1,2,3 and TRPP2. However, CRC cells also showed the enhanced expression of a few TRP channels, including the TRPC5, TRPV6, and TRPP1 channels, that may have contributed to the appearance of non-selective store-operated currents characteristic of CRC cells [16]. qPCR data confirmed these results for TRPV6 and TRPP1 but not for TRPC5. This finding for TRPC5 could have been due to the extremely low number of copies of TRPC5 transcripts. Again, polyamine synthesis inhibition had no effect on the transcription of TRP channels in normal cells. In contrast, in CRC cells, polyamine synthesis inhibition decreased the expression of the TRPC1 and 5, TRPV6, and TRPP1 channels, whereas it increased TRPP2 gene transcription. The pPCR data also confirmed the effects of DFMO on the expression of TRPV6 and TRPP1. We reported previously that DFMO treatment decreased the expression of TRPC1 in HT29 cells at the protein level [19]. Further research is required to validate these additional changes in the TRP channel expression at the protein level. The data on TRP channel remodeling induced by DFMO treatment strongly suggested that polyamines may influence cancer hallmarks acting on the transcriptional level of these TRP channels. For instance, TRPV6, the epithelial Ca2+ channel modulated by the vitamin D receptor, has also been previously related to CRC and other forms of cancer [40]. The cases of TRPP1 and TRPP2 are intriguing, as these are channel complexes involved in sensing shear stress in epithelia that are present in the ER. The results indicated an increased TRPP1/TRPP2 ratio in CRC cells, a feature that was also reversed by polyamine synthesis inhibition. Further research is required to understand the role of the TRPP1 and 2 channels in colon cancer and their relation to polyamine synthesis. The Ca2+ release channels of the ER, including IP3 and ryanodine receptors, were differentially expressed in cancer cells and may also contribute to cancer hallmarks. However, none of these Ca2+ release channels were modulated by polyamine synthesis inhibition, either in normal or CRC cells, at least at the transcriptional level, thus excluding the possibility that changes in the transcription of these channels are mediated by ODC overexpression. Interestingly, as mentioned above, the TRPP1 and TRPP2 channels can also be found at the ER membranes, where they could play a role as leak channels. Specifically, TRPP2 is a calcium-permeant transient receptor potential (TRP) cation channel expressed primarily on the ER membrane and primary cilia of all cell and tissue types [41]. TRPP2 mutations lead to autosomal-dominant polycystic kidney disease. Recent data indicate that TRPP2 is involved in susceptibility to cell death induced by stress [41]. Accordingly, changes in the TRPP1/TRPP2 ratio related to CRC and polyamine synthesis could be involved in Ca2+ store content, Ca2+ transfer from the ER to mitochondria, and sensitivity to stress. Again, further research is required to validate this possibility. Regarding Ca2+ extrusion systems such as Ca2+ pumps and transporters, the data showed the differential expression of several transport systems, including the enhanced expression of the pumps PMCA1 and SPCA2 and the exchanger NCX2, along with the decreased transcription of the pumps PMCA4 and SPCA1. Again, polyamine synthesis inhibition had no effect on the transcription of any of these calcium extrusion systems in normal cells. However, in CRC cells, polyamine synthesis inhibition reversed the changes in the transcription of PMCA4 and SPCA2 linked to CRC. These results were confirmed by qPCR analysis. As stated above, SPCA2 may interact and modulate Orai1 to activate Orai1 independently of both Stim1 and Ca2+ store depletion, thus leading to store-independent Ca2+ entry. PMCA4, the plasma membrane Ca2+ ATPase linked previously to CRC [42], was downregulated in CRC, likely contributing to enhanced resting intracellular [Ca2+]. This gene returned to normal levels after DFMO treatment, thus probably contributing to decreased resting Ca2+ levels in treated cells. Finally, mitochondrial Ca2+ transport systems were differentially expressed in CRC cells. Most changes involved the decreased expression of the molecular players involved in the control of the mitochondrial Ca2+ uniporter (MCU) and the Ca2+ channel involved in mitochondrial Ca2+ uptake. Once more, polyamine synthesis inhibition had no effect on the transcription levels of the mitochondrial Ca2+ transport systems in normal cells. However, in cancer cells, polyamine synthesis inhibition increased the expression of both MCU and VDAC3. qPCR analysis also confirmed the increased expression of VDAC3 in DFMO-treated HT29 cells. We used western blotting to confirm that MCU expression was also enhanced at the protein level in HT29 cells treated with DFMO (Supplementary Figure S3). Therefore, the modulation of the transcription of these two particularly relevant mitochondrial channels in CRC could be mediated by ODC and polyamines. Interestingly, these changes may contribute to explaining the reversal of the cancer hallmarks related to susceptibility to apoptosis. In other words, the enhanced resistance to apoptosis characteristic of CRC cells could be mediated, at least partially, by changes in the expression of MCU and VDAC3 due to ODC activation. In summary, our results indicated that a few genes involved in Ca2+ transport appeared to be specifically modulated by polyamines in CRC cells and could be responsible for most changes in intracellular Ca2+ homeostasis in CRC cells. Thus, an excess of polyamines could induce the downregulation of SOCE modulatory genes (STIM1,2), the plasma membrane Ca2+ ATPase PMCA4 Ca2+ pump, and the mitochondrial Ca2+ channels MCU and VDAC3, contributing to dysregulated SOCE and enhanced resting Ca2+ and mitochondrial Ca2+ uptake in CRC. Conversely, polyamines could also induce the overexpression of TRPV6 channels, the Orai1 activator SPCA2, and the exchange of TRPP1 channels by TRPP2, thus likely contributing to enhanced Ca2+ entry and decreased Ca2+ store content in CRC. These findings may provide new insights into the role of polyamines in Ca2+ remodeling in cancer. Further research in animal models and/or tumor samples from patients is required to validate our findings. We conclude that polyamine synthesis inhibition may partially reverse changes in intracellular calcium homeostasis hypothetically associated with CRC, including a decrease in resting intracellular Ca2+ and store-operated Ca2+ entry, as well as an increase in Ca2+ store content. These effects were observed in CRC cells but not in normal colonic cells. Analogously, polyamine synthesis inhibition induced the differential expression of 25% of the whole transcriptome in cancer cells but only about 0.25% of the transcripts in normal colonic cells. We also conclude that polyamine synthesis inhibition reversed the changes in the differential expression of transcripts associated with cancer. Specifically, polyamine synthesis inhibition decreased the expression of the TRPV6 and TRPP1 channels, as well as the Orai1 positive modulator SPCA2, probably contributing to decreased Ca2+ influx in DFMO-treated tumor cells. In addition, polyamine depletion enhanced the transcription of the plasma membrane PMCA4 Ca2+ pump and the mitochondrial channels MCU and VDAC3, thus probably contributing to enhanced Ca2+ extrusion in DFMO-treated cells. Collectively, these results may provide a transcriptional basis for the hypothetical calcium remodeling in CRC and its reversal by DFMO. Further research in animal models and/or tumor samples from patients is required to validate our findings.

PMC10000433

Marjaana J. Häyrinen,Jenni Kiiskilä,Annamari Ranki,Liisa Väkevä,Henry J. Barton,Milla E. L. Kuusisto,Katja Porvari,Hanne Kuitunen,Kirsi-Maria Haapasaari,Hanna-Riikka Teppo,Outi Kuittinen

The Transcription Factor Twist1 Has a Significant Role in Mycosis Fungoides (MF) Cell Biology: An RNA Sequencing Study of 40 MF Cases

28-02-2023

cutaneous T-cell lymphoma,mycosis fungoides,Twist1,Zeb1,RNA sequencing,DNA methylation,laser capture microdissection

Simple Summary Mycosis fungoides (MF) is the most common variety of cutaneous T-cell lymphoma. Our previous studies showed that the epithelial–mesenchymal transition (EMT) transcription factors (TFs) Twist1 and Zeb1 have prognostic value in MF. The main objective of the present study was to gain better knowledge about the biological mechanisms behind this phenomenon. The RNA of 40 skin tumor biopsies (from 40 patients) was sequenced and analyzed. Twist1 protein expression seemed to classify MF cases into different groups based on their global RNA expression. Additionally, high Twist1 protein expression was associated with several genes and pathways known to have roles in aggressive tumor biology. For Zeb1, similar results were not found. Our results suggest Twist1 to be a central transcription factor and pathway regulator in the disease progression of MF. Twist1 might be an interesting object for developing targeted therapies for MF. Abstract The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I–IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

The Transcription Factor Twist1 Has a Significant Role in Mycosis Fungoides (MF) Cell Biology: An RNA Sequencing Study of 40 MF Cases Mycosis fungoides (MF) is the most common variety of cutaneous T-cell lymphoma. Our previous studies showed that the epithelial–mesenchymal transition (EMT) transcription factors (TFs) Twist1 and Zeb1 have prognostic value in MF. The main objective of the present study was to gain better knowledge about the biological mechanisms behind this phenomenon. The RNA of 40 skin tumor biopsies (from 40 patients) was sequenced and analyzed. Twist1 protein expression seemed to classify MF cases into different groups based on their global RNA expression. Additionally, high Twist1 protein expression was associated with several genes and pathways known to have roles in aggressive tumor biology. For Zeb1, similar results were not found. Our results suggest Twist1 to be a central transcription factor and pathway regulator in the disease progression of MF. Twist1 might be an interesting object for developing targeted therapies for MF. The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I–IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF. Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of non-Hodgkin’s lymphomas with no evidence of extracutaneous spread at the time of diagnosis. Mycosis fungoides (MF) is the most common CTCL, accounting for about 60% of all CTCL cases [1]. Usually, patients are adults, and most of the patients are males. Sézary syndrome (SS) is a leukemic variant of CTCL. The clinical course of MF is variable. The disease starts with localized or disseminated patches or plaques that can remain skin-limited for years. However, in a subset of patients (10–20%) [2] the disease evolves into the tumor or erythroderma stages, including extracutaneous spread with poor prognosis. Although the treatment modalities for MF have developed over the years, the disease remains still incurable, indicating the need to understand the biology of this disease better. Epithelial-to-mesenchymal transition (EMT) is an essential process in embryonic development and common in cancer progression [3,4,5]. During EMT, the epithelial cells obtain the mesenchymal phenotype and convert to a more invasive, motile phenotype and acquire resistance to apoptosis. The role of EMT in cancer progression, dissemination, and therapy resistance has been well recognized in epithelial tumors, but in the case of hematopoietic malignancies, the significance of EMT is less well studied. One basic difference is that the cells of hematological malignancies already have a mesenchymal phenotype since they arrive from blood cells derived from the embryonic mesoderm. However, some of the EMT-controlling transcription factors (EMT–TFs), including Twist1 and Zeb1, control the differentiation of hematopoietic cells and have been associated with the progression of hematological malignancies [6]. Twist1 is a T-cell oncoprotein that belongs to the basic helix–loop–helix (bHLH) protein family. Twist1 regulates the inflammatory processes and is involved in lymphocyte function and maturation, working as a key regulator of immune cells, especially T helper (Th) cell activation [7,8]. Zeb1 is a protein-coding gene that suppresses hematopoiesis and downregulates the expression of CD4 during T-cell maturation [9]. In the study of vanDoorn et al. (2004) [10], Twist1 was highly overexpressed among SS patients. Goswami et al. (2012) [11] showed that Twist1 expression was correlated with MF and SS stages. They also observed an association between increased Twist1 and c-Myc expression and abnormal p53 expression. Our earlier studies showed that IHC Twist1+ is associated with worse prognosis and Zeb1+ with better prognosis in patients with MF [12]. In this study, our aim was to investigate both the up- and downstream regulation of Twist1 and Zeb1 to understand the biology behind their prognostic value. Our methods included Twist1 and Zeb1 IHC and RNA sequencing from 40 MF cases. The methylation level of the Twist1 promoter was analyzed from 28 MF samples. The retrospective patient material consisted of 40 biopsies from 40 MF patients with stage I–IV disease from the Helsinki University Hospital obtained during the years 2015–2019. 21 formalin-fixed and paraffin-embedded (FFPE) samples were taken at the time of the diagnosis and 19 from patients with relapsed disease. Patient data were collected from hospital records. These clinical variables included gender, age, WHO-EORTC stage, plasma lactate dehydrogenase (LDH) level, treatments, data on follow-up or relapses, progression, and mortality. Twist1 and Zeb1 immunohistochemical staining was performed as previously described in Lemma et al. (2013) [13]. In the IHC analysis, the cut-off point for low and high expression of Twist1 was 17.6% and that for Zeb1 was 37%, defined by using a receiver operating characteristic (ROC) curve. Morphologically assessed neoplastic cells were counted as positive in the IHC analysis. Tumor cell count was estimated from the hematoxylin–eosin-stained samples identifying the hyperchromatic small to medium-sized haloed lymphocytes with hyperconvoluted nuclei as a percentage of the surrounding reactive lymphocytic cell infiltrate. The tumor cell count was estimated by an experienced hematopathologist. Additionally, the proportion of the entire lymphocytic infiltrate was estimated from the sample area. The time from diagnosis to the initiation of systemic therapy or to the last follow-up date (TTST) was calculated and used to perform a Kaplan–Meier analysis. A chi-squared test was used as a statistical method, and statistical significance was evaluated with the log rank. p-values < 0.05 were considered statistically significant. IBM SPSS Statistics for Macintosh, Version 28.0. (Armonk, NY: IBM Corp.) was used for ROC curves and Kaplan–Meier analysis. In total, 8–13 sequential paraffin-embedded slide sections with a thickness of 5 µm were prepared and mounted on pet (polyethylene terephthalate) slides (Leica AS LMD; Leica Microsystems Ltd., Wetzlar, Germany). Paraffin was removed by soaking in xylene twice for 10 min, and sections were stained with hematoxylin. One section was stained with CD3 antibody (NCL-L-CD3-565), which was used as a guide to differentiate between lymphocytic and epithelial cells (Supplementary Figure S1). Laser capture microdissection (LCM) was performed using a ZEISS PALM MicroBeam Microsdissection system (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and adhesive collection caps. After microdissection, the samples were placed into collection vessels containing appropriate volumes of Depaffinization Solution (#19093, Qiagen GmbH, Hilden, Germany), and RNA was extracted using an MiRNeasy FFPE extraction kit (#217504, Qiagen GmbH, Hilden, Germany) generally according to the manufacturer’s instructions. However, after adding proteinase K solution, the samples were incubated overnight at 56 °C with gentle shaking for a better yield. After LCM, DNA was isolated using a QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). DNA concentrations were measured using a Qubit 4 fluorometer, and bisulfite treatment was carried out using an EpiTect Fast DNA Bisulfite kit (Cat. No. 59824, Qiagen). The promoter area of the TWIST1 gene (Entrez Gene ID: 7291) was then amplified using a PyroMark CpG Assay (GeneGlobe Cat. no: PM00030121), PyroMark PCR kit, and RotorGeneQ device (Qiagen). The specificity of biotin-labeled amplification products was confirmed on agarose gel and purified for pyrosequencing with Streptavidin Sepharose beads (Cytiva) using a PyroMark Q24 Vacuum Workstation. Single-stranded DNA on a sequencing plate was annealed with the sequencing primer at 80 ºC (2 min) and cooled at room temperature (15 min). Then, the plate was processed using the PyroMark Q24 Instrument with compatible Gold Q24 reagents. Finally, the sequencing run was analyzed via the PyroMark Q24 software version 2.0.6. (Qiagen, Hilden, Germany). The quality and quantity of the extracted RNA samples were analyzed with a LabChip GX Touch HT RNA Assay Reagent Kit (PerkinElmer, Waltham, MA, USA) and Qubit RNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA). For genomic DNA contamination measurement, a Qubit DNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. Dual-indexed mRNA libraries were prepared from 150 ng of total RNA with a QuantSeq 3′ mRNA–Seq Library Prep Kit FWD (Lexogen Gmbh, Vienna, Austria) according to user guide version 015UG009V0251. During second strand synthesis, 6 bp Unique Molecular Identifiers (UMIs) were introduced with the UMI Second Strand Synthesis Module (Lexogen Gmbh, Vienna, Austria) for detection and removal of PCR duplicates. The quality of the libraries was measured with a LabChip GX Touch HT DNA High Sensitivity Reagent Kit (PerkinElmer, Waltham, MA, USA). Sequencing was performed with a NovaSeq 6000 System (Illumina, San Diego, CA, USA) with read length of 2 × 101 bp and target coverage of 10 M reads for each library. QuantSeq 3′ mRNA–Seq Integrated Data Analysis Pipeline version 2.3.1 FWD UMI (Lexogen Gmbh, Vienna, Austria) on Bluebee® Genomics Platform was used for primary quality evaluation of the RNA sequencing data. For visual exploration of the data, the read counts were normalized using the variance stabilizing transformation (VST) method implemented in the DESeq2 (version 1.30.1) package [14] in R (version 4.0.3) [15], which transforms the count data in a way that minimizes differences between samples for rows with small counts and normalizes the data with respect to library size, with large values approximating a log2 scale. Visual inspection of the samples was performed using principal component analysis (PCA) implemented in the ‘prcomp’ function in R, applied to the normalized read counts of the top 500 genes according to variance. Additionally, the same data were used to generate a Pearson’s correlation heat map from all pairwise comparisons of samples, using the ‘pheatmap’ package (version 1.0.12) [16] in R. Data normalization and differential expression (DE) analysis were performed using the DESeq2 package [14] in R. Genes with an absolute log2 fold change > 0.58 (absolute fold change of 1.5) and an adjusted p value < 0.05 (adjusted for multiple testing using the Benjamini–Hochberg procedure [17]) were considered to be significantly differentially expressed. Initially, two contrasts were made using only diagnostic samples: first, high-Twist1-expression (Twist+) samples against low-Twist1-expression (Twist1-) samples, followed by high-Zeb1-expression (Zeb1+) samples against low-Zeb1-expression (Zeb1-) samples. A second round of DE analysis was performed between Twist1+ and Twist1- samples, this time including both diagnostic and follow-up samples. The results of the two sets of Twist1 expression contrasts were compared using Pearson’s correlations. The full results table from the DESeq2 analysis of Twist1+ versus Twist1-, using both diagnostic and follow-up samples, was read into the Ingenuity Pathway Analysis (IPA) software [18]. Then an IPA core analysis was run with the following analysis settings: the reference was set to ‘User Dataset’, the confidence level was set to ‘Experimentally Observed’, the species was set to ‘Human’, the log2 fold-change filter was set to <−0.58 and >0.58, and the adjusted p value filter was set to <0.05. The significance threshold for the identified pathways and regulators was also set to an adjusted p value of 0.05. To identify hub genes, a protein–protein interaction (PPI) network was constructed from the genes that were significantly differentially expressed between high and low Twist1 expression groups (all samples) using the STRING database [19] through the StringApp [20] within Cytoscape [21]. The connectivity of the nodes in the network was assessed using the cytoHubba plugin [22] and nodes with a degree of connectivity of 10 or more were said to be hub genes. Patient demographics are presented in Table 1. The median age was 63 years (range 19–86 years), and most of the patients were male (70%). The median follow-up time was 32.2 months (range: 6.28–203 months). For Twist1, there were 20 high expression and 20 low expression cases. For Zeb1, there were 4 high expression and 36 low expression cases. Twist1 expression was not associated with tumor cell percentage or lymphocyte cell proportion, or with the clinical stage. There were no significant correlations between the collected clinical variables and Twist1 protein expression. However, among the patients with diagnostic samples, there was a trend for the cases with high Twist1 protein expression to require systemic therapy sooner than the cases with low expression (p value = 0.133, Figure 1). The methylation levels of four CpG islands of 28 cases were analyzed: CpG1, CpG2, CpG3, and CpG4. The means and standard deviations for GpG islands 1, 2, 3, and 4 were M1 = 3.96 (SD1 = 2.25), M2 = 3.36 (SD2 = 2.22), M3 = 12.31 (SD3 = 3.76), and M4 = 1.58 (SD4 = 0.902), respectively. For the total methylation level, the mean was 20.95 and SD was 6.88. The methylation levels did not correlate with the IHC or RNA expression of Twist1. To see how well the RNA expression for Twist1 and Zeb1 corresponded to high/low classification based on IHC expression, the normalized expression for each gene in each sample was plotted on a heat map (Figure 2). Generally, Twist1 RNA expression agreed with the high/low IHC expression classification of the samples, while Zeb1 RNA expression did not correlate well with the classification. A Pearson correlation coefficient was conducted to examine these correlations. Twist1 RNA expression correlated positively to IHC expression (r (38) = 0.46, p value < 0.01), while no correlation could be seen between Zeb1 RNA and protein expression (r (38) = −0.070, p value = 0.67). For Twist1, there were also cases that did not correlate; for example, the case of MH37 had high Twist1 protein expression but the lowest RNA expression of the whole series. To investigate whether the samples clustered according to their Twist1 and Zeb1 expression, we performed PCAs on the normalized read counts both for all samples combined and diagnostic samples individually (see Methods). The samples were observed to separate along PC1 according to their Twist1 expression category in both analyses. PC1 explained 26% of the variation for all samples (Figure 3a) and 32% of the variation for diagnostic samples (Figure 3b). A similar pattern was not seen for Zeb1 expression categories. Furthermore, there appeared to be no separation between the diagnostic and follow-up samples (Supplementary Figure S2). Differential expression (DE) analysis between Twist1+ and Twist1- diagnostic samples only returned 11 significantly (adjusted p value <= 0.05, absolute log2 fold change > = 0.58) differentially expressed genes: OAS2, ENSG00000201329, FCER1G, LGALS9, LYZ, LITAF, HLA-DRA, HLA-A, IGHM, NDUFA4 and RPGR (Figure 4). The corresponding analysis for Zeb1 expression yielded no significant genes. Considering the low statistical power when analyzing only diagnostic samples and given that there seemed to be little separation between diagnostic and follow-up samples in terms of gene expression (Supplementary Material, Figure S2), the DE analysis between Twist1+ and Twist1- samples was repeated for the diagnostic and follow-up samples combined. This analysis yielded 321 significant genes (adjusted p value < = 0.05, absolute log2 fold change > = 0.58). Expression of the top 100 significant genes (according to the adjusted p value) is visualized in the heatmap in Figure 5, where the samples are clustered largely by their Twist1 categories. The top genes generally have higher expression in the Twist1+ samples than in the Twist1- samples. Furthermore, diagnostic and follow-up samples appeared to be intermixed, irrespective of the clinical staging. The results of the DE analysis with all samples combined correlated well with those from the analysis using only diagnostic samples for both the adjusted p value (Pearson’s r = 0.63, p << 0.01, Figure 6a) and the log2 fold change (Pearson’s r = 0.85, p << 0.01, Figure 6b). Seven of the significant genes from the first analysis were also identified in the second analysis (OAS2, ENSG00000201329, LGALS9, LITAF, HLA-DRA, IGHM and NDUFA4). The results of the differential expression analysis were used as input for Ingenuity Pathway Analysis (IPA). This resulted in three significant pathways (adjusted p value < 0.05): the ‘GP6 Signaling Pathway’, ‘Hepatic Fibrosis/Hepatic Stellate Cell Activation’, and ‘B Cell Development’. Prior to correcting for multiple testing, there were 35 significant pathways (p value < 0.05), as shown in Supplementary Figure S3. The IPA core analysis also identified 228 significant upstream regulators (adjusted p value < 0.05) and 177 significant master regulators/causal networks (adjusted p value < 0.05) (Supplementary Tables S1 and S2). Analysis of the protein–protein interaction network constructed from the results of the Twist1 differential expression analysis resulted in 28 identified hub genes (connectivity degree >= 10), as shown in Figure 7. In previous studies, the protein expression of the EMT TFs Twist1 and Zeb1 was shown to have prognostic relevance in MF [12]. Here, we sought to explore the biology of these differences more deeply. Through RNA sequencing, we found that most of the regulation of Twist1 expression occurs at the translational level, while no correlations were found between Zeb1 protein and the mRNA level. In the PCA, Twist1 expression was found to classify MF cases into different clusters according to their global RNA expression. Several genes and pathways known to be associated with aggressive tumor biology were found to be overexpressed among high Twist1 cases. For Zeb1, similar associations were not observed. MF is the most frequent cutaneous T-cell lymphoma, originating from the peripheral epidermotropic T-cells. Despite the many available treatment options, MF is still considered incurable, except for allogenic stem cell transplantation. The intricate molecular mechanisms behind the MF transition from an indolent to a progressive disease are not completely understood. Currently, it is anticipated that alterations in defined signaling networks promote the proliferation, survival, and migration of malignant T-cells, as well as the suppression of their immune regulation, resulting in changes to the tumor microenvironment that enables disease progression [23]. In our previous study [12], we found that the IHC detection of Twist1 and Zeb1 have prognostic value in MF: Twist1+ and Zeb1- identified patients with a worse prognosis. The results of the IHC analysis of the present study were in line with previous results, but statistical significance could not be shown, likely due to the limited sample size. Other studies have also proposed that Twist1 protein may be one of the key regulators in MF progression. Dobos et al. [24] studied the prognostic value of the expression levels of several proteins of peripheral blood leukocytes in MF and SS using the multiomics method. The authors found T-plastin, Twist1, and KIR3DL2 to bear the highest prognostic relevance. On the other hand, Song et al. [25] demonstrated core oncogenic processes behind large cell transformation of MF. These processes included metabolic reprogramming, cellular plasticity, upregulation of myelocytomatosis oncogene (MYC) and E2 promoter binding factor (E2F) activities, and downregulation of major histocompatibility complex 1 (MHC1). One of the key elements in cellular plasticity is the upregulation of Twist1 protein expression through gene amplification [25]. The regulation of Twist1 expression in cancer is a complicated process including modulation at many levels and depending on the cancer type and tissue context [26]. Transcriptionally, Twist1 can be upregulated via multiple signal transduction pathways such as the tumor necrosis factor receptor (TNFR), receptor tyrosine kinase (RTK), frizzled (FZD), tumor growth factor beta (TGFß), NOTCH, and epidermal growth factor receptor (EGFR) pathways [26]. The most important intracellular regulators include mitogen activated protein kinase (MAPK), protein kinase B (Akt), nuclear factor-κB (NF-κB), muscle segment homeobox 2 (MSX2), ß-catenin, Fibulin 5 (FBLN5), mothers against decapentaplegic homolog 2 (Smad), high-mobility group AT-hook 2 (HMGA2), signal transducer and activator of transcription 3 (STAT3), and hypoxia inducible factor-1 (HIF-1α) [26]. MAPK, Akt, and casein kinase (CK2) are important Twist1 phosphorylating kinases that participate in the post-translational regulation of Twist1 [21]. We found an association between Twist1 protein and RNA expression levels. In contrast to a study by Galvan et al. [27], we did not detect a clear connection between promoter methylation and the RNA levels of TWIST1/Twist1, indicating that this is likely not a major reason for TWIST1 overexpression in MF. We also did not observe overexpression of other known positive TWIST1 regulators, thereby leaving TWIST1 overexpression largely unexplained. Despite a robust correlation between TWIST1 mRNA and protein levels, there were also cases with deviant results, indicating that translational/posttranslational regulation also plays a role. For example, beta-transducing repeat containing protein (β-TRCP) was shown to play a role in Twist1 degradation [28]. For Zeb1, the protein and RNA amounts did not correlate with each other, suggesting that most regulation takes place in the posttranscriptional level. In the PCA, Twist1 expression partly explained the clustering of the MF cases along the first principal component. This result illustrates that Twist1 is an important modulator of MF biology. This implication seems reasonable considering Twist1’s integral role in T-cell differentiation. However, in this study setting, we were only able to demonstrate association between these two factors, which does not always imply causality. For Zeb1, the PCA did not reveal any grouping of samples according to their Zeb1 expression. The small number of cases with high Zeb1 expression might explain why we could not detect a function of Zeb1 in MF. Differential expression analysis between Twist1+ and Twist1- diagnostic MF samples revealed 11 significantly differentially expressed genes; OAS2, ENSG00000201329, FCER1G, LGALS9, LYZ, LITAF, HLA-DRA, HLA-A, IGHM, NDUFA4 and RPGR. High Twist1 protein expression was associated with overexpression of RPGR and ENSG0000020132. The rest of the genes were downregulated when Twist1 protein expression was high. Most of these genes are associated with adverse biological features in different malignancies. High OAS2 expression was shown to associate with better prognosis in breast [29], bladder [30] and colorectal cancer [31]. In acute myeloid leukemia (AML), OAS2 expression was shown to induce chemoresistance [32]. Galectin-9 was previously shown to correlate with disease severity and decreased CD8 cell infiltration in CTCL [33]. Moreover, anti-Gal-9 therapy selectively expands intratumoral T-cell immunoglobulin and mucin-domain containing-3 positive (TIM-3+) cytotoxic CD8 T-cells, as well as immunosuppressive regulatory cells [34]. Previous studies have confirmed the role of FCER1G in several cancers [35]. FCER1G takes part in promoting squamous carcinogenesis (SCC) progression [36], and predicts poor prognosis in gliomas and clear cell renal cell carcinomas (RCC) [37,38]. In multiple myeloma, FCER1G predicts better prognosis [39,40]. According to the previous research, LITAF may be considered as a tumor suppressor [41,42]. In AML, LITAF was shown to increase cell apoptosis and differentiation [43]. In colorectal cancer, HLA-A is associated with a favorable prognosis [44]. The downregulation of NDUFA4 was detected in RCC [45]. The ingenuity pathway analysis of Twist1 overexpression resulted in 35 pathways before correcting for multiple testing. When Twist1 was overexpressed, the glycol protein VI (GP6) signaling pathway was one of the most strongly upregulated pathways. GP6 is part of the immunoglobulin superfamily and is expressed in the platelets and megakaryocytes taking part in their activation. Along with their coagulative functions, platelets have an active role in regulating immune phenomena and in tumor cells immune escape. On the other hand, platelets also induce EMT in tumor cells. It was proposed that platelets play a role in tumor progression and metastasis by reducing natural killer (NK) cell antitumor activity. Kopp et al. [46] showed that when coating tumor cells with platelet-derived soluble factors from stimulated platelets, the functions of NK cells were impaired. Additionally, the platelet coat might protect tumor cells from immunosurveillance [28]. In a study by Yavadav et al. [47], the GP6 signaling pathway was associated with endometrial cancer progression. The other upregulated pathways included “regulation of the EMT in development pathway”, “actin cytoskeleton signaling”, “pulmonary fibrosis idiopathic signaling pathway”, and “integrin linked kinase (ILK) signaling”. Since Twist1 functions as an EMT inducer, it is a logical consequence that this pathway is upregulated. Actin filament modulation has also been closely associated with EMT [48] and the actin cytoskeleton has a vital role in completing EMT-induced alterations in the cells [49,50]. The transformations in the cell cytoskeleton are significant in several cancers. For example, changes to the actin cytoskeleton can promote metastasis [50]. Twist1 was shown to modulate the actin cytoskeleton in human glioblastoma [51]. Upregulation of the pulmonary fibrosis pathway seems reasonable since EMT also plays a role in pulmonary fibrosis [52]. ILK participates in many cell functions such as cell-extracellular matrix interactions, cell cycle, apoptosis, cell proliferation, and cell motility. ILK also has multiple functions in different cancers, such as inducing EMT [53,54]. Twist was proven to activate ILK, while in phyllodes breast tumors, ILK was shown to transmit its effects via the Twist pathway [35]. ‘Hub genes’ are genes with a high degree of connectivity in the protein–protein interaction network that are significantly enriched in transcriptional regulation. From our differential expression analysis, we were able to highlight 28 genes that share a known protein to protein interaction. In “high Twist1”- samples, the transcriptionally downregulated molecules included the B-cell lineage markers PAX5, CD19, CD22, CD20 (MS4A1) CD79a, B-cell activator cytokine TNFSF13B, and the antigen presentation marker CD40, whereas transcriptionally upregulated molecules included cell–cell interaction molecules tight junction protein 1 (TJP1) and gap junction alpha-1 protein (GJA1), cell–matrix interaction molecule integrin alpha 1 (ITGA1) with multiple extracellular matrix proteins collagen type V alpha 2 (COL5A2), decorin (DCN), fibrillin (FBN1), transgelin (TAGLN), basement membrane protein laminins (LAMA, LAMB, LAMC), and extracellular matrix cross linker lysyl oxidase (LOX). Very few studies are available about cell interaction and matrix molecules in mycosis fungoides [55,56,57]. The observed downregulation of B-cell markers contrasts with previous papers reporting a trend towards worse prognosis in the presence of over 50% of CD20 positive cells [58] and upregulation of the CD20 gene (MS4A1) in MF disease progression [59]. The downregulation of B-cell markers could result from a paucity of reactive intratumoral B-lymphocytes or true gene expression downregulation in such cells. One of the most interesting, downregulated pathways was the “Th 1 pathway”. During MF progression, the amount of Th1 cells decreases while the amount of Th2 cells increases [23,60,61]. This change in the predominance between Th1 and Th2 cells also changes the cytokine milieu of the tumor. Malignant T-cells produce immunoregulatory cytokines that repress Th1 responses and activate signaling pathways related to altered immune responses in the tumor microenvironment, further enhancing disease progression [17]. Based on hub gene analysis, one of the most strongly upregulated genes in the Twist1 high group is CD73 (NT5E), which is an integral protein in immune suppression [62]. The expression of CD22, a molecule that prevents autoimmune reactions, was reported to be expressed in MF [63] but our results were different. Surprisingly, the extracellular-signal-regulated kinase (ERK)/MAPK signaling pathway was downregulated when Twist1 expression was high. Hyperactivation of this signaling pathway was detected in cancer development and progression [64]. Previously, the upregulation of MAPKs was associated with Twist1 overexpression in breast cancer [65] and melanoma [66]. The hub gene analysis highlighted the upregulation of gap-junction protein (GJA1), which is a limiting factor in MAPK/ERK signaling [55]. In MF, malignant cells form gap junctions with Langerhans cells [67]. Other downregulated pathways included “phosphoinositide 3–kinase (PI3K) signaling in B-lymphocytes”, “ribosomal protein S6 kinase beta 1 (p70S6K) signaling”, and “triggering receptor expressed on myeloid cells 1 (TREM1) signaling systemic lupus erythematosus in B-cell-signaling pathway”. The PI3K–Akt pathway is an intracellular signal transduction pathway that promotes metabolism, proliferation, cell survival, growth, and angiogenesis in response to extracellular signals. The PI3K/Akt signaling pathway has a connection with EMT, having the ability to influence tumor aggressiveness by affecting EMT [68]. Indeed, the hub gene analysis highlighted differentially expressed EMT related adhesion and matrix proteins, which is no surprise as the DE analysis was set against high and low expression of EMT transcription factor Twist1, and additionally, they are essential to the microdissected area. In conclusion, Twist1 overexpression seems to be associated with several proteins and pathways involved in immunoregulation and lymphocyte differentiation. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. However, there were only four cases with strong Zeb1 protein expression, a fact that precludes drawing any firm conclusions from these data. One limitation of our study is that we used both diagnostic and follow-up samples. However, these two groups did not considerably differ in terms of their gene expression. In addition, the results of the DE analysis with all samples combined correlated well with those from the analysis using only diagnostic samples, thus indicating that this limitation likely did not have a major impact on the results. Additionally, the number of cases was limited; especially regarding the analyses of Zeb1 expression, limited number of cases may have hindered the detection of some existing biological differences. Additionally, using paraffin-embedded tissue may have interfered with the sensitivity of the method. The strength of our study is in the high standard data analyses as well as the use of laser-captured microdissected samples, which decreased the bias caused by non-malignant stromal and epithelial cells. However, we were not able to fully rule out the impact of dilution of genes of interest. Could there, with this approach, be a dilution of genes of interest in early-stage disease as the microdissected area represents a much larger percentage of stromal and non-neoplastic T-cells compared to MF in advanced-stage disease? We find this unlikely, since Twist1 expression did not correlate with MF staging or tumor cell density or percentage of reactive lymphocyte. Although we microdissected CD3–positive cells from the FFPE sections, cell populations with different backgrounds were inevitably collected during RNA extraction. Considering the present results compared to recent literature, we anticipate Twist1 to be a central transcription factor and pathway regulator in the disease progression of MF. Naturally, in this kind of experiment setting, we were not able to confirm causality, and our results still need to be validated in cell culture or animal models with Twist1 knockout. Nevertheless, these results suggest that Twist1 is an interesting object for developing targeted therapies for MF.

PMC10000435

Javier Feito,Carlos Araújo,Sara Arbulu,Diogo Contente,Beatriz Gómez-Sala,Lara Díaz-Formoso,Estefanía Muñoz-Atienza,Juan Borrero,Luis M. Cintas,Pablo E. Hernández

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae

02-03-2023

bacteriocins,garvicin,nisin,Lactococcus garvieae,lactic acid bacteria (LAB),heterologous production,Lactococcus lactis,probiotics,paraprobiotics,postbiotics

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains. Bacteriocins produced by lactic acid bacteria (LAB) have been largely valued as potential food preservatives, and the LAB producers of bacteriocins (bacteriocinogenic strains) have been valued as potential starter, protective, probiotic, paraprobiotic and postbiotic cultures [1,2,3]. Moreover, concerns regarding the increase in antimicrobial resistances (AMRs) confer bacteriocins and the bacteriocinogenic LAB unlimited possibilities for applications in the food industry, human and veterinary medicine and the animal production field [4,5,6]. Microbial-derived biotics, including bacteriocins, are recognized as functional components of natural and bioengineered probiotic, paraprobiotic and postbiotic cultures [2,7,8]. Bacteriocins, including nisin A (NisA) and nisin Z (NisZ), drive the apoptosis of cancer cells and show low toxicity toward normal cells, making them promising anticancer candidates to replace or be combined with conventional therapeutic agents [9,10,11]. Bacteriocinogenic LAB and the bacteriocins they produce may also have an impact in the modulation of the microbiota and immune system of their host [12,13]. Most LAB bacteriocins are synthesized as biologically inactive precursors that contain an N-terminal extension, whereas the mature peptides are often cationic, amphiphilic, membrane-permeabilizing molecules. The N-terminal extensions of most bacteriocins are of the so-called double-glycine type (leader sequence) and are cleaved off concomitantly with export across the cytoplasmic membrane by dedicated, ATP-binding cassette transporters (ABC-transporters) and their accessory proteins [14]. However, a few bacteriocins contain N-terminal extensions of the Sec-type (signal peptide), which are proteolytically cleaved concomitantly with peptide externalization by the general secretory pathway (GSP) or the Sec-dependent pathway [15]. Several bacteriocins have been shown to be synthesized without N-terminal sequences and represent bacteriocins with a novel secretion mechanism [16]. The mature bacteriocins are often classified into three main classes: class I, or lantibiotics, which have post-translationally modified amino acid residues, class II bacteriocins, which have unmodified amino acid residues, and class III large, heat-labile bacteriocins [17,18]. However, the class I bacteriocins are currently included in the group of ribosomally synthesized and post-translationally modified peptides (RiPPS) [18,19,20]. The class II bacteriocins can be further divided into the subclasses pediocin-like (class IIa), two-peptide (class IIb), leaderless (class IIc) and the non-pediocin single (class IId) bacteriocins [18,21]. In most cases, bacteriocin production appears to be regulated and involves a quorum-sensing mode of regulation [22,23]. The production of nisin is autoregulated by NisK, a sensor kinase protein, and NisR, a response regulator, which regulate transcription via signal transduction by a two-component regulatory system. The induction of NisA or NisZ is likely to be dependent on their interaction with NisK [23]. The NisI and the ATP-binding cassette transporter NisEFG support the immunity of the producer cells to nisin [24]. The presence of LAB in many ecological niches, including fish and their rearing environment, is well characterized, and the antimicrobial activity and the probiotic potential of the isolated strains is emphasized [25,26,27,28,29]. However, despite the beneficial role of most LAB, the species Lactococcus garvieae has been identified as the aetiological agent of lactococcosis, a major, re-emerging and widely distributed ichthyopathology that causes a hyperacute haemorrhagic septicaemia and relevant economic losses in rainbow trout (Oncorhynchus mykiss, Walbaum) farming [25,30]. The reported human cases of pathologies associated with this bacterium are increasing, and its zoonotic potential has been accepted [31,32,33]. Bacteriocinogenic L. garvieae that demonstrate an antagonistic activity against virulent L. garvieae strains have been isolated. Their bacteriocins have been characterized, such as garvicin A [34], garvicin AG1 and garvicin AG2 [35], garvicin KS [36], garvicin L1-5 [37], garvicin ML [38] and garvieacin Q [39]. GarA, produced by L. garvieae 21881, is encoded in the plasmid pGL5, which holds the structural gene of GarA (lgnA), its putative immunity protein (lgnI) and the ABC-transporter and its accessory proteins (lgnC and lgnD). GarA is synthesized as a 63 amino acid precursor with a typical double-glycine leader peptide. Once processed, this peptide results in a 43-aa mature peptide with a theoretical molecular mass of 4645.5 Da. GarA has a narrow activity spectrum and is limited only to other L. garvieae strains, suggesting that its mechanism of action is based on the inhibition of cell division, most likely by inhibiting septum formation in target cells [34]. GarQ is produced by L. garvieae BCC 43588 and is encoded as the GarQ structural gene (garQ), its putative immunity protein (garI) and the ABC-transporter (garT). GarQ shows a wide spectrum of antimicrobial activity against LAB and other potentially foodborne pathogenic strains [39]. GarQ is synthesized as a 70-aa precursor with a double-glycine cleavage site, resulting in mature GarQ of 50-aa and a theoretical molecular mass of 5340 Da. GarQ utilizes the IID and IIC subunits of the mannose phosphotransferase system (Man-PTS) as a receptor. It kills target bacteria by disrupting the membrane integrity, mainly locking the Man-PTS into a conformation that leads to the formation of a constitutively open pore [40,41]. NisZ, a 34 amino acid pentacyclic peptide naturally produced by L. lactis, exhibits antimicrobial activity against several Gram-positive and Gram-negative bacteria [42], and the bacteriocin exerts its antimicrobial activity by both pore formation and the inhibition of cell wall synthesis through specific binding to lipid II, which is an essential precursor of the bacterial cell wall [43]. The cloning and heterologous expression of bacteriocins by LAB, particularly Lactococcus lactis, has proven to be a promising approach for obtaining microbial cell factories with a potent antimicrobial activity [44,45,46,47,48]. Moreover, the simultaneous production of bacteriocins of different classes and/or subclasses and distinct modes of action may not only improve their antimicrobial activity and spectrum in a synergistic fashion but may also reduce the presence of bacteria that are resistant to their antagonistic activity [49,50,51]. In this study, we have proceeded to the design and expression in different L. lactis strains of up to three different bacteriocins with antimicrobial activity against virulent L. garvieae. These bacteriocins, namely, GarA, GarQ and NisA/NisZ, show different modes of action and other well-described beneficial effects, such as the anticarcinogenic effect of NisA/NisZ and its ability to modulate the microbiota and regulate the immune system of its host. Thus, synthetic genes that encode the signal peptide of the lactococcal secreted protein Usp45 (SPusp45), fused to either mature GarA (lgnA) with its putative immunity gene (lgnI) and/or to mature GarQ (garQ) with its immunity gene (garI), were cloned into the protein expression vectors pMG36c which encodes the P32 constitutive promoter and in plasmid pNZ8048c, under control of the inducible PnisA promoter. Recombinant L. lactis strains were then obtained, and their antimicrobial activity against virulent L. garvieae was determined. The bacterial strains and plasmids used in this study are listed in Table 1. The L. lactis strains were grown at 30 °C in M17 broth (Oxoid Ltd., Basingstoke, UK) supplemented with 0.5% (w/v) glucose (GM17). Pediococcus damnosus CECT4797 was grown in MRS broth (Oxoid Ltd.) at 30 °C. Escherichia coli JM109 (Promega, Madison, WI, USA) was grown in Luria–Bertani (LB) broth (Oxoid Ltd.) at 30 °C with shaking. Chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) was added at 20 µg/mL to select growth of E. coli and at 5 µg/mL for the selection of the recombinant lactococcal strains. The cell dry weights of the late exponential phase cultures were determined gravimetrically. Agar plates were made by the addition of 1.5% (w/v) agar (Oxoid) to the liquid media. Synthetic gene fragments were designed from the described amino acid sequence of the bacteriocin GarA (lgnA) and GarQ (garQ), as well as those from their putative immunity proteins GarAI (lgnI) and GarQI (garI), respectively. In addition, the leader peptide of the native bacteriocins was replaced by the signal peptide of the secreted protein Usp45 (SPusp45), a Sec-dependent protein produced by L. lactis MG1363 [45,57]. Similarly, additional sequences containing the SacI cleavage site and the P32 ribosome binding site (RBS) as well as the SacI/HindIII or the BspHI/HindIII enzyme restriction cleavage sites were added at the 5′ and 3′ ends, respectively, of the designed synthetic gene fragments. Their codon usage was adapted for its expression by L. lactis. GeneArt® supplied the synthetic genes into the carrier plasmid pMA-T (Life Technologies S.A., Madrid, Spain). The protein expression vectors pMG36c and pNZ8048c were purified from E. coli JM109 by using the NucleoSpin Plasmid Kit (Macherey-Nagel, Düren, Germany). DNA restriction enzymes were supplied by New England Biolabs (Beverly, MA, USA). Ligations were performed with the T4 DNA ligase (Invitrogen, Walthman, MA, USA). Electrocompetent L. lactis subsp. cremoris NZ9000, L. lactis subsp. cremoris WA2-67, L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 cells were obtained after successive growth in the SGGM17 medium, which consisted of of M17 (Oxoid Ltd.) supplemented with 0.5 M sucrose, glucose (0.5%; w/v) and glycine (2%; w/v). The cultures were centrifuged and resuspended in a cold wash buffer containing glycerol (20%; v/v) and 0.5 M sucrose. Aliquots of 50 µL were stored at −80 °C until further use. The primers and PCR products used for the construction of the pMG36c-derived vectors are listed in Table S1. PCR product amplifications were performed in 50 μL reaction mixtures that contained 20 ng of the synthetic gene fragments included in the carrier pMA-T vectors, 70 pmol of each primer, 1 U of Velocity DNA polymerase (Bioline Reagents, Ltd., London, UK), 10 µL of Hi-Fi buffer and 1.5 µL of 30 mM dNTP’s mix in a MJ Mini Gradient Thermal Cycler (BioRad Laboratories). PCR cycling conditions were as follows: denaturation at 98 °C (2 min), 35 cycles of denaturation–annealing–extension (98 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, respectively) and a final extension step at 72 °C (5 min). The PCR-generated fragments were purified using a NucleoSpin® Gel and PCR clean-up kit (Macherey-Nagel) for cloning and nucleotide sequencing. When required, PCR amplifications were sequenced using the ABI PRISM® BigDye® Terminator cycle sequence reaction kit and the automatic DNA sequencer ABI PRISM, model 377 (Applied Biosystems, Foster City, CA, USA) at the Unidad de Genómica (CAI Técnicas Biológicas, UCM, Madrid, Spain). Digestion of the amplified PCR products with SacI/HindIII permitted the ligation of the resulting restriction fragments into pMG36c, which was digested with the same enzymes. The resulting pMG36c-derived vectors were transformed into competent lactococcal hosts and electrotransformed with a Gene PulserTM and Pulse Controller apparatus (Bio-Rad Laboratories, Hercules, CA, USA), according to a previously described procedure [58]. Transformed cells containing the pMG36c-derived vectors pJFAI, pJFQI, pJFAIQI and pJFQIAI (Table 1), were selected for their growth with chloramphenicol and evaluated for their bacteriocinogenity. The total bacterial DNA from the transformed lactococcal strains was purified using the InstaGene Matrix (BioRad Laboratories). It was then submitted to PCR using the primers MGPJ-F and MGPJ-R. The sequencing of the generated PCR products was performed at the Unidad de Genómica (CAI Técnicas Biológicas, UCM). The primers and PCR products used for the construction of the pNZ8048c-derived vectors are listed in Table S1. The initial PCR amplification of the designed gene fragments into carrier pMA-T vectors was performed with primers GARF-BSPHI and GARAIM-R, which were designed to provide the restriction cleavage site for BspHI/HindIII. The digestion of the amplified PCR products with BspHI/HindIII permitted the ligation of the resulting restriction fragments into pNZ8048c, which was digested with NcoI and HindIII. However, it should be highlighted that construction of the pNZ8048c-derived vectors carrying GarQ or GarQ and GarA and their respective immunity proteins was achieved using a novel, PCR-based, restriction-enzyme-free cloning, or ABC cloning, method [59]. Briefly, the procedure implies the PCR amplification of three overlapping fragments, two from the pNZ8048c vector and one from the previously designed synthetic gene fragments, to generate a single, circular, pNZ8048c-derived vector by using a pair of overlapping primers. For the amplification of the appropriate gene fragments, 50 µL PCR reactions containing 100 ng of plasmid pNZ8048c or the synthetic gene fragments included in the carrier pMA-T vectors, 0.5 µmol of each primer and 25 µL of Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific, Waltham, MA, USA) were used. PCR cycling conditions were as follows: one initial denaturation step at 98 °C (30 s), 30 cycles of denaturation–annealing–extension (98 °C for 10 s, 49.1–65.8 °C for 20 s and 72 °C for 25 s, respectively) and a final extension step at 72 °C for 5–10 min). Overlapping PCRs were carried out using the three corresponding fragments as templates. Specifically, 1.5 × 1010 copies of fragments derived from amplification of pNZ8048c and 3 × 1010 copies of fragments obtained from amplification of the designed synthetic genes were used. Agarose gel electrophoresis, visualization and sequencing of the generated PCR products were performed essentially as described for the construction of the pMG36c-derived vectors. The resulting pNZ8048c-derived vectors were transformed into competent lactococcal hosts, and the transformed cells containing the pNZ8048c-derived vectors pNJFAI, pNJQI and pNJFQIAI (Table 1) were selected for their growth with chloramphenicol and evaluated for their bacteriocinogenicity. Bacterial DNA from the lactococcal transformed cells was submitted to PCR amplification with the primers NZPJ-F and NZPJ-R. The sequencing of the generated PCR products was performed at the Unidad de Genómica (CAI Técnicas Biológicas, UCM). The direct antimicrobial activity of colonies from the recombinant lactococcal strains was examined by a stab-on-agar test (SOAT) as previously described [60]. When appropriate, cultures were induced with nisin A (Sigma-Aldrich) at a final concentration of 10 ng/mL for the production of the cloned bacteriocins. Cell-free culture supernatants (CFS) were obtained by the centrifugation of cultures at 12,000× g at 4 °C for 10 min, adjusted to pH 6.2 with 1 M NaOH, filtered through 0.22 μm pore-size syringe filters (Sartorius, Göttingen, Germany) and stored at −20 °C until further use. The antimicrobial activity of the supernatants was determined by an agar diffusion test (ADT). It was further quantified by a microtiter plate assay (MPA) as previously described [60]. For the MPA, the growth inhibition of sensitive cultures was measured spectrophotometrically at 620 nm with a FLUOstar OPTIMA (BMGLabtech, Ortenberg, Germany) plate reader. One bacteriocin unit (BU) was defined as the reciprocal of the highest dilution of the bacteriocin that caused a growth inhibition of 50% (50% of the turbidity of the control culture without bacteriocin). Bacteriocins were purified as previously described using a multi-chromatographic procedure [60]. Briefly, 1 L supernatants from early stationary cultures of the recombinant lactococci were precipitated with (NH4)2SO4 (50%; w/v), desalted by gel filtration (PD-10 columns) and subjected to a cation-exchange (SP Sepharose Fast Flow), followed by a hydrophobic interaction (Octyl-Sepharose CL-4B) and reverse-phase chromatography in an ÄKTA purifier Reverse Phase Fast Protein Liquid Chromatography system (RP-FPLC), using the PepRPC HR 5/5 column. Fractions exhibiting the highest bacteriocin activity were pooled and re-chromatographed on the same column until chromatographically pure bacteriocin peptides were obtained. All chromatographic columns and equipment were obtained from GE Healthcare Life Sciences (Barcelona, Spain). Purified RP-FPLC fractions from the supernatants of the recombinant lactococcal strains were subjected to matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) and multiple reaction monitoring liquid chromatography–electrospray ionization tandem mass spectrometry (MRM-LC-ESI-MS/MS) analyses at the Unidad de Proteómica (CAI Técnicas Biológicas, UCM). Briefly, 1 μL of eluted fractions were spotted onto a MALDI target plate and allowed to air-dry at room temperature. Then, 0.8 μL of a sinapic acid matrix (Sigma-Aldrich) in 30% acetonitrile and 0.3% trifluoroacetic acid was added and allowed to air-dry at room temperature. MALDI-TOF MS analyses were performed using a 4800 Plus Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada). For the identification of bacteriocins, the MRM method evaluates a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray MS. Briefly, the purified RP-FPLC fractions of interest were dried in Speed-vac and resuspended in 20 μL of 8 M urea. The samples were reduced by adding 10 mM of dithiothreitol for 45 min at 37 °C and alkylated with 55 mM of iodacetamide for 30 min in the dark. The urea was then diluted with 25 mM of ammonium bicarbonate to obtain a molarity of less than 2. When the pH was 8.5, digestion was performed by adding recombinant sequencing grade Trypsin (Roche Molecular Biochemicals, Branchburg, NJ, USA) 1:20 (w/w) and incubating at 37 °C. After 60 min, an aliquot was taken for the partial digestion of the sample. The rest was incubated overnight. The produced peptides were dried in Speed-vac and resuspended in 2% acetonitrile and 0, 1% formic acid. Skyline (64-bit), version 20.1, was used to build and optimize the MRM for the detection of the peptides of interest [61]. All analyses were performed on a LC-MS/MS Eksigent Nanoflow LC system coupled to a hybrid triple quadrupole/ion trap mass spectrometer, 5500 QTRAP (AB Sciex, Foster City, CA, USA), equipped with a nano electrospray interface operating in the positive ion mode. The MS/MS data were analyzed using Protein Pilot 4.5 software (AB Sciex) or MASCOT 2.3 (MatrixScience, London, UK) to identify the peptides against an in-house DataBase with the fasta sequences of the targeted proteins. The searches were performed assuming a digestion with trypsin with a maximum of 2 missed cleavages, a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 0.15 Da. Peptide identifications based on the MS/MS data were accepted if they could be established at a CI of greater than 95% (p < 0.05). Data were then processed against the MRM-library on Skyline to ensure consistency between the transitions detected and the sequences of the peptides searched. In this work, synthetic genes containing the protein SPusp45, fused to mature GarA (lgnA) and its putative immunity protein GarAI (lgnI) (AI) encoded by L. garvieae 21881 [34], synthetic genes containing the protein SPusp45 fused to mature GarQ (garQ) and its putative immunity protein GarQI (lgnI) (QI) encoded by L. garvieae BCC43578 [39], and synthetic genes containing the genetic fusions SPusp45::lgnA+lgnI+garQ+garI (AIQI) and SPusp45::garQ+garI+lgnA+lgnI (QIAI), were designed for cloning into the protein expression vectors pMG36c which carries the P32constitutive promoter, and in pNZ8048c with the inducible PnisA promoter. PCR-based amplifications of the synthesized gene fragments allowed for the generation of the PCR products shown in Table S1. Cloning the PCR products A, B, C and D in pMG36c resulted in the pMG36c-derived vectors pJFAI, pJFQI, pJFAIQI and pJFQIAI, and cloning the PCR product E in pNZ8048c resulted in the pNZ8048c-derived vector pNJFAI (Table 1). Similarly, the use of a novel, PCR-based, restriction-enzyme-free cloning method [59] for cloning fragments in plasmid pNZ8048 allowed for the construction of the pNZ8048c-derived vectors pNJFQI and pNJFQIAI (Table 1). In this study, no efforts were made to omit the presence of hypothetically redundant genes that encode putative immunity proteins in the designed synthetic gene fragments. The transformation of recombinant plasmids into L. lactis subsp. cremoris NZ9000 showed that while the control NZ9000 (pMG36c) and NZ9000 (pNZ8048c) cells showed no antimicrobial activity against L. garvieae CF00021 or P. damnosus CECT4797, all the recombinant NZ9000-derived strains exhibited a measurable antagonistic effect against L. garvieae CF00021 (Table 2). Interestingly, results from both the SOAT and ADT tests showed that the recombinant strains NZ9000 (pJFAI) and NZ9000 (pNJFAI), which only encode the production of GarA, showed no antimicrobial activity against P. damnosus CECT4797. However, the recombinant strains derived from the native NisZ producer (L. lactis subsp. cremoris WA2-67) and the native NisA producers (L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24) showed a higher antimicrobial activity against P. damnosus CECT4797 than L. garvieae CF00021. The strains that only encoded the production of GarA and/or GarQ but not NisZ or NisA (NZ9000 derivatives) showed a weaker antimicrobial activity against P. damnosus CECT4797. A closer evaluation of the antimicrobial activity of the NisZ-producing strain L. lactis subsp. cremoris WA2-67 suggested that the strains WA2-67 (pJFQI) and WA2-67 (pJFQIAI) displayed slightly larger halos of inhibition against both indicator strains than the rest of the recombinant strains evaluated (Table 2). Furthermore, the evaluation of the diluted supernatants of L. lactis subsp. cremoris WA2-67, which were transformed with pMG36c or the pMG36c-derived vectors, showed that the antimicrobial activity of the recombinant strains WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI) and WA2-67 (pJFQIAI) was 2-, 8-, 2- and 16-fold higher, respectively, than the antimicrobial activity of L. lactis subsp. cremoris WA2-67 (pMG36c) (Figure 1). The evaluation of the antimicrobial activity of the NisA-producing strain L. lactis subsp. lactis DPC5598 showed that recombinants encoding the production of either GarAI, GarQI, GarAI+GarQI or GarQI+GarAI displayed similar halos of inhibition to the control strains, DPC5598 (pMG36c) and DPC5598 (pNZ8048c), against both P. damnosus CECT4797 and L. garvieae CF00021. However, supernatants of the recombinant strains, derived from the NisA producer L. lactis subsp. lactis BB24, showed slightly larger inhibition halos against L. garvieae CF00021 than the control BB24 (pMG36c) and BB24 (pNZ8048) cells (Table 2). The antimicrobial activity of supernatants from all lactococcal strains was quantified against different virulent L. garvieae strains by using a more sensitive microtiter plate assay (MPA). Again, L. lactis subsp. cremoris NZ9000 (pMG36c) showed no antimicrobial activity against any of the L. garvieae strains evaluated, while the recombinant NZ9000-derived strains transformed with the constitutive pMG36c-derived vectors showed a weak antimicrobial activity against the virulent L. garvieae strains (Table 3). However, the NisZ-producing L. lactis subsp. cremoris WA2-67 (pMG36c) showed a much higher antimicrobial activity against L. garvieae. Remarkably, the antimicrobial activity determined for the recombinant WA2-67-derived strains transformed with the pMG36c-derived vectors to generate the WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI) and WA2-67 (pJFQIAI) bacteriocin producers was 1.0- to 1.6-fold, 5.1- to 10.7-fold, 0.9- to 1.6-fold, and17.3- to 68.2-fold higher, respectively, than that of the control strain WA2-67 (pMG36c) (Table 3). The antimicrobial activity of the NisA producer L. lactis subsp. lactis DPC5598 recombinants was 0.5- to 1.2-fold higher than that of the control DPC5598 (pMG36c) strain against L. garvieae, while the antagonistic activity of the NisA producer L. lactis subsp. lactis BB24 recombinants was 1.3- to 3.7-fold higher than that of the control BB24 (pMG36c) strain against the same L. garvieae indicator strains (Table 3). When the antimicrobial activity of the lactococcal strains that were transformed with the inducible pNZ8048c-derived vectors was determined, L. lactis subsp. cremoris NZ9000 (pNZ8048c) showed no antimicrobial activity against any of the L. garvieae strains, while the recombinant NZ9000 (pNJFAI), NZ9000 (pNJFQI), and NZ9000 (pNJQIAI) strains showed a 12.8- to 18.9-fold, 7.4- to 18.1-fold and 6.9- to 14.4-fold higher antimicrobial activity, respectively, than the cells transformed with the pMG36c-derived vectors (Table 4). However, the antimicrobial activity of the L. lactis subsp. cremoris WA2-67 (pNZ8048c) cells and their recombinant WA2-67 (pNJFAI), WA2-67 (pNJFQI) and WA2-67 (pNJFQIAI) strains showed a 0.7- to 0.9-fold, 0.5- to 0.8-fold, 0.1- to 0.2-fold and 0.01- to 0.08-fold lower antimicrobial activity, respectively, against the L. garvieae strains than the cells transformed with the pMG36c-derived vectors (Table 4). The antimicrobial activity of the NisA-producing L. lactis subsp. lactis DPC5598 transformed with the pNZ8048c-derived vectors was only slightly (0.9- to 2.0-fold) higher than the antimicrobial activity of the cells transformed with the pMG36c-derived vectors. Similarly, the antagonistic activity of the NisA producer L. lactis subsp. lactis BB24 recombinants transformed with the pNZ8048c-derived vectors was only slightly (0.6- to 1.9-fold) higher than cells transformed with the pMG36c-derived vectors (Table 4). The results regarding the purification to homogeneity of the bacteriocins in supernatants of the selected L. lactis subsp. cremoris recombinants are summarized in Table 5. The evaluation of the most active antimicrobial fractions after the first reversed-phase chromatography step (RP-FPLC) permitted the identification of two active fractions during the purification of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and three active fractions during the purification of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQIAI). Although the antimicrobial activity of the eluted fractions was low, a significant increase in their specific antimicrobial activity was observed. MALDI-TOF MS analysis of fraction 8 and fraction 7, eluted during the RP-FPLC step of the purification of supernatants from L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI), showed major peaks of 3331.4 Da and 3331.3 Da (Figure S1), respectively, matching the molecular mass described for NisZ. In addition, second peaks of 3349.3 Da and 3348.8 Da, respectively, likely correspond to the oxidation of the lanthionine ring of NisZ (Figure S1). However, MALDI-TOF MS analysis of the eluted fraction 14 from the L. lactis subsp. cremoris WA2-67 (pJFQI), which encoded GarQ and NisZ, in addition to an analysis of fractions 9 and 12 from the L. lactis subsp. cremoris WA2-67 (pJFQIAI), which encoded GarA, GarQ and NisZ, could not identify the presence of the bacteriocins GarA and GarQ with predicted molecular masses of 4645.2 Da and 5340 Da, respectively, in the eluted fractions. Since this was a totally unexpected result, the fractions were subjected to MRM-LC-ESI-MS/MS analysis to determine the presence of the expected bacteriocins in the samples. In the MRM method, a series of target tryptic peptides and their associated transitions (fragments m/z) were predicted from the molecular masses and amino acid sequences of the bacteriocins GarA and GarQ. Each targeted peptide has a set of accompanying transitions which are then selectively detected in the second stage of the MS. A summary of the results obtained is shown in Table 6. MRM transitions were established and validated by tandem mass spectrometry (MS/MS). For each bacteriocin, two encrypted peptides were confidently detected in duplicate runs. Virulent L. garvieae are the etiological agents of a hyperacute hemorrhagic septicemia in fish, known as lactococcosis. They are also responsible for human pathologies due to their zoonotic character and potential presence in foods [31,33]. Bacteriophages and bacteriocins have potential as complementary strategies for combating L. garvieae in foods and fish [26,27], while bacteriocinogenic LAB could be evaluated for their potential use as probiotics, paraprobiotics and postbiotics in food, feed and other biotechnological applications [7,53,62]. The optimization of bacteriocin gene synthesis, expression and production helps the development of LAB as cell factories for the production and delivery of multiple bacteriocins [45,46,63]. The use of synthetic genes that match the codon usage of the producer organisms has a significant impact on gene expression levels and protein folding [47,64]. In this work, the transformation of L. lactis subsp. cremoris NZ9000 with pMG36c- or pNZ8048c-derived vectors demonstrated that the NZ9000 (pJFAI) and NZ9000 (pNJFAI) recombinant cells, which encode GarAI, showed antimicrobial activity against L. garvieae CF00021 but not against P. damnosus CECT4797 (Table 2), confirming their production of GarA and previous observations that this bacteriocin was only active against L. garvieae [34]. However, differences in the antimicrobial activity of recombinants derived from the NisZ producer L. lactis subsp. cremoris WA2-67, which was transformed with the pMG36c-derived vectors but not pNZ8048c-derived vectors, were observed against both indicator strains. The obtained results showed that the WA2-67 (pJFQI) cells exhibited larger halos of inhibition than the WA2-67 (pJFAI) cells, and that the WA2-67 (pJFQIAI) cells displayed the largest observed halos (Figure 1). These results suggest that the constitutive expression of GarQI is higher than GarAI or that the specific antimicrobial activity of GarQ is higher than that of GarA. Perhaps the transcription, processing and secretion from genes encoding GarQI+GarAI are more effective than from genes encoding GarAI+GarQI. On the other hand, no remarkable differences were found in the antimicrobial activity of the recombinants derived from the NisA producers transformed with the pMG36c-derived or the pNZ8048c-derived vectors, L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 (Table 2). Due to the increasing interest that L. garvieae is attracting as not only a relevant bacterial pathogen but also as a zoonotic agent [29,65,66], the antimicrobial activity of the L. lactis recombinants was further evaluated and quantified against different virulent L. garvieae strains by using a more sensitive microplate assay (MPA). The obtained results showed that the L. lactis subsp. cremoris NZ9000 cells transformed with the pNZ8048c-derived vectors showed a 6.9- to 18.9-fold higher antimicrobial activity than the recombinant cells bearing the pMG36c-derived vectors (Table 3 and Table 4). The enhanced antimicrobial activity in cells with the nisin-inducible constructs may be due to copy number differences between pNZ8048c and pMG36c, but is more likely caused by the promoters used to drive gene expression [67,68]. Plasmid pNZ8048c contains the high-copy number heterogramic replicon of the lactococcal plasmid pSH71 with a unique NcoI cleavage site downstream of the nisA ribosome binding site (RBS), which is used for translational fusions inducible by NisA [23,69]. To optimize protein production, inducible systems are usually considered superior to constitutive expression systems since the former allow for the achievement of a sufficient biomass before the initiation of target protein expression [70]. The increased antimicrobial activity observed with the NisA-induced cells may also be ascribed to the short induction time for the production of GarA and/or GarQ (3 h), which most likely prevented the secreted bacteriocins from attaching to cell walls to form aggregates and/or to undergo protease degradations. Moreover and quite remarkably, the antimicrobial activity of L. lactis subsp. cremoris WA2-67 (pMG36c) and the pMG36c-derived WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI), and WA2-67 (pJFQIAI) strains showed a 1.0- to 1.6-fold, 5.1- to 10.7-fold, 0.9- to 1.6-fold and 17.3- to 68.2-fold higher antimicrobial activity, respectively, than the control WA2-67 (pMG36c) strain (Table 3). These results also indicate that the expression of QI increases the antimicrobial activity of the producer cells; however, the expression of AI has a much lower effect. Additionally, no increase in antimicrobial activity is observed when QI is expressed as the second of the two modules (AIQI). However, when QI is expressed as the first module (QIAI), a synergistic effect of both modules seems to occur regarding the very high antimicrobial activity of the producer cells. Thus, the AI in the AIQI module appears to prevent the QI from becoming active. However, when QI is expressed first in the QIAI module, the AI appears to synergistically increase the QI activity (Table 3). The pMG36c vector is a shuttle vector. It is based on the low-copy replication origin of pWV01 and is able to replicate in Escherichia coli, Bacillus subtilis and LAB, whereas the strong P32 promoter drives the constitutive transcription of inserted genes into the multicloning site (MCS) of pUC18 [56]. From the results obtained, it may occur that, as previously suggested, the specific antimicrobial activity of GarA against L. garvieae is lower and/or its production and stability is less than that of GarQ. Additionally, besides the choice of vector and promoters, other factors such as the activation of quality control networks involving folding factors and housekeeping proteases, the oxidation of methionine to methionine-sulfoxide, bacteriocin self-aggregation and mRNA stability may affect bacteriocin production and activity from the recombinant hosts [46,71]. The coexpression of putative immunity genes may also increase the production of bacteriocins in heterologous hosts. These immunity proteins can act by either affecting bacteriocin pore formation or by perturbing the interaction between the bacteriocin and a membrane-located bacteriocin receptor, thereby preventing producer cells from being killed [16]. The expression in AIQI of LgnI before the expression of GarI could also affect producer protection against GarQ, thereby affecting growth and bacteriocin production by the producer cells. Importantly, L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) showed the highest antimicrobial activity against all virulent strains of L. garvieae evaluated (Table 3). However, the antimicrobial activity of L. lactis subsp. cremoris WA2-67, which was transformed with the pNZ8048c-derived vectors, produced WA2-67-derivatives that showed a 0.01- to 0.9-fold lower antimicrobial activity, respectively, than the cells transformed with the pMG36c-derived vectors (Table 4). This was an unexpected result since, as previously described, inducible systems are often considered superior to constitutive expression systems for the optimization of protein production. Perhaps NisK and NisR, the two component signal transduction systems for the regulation of NisZ synthesis in L. lactis subsp. cremoris WA2-67, do not fully activate transcription of the PnisA present in pNZ8048c. It could be also possible that levels of phosphorylated NisR may be not enough to drive the activation of two independent Pnis promoters which, in addition, derive from two different Lactoccocus lactis subspecies: cremoris and lactis, respectively. Alternatively, NisZ may not be as efficient as NisA for an interaction with the NisK produced by L. lactis subsp. cremoris WA2-67, thus constraining the induction of transcription of PnisA in pNZ8048c by blocking NisZ with NisI and NisEFG. The antimicrobial activity of the NisA-producers L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 was slightly higher for the cells transformed with the pNZ8048c- than the pMG36c-derived vectors, suggesting that NisA is a better inducer than NisZ for the activation of the transcription of PnisA in pNZ8048c (Table 3 and Table 4). However, the antimicrobial activity of the DPC5598-derived recombinants under constitutive or inducible conditions was lower during multi-bacteriocin production. This was probably due to the high energy and metabolic cost linked to plasmid maintenance and replication, to the secretion stress associated with bacteriocin overproduction and/or to the synthesis of proteinases for the elimination of misfolded proteins [72,73,74]. Differences in the antimicrobial activity of these strains and those from the L. lactis subsp. cremoris WA2-67 transformed with the pMG36c-derived vectors may be also ascribed to yet-unknown genetic and/or metabolic differences between the strains. L. lactis subsp. lactis DPC5598 was selected as a potential multi-bacteriocin-producing host because it is a plasmid-free derivative of an industrial strain that is extensively used in fermented dairy products due to its phage insensitivity and fast acid-producing ability [54]. L. lactis subsp. lactis BB24 is a fermented, meat-derived isolate widely used as an efficient host for the heterologous production of bacteriocins [45,55]. Both multi-bacteriocin producers should be considered for their potential evaluation as probiotics, paraprobiotics and/or postbiotics reducing the increasing presence of virulent and zoonotic L. garvieae in selected milk and meat substrates, respectively [31,75]. The MALDI-TOF MS analysis of purified eluted fractions from supernatants of the most active antimicrobial strains, L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) (Table 5), allowed for the detection of NisZ in supernatants of the producer strains (Figure S1), suggesting that this bacteriocin is appropriately processed and transported out of the producer cells. However, the presence of GarA and GarQ could not be detected, suggesting interactions of the bacteriocins with unknown biological compounds, or a low amount and recovery of the bacteriocins during their purification to homogeneity [47,64]. However, bacteriocins in the purified fractions were suitable for MRM evaluation and data analysis, which is an emerging targeted proteomics workflow and a highly selective and sensitive method for detecting peptides in the low ng/mL to sub-ng/mL range concentrations [64,76,77]. When the purified fractions were subjected to MRM-LC-ESI-MS/MS analysis, two encrypted peptides were confidently (99%) detected. The detected peptides were confirmed by MS/MS, and at least four transitions were identified for each (Table 6). The peptide fragments covered 44% of the sequence of GarQ, while the coverage was 21% for GarA. These relatively low-coverage percentages are related to the reduced presence of lysine and arginine (trypsin cleavage sites) in GarA and GarQ, which significatively reduces the number of potentially identifiable target peptides. Previous studies by our research group identified probiotic features of the native or wild-type NisZ producer L. lactis subsp. cremoris WA2-67 such as a potent antimicrobial activity against ichthyopathogens, survival in fresh water and the gastrointestinal tract of trout, a resistance to bile and low pH, and an improved colonization ability with respect to the intestinal trout mucosa [26,53,78]. Further in silico analyses of the whole-genome sequence (WGS) of this strain also identified other potential probiotic traits such as the production of vitamins and amino acids, adhesion/aggregation and stress resistance factors and the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities or potential virulence factors [79]. Other studies performed by our group demonstrated the effectiveness of L. lactis subsp. cremoris WA2-67 to protect rainbow trout in vivo against infection of the virulent L. garvieae and the relevance of NisZ production as an anti-infective mechanism [53]. The work described in this study constitutes the first report on the design of multi-bacteriocinogenic L. lactis subsp. cremoris WA2-67 strains with a high antimicrobial activity against virulent L. garvieae and a promising role as probiotics, paraprobiotics and/or postbiotics in food, feed and other biotechnological applications. The evaluation of bioengineered strains as probiotics is subjected to approval by regulatory authorities and is performed under strict biological conditions. However, the number of reports on the evaluation of bioengineered bacterial strains as probiotics (live cells), paraprobiotics (dead, non-viable cells) and postbiotics (physical-, chemical- or enzymatic-lysis of probiotic cells) is increasing [7,80,81]. Accordingly, experiments are being planned to evaluate the in vitro effect of L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) on rainbow trout intestinal epithelial cells (RTgutGC) for a transcriptional analysis of several immune, intestinal, barrier-integrity and homeostasis genes and the induction of antimicrobial peptides (AMPs), as well as for their effect on the in vivo modulation of the intestinal microbiota and immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) and turbot (Scophthalmus maximus). The design of synthetic genes and their cloning into protein expression vectors bearing constitutive or inducible promoters has allowed for the production and functional expression of GarA and/or GarQ by L. lactis subsp. lactis and L. lactis subsp. cremoris strains. Most importantly, L. lactis subsp. cremoris WA2-67, transformed with the pMG36c-derived vectors, allowed for the obtention of L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, with a much higher antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae than the rest of the L. lactis strains evaluated. The concerted use of a sensitive microtiter plate assay (MPA) for the quantification of the antimicrobial activity of supernatants, the use of a multi-chromatographic procedure for the purification of bacteriocins to homogeneity, and the use of a MALDI-TOF MS multiple reaction monitoring (MRM-LC-ESI-MS/MS) analysis of the purified bacteriocins are unavoidable and possibly irreplaceable tools for the identification and characterization of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI).

PMC10000437

Xiaojing Wei,Leilei Yu,Chuan Zhang,Yongqing Ni,Hao Zhang,Qixiao Zhai,Fengwei Tian

Genetic-Phenotype Analysis of Bifidobacterium bifidum and Its Glycoside Hydrolase Gene Distribution at Different Age Groups

22-02-2023

glycoside hydrolase (GH),Bifidobacterium bifidum,6’-sialyllactose,comparative genomics,genotype,phenotype

Human gut microbiota interfere with host development and aging. Bifidobacterium is a microbial genus found in the human digestive tract that has probiotic activities such as improving constipation and enhancing immunity. The species and numbers present change with age, but there has been limited research on probiotic gut microbiota at specific ages. This study analyzed the distribution of 610 bifidobacteria in subjects in several age groups (0−17, 18−65, and 66−108 y) using 486 fecal samples and determined the distribution of glycoside hydrolases based on genetic analysis of strains representing 85% of the Bifidobacterium species abundance in each age group. 6’-Sialyllactose is a major component of acidic breast milk oligosaccharides, which can promote human neurogenesis and bifidobacteria growth. Using genotypic and phenotypic association analysis, we investigated the utilization of 6’-sialyllactose by six B. bifidum strains isolated from subjects 0–17 and 18–65 y. A comparative genomic analysis of the six B. bifidum strains revealed differences in genomic features across age groups. Finally, the safety of these strains was evaluated by antibiotic gene and drug resistance phenotype analysis. Our results reveal that the distribution of glycoside hydrolase genes in B. bifidum varies with age, thus affecting the phenotypic results. This provides important insights for the design and application of probiotic products for different ages.

Genetic-Phenotype Analysis of Bifidobacterium bifidum and Its Glycoside Hydrolase Gene Distribution at Different Age Groups Human gut microbiota interfere with host development and aging. Bifidobacterium is a microbial genus found in the human digestive tract that has probiotic activities such as improving constipation and enhancing immunity. The species and numbers present change with age, but there has been limited research on probiotic gut microbiota at specific ages. This study analyzed the distribution of 610 bifidobacteria in subjects in several age groups (0−17, 18−65, and 66−108 y) using 486 fecal samples and determined the distribution of glycoside hydrolases based on genetic analysis of strains representing 85% of the Bifidobacterium species abundance in each age group. 6’-Sialyllactose is a major component of acidic breast milk oligosaccharides, which can promote human neurogenesis and bifidobacteria growth. Using genotypic and phenotypic association analysis, we investigated the utilization of 6’-sialyllactose by six B. bifidum strains isolated from subjects 0–17 and 18–65 y. A comparative genomic analysis of the six B. bifidum strains revealed differences in genomic features across age groups. Finally, the safety of these strains was evaluated by antibiotic gene and drug resistance phenotype analysis. Our results reveal that the distribution of glycoside hydrolase genes in B. bifidum varies with age, thus affecting the phenotypic results. This provides important insights for the design and application of probiotic products for different ages. Billions of bacteria colonize the human gut, with effects that are strongly linked to human health [1]. Different microbial communities colonize different parts of the human body [2], and the most densely colonized body part is the intestinal tract [3]. As humans age, the gut microbiota proceed through a series of phase changes. In early childhood, there is a dynamic gut microbial composition, while adults have a relatively stable flora, and the numbers of the intestinal microbiota in the elderly gradually decrease and health-promoting functions decline [4,5]. There is a close association between age-related changes in immunity, dysbiosis, disease, and probiotic-based products, and utilizing this association in combating age-related disease is an attractive proposition [6]. Some studies have proposed modifying the gut microbiome with live microbes, and clinical trials have demonstrated age-dependent beneficial effects of the consumption of LcZ [7,8]. Given the importance and complexity of the gut microbiota, it is critical to gain an understanding of compositions, patterns, and the laws of intestinal microecology to better understand human health and disease. Intestinal physiological changes due to age-related factors have an inevitable impact on the structure of the gut microbiota. Bifidobacteria are dominant microorganisms colonizing the gut in early life, with a strong competitive advantage due to an ability to break down human milk oligosaccharides (HMOs) [9,10]. The predominant Bifidobacterium species in the early infant gut is Bifidobacterium breve, along with B. bifidum and B. longum subsp. infantis [11,12]. Macrogenomic sequencing revealed a high abundance of B. longum subsp. longum and B. pseudobulbarum in the gut of breastfed infants [13,14]. With a decrease in breastfeeding and a gradual increase in solid food intake, the composition of the infant intestinal microbiota begins to shift toward that of the adult gut [15]. The composition of Bifidobacterium species in the adult gut is more complex. Among the Bifidobacterium species present are B. longum subsp. longum, B. adolescentis, and B. catenulatum, as well as a lower-abundance B. bifidum and B. breve [16]. The numbers of bifidobacteria in the intestinal microbiota of the elderly are generally lower than in that of non-elderly adults, and the diversity of bifidobacteria is significantly reduced primarily due to a loss of B. adolescentis, B. longum subsp. longum, and B. angulatum [17]. Bifidobacterium uses non-digestive carbon sources and has cross-intertrophic effects on nutrients with other intestinal microbiota to maintain gut homeostasis. More than 13% of the genes in the bifidobacteria genome are involved in carbohydrate metabolism, with glycoside hydrolase being the most important component. Given the various glycoside hydrolases carried by different species, the types of carbohydrates in the gut have a significant impact on Bifidobacterium composition [18]. Through the analysis of bacterial comparative genomics, the relationship between their genomic and phenotypic features can be determined, thus facilitating further examination of the related molecular mechanisms. The whole genome sequencing and bioinformatics analysis of B. bifidum PRL2010 published in PNAS in 2010 revealed the unique degradation and utilization mechanism of B. bifidum mucin oligosaccharides, helping people understand the unique mechanism of B. bifidum intestinal adaptation [19,20]. Currently, the available bifidobacteria genome in the NCBI database allows us to conduct genomic analyses of strain evolutionary relationships, functional conservation, and variability. Arboleya et al. [21] reported genomic data for B. longum, revealing a rich complement of glycosyl hydrolase genes leading to strong polysaccharide utilization ability. Using comparative genomics, Duranti et al. [22] demonstrated a lack of genes for metabolism of host-derived polysaccharides (such as HMOs or mucin) in the B. adolescentis genome, explaining at a genetic level why B. adolescentis is abundant in the adult intestine. Using comparative genomic analysis, Lu et al. [23] found that core enzymes isolated from B. bifidum strains from different ecological niches accounted at a molecular level for an ability to metabolize host-derived polysaccharides. In contrast to other Bifidobacterium species, B. bifidum has extracellular glycosidases that can degrade many host-derived glycans, including HMOs, glycan chains of high-molecular-weight glycoproteins, and glycosphingolipids [24,25]. Sialic acids are constituents of HMOs and include sialyloligosaccharides [26] such as 6′-sialyllactose (6′-SL) and 3′-SL as well as N-acetylneuraminic acid (Neu5Ac). These compounds play an important role in nerve development. The extracellular degradation of sialylated HMOs by B. bifidum JCM1254 is one example of a broader metabolic activity of bifidobacteria [27]. B. bifidum not only utilizes polysaccharides in the gastrointestinal tract through its own GH activity but also benefits the growth of other microorganisms by breaking down polysaccharides into monosaccharides. Comparative studies of the genomes of B. bifidum strains require additional strains for validation. Existing studies on strain–age relationships are also limited. Various studies have shown that an imbalance in human intestinal bifidobacteria can have adverse effects on health. As the ability of bifidobacteria to metabolize plant- and host-derived carbon sources is the basis of their long-term colonization of the intestinal tract, it is critical to investigate the carbohydrate utilization abilities of bifidobacteria present at different stages of human life. The data obtained can be used as a reference for targeted intervention to assist the intestinal growth of Bifidobacterium and to provide new ideas for designing probiotic products for different age groups. There is a need to collect more genome-level information on Bifidobacterium and conduct in-depth comparative analyses to better understand the relationship between the intestinal microbiota, host age, and health. The distribution of 610 bifidobacteria from three age groups in the strain library was counted (Table 1). Reference strains were from the food biotechnology research center of Jiangnan University. The collection of fecal samples was approved by the Ethics Committee of Jiangnan University, China (SYXK 2012–0002). Based on the occurrence of Bifidobacterium spp. in each age group, we selected strains representing a summed abundance of bifidobacteria in each age group of >85%, comprising five species each for the age 0–17 y and 18–65 y groups, and four species for the age 66–108 y group. Three bifidobacterial strains were selected at random for each species, comprising a total of 42 strains. Strains preserved at −80 °C were thawed and inoculated onto 2% mMRS culture medium and grown in an anaerobic incubator for 24–48 h. Purified single colonies were taken up in 0.1 mL medium, incubated for 48 h, then grown in mMRS liquid medium supplemented with 0.5% L-cysteine for 24–48 h. Samples (1 mL) were centrifuged (6000 rpm for 3 min). The supernatants were discarded, and pellets were washed twice with 1.5 mL sterile water. Pellets were resuspended in 1 mL sterile water, and samples were used as templates for strain identification. 16S rRNA PCR amplification was performed as previous reported [28]. Amplified products were subjected to nucleic acid electrophoresis, and the single band on the agarose gel was excised and sent to GENEWIZ Co., Ltd. (South Plainfield, NJ, USA). for sequencing. 16S rDNA sequences were used for NCBI BLAST alignment. Strains were cultured in mMRS liquid medium at 37 °C for 24–48 h and then pelleted for genomic DNA extraction. Genomic DNA was extracted using a bacterial DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The extracted genomic DNA was tested for quality using agarose gel electrophoresis (1% gel concentration), purity using a UV spectrophotometer, and concentration using a QubitTM 4 fluorometer and a Qubit DNA Assay Kit (Life Technologies, Carlsbad, CA, USA). The genomes of 42 Bifidobacterium strains were sequenced at Majorbio BioTech Co., Ltd. using the Hiseq X Ten platform (Illumina, San Diego, CA, USA). Qualified bacterial genomic DNA (700 ng) was used for the construction of sequencing libraries using an NEB Next Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). After library construction, library sequences were clustered using the HiSeq 4000 PE Cluster Kit (Illumina) and sequenced using the Hiseq 4000 platform (Illumina). A paired-end read of 150 bp was selected for the sequencing library. High-quality paired-end reads were spliced using SOAPdenovo2, and internal gaps were filled using GapCloser [29]. The open reading frame (ORF) of Glimmer 3.02 was used, and gene sequences were translated into amino acid sequences using the Transeq tool in EMBOSS6. Following the method of Pan et al. [30], functional gene annotation of predicted genes (COG and NR databases) was performed using Prokka or BLASTX. Pan-genome and core gene analyses were performed using PGAP-1.2.1. Glimmer 3.02 and GeneMarkS [31] were used to obtain the GC content, the raw amino acid sequence (.faa), and the nucleotide sequence (.fna) of each genome. Results were base-annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), and Swiss-Prot databases. Protein sequences for all strains were obtained using the dbcan2 metagenome tool (http://bcb.unl.edu/dbCAN2/, accessed on 18 March 2021). Carbohydrate-active enzyme annotations were performed using Hmmer. Annotated glycoside hydrolases were coded and classified using EC data (http://www.ebi.ac.uk/, accessed on 18 March 2021). Frozen bifidobacterial cultures (containing carbohydrate-active enzymes in the GH33 family) were added to 5 mL mMRS medium (2% (v/v) inoculation), placed in an anaerobic workstation, and cultured at 37 °C for 24–36 h. After 3 generations of continuous subculturing, bacteria were collected via centrifugation, washed, and resuspended in an equal volume of sterilized normal saline. The bacterial suspension was inoculated into culture medium with unique carbon sources and the resuspended cultures were added to three 96-well plates (2 mL capacity) and incubated in a fully automated enzyme labeling instrument in an anaerobic workstation at 37 °C for 40 h. OD600 values were measured at every other period. The experiment was conducted in triplicate. 6′-Sialyllactose used in the carbon source medium was purchased from Shanghai HuicH Biotechnology Co., Ltd. (Shanghai, China) and had a purity of 98% by HPLC and a degree of polymerization of 23, 6′-Sialyllactose was added to the medium at a final concentration of 0.5% (w/v) [21]. Orthologs from six B. bifidum strains were defined using OrthoMCL with default parameters [32]. The core gene sequences chosen were aligned using PGAP-1.2.1. Pairwise average nucleotide identity (ANI) values of B. bifidum genomes were calculated and visualized using R (Version 3.6.1). Orthologous proteins, carbohydrate-active enzymes, virulence factors, and antibiotic factors were annotated using the COG, CAZyme, Gene Ontology (GO), ANI Calculation, comprehensive antibiotic research (CARD), and Virulence Factor (VFDB) databases using BLAST with default parameters. Information about the six B. bifidum strains and quality data for each genome (genome size, GC content, genomic level, and gene number) [33]. Antibiotic resistance genes were predicted to be present in the B. bifidum genome by comparing the amino acid sequences of six B. bifidum strains to those registered at CARD (http://arpcard.mcmaster.ca, accessed on 25 April 2021) [34]. The antibiotic susceptibility of B. bifidum strains was determined using a disk diffusion method, as recommended by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2012). Drugs and concentrations were taken from the experimental method by Jin et al. [35], and all drugs were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). B. bifidum strains were categorized as resistant, susceptible, or intermediate, according to inhibition zone diameters. Growth curve data were analyzed and visualized using GraphPad Prism 9.1. Excel 2021 was used to analyze the distribution of bifidobacteria. R (Version 3.6.1) software was used to compare genomic data. The heat map of Bifidobacterium glycoside hydrolase gene distributions was constructed using Helm V1.0.s [36]. The identification of gut microbiota using 16S rRNA sequencing is only accurate at a genus level, while GroEL sequencing can distinguish bifidobacteria at a species or subspecies level. In this study, 610 strains of bifidobacteria were identified in 446 fecal samples using GroEL sequencing. The numbers of bacterial strains in subjects from age groups 0–17, 18–65, and 66–108 y was 260, 244, and 106, respectively. A total of nine species of Bifidobacterium was identified across all samples (Figure 1a), of which 250 B. longum strains included two clearly identified subspecies: B. longum subsp. infantis and B. longum subsp. longum; while unidentified subspecies of B. animali and B. augulatum were not isolated from the 66–108 age group but were present in the other two age groups. B. breve was the most abundant bifidobacterium in the 0–17 y age group, accounting for 33% of all Bifidobacterium species in this group. Other species with high abundance were B. bifidum, B. longum subsp. longum, B. longum subsp. infantis, and B. pseudocatenulatum. The most abundant species in the 18–65 y age group was B. longum, accounting for 49% of the bifidobacteria in this group (including two well-identified subspecies, B. longum subsp. infantis and B. longum subsp. longum, as well as an unknown subspecies), followed by B. adolescentis (18%), B. pseudocatenulatum, and B. bifidum. The most abundant species in the 66–108 y age group was B. longum subsp. longum, accounting for 65% of the bifidobacteria isolated from subjects in this age group, which was followed by B. pseudocatenulatum (14%), B. bifidum, and B. breve (Figure 1b). An ability to utilize carbon sources in the gut is an important factor in determining the abundance of gut microbes. The hydrolysis of carbohydrates in the gut by GHs is a key step allowing utilization by microbes, whereby the substrate specificities and numbers of glycoside hydrolases encoded by microorganisms determine the range of available carbon sources. To evaluate the carbohydrate utilization ability of bifidobacteria from subjects in different age groups, we selected strains representing a summed abundance of bifidobacterial species in each age group of >85%, including five species from the 0–17 and 18–65 y age groups and four species from the 66–108 y age group. A total of 42 strains of Bifidobacterium were selected. We used the dbCAN server to assess the presence of genes encoding glycosyl hydrolases and annotated genes based on GH hidden Markov models (HMMs) generated from data from the CAZy database. Forty-nine families of glycoside hydrolase were found encoded in the genome. Heat maps are shown in Figure 2. The heart icon represents the 0–17 y age group, the oval icon represents the 18–65 y age group, and the firework icon represents the 66–108 y age group. Red represents B. breve, green represents B. bifidum, black represents B. pseudocatenulatum, yellow represents B. longum subsp. infantis, blue represents B. adolescentis, and purple represents B. longum subsp. longum. Statistical analysis showed that there was no significant difference in the average number of glycoside hydrolase genes among species isolated from different age groups. However, it is of interest to note that B. pseudocatenulatum has the highest average number of carbohydrase genes among species taken from all age groups, which is followed by B. longum subsp. longum. The average number of carbohydrase genes in B. bifidum was the lowest among species taken from all age groups, being 38, 34, and 36 in samples from subjects in the 0–17, 18–65, and 66–108 y age groups, respectively. The genomes of all strains encoded the GH2, GH13, GH36, GH42, and GH77 families of glycosidase hydrolases, and they were predicted to be primarily amylases as well as some β-galactosidases that are involved in degrading most of the non-digestible carbon sources in staple foods and dairy products. These glycosidases are essential for the proliferation of Bifidobacterium and can be considered core bifidobacterial glycoside hydrolases. Bifidobacterium genomes also encode glycoside hydrolases associated with the degradation of host-derived carbon sources, and these are encoded in the largest number in the genomes of B. bifidum and B. longum subsp. infantis. For example, B. bifidum and B. longum subsp. infantis encode an average of four β-hexosaminidase genes in the GH20 family, but B. adolescens does not possess genes for this enzyme. α-Fucosidases of the GH95 family are encoded in the genomes of B. bifidum, B. longum subsp. infantis, B. breve, and some strains of B. pseudocatenulatum, and they are involved in the degradation of 2′-fucosyllactose. B. longum subsp. infantis encodes an average of two sialidases from the GH33 family, which is possibly related to the degradation of sialyllactose or other sialic-acid-modified glycans in HMOs. The three strains of B. bifidum encode an average of two sialidases from the GH33 family in ages 0–17 y, but B. bifidum from subjects of age 18–65 y does not encode these enzymes, and there is only an average of one sialidase from the GH33 family in samples from the 66–108 y age group. Different glycoside hydrolases endow Bifidobacterium with specific carbon source utilization abilities, provide a unique ecological adaptation mechanism, and affect interactions with other species. 6’-Sialyllactose is primarily found in human milk and mammalian tissues. It has many biological functions and is an important component of glycoproteins and glycolipids with functions in cell recognition and immune responses. In this study, 6’-sialyllactose (structure in Figure 3b) was used as a sole carbohydrate source to evaluate the utilization phenotype for 6’-SL utilization of different Bifidobacterium species containing the GH33 gene (Figure 2). The results of the phenotypic experiments are given in Table 2. All six strains of B. breve were able to utilize 6’-SL, although strain FBJCP1M6 was less able to use it than the other strains. The six strains of B. longum subsp. infantis were able to grow well in the medium with 6’-SL as a sole carbon source. The phenotypic utilization of 6’-SL by B. bifidum in different age groups was mainly studied. Six B. bifidum strains from the 0–17 and 65–108 y age groups were able to use this acidic oligosaccharide, but the three B. bifidum strains from subjects in the 18–65 age group showed no obvious growth consistent with the genotype of B. bifidum in terms of glycoside hydrolases from the GH33 family, as shown in Figure 3a. The corresponding growth curves of these strains are shown in Figure 3c. Therefore, further investigation was conducted to determine whether three B. bifidum strains, FJSNJ1M3, FZJHZD4M4, and FAHWH21M3, from subjects in the 0–17 y age group (encoding an average of two sialidases from the GH33 family) and three B. bifidum strains from subjects in the 18–65 age group that lacked genes for the GH33 family of sialidases, were different at a genomic level. Comparative genomics was used to explore differences in the genomes of six strains of B. bifidum strains from subjects in the 0–17 and 18–65 y age groups. As shown in Table 3, the average genome size for the six bifidobacteria is 2.14 MB, and the average GC content is 62.6%. GC content and genome size are considered to be closely related to bacterial genome evolution and energy metabolism. Changes in the bacterial genome size may explain changes in bacterial carbohydrate metabolism and amino acid metabolism. The genomes of six B. bifidum strains were analyzed for pan- and core genes (Figure 4a). The results showed that the number of pan-genes increased with the number of strains, while the number of core genes tended to stabilize. When the sixth strain was added, the number of pan-genes stabilized at 2488 and the number of core genes reached 1533. The asymptotic trend of the pan-genomic curve may indicate that B. bifidum has an open pan-genome. The specific core genes and homologous core genes of B. bifidum strains were determined, and a Wayne diagram was drawn (Figure 4b). The analysis using OrthoMCL showed that the six strains had 1533 common core genes with numbers of unique specific genes, ranging from 16 to 124. Although there are some differences in age-specific genes, the results showed that the specific genes in the three strains of B. bifidum strains from subjects in the 0–17 y age group were present in significantly lower numbers than those in B. bifidum from subjects in the 18–65 age group. Between-strain ANI values can determine the similarity of two genetic sequences at a genomic level. ANI values for the six B. bifidum strains were calculated, and it was found that the distribution of ANI values within the B. bifidum group was consistent, ranging from 99% to 100%. As shown in Figure 4c, ANI values were more similar within a single age group. The results show that B. bifidum genomes from subjects in the 0–17 y age group are more similar to each other than to those of B. bifidum strains in the 18–65 age group. To distinguish whether the distribution of functional genes in the B. bifidum genome differed among samples from the different age groups, the six B. bifidum genomes were functionally annotated using the COG database and analyzed statistically for the two age groups (Figure 5a). By comparing the distribution of COG genes in the two age groups, it was found that gene classes J (Translation, ribosomal structure), G (Carbohydrate transport), M (Cell wall biogenesis), K (Transcription), and T (Signal transduction) were higher in the genome of B. bifidum from subjects of age 0−17 y than from subjects of age 18−65 y, while gene classes A (RNA processing), N (Cell motility), U (Intracellular trafficking), O (protein turnover), and L were lower in subjects of age 0–17 y than in those of ages 18−65 y. Carbohydrate metabolic pathway expression was significantly greater in B. bifidum from subjects aged 0–17 y than in B. bifidum from subjects aged 18–65 y. B. bifidum in the two age groups showed no significant differences in GO enrichment. Most of the differentially distributed genes had catalytic activity and metabolism-related functions (Figure 5c). Six B. bifidum strains encoding a large number of carbohydrate-active enzymes (CAZymes), including GTs, GHs, CEs, and CBMs, of which, GHs were the most abundantly distributed (Figure 5b). Antibiotic tolerance is important in maintaining the abundance of gut symbiotic bacteria, but the horizontal transfer of resistance genes between gut microbes may lead to the generation of deleterious antibiotic-resistant pathogenic bacteria. Using antibiotic resistance testing, this study evaluated the safety of six strains of B. bifidum obtained from subjects of different ages, providing a reference for future application in the probiotics industry. The B. bifidum genomes were annotated using the CRAD database to determine whether they contained potential antibiotic resistance genes. As shown in Figure 6a, each strain carried an average of 97 antibiotic resistance genes of 73 different types, including a macrolide resistance gene (macB), which had the highest content in all strains, which was followed by the lincosamide resistance genes (lmrB and lmrD), aminoglycoside resistance genes (baeS), and glycopeptide resistance genes (vanHF and vanHM). By comparing the total numbers of resistance genes in six B. bifidum isolates from subjects of different ages, the result showed that the resistance genes in B. bifidum isolates from the 0–17 y group were higher than those from the 18–65 age group (Figure 6b). Among these, the number of resistance genes in B. bifidum FZJHZD4M4 and B. bifidum FJSNJ1M3 was the highest (99), and that in B. bifidum FGSZY50M8 was the lowest (94). Table 4 shows the antibiotic susceptibility of the six B. bifidum strains to 18 antibiotics. In this study, the tested strains were resistant to ciprofloxacin, trimethoprim, neomycin, kanamycin, streptomycin, aztreonam, and erythromycin but were fully or moderately susceptible to penicillin, ampicillin sodium, amoxicillin, chloramphenicol, tetracycline, rifampicin, ceftizoxime, teicoplanin, vancomycin, and oxacillin. These was semi-tolerance to other antibiotics. The results indicated that B. bifidum strains had different responses to the antibiotics; however, most of them were susceptible to ten different antibiotics. The antibiotic resistance phenotypes were highly consistent with their genotypes. Bifidobacterium species are among the earliest microorganisms to colonize the human intestine and play important roles in maintaining host health. Bifidobacterium species can affect various disease states, for example, by exercising anti-tumor effects. In a recent study, oral administration of Bifidobacterium to mice achieved an anti-cancer effect of similar magnitude to that of anti-PD-L1 therapy, while combined treatment almost completely inhibited tumor growth [37]. Bifidobacterium and derived preparations have been reported to regulate inflammatory bowel disease by altering the diversity of the intestinal microbiota, regulating the intestinal immune response, and secreting anti-pathogenic substances [38]. The establishment of Bifidobacterium in the intestine can lead to an increase in lactic acid production and reduce the pH of the intestine, inhibiting the propagation of harmful bacteria such as Escherichia coli and Clostridium spp. They can also optimize the physical and chemical environment of the intestine and inhibit the initiation and development of colon cancer induced by azomethane oxide [39]. Bifidobacterium spp. are also capable of reducing blood lipid levels, regulating the intestinal environment, exercising anti-aging effects, and assisting defecation [40]. Bifidobacterium is mainly passed via mother-to-child vertical transmission at birth [41]. Common bifidobacteria in the intestinal tracts of infants include B. bifidum, B. longum subsp. infants, and B. breve [42], while the common Bifidobacterium species in the adult gut are B. adolescentis, B. pseudobulbarum, and B. longum subsp. longum [43]. Common bifidobacteria species in the intestine of the elderly are B. longum subsp. longum, B. pseudobulbarum and B. bifidum. Aging, which may be accompanied by a change in carbon source intake, is one of many factors that can influence the species composition of Bifidobacterium in the human intestinal tract [44]. According to the World Health Organization, human life can be divided into five stages: 0 to 17 years, 18 to 65 years, 66 to 79 years, 80 to 99 years and over 100 years. In this work, 610 Bifidobacterium strains were divided into three groups based on subject age. The results showed that there were 260 Bifidobacterium strains from subjects in the 0−17 y age group, with B. breve, B. bifidum, B. longum subsp. longum, B. longum subsp. infantis, and B. pseudobulbarum having the highest abundance. From subjects of age 18–65 y, we isolated 244 Bifidobacterium strains, with B. adolescentis, B. pseudobulbarum, B. longum subsp. longum, and B. longum subsp. infantis having the highest abundance. A total of 106 strains were found in fecal samples from subjects in the 66−108 y age group, of which the abundance of B. longum subsp. longum accounted for 65%, which was followed by B. pseudobulbarum and B. bifidum. These findings are consistent with previous results [45]. More than 13% of the homologous gene family clusters in the bifidobacterial genome are associated with carbohydrate metabolism [20]. This glycemic genotype allows Bifidobacterium to metabolize various carbohydrates that cannot be digested by host enzymes [46], providing a competitive advantage for Bifidobacterium in colonizing the complex intestinal environment. Glycoside hydrolases are enzymes that catalyze carbohydrate hydrolysis. In recent years, the number of GHs represented in the CAZy database has increased almost exponentially [47]. When He et al. [48] studied the microbial samples in the rumen of sheep using macrotranscriptional sequencing, it was found that more than half of the GHs were located in the CAZymes group. B. bifidum has many extracellular glycosidases that can degrade host-derived glycans, including human milk oligosaccharides and high-molecular-weight carbohydrate chains [24]. Bifidobacterium enzymes can be used in the food industry, especially in the production of glycosylated products. For example, Bifidobacterium can be added in the preparation of yogurt to directly synthesize oligomeric galactose using lactose as a substrate [49]. The present study focused on carbohydrate-active enzymes in Bifidobacterium from subjects in different age groups and the annotation and distribution of the abundance glycoside hydrolase genes. The results showed no obvious differences in the glycoside hydrolase gene distribution between samples from subjects of different ages, but there were significant differences in Bifidobacterium species distribution. Across all age groups, B. pseudobulbarum had the largest number of glycoside hydrolase genes, with an average of 58 genes, which was followed by B. longum subsp. longum (55) and B. breve (51). The B. bifidum genome coded for the smallest number of GH genes, with an average of 36. B. bifidum is one of the earliest bacteria to colonize the intestinal tracts of infants, and it has high abundance in the infant intestinal tract. Some specific mechanisms in B. bifidum may be responsible for the highly competitive nature of this taxon in the infant intestine, allowing it to persist in this special environment. Previous studies have found that the combined effects of N-acetylneuraminic lyase (nanA), N-acetylmannosamine kinase (nanK), and N-acetylmannosamine isomerase (nanE) affect the degradation of sialic acid in some Bifidobacterium species [50]. The presence of sialidase was confirmed in the genomes of strains including B. longum subsp. infantis ATCC15697, B. breve UCC2003, and B. bifidum PRL2010 [51]. Kiyohara et al. [27] first reported that the B. bifidum JCM1254 exosialidase SiaBb2, an extracellular enzyme located on the membrane and belonging to the GH33 family, can act on sialylated oligosaccharides and promote release of free sialic acid. Yu et al. [52] showed that B. longum subsp. infantis JCM7009 and JCM7011 can efficiently utilize 3′-SL and 6′-SL using neuraminidase and ultimately produce lactate and short-chain fatty acids. Other results showed that B. bifidum extracellular sialidase promotes the utilization of sialylated carbohydrates with cross-feeding of free sialic acid to other Bifidobacterium strains. Bifidobacterium can grow in media containing SL as the main carbon source as it has sialidase- and galactosidase-encoding genes that allow for the cleavage of the relevant glycosidic bonds [53]. To assess this, we selected 21 Bifidobacterium strains from subjects of different ages, including B. breve (6 strains), B. longum subsp. infantis (6), and B. bifidum (9) and assessed the gene distribution of the GH33 family of glycosidases. All the strains were subjected to an in vitro 6′-SL utilization test. The results showed that all the B. longum subsp. infantis and B. breve samples grew well with 6′-SL as sole carbon source, and most of the B. bifidum strains (other than those isolated from subjects in the 18–65 age group) could also utilize 6′-SL (Table 2), which is consistent with previous reports [52]. By correlating the presence or absence of genes and the growth or nongrowth patterns of 21 Bifidobacterium strains with 6′-SL as sole carbon source, we found that the phenotypic experiment results were consistent with the genotypic predictions. The distribution of genes in the GH33 family of sialidases varied for B. bifidum across samples from different age groups, with no GH33 genes detected in strains isolated from the 18–65 y age group, and only a few genes detected in strains from the 0–17 y (2) and 66–108 y (1) age groups. The development of genomic tools provides strong support for understanding the diversity and functional characteristics of bacterial strains. We performed comparative genomic analyses on six strains of B. bifidum from subjects in the 0–18 y and 18–65 y age groups and determined the carbohydrate metabolic capacity. The average genome size for the six B. bifidum strains was 2.17 Mb, which is consistent with previous reports [54]. The pan-genome and core genome results suggested that the six B. bifidum strains have an open pan-genome. Average nucleotide identity is a standard method to determine whether a particular strain belongs to a reference species or whether a subspecies exists, whereby a threshold of 96% is used as a species boundary [55,56]. The heatmap of ANI values showed that the average nucleotide identity was higher in strains obtained from the same age group (Figure 4c). Furthermore, by annotating the core genes, the functions of transcription, defense mechanisms, and general function prediction of B. bifidum were revealed. We found differences between the six B. bifidum strains obtained from subjects in the 0−17 and 18−65 y age groups in terms of core and pan-genomes, ANI, carbohydrate utilization enzymes, COG, and antibiotic resistance. GO analysis, antibiotic resistance genes, and antibiotic resistance phenotypes did not show differences with age. Even so, age-related factors may be important in developing appropriate probiotics. In contrast to the other Bifidobacterium species, B. bifidum has been shown to utilize host-derived carbohydrates, especially human milk oligosaccharides [57] and mucin [58]. In vitro and animal studies support the proposal that various extracellular proteins produced by B. bifidum are crucial for the interaction between strain and host. In recent years, numerous studies have confirmed the influence of gut microbiota on healthy aging and shown that the ability of intestinal microbiota to assist in fighting disease gradually decreases with age. This study found a loss of the GH33 family of glycosidase genes in strains isolated from the 18–65 y age group, possibly indicating a partial loss of metabolic capacity with age. Probiotics with particular functions may be useful as supplements for populations with specific needs. At present, the knowledge of the B. bifidum genome is limited, and more strains should be studied. Species numbers, subject ages, geographic regions, and other potentially relevant factors in existing studies remain limited. It is urgent to investigate the potential of age-specific probiotics at a genetic level. This study provides a reference for future application in probiotic development, taking into account factors including glycoside hydrolase types, phenotype–genotype relationships, and subject age. In this study, whole genome sequencing was used to reveal the distribution of glycoside hydrolases in Bifidobacterium, with the results revealing differences in the distribution of the sialidase GH33 family in B. bifidum at different ages. Comparative genome and phenotypic studies were conducted, and the studies confirmed that the distribution of glycoenzyme genes in B. bifidum varied with age, affecting the phenotypic outcomes. It is critical to investigate the genomic and carbohydrate utilization properties of Bifidobacterium present in the digestive tract at various ages, as the data can be used as a reference for targeted intervention addressing intestinal Bifidobacterium composition as well as providing new ideas for probiotic products targeting various age groups.

PMC10000440

Xiaoxiu Tan,Linfeng Xu,Xingxing Jian,Jian Ouyang,Bo Hu,Xinrong Yang,Tao Wang,Lu Xie

PGNneo: A Proteogenomics-Based Neoantigen Prediction Pipeline in Noncoding Regions

01-03-2023

neoantigen,noncoding regions,proteogenomics,prediction pipeline,tumor immunotherapy

The development of a neoantigen-based personalized vaccine has promise in the hunt for cancer immunotherapy. The challenge in neoantigen vaccine design is the need to rapidly and accurately identify, in patients, those neoantigens with vaccine potential. Evidence shows that neoantigens can be derived from noncoding sequences, but there are few specific tools for identifying neoantigens in noncoding regions. In this work, we describe a proteogenomics-based pipeline, namely PGNneo, for use in discovering neoantigens derived from the noncoding region of the human genome with reliability. In PGNneo, four modules are included: (1) noncoding somatic variant calling and HLA typing; (2) peptide extraction and customized database construction; (3) variant peptide identification; (4) neoantigen prediction and selection. We have demonstrated the effectiveness of PGNneo and applied and validated our methodology in two real-world hepatocellular carcinoma (HCC) cohorts. TP53, WWP1, ATM, KMT2C, and NFE2L2, which are frequently mutating genes associated with HCC, were identified in two cohorts and corresponded to 107 neoantigens from non-coding regions. In addition, we applied PGNneo to a colorectal cancer (CRC) cohort, demonstrating that the tool can be extended and verified in other tumor types. In summary, PGNneo can specifically detect neoantigens generated by noncoding regions in tumors, providing additional immune targets for cancer types with a low tumor mutational burden (TMB) in coding regions. PGNneo, together with our previous tool, can identify coding and noncoding region-derived neoantigens and, thus, will contribute to a complete understanding of the tumor immune target landscape. PGNneo source code and documentation are available at Github. To facilitate the installation and use of PGNneo, we provide a Docker container and a GUI.

PGNneo: A Proteogenomics-Based Neoantigen Prediction Pipeline in Noncoding Regions The development of a neoantigen-based personalized vaccine has promise in the hunt for cancer immunotherapy. The challenge in neoantigen vaccine design is the need to rapidly and accurately identify, in patients, those neoantigens with vaccine potential. Evidence shows that neoantigens can be derived from noncoding sequences, but there are few specific tools for identifying neoantigens in noncoding regions. In this work, we describe a proteogenomics-based pipeline, namely PGNneo, for use in discovering neoantigens derived from the noncoding region of the human genome with reliability. In PGNneo, four modules are included: (1) noncoding somatic variant calling and HLA typing; (2) peptide extraction and customized database construction; (3) variant peptide identification; (4) neoantigen prediction and selection. We have demonstrated the effectiveness of PGNneo and applied and validated our methodology in two real-world hepatocellular carcinoma (HCC) cohorts. TP53, WWP1, ATM, KMT2C, and NFE2L2, which are frequently mutating genes associated with HCC, were identified in two cohorts and corresponded to 107 neoantigens from non-coding regions. In addition, we applied PGNneo to a colorectal cancer (CRC) cohort, demonstrating that the tool can be extended and verified in other tumor types. In summary, PGNneo can specifically detect neoantigens generated by noncoding regions in tumors, providing additional immune targets for cancer types with a low tumor mutational burden (TMB) in coding regions. PGNneo, together with our previous tool, can identify coding and noncoding region-derived neoantigens and, thus, will contribute to a complete understanding of the tumor immune target landscape. PGNneo source code and documentation are available at Github. To facilitate the installation and use of PGNneo, we provide a Docker container and a GUI. Neoantigens are considered to be promising therapeutic targets owing to their tumor specificity, and to their neither being affected by pre-existing immune tolerance nor generating autoimmunity [1]. Thus, neoantigens can be used as potential targets for therapeutic vaccines. A neoantigen vaccine is designed to trigger de novo T cell response and broaden the endogenous repertoire of tumor-specific T cells [2]. A phase-I trial of a neoantigen-based peptide vaccine showed that four patients with stage III melanoma induced CD4+ T cell and CD8+ T cell responses after vaccination and remained disease-free for a median follow-up of 25 months after vaccination. This demonstrates the potent tumor-specific immunogenicity and antitumor activity of neoantigen vaccines [3]. However, one major challenge in neoantigen vaccine design is the rapid and accurate identification of neoantigens that can induce T cell responses in patients. The advent of next-generation sequencing has provided opportunities to efficiently identify tumor-specific antigens in individual patients, leading to the exploration of clinical target therapies. In fact, several pipelines have been developed to predict neoantigens, such as pVACtools [4], NeoPredPipe [5], Neopepsee [6], etc. Although the development of these tools has opened the way for identifying potentially immunogenic neoantigens [7], limitations to these tools exist. First, most traditional prediction pipelines were developed based on genomic and transcriptomic data. Many false-positive neoantigens inevitably occur, due to the large number of mutations in individual patients and the limited performance of MHC ligand binding prediction [8]. In addition, studies have shown that the mRNA measurements of many genes correlate poorly with protein expression [9,10]. With advances in mass spectrometry (MS)-based proteomics, the combination of proteomics and genomics, i.e., proteogenomics, has been a major force in driving personalized neoantigen vaccine identification [11,12,13]. It allows the presence verification of those peptides that are most likely to generate an immune response based on neoantigen prediction pipelines; thus, such peptides may be moved into subsequent functional selection processes. Proteogenomics has greatly reduced the number of false positives for predicted neoantigens and has eased the burden of experimental validation. Our group previously developed proteogenomics neoantigen prediction pipelines, ProGeo-neo [14] and ProGeo-neo2.0 [15], and WEN B et al. developed NeoFlow [16]. Another limitation of the currently existing neoantigen prediction pipelines is that they almost all focus on genomic coding regions. While variants in protein-coding regions have received the most attention, numerous studies have noted the importance of noncoding variants in cancers [17]. Exomes only account for 2% of the human genome, whereas up to 75% of the genome has been shown to be transcribed and potentially translated [18]. Therefore, many allegedly noncoding regions are actually protein-coding. For example, long noncoding RNAs (LncRNA) are a type of noncoding RNA with a length of more than 200 nt, lacking a protein-coding function due to the lack of a complete open read frame (ORF) [19]. Intriguingly, several recent studies have noted LncRNAs as a source of new peptides [20,21]. Of particular relevance to tumor neoantigen discovery, 99% of cancer mutations are located in noncoding regions [17]. Therefore, focusing on the exome as the only source of tumor neoantigens is very restrictive. Notably, peptides derived from the noncoding regions have been shown to bind to MHC molecules, some of which were identified as the targets of T cells [22,23,24]. Subsequently, landmark studies demonstrated that the noncoding regions are the main source of targetable tumor-specific antigens [25,26]. However, an efficient and easy-to-use tool to predict and investigate the personalized neoantigens from noncoding regions is still lacking. Herein, we present PGNneo, an integrated computational pipeline to predict noncoding neoantigens from RNA-seq and MS data. We demonstrated the effectiveness of PGNneo and validated our methodology in two real-world hepatocellular carcinoma (HCC) cohorts. In addition, we applied PGNneo to a colorectal cancer (CRC) cohort, demonstrating that the tool can be extended and verified in other tumor types. PGNneo is an efficient tool to identify noncoding neoantigens and can be easily installed and deployed at https://github.com/tanxiaoxiu/PGNneo. To be more user-friendly, we also provide a Docker version at (https://hub.docker.com/r/xiaoxiutan/pgnneo) and a GUI. The paired-end sequencing data of lncRNA from 5 HCC patient-derived xenograft (PDX) samples, including tumor and normal tissues, were obtained from Hu et al. [27]. The proteomics datasets of the HCC cell line were downloaded from the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) (accessed on 21 September 2020) with the identifier, PXD000529. This cohort is hereinafter referred to as HCC_HF. Another HCC cohort (hereinafter referred to as HCC_HT) from a previous collaboration with Jiang et al. [28] included RNA-seq files and MS raw data from 10 patients (early stage of HCC, subtype 3) were downloaded from the Gene Expression Omnibus (GEO) (accession number GSE124535) (accessed on 15 November 2021) and iProX database (http://www.iprox.org, accession number IPX0000937000) (accessed on 15 November 2021), respectively. Detailed sample information is provided in Table S1 in the Supplementary Materials. In addition, we collected a CRC cohort [29], including RNA-seq data and MS raw data from three CRC cell lines and RNA-seq data from one normal fetal small intestine cell line. This can be downloaded from the GEO (accession number GSE195985) (accessed on 2 October 2022) and the ProteomeXchange Consortium (Identifier PXD028309) (accessed on 2 October 2022), respectively. Detailed sample information of this cohort is presented in Table S12 in the Supplementary Materials. The human reference genome (hg38) and Proteome (version 101) were downloaded from UCSC (http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz) (accessed on 1 September 2020) and the Ensembl database (http://www.ensembl.org/) (accessed on 1 September 2020), respectively. Contaminated protein sequences were available in a FASTA format from the common repository of adventitious proteins (cRAP) (http://www.thegpm.org/crap/) (accessed on 1 September 2020). RNA-seq raw data were cleaned by Trimmomatic (v0.39) (Max Planck Institute of Molecular Plant Physiology, Golm, Germany) [30], with the standard adapters trimmed and low-quality reads filtered. All clean reads were aligned to the human reference genome using the Burrows–Wheeler alignment tool (BWA, v0.7.17) (Wellcome Trust Sanger Institute, Cambridge, UK) [31] with the default parameters. The resulting .sam file was converted to .bam, sorted, and indexed using samtools [32]. The Picard [33] tool, MarkDuplicates (Broad Institute, Cambridge, MA, USA), was used to identify duplicates. To correct as many systematic errors in the sequencing process as possible, we performed base quality score recalibration. The Picard AddorReplacereAdgroups tool (Broad Institute, Cambridge, MA, USA) was used to modify the headers of BAM files for subsequent processing. Somatic single nucleotide variants (SNVs), and insertions and deletions (Indels), were detected by GATK Mutect2 (v4.1.9) (The Broad Institute of Harvard and MIT, Cambridge, MA, USA) [34] from the BAM files of paired tumor and normal samples. The GATK FilterMutectCalls (The Broad Institute of Harvard and MIT, Cambridge, MA, USA) tool was used to filter somatic mutations using default parameters, with true positive mutations marked with “PASS” and we selected “PASS” mutations. Since affinity predictions for the peptide-MHC interface are MHC-specific, it is critical to know the patient HLA types. HLA alleles in each sample were inferred from trimmed RNA-seq data using OptiType (University of Tübingen, Tübingen, Germany) with the default settings, which has been demonstrated to achieve HLA typing with ~97% accuracy [35,36]. The annotation of mutations and the extraction of the peptide are shown in Figure 1. Somatic mutation data that were obtained based on RNA-seq data were annotated using Annovar [37] to identify noncoding-region somatic mutations, including SNVs and indels. The noncoding regions that we used specifically included: “downstream”, “intergenic”, “intronic”, “ncRNA_exonic”, “ncRNA_intronic”, “ncRNA_splicing”, “splicing”, “upstream”, “UTR3”, and “UTR5”. After mutation filtering, nucleotide sequences with a set interval length were obtained according to the mutation sites and reference genome using Bedtools (University of Virginia School of Medicine, Charlottesville, VA, USA) [38]. Specifically, 100 nucleotide sequences were taken from along each side of the mutation site. To generate the nucleotide sequences containing the mutation, we replaced the reference bases with mutant bases, i.e., we replaced the “REF” column with the “ALT” column in the mutation table. For the translation of nucleotide sequences in the genome, six-frame translation is the classical method. Six-frame translation has the advantage of being independent of any a priori annotation of the nucleotide sequence [39,40]. Thus, according to the 64 codons, the mutant nucleotide sequences were translated into novel proteins via six-frame translation. The termination codons were replaced with “*” and the protein sequences were cleaved into short peptides according to “*”. The short peptides that did not contain the mutations were then filtered out. Finally, we obtained tumor protein sequences containing mutations from noncoding regions. Identifying a mutant peptide expressed at the protein level is a crucial step. In this study, MaxQuant (Max-Planck Institute for Biochemistry, Martinsried, Germany) [41], a proteomics identification quantitative tool, was used to identify the peptides. To search the proteomics data, we first constructed a customized database for each individual tumor sample, including human reference proteins, common contaminant protein sequences in the laboratory (cRAP), and cancer-specific proteomes. Then, to filter for true peptides, all MS/MS spectra were searched using MaxQuant in the customized peptide database. A separate target-decoy search strategy was used. Decoy peptides were generated from the peptides of corresponding target databases using a reversed tryptic approach. The parameters of MaxQuant were set as follows: (1) the variable modifications included protein N-terminal acetylation with methionine oxidation; (2) strict trypsin specificity was required, allowing up to two missed cleavages; (3) the carbamidomethylation of cysteine was set as a fixed modification. In addition, false discovery rate (FDR) thresholds for protein peptides were specified at 1%. The minimum required peptide length was set to 7 amino acids. Finally, we extracted the cancer-specific mutant peptides identified by MS data and provided evidence in terms of protein expression level. PGNneo uses NetMHCpan 4.1 (Department of Bio and Health Informatics, Technical University of Denmark, Kgs. Lyngby, Denmark) [42] to calculate the binding affinity of peptides to patient-specific HLA alleles. To match the length of the peptides bound by MHC-I molecules, the peptides that were filtered by mass spectrometry were cleaved into short peptides containing mutated 8–11 mers. The percentile rank score was proven to exhibit higher sensitivity and less bias in HLA-peptide identification than the half-maximum inhibitory concentration (IC50) [43]. Therefore, the percentile rank (%rank) value was used as the metric of HLA-peptide binding prediction, and peptides with a %rank < 2 were considered to be candidate neoantigens. Similar sequences often originate from a common ancestral sequence and they are likely to have similar spatial structure and biological function; in fact, tumor-infiltrating T cells were found to exhibit a cross-reactivity that recognizes both tumor neoantigens and homologous non-tumor microbial antigens [44]. Therefore, to filter candidate neoantigens, sequence similarity analysis was performed using the basic local alignment search tool (BLAST) (National Center for Biotechnology Information, Bethesda, MD, USA) [45]. In total, 746 experimentally immunogenic neoantigens, collected from an in-house database, dbPepNeo2.0 [46], were used to build the target sequence database, while candidate neoantigens were treated as retrieval sequences. Then, BLASTp was used to identify homologous sequences so that the degree of homology between candidate neoantigens and immunogenic neoantigens could be established. We adjusted several default options to increase the sensitivity of BLAST searches that were performed with short input sequences. The peptides were reported to have sequence identity to immunogenic neoantigens if the percentage of identical matches was above 60%. PGNneo is open-source software written in Python, shell, and R. The software is divided into four toolkits, based on four modules. The user needs to configure the path and parameters of the software before applying the toolkits. After completing the configuration, the pipeline can be run by executing the command line. A more detailed tutorial on the use of PGNneo is available in the User’s Manual. The PGNneo source code and documentation are available at https://github.com/tanxiaoxiu/PGNneo. To facilitate the installation and use of PGNneo, we provide a Docker container (Docker: https://hub.docker.com/r/xiaoxiutan/pgnneo) and a GUI. Here, as shown in Figure 2, a versatile and comprehensive workflow, PGNneo, is presented to identify neoantigens in the noncoding region. In PGNneo, several input datasets are required, including RNA-seq profiles and MS datasets. First, the RNA-seq profiles from the paired tumor and normal samples are used to screen for somatic mutations in the noncoding regions, and the amino acid sequences containing mutant sites in the noncoding region can thus be identified. Then, those expressed sequences can be filtered using MS datasets. Eventually, the resulting peptides are used for neoantigen prediction and selection by using MHC affinity and the database dbPepNeo2.0 [46]. The general computational framework of PGNneo consists of the following modules. (1) Noncoding somatic variant calling and HLA typing. Identifying somatic mutations in tumor cells is a key step in the neoantigen presentation pathway. For this purpose, paired tumor and normal samples were used for somatic variant calling. Prior to annotation, we removed any low-quality somatic mutations. Eventually, the noncoding mutations were extracted. The prediction of HLA typing was performed, based on the RNA-seq data from tumor samples. (2) Peptide extraction and customized database construction. Working according to the mutation information, the nucleotide sequences were obtained and were then translated into proteins by six-frame translation. The process of extraction of the peptide is shown in Figure 1. Eventually, tumor mutant peptides were obtained. Subsequently, these mutant protein sequences and reference proteins were combined to construct a customized protein database. (3) Variant peptide identification. The resulting peptides were filtered using MS datasets. The proteomic data provided evidence not only for the presence of peptides at protein levels but also for the binding of peptides to MHC molecules. (4) Neoantigen prediction and selection. Candidate neoantigens were predicted, according to peptides and HLA types, using NetMHCpan 4.1. The candidate neoantigens were filtered using the database dbPepNeo 2.0, which is an in-house dataset using 746 experimental immunogenic peptides as a reference. The resulting datasets at different filtering stages could then be obtained and downloaded according to the user’s preference. Table 1 summarizes the software used in PGNneo. To evaluate the performance of PGNneo, we performed sequence similarity analysis using two independent datasets. The positive dataset comprises 746 experimentally validated immunogenic neoantigens (Positive) collected from the dbPepNeo2.0 dataset, and the background control dataset contains 6400 mutant peptides (Random) of length 8–11 residue [48]. We performed sequence similarity analysis on the candidate neoantigens (unfiltered) that were obtained from the two cohorts of HCC_HF and HCC_HT with the positive dataset and random dataset, respectively. Two sequences exhibiting more than 60% of identical matches and an E-value of less than 0.5 are considered to be similar. Figure 3 shows that the results obtained from the positive and random datasets were significantly different in both the HCC_HF and HCC_HT cohorts, with p-values of 0.0278 (<0.05) and 0.01704 (<0.05), respectively (Wilcoxon test). The results indicate that the candidate neoantigens predicted by our method are more likely to have immunogenic potential. Therefore, filtering using the positive datasets was incorporated into the pipeline. This can be considered as an in silico verification step in the pipeline for the prediction of neoantigens. PGNneo was applied to two independent HCC cohorts. We statistically analyzed the number of key steps in the pipeline for each sample (Figure S1 in the Supplementary Materials). In the HCC_HT cohort, one sample deviated significantly from other samples, possibly due to data quality problems, so this sample was deleted; finally, for this cohort, 9 samples were retained. The average number of key steps in the pipeline on the HCC_HF and HCC_HT cohorts are shown in Figure 4A. After filtering and annotation, an average of 3260 and 1178 noncoding mutations were obtained (Tables S2 and S3 in the Supplementary Materials). At the protein level, an average of 2339 and 610 peptides were identified by MS data analysis. HLA genotypes were predicted from the RNA-seq fastq file using the Optitype, and MHC-I binding predictions for the filtered peptides were predicted with netMHCpan4.1. As a result, an average of 3518 and 932 candidate neoantigens were obtained in the two cohorts, respectively (Tables S4 and S5 in the Supplementary Materials). After screening for HCC_HF, an average of 77 noncoding high-confidence neoantigens were eventually identified in each sample, ranging from 37 to 147 (Table S6 in the Supplementary Materials). However, in the HCC_HT cohort, an average of 13 noncoding high-confidence neoantigens were identified per sample, ranging from 4 to 23 (Table S7 in the Supplementary Materials). Upon comparing the results of the two cohorts, we found 403 overlapping non-coding mutations and 189 overlapping candidate neoantigens in the two cohorts (Figure 4B,C); however, no overlap was found in the high-confidence neoantigens. The results show that neoantigens are unique and the number of neoantigens varies greatly between different datasets. In addition, 26 and 28 unique HLA alleles were predicted for the two cohorts, respectively (Table S8 in the Supplementary Materials). We further calculated the frequency of HLA alleles in the sample population using the getHlaFrequencies function in the midasHLA package of R [49]. Then, the mean value of neoantigens bound by each HLA allele was calculated, based on the count of HLA alleles, thus normalizing the number of neoantigens. The frequency of HLA alleles and the number of corresponding neoantigens are given in Table S9 in the Supplementary Materials. Based on the ranking of the normalized number of neoantigens, the 10 most frequent binding HLA alleles that matched with candidate neoantigens in two cohorts are shown in Figure 5A and Figure 5B, respectively. The results showed that HLA alleles exhibited a preference for the binding of neoantigens, while HLA-A33:03 and HLA-A23:01 accounted for the largest binding proportion in the HCC_HF and HCC_HT cohorts. Moreover, the same candidate neoantigen can bind to different HLA alleles; this neoantigen is more likely to be present and may be applicable to a wider range of individuals. We further analyzed the overlapping neoantigens and their corresponding genes in the two cohorts. In the HCC_HF cohort (lncRNA-seq data from 5 HCC PDX samples and MS data from the HCC cell line), 10 neoantigens were found to be in common in at least 2 patients (Table S10 in the Supplementary Materials). These overlapping neoantigens are called “shared neoantigens” and have the potential to be designed as shared neoantigen vaccines. Conversely, neoantigens across the 5 patients were mapped to 118 unique genes, and 6 of these genes were observed in at least 2 patients (Table S10 in the Supplementary Materials). These overlapping genes are “hot-spot mutations” where the corresponding neoantigens may be in common among multiple patients. Unfortunately, no overlapping neoantigens or genes were found in the HCC_HT cohort (with paired RNA-seq data and MS data from 10 human HCC samples). This may be because the HCC_HF cohort dataset comprises unpaired lncRNA-seq data and MS data; the MS data is cell line data with lower heterogeneity, so that shared neoantigens can be found, while the HCC_HT cohort is of paired RNA-seq data and MS data from patients with a higher degree of individualization. To some extent, this explains the individualization of neoantigens in real patients. Therefore, this also reinforces the necessity for more research on hot-spot mutations for building up data resources for shared neoantigens. In order to correlate the predicted neoantigens with the clinical information garnered from patients, we explored the association between the predicted neoantigens and the pathogenesis of HCC. Rao et al. [50] summarized the frequently mutated genes and their functions that are associated with HCC. We compared candidate neoantigen-associated genes in the HCC_HF cohort and HCC_HT cohort with the frequently mutated genes associated with HCC. The TP53, WWP1, ATM, KMT2C, and NFE2L2 mutant genes associated with HCC were identified in two cohorts and corresponded to 98 neoantigens (Table S11 in the Supplementary Materials). Table 2 only shows information about the 26 candidate neoantigens that bind most strongly to HLA. Among them, TP53 is one of the most studied tumor suppressors, with multiple functions, and is associated with DNA damage checkpoints and repair defects. WWP1 is associated with the activation of oncogenic pathways in HCC; the overexpression of WWP1 promotes tumorigenesis in HCC patients and predicts poor prognosis. The loss of ATM reduces hepatocyte apoptosis and fibrosis, suggesting that the activation of ATM in response to oxidative stress plays a role in hepatic fibrosis development. KMT2C and KMT2D are functionally similar and may be involved in chromatin localization and genomic instability. NFE2L2 deficiency may render cells susceptible to oxidative stress-mediated DNA damage. Genes that are highly mutated in HCC may be attractive potential therapeutic targets. In addition, we applied PGNneo to another tumor type with moderate TMB, colorectal cancer (CRC) [29]. Firstly, 4206, 3664, and 5823 non-coding mutations were obtained on COLO205, SW620, and HCT116 cell line data, respectively, and further predictions yielded 217, 330, and 291 candidate neoantigens. In addition, to evaluate the potential immunogenicity of candidate neoantigens, we obtained high-confidence neoantigens based on the filtering of an experimentally validated immunogenic neoantigen database constructed by our group. Detailed results on sample information, noncoding region mutations, HLA typing, candidate neoantigens, and high-confidence neoantigens are provided in Table S12 in the Supplementary Materials. The results demonstrate that our pipeline can be applied to multiple tumor types. The biological mechanisms of noncoding neoantigens may be cross-verified as the application of PGNneo expands. To complete the identification and comparison of neoantigens from both coding and noncoding regions, we applied four other common neoantigen prediction tools, including ProGeo-neo [14], pVACtools [4], Neopredpipe [5], and TSAFinder [51], to compare their performance with our own tool, PGNneo. pVACtools, Neopredpipe, and TSAFinder require fastq data for RNA-seq and/or VCF data for mutations as input, and ProGeo-neo requires the additional input of MS data. For two HCC cohorts, we randomly selected three patient samples, HCC42, HCC67, and HCC1076, for comparison across five neoantigen prediction tools. Since most neoantigens are composed of 9 amino acids, we only compared the prediction of 9-mer neoantigens [52]. The number of candidate neoantigens obtained by the five tools is shown in Figure 6. It is worth noting that PGNneo introduces MS data and shows candidate neoantigens after MS filtering so that the results of PGNneo are more stringent. Although ProGeo-neo also has a module for MS data filtering, unfortunately, no neoantigens were obtained after MS data identification. This is consistent with studies on neoantigens in the coding region of HCC, which suggested that the tumor mutational burden (TMB) of HCC is relatively low and neoantigens in coding regions are scarce [53]. In contrast, the other tools do not have an MS filtering step, and we only show their candidate neoantigens as predicted by peptide-MHC binding affinity (Table S13 in the Supplementary Materials). In addition, PGNneo sets up a module for secondary filtering by using 746 experimentally validated neoantigens, resulting in high-confidence neoantigens. Twenty, three, and one high-confidence 9-mer neoantigens were obtained in samples HCC1076, HCC42, and HCC67, respectively (Table S13 in the Supplementary Materials). Furthermore, we explored the association between these high-confidence neoantigens and the pathogenesis of HCC. The high-confidence neoantigen genes TNFSF14, GAD1, STARD1, and DHRS4-AS1 have been reported in the literature to be closely associated with HCC [54,55,56]. Specifically, TNFSF14 and GAD1 are highly expressed in HCC [54]; STARD1 promotes the progression of non-alcoholic steatohepatitis to HCC via bile acids [55]; DHRS4-AS1 ameliorates HCC by suppressing proliferation and promoting apoptosis via the miR-522-3p/SOCS5 axis [56]. Moreover, we analyzed the overlap of the candidate neoantigens predicted by different tools. For three samples, HCC1076, HCC42, and HCC67, the number of neoantigens identified by at least three tools was 411, 80, and 3, respectively (Table S14 in the Supplementary Materials). We recommend using multiple tools to predict neoantigens, which may yield more reliable results. Our investigation demonstrates that for cancer types with a low TMB, the source of neoantigens may be enriched when the noncoding region is taken into consideration. Therefore, PGNneo aims to expand the scope of neoantigen prediction and provide a richer neoantigen reference for some cancer types with a low TMB in the coding region. Although some algorithms and tools have been developed to tackle the problem of neoantigen prediction, most are based on coding regions. However, in the human genome, 98% of the sequence involves noncoding regions, and most DNA sequence variants occur in the noncoding regions [17]. The general properties of sequence variants are also applicable to noncoding variants, such as SNVs and Indels. What is more, noncoding variants can also generate neoantigens. However, there are few prediction tools that operate on noncoding regions; the majority of tools focus on exonic variant calling, which is based on genomic data rather than transcriptomic data. For this reason, we developed a proteogenomics-based pipeline, PGNneo, to identify those neoantigens derived from noncoding regions, based on transcriptomic data from the human genome. Furthermore, we successfully validated the effectiveness of PGNneo through its application in two HCC cohorts. A total of 386 and 113 high-confidence neoantigens were identified in the two HCC cohorts, respectively. In addition, we applied PGNneo to a CRC cohort, demonstrating that the tool can be extended to multiple tumor types. Compared with the traditional neoantigen prediction pipeline, PGNneo has several advantages. First, it focuses on neoantigens in noncoding regions, which provides a new source of neoantigens for low-TMB tumor types. Studies have shown that for cancer types with a low TMB, such as liver cancer, the source of neoantigens should be extended to noncoding regions for better applicability to immunotherapy [53]. Second, it combines transcriptomics and proteomics data, furthering the proteogenomics neoantigen prediction pipelines for coding regions in our research group [14,15]. Most of the previously developed neoantigen prediction tools are based on genomic data only, and the predicted false-positive rate of neoantigens is relatively high. Combined with proteomic data, the accuracy of neoantigen prediction can be improved. Moreover, proteogenomics holds the promise of providing deeper mechanistic insights to enable the better matching of patients to targeted therapies than when analyzing each type of omics data separately. In addition, our pipeline uses RNA-seq data from paired tumor and normal tissues to call somatic mutations. Mutations in the RNA-seq data provide a better reference for a proteomics dataset than WES, mainly because of the ability of RNA-seq to identify novel somatic variants, while for oncogenes that are highly expressed in cancer, RNA-seq provides higher sequencing coverage than WES and, therefore, has higher statistical confidence in detecting variants [57]. Thus, in our pipeline, the customized searchable peptide database was derived from tumor RNA-seq data. Compared to the coding-region-based proteogenomic prediction pipeline established by our group, in terms of data requirements, ProGeo-neo requires data at the genomic, transcriptomic, and proteomic levels of the patients, while PGNneo only requires transcriptomic and proteomic data from patients, and not WES/WGS data. This is because the neoantigen prediction step in ProGneo-neo is performed based on the mutations detected by WES/WGS, whereas in PGNneo, the step is based on RNA-seq data. Therefore, the application scenario of the PGNneo can be further expanded. There are still some limitations to our study, so we will expand on the following aspects. To further enrich the types of neoantigens, we may later update the tool to predict those neoantigens derived from gene fusion and RNA splicing, which will provide more potential neoantigen targets for developing therapeutic cancer vaccines. In addition, considering the complementary roles of coding-region-based and noncoding-region-based pipelines in identifying neoantigens, we will integrate neoantigen prediction tools such as ProGeo-neo that have already been developed within the group to further facilitate their use by researchers. Moreover, as noncoding neoantigens have been shown to have strong tumor specificity, more relevant studies have since emerged in the field. Recently, Cai et al. [58] developed IEAtlas, an atlas of HLA-presented immune epitopes derived from noncoding regions, which provides a valuable data resource for studying the immunogenicity of noncoding epitopes. Therefore, we will combine the data from IEAtlas to further improve the predictive power of PGNneo. Even though the subsequent experimental validation of candidate neoantigens is essential for real-world clinical application, our computational methods can narrow down the range of neoantigens to a certain extent and thereby pave the way to improved preclinical vaccine design. Therefore, PGNneo may prove to be a useful tool for cancer researchers and clinicians. In this study, we developed a proteogenomics-based pipeline to predict neoantigens in noncoding regions, namely, PGNneo. PGNneo is a pipeline that integrates state-of-the-art computational tools. It mainly includes four modules: (1) noncoding somatic variant calling and HLA typing; (2) peptide extraction and customized database construction; (3) variant peptide identification; (4) neoantigen prediction and selection. PGNneo can be easily applied to RNA-seq data and MS data drawn from patients of different cancer types. In summary, PGNneo can specifically detect the neoantigens generated by noncoding regions in tumors, providing guidance for cancer types with a low TMB in coding regions. PGNneo, together with our previous tool, can improve the identification of coding and noncoding region-derived neoantigens and will contribute to a more complete understanding of the tumor immune landscape. This capability holds promise for broadening the repertoire of candidates for therapeutic cancer vaccination and T cell-based therapy and may ultimately extend the neoantigen clinical benefits of immunotherapy.

PMC10000455

Luogen Peng,Yuchan Li,Sha Yao,Jochen Gaedcke,Victor M. Baart,Cornelis F. M. Sier,Albrecht Neesse,Volker Ellenrieder,Hanibal Bohnenberger,Frieder Fuchs,Julia Kitz,Philipp Ströbel,Stefan Küffer

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas

03-03-2023

pancreatic cancer,uPAR,KRAS,FAK,MEK,ERK,dormancy,gemcitabine

Simple Summary Pancreatic ductal adenocarcinomas (PDACs) with gene amplification or overexpression of urokinase-type plasminogen activator receptor (uPAR) have a particularly dismal prognosis. We show here that uPAR reinforces the MEK/ERK signaling pathway in tumor cells with a KRAS mutation with the suppression of FAK and CDC42 signaling. This synergy keeps tumor cells in a mesenchymal state that favors cell migration and proliferation, but also sensitizes them towards gemcitabine. These observations highlight a potential therapeutic dilemma that applies to KRAS and uPAR as emerging targets. Treatments targeting either KRAS or uPAR could induce cellular dormancy and render the tumor more resistant to chemotherapy (such as gemcitabine). The clinical benefit of adding autophagy inhibitors such as chloroquine in this situation remains to be shown. Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup’s biology better. Methods: A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. Results: High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. Conclusions: Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas Pancreatic ductal adenocarcinomas (PDACs) with gene amplification or overexpression of urokinase-type plasminogen activator receptor (uPAR) have a particularly dismal prognosis. We show here that uPAR reinforces the MEK/ERK signaling pathway in tumor cells with a KRAS mutation with the suppression of FAK and CDC42 signaling. This synergy keeps tumor cells in a mesenchymal state that favors cell migration and proliferation, but also sensitizes them towards gemcitabine. These observations highlight a potential therapeutic dilemma that applies to KRAS and uPAR as emerging targets. Treatments targeting either KRAS or uPAR could induce cellular dormancy and render the tumor more resistant to chemotherapy (such as gemcitabine). The clinical benefit of adding autophagy inhibitors such as chloroquine in this situation remains to be shown. Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup’s biology better. Methods: A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. Results: High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. Conclusions: Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism. Pancreatic ductal adenocarcinomas (PDACs) are among the human tumors with the worst prognosis. Most PDAC patients are already at an advanced stage at diagnosis, and resection as the most effective treatment is only feasible in 20% of patients [1]. With gemcitabine as a baseline combined with FOLFIRINOX, next to albumin-bound paclitaxel, therapeutic options are limited [2,3,4]. The current clinical staging of PDAC cannot fully predict tumor behavior, and the prognosis of patients receiving the same treatment varies considerably. Therefore, it is essential to develop robust molecular classifications of PDAC for more tailored therapeutic approaches [5]. An increasing number of molecular and histological subtypes already define subtype-specific therapeutic vulnerabilities and provide the opportunity to supplement current pathological classifications. Recent studies discovered many PDAC subtype-specific markers connected to different clinical behavior; however, the three main subtypes remain classical, quasi-mesenchymal (QM-PDA), and exocrine-like [6]. Nevertheless, there is now good evidence that cancer cells preserve cellular plasticity [7,8]. Increased levels of urokinase-type plasminogen activator receptor (uPAR) are associated with early invasion, metastasis, and poor prognosis in many solid and hematological tumors, including PDAC [9,10,11]. uPAR is a GPI-anchored cell membrane receptor without an intracellular domain that mediates the degradation of extracellular matrix (ECM) components [12], including fibronectin and vitronectin [13]. It locally increases plasmin activity that facilitates cell migration. Interaction of uPAR with integrins occurs indirectly through stabilized binding to vitronectin [14]. This leads to intracellular activation of the Ras pathway, the focal adhesion kinase (FAK), and the Rho family small GTPase Rac (reviewed in [15]). PDAC is also one of the tumors with the highest frequency of KRAS mutations. KRAS has not only been shown to activate cell proliferation through RAF/MEK/ERK [16]; it has also been reported to regulate uPAR expression by AP1-dependent transactivation of the uPAR promoter [17]. Downregulation or blocking of uPAR causes activation of FAK, Src, CDC42, and p38, resulting in cell-cycle arrest and dormancy [18,19]. We have previously shown that 50% of PDACs show overexpression of uPAR due to low or high-level amplifications of the uPAR gene PLAUR. These tumors are associated with an inferior prognosis [20]. In this study, we functionally studied the role of uPAR in cell lines and validated the results in a cohort of 67 PDAC patients with clinical follow-up supplemented by TCGA data of 316 PDAC patient samples. Tumor samples from 67 PDAC patients organized on a multi-tissue array (TMA) were used for immunohistochemical staining (clinical data are summarized in Table 1). The patient sample collection was approved by the ethics committee of the University Medical Center Göttingen (GÖ 912/15). Immunohistochemical staining (IHC) of 2 µm paraffin sections was performed according to standard methods. Briefly, after deparaffinization in serially diluted alcohol and blocking endogenous peroxide in 0.3% hydrogen peroxide in PBS, antigen retrieval was performed at 95 °C in either a low or high-pH Envision FLEX target retrieval solution (Agilent, Santa Clara, CA, USA) using PT Link (Agilent). Subsequently, the stainings were incubated for 1 h with primary antibodies, followed by washing in PBS and incubation with the appropriate detection system for 30 min (Envision, Agilent). Antibodies were used at predetermined optimal dilutions (Supplementary Table S3) with the proper positive and negative controls. Staining was visualized by 3,3-diaminobenzidine tetrahydrochloride solution, counterstained with hematoxylin, dehydrated, and mounted in Pertex. Using an H-score, all tissue samples were evaluated for nuclear staining of p-p38, uPA, uPAR, and PAI1. The H-score was calculated by 3 × the percentage of the strongest staining signal + 2 × the percentage of a moderate signal + the percentage of a weak signal, resulting in a value range from 0 to 300. HNF1A and KRT81 were graded for “low” or “high” expression according to signal intensity. The optimal levels for the discrimination between high and low signals of uPAR, HNF1A, and KRT81 were determined using the cutoff finder [21]. The human pancreatic cancer cell lines BxPC-3, AsPC-1, CAPAN-2, MIA PaCa-2, PATU8988T, and PANC-1 were obtained from the American Type Culture Collection (ATCC) (Supplementary Table S1). All cells were grown in RPMI-1640 medium (Gibco, Waltham, MA, USA), supplemented with 10% FCS (Gibco), 1% L-glutamine (Gibco), and 1% Penicillin/Streptomycin (Gibco) under humidified conditions at 37 °C and 5% CO2. PANC-1 was transfected with the pCMV-AC-GFP vector PLAUR (NM_002659) human-tagged ORF clone (Origene, Rockville, MD, USA), and BxPC-3 with the pCMV6-Entry-KRASG12C vector (Origene Technologies Inc., Rockville, MD, USA) using the X-tremeGENE HP DNA transfection reagent (Merck, Darmstadt, Germany). Transfected cells were selected with G418 (400 ng/µL). uPAR protein levels were tested by ELISA as described above. KRASG12C-expressing cells were selected using 2µg/mL puromycin. The uPAR CRISPR/Cas9 knockout strategy is shown in Supplementary Figure S1. Cells were transfected with two CRISPR/Cas9 constructs, pCMV-Cas9-RFP (target site: 5′-GGACCCTGAGCTATCGGACTGG-3′), and pCMV-Cas9-GFP (target site: 5′-AGGTAACGGCTTCGGGAATAGG-3′) (Sigma-Aldrich, Darmstadt, Germany) using the X-tremeGENE HP DNA transfection reagent (Merck, Rahway, NJ, USA) according to the manufacturer’s instructions. After transient CRISPR/Cas9 activation, fluorescence-activated cell sorting (FACS) of GFP/RFP double-positive cells was performed for clone selection. PCR-screened clones for the gRNA target site or a potential deletion, as described later (Supplementary Figure S1). Clones that were heterozygous for the deletion were further screened for specific gRNA target site mutations by Sanger sequencing (Supplementary Figure S2). The gRNA target sites were amplified with the primers GFP F: 5′-CTGTCCCCATGGAGTCTCAC-3′, GFP R:5′-CATCCAGGCACTGTTCTTCA-3′, RFP F: 5′-CTGGAGCTGGTGGAGAAAAG-3′, and RFP R: 5′-GGATTGGGATGATGATGAGG-3′ using MyTaq™ Mix (Bioline, London, UK) and the PCR products were analyzed via QIAxcel (Qiagen, Hilden, Germany). The PCR product was purified with ExoSAP-ITTM (Applied Biosystems, Foster City, CA, USA), and sequenced according to Sanger sequencing using the BigDye® terminator v3.1 cycle sequencing kit (Applied Biosystems, Waltham, MA, USA). Sequences were analyzed using an ABI 3500 genetic analyzer (Applied Biosystems). The CellTiter 96® AQueous one-solution cell-proliferation assay (MTS, Promega, Madison, WI, USA) was performed according to the manufacturer’s recommendations. In brief, 1 × 104 cells were grown in a 96-well format in 100 µL/medium and treated with indicated conditions over different periods, as described under results. Then, 20 µL of the MTS reagent was added and incubated for 1–3 h at 37 °C, and the absorbance was measured at 490 nm and 655 nm. Relative cell viability after treatment was calculated by normalizing each value by the mean of the untreated control replicates. Unless stated otherwise, all experiments were conducted by pretreating cells with 80 nM of the specific siRNA or inhibitors for 24 h and subsequent treatment with 0.1 µM gemcitabine for 72 h. siRNA or mock-transfected cells were grown to almost 100% confluency before synchronizing the cells by decreasing FCS to 1% for 24 h. Wounds were created by scratching the cell monolayer with a 100 μL sterile pipette tip. Wound healing was monitored at 0, 24, and 48 h. Relative wound healing was calculated by measuring the mean distance at three defined positions of the scratch expressed as a percentage of the 0 h control. siRNA transfection was performed using HiPerFect transfection reagent (Qiagen) as described elsewhere [22]. In brief, 80 nM of gene-specific or negative control siRNA (all Star Negative Control, Qiagen) was incubated with 12 µL HiPerFect in 100 µL transfection medium containing serum-free RPMI at RT for 20 min and added to freshly seeded cells (3 × 105 cells). After 24 h or 48 h incubation, cells were further processed as indicated. siRNAs used were purchased from Qiagen and are summarized in Supplementary Table S2. Cells at 60–70% confluency were treated as indicated in the results section. Cells were washed in PBS and scraped in a 100 µL RIPA lysis buffer containing protease inhibitor cOmplete (Roche, Mannheim, Germany), PMSF (1 mM), and orthovanadate (1 mM). Total protein was quantified using a DC™ protein assay (Bio-Rad, Hercules, CA, USA). A total of 15 µg of proteins was separated using gradient SDS gels (4–20%, Bio-Rad) and blotted on nitrocellulose membranes by a Turbo Blot (Bio-Rad). Gene signals were detected as described before [23]. uPAR protein levels were determined by ELISA (DUP00, R&D Systems, Minneapolis, USA) according to the manufacturer’s protocol. In brief, cell lysates from 105 to 106 cells were 10-fold diluted in a RIPA lysis buffer, and 50 μL of cell lysates or standard was added to 100 μL of assay diluent RD1W solution. The samples were incubated for two hours at RT and washed four times with a 400 μL wash buffer. A total of 200 μL of human uPAR conjugate was added and incubated for 2 h at RT. After four washing steps, 200 μL of substrate solution was added and incubated for 30 min at RT protected from light before adding 50 μL of stop solution. The optical density was measured at 450 nm with a reference of 540 nm on a Tecan reader Infinite 200 Pro. uPAR concentrations were calculated for 106 cells. KRAS activity was quantified using the STA-400-K-T assay (Cell Biolabs) following the manufacturer’s instructions. In brief, 1 mg protein was subjected to raf1 RBD agarose beads and incubated at 4 °C for one h. Beads were pelleted, washed, and resuspended in 4× Laemmle buffer. KRAS activity was quantified by Western blotting of 20 µg supernatant protein. Statistical analysis and AUC estimation were performed using GraphPad 8.3.0. Data are shown as mean ± SEM. Two group comparisons were performed using Student’s t-test. Two-way ANOVA was applied to compare cell growth and resistance analyses. Survival was analyzed using the Kaplan–Meier test and significance was evaluated using the log-rank (Cox–Mantel) test. A p-value of < 0.05 was considered significant (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). Our previous study showed that uPAR gene amplification in PDAC correlates with poor prognosis [24]. Immunohistochemical (IHC) staining for uPAR, its ligand uPA, and the inhibitor PAI1 in a clinical cohort of 67 patients (Figure 1, Table 1) also confirmed a prognostic relevance of uPAR on the protein level. Patients with high uPAR expression (n = 46) had significantly shorter overall survival (OS) than patients with low uPAR levels (n = 23) (median survival 320 days in uPARhigh vs. 603 days in uPARlow patients, log-rank (Cox–Mantel) test, p = 0.0273) (Figure 1b) [25]. Using gene expression data from two TCGA datasets including 312 PDAC patients [26,27], patients with high uPAR mRNA expression had a significantly reduced OS compared to patients with tumors of low expression (log-rank (Cox–Mantel) test, p = 0.0099) (Figure 1c). IHC did not reveal any significant difference in OS for uPA and PAI1 (Supplementary Figure S1b,c); however, on the transcriptional level, high expression of both uPA and PAI1 showed a significantly decreased OS (Supplementary Figure S1d,e). Next, we wanted to investigate the molecular function of uPAR in PDAC cells. Therefore, we measured the uPAR protein expression levels by ELISA in six PDAC cell lines with known gene mutation status of KRAS, TP53, and PIK3CA as described in the Material and Methods section (Supplementary Figure S2a,b and Supplementary Table S1). We then generated uPAR knockout clones of the cell line with the highest uPAR expression (AsPC-1), using two gRNAs directed against uPAR exons 3 and 4 (Supplementary Figure S2c). Two clones with homozygous functional uPAR knockout (KO#1 and KO#2), carrying a deletion on one allele and a gRNA target-site-specific frameshift mutation on the other, revealed a virtually absent uPAR protein (Supplementary Figure S2d–g). Functional roles of uPAR have been described in cell proliferation, migration, and cellular plasticity [28,29,30,31]. Both AsPC-1 uPAR−/− clones showed a significant decrease in growth and migration capacity compared to the AsPC-1 WT controls (Figure 2a,b). To evaluate the role of uPAR in cellular plasticity, we immunoblotted nine markers involved in epithelial–mesenchymal transition (EMT) (Figure 2c). Western blot revealed a marked upregulation of epithelial markers E-cadherin and β-catenin. While the transcription factor Slug was slightly upregulated, Snail and TCF8/ZEB1, together with claudin and ZO1, showed a decreased expression, further indicating the mesenchymal to epithelial transition (MET) in uPAR−/− clones compared to AsPC-1 WT (Figure 2c). In accordance with this phenotype, we detected a marked increase in chemoresistance against up to 1 µM gemcitabine in uPAR−/− cells (Figure 2d). uPAR signaling has been described to involve FAK, Src, CDC42, p38, autophagy, and RAS signaling [32]. In addition, Wu et al. reported that FAK signaling contributes to intrinsic gemcitabine chemoresistance in pancreatic cancer cell lines [33]. By immunoblotting, we detected the activation of FAK, CDC42, p38, and LC3B, while ERK was inactivated in AsPC-1 uPAR−/− cells (Figure 3a). The influence of FAK on Ras signaling has been described before [34]. However, in cells with aberrant KRAS activation, FAK-Ras regulation seems to be disturbed. Knockdown of FAK in uPAR−/− cells using siRNAs led to decreased phosphorylation of CDC42, p38, and LC3B, and reactivation of ERK (Figure 3b). The diminished FAK activity also partially restored the sensitivity towards gemcitabine (Figure 3c and Supplementary Figure S3a). Knockdown of CDC42 and p38 also reactivated ERK, decreased LC3B, and increased gemcitabine sensitivity (Figure 3d–g and Supplementary Figure S3b,c). This indicates that CDC42 and p38 suppress ERK activity downstream of KRAS in the absence of uPAR. To evaluate whether uPAR re-expression could restore the WT phenotype, uPAR−/− cells were transfected with a human uPAR gene expression vector as described in the Material and Methods section. This recovered uPAR protein levels (Supplementary Figure S2h) and significantly enhanced migratory capacity (Figure 3h). uPAR re-expression also recovered gemcitabine sensitivity and induced resistance against the p38 inhibitor JX401 (Figure 3i and Supplementary Figure S3d). Pharmacological inhibition of ERK with SCH772948 reduced gemcitabine sensitivity only in uPAR WT but not in AsPC-1 uPAR−/− cells (Figure 3j and Supplementary Figure S3e). Together, this indicates that uPAR mediates gemcitabine sensitivity in an ERK-dependent manner. The autophagy marker LC3B was induced in AsPC-1 uPAR−/− cells. Autophagy promotes tumor cell survival and contributes to chemoresistance [35]. Increased autophagy has been described to be responsible for the resistance of PDAC to gemcitabine that could be partially reversed by specific inhibitors [36]. To investigate whether increased autophagy in uPAR−/− clones was responsible for the observed gemcitabine resistance, we inhibited autophagy with 3-methyladenine (3-MA) or chloroquine (CQ). Both inhibitors significantly restored sensitivity towards gemcitabine in AsPC-1 uPAR−/− but not in AsPC-1 WT (Figure 3k and Supplementary Figure S3f). To evaluate the interplay of uPAR and mutated KRAS in response to gemcitabine, we used the KRAS WT cell line BxPC-3 (uPAR high), the KRAS mutant cell line AsPC1 (uPAR high), and the KRAS mutant cell line PANC-1 (uPAR low) (Figure 4a and Supplementary Figure S2a). AsPC1 responded best towards gemcitabine, PANC-1 showed a medium response, and BxPC3 was the most resistant cell line (Figure 4b). KRAS has been described to induce uPAR expression [17]. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly (Figure 4c). Silencing of KRAS in AsPC1 reduced the response towards gemcitabine whereas the expression of mutated KRAS in BxPC-3 cells increased gemcitabine sensitivity. Transfection of uPAR in PANC-1 likewise increased gemcitabine sensitivity (Figure 4d). uPAR and mutated KRAS switched cells to a mesenchymal phenotype (Figure 4e), at the same time promoting activation of MEK and ERK and suppressing FAK and CDC42 signaling (Figure 4f). Noll et al. [8] published HNF1A as a surrogate marker for the exocrine-like PDAC subtype and expression of keratin 81 (KRT81) as a marker for the quasi-mesenchymal (QM) type. Tumors negative for both markers (DN) were enriched for the classical PDAC subtype. We wanted to know if tumors with high uPAR expression segregate with one of these subtypes. In our own cohort of 57 patients with clinical follow-up, n = 31 (54%) showed expression of HNF1A, n = 19 (33%) were positive for KRT81, and n = 7 (12%) were DN. Because the DN group was too small, we excluded it from further analysis. The exocrine-like group consisted of 21 uPAR low and 10 uPAR high cases, and the QM group contained 9 uPAR low and 10 uPAR high cases. Survival analysis was supplemented by gene expression data from the two TCGA cohorts (n = 82 cases HNF1A high vs. n = 85 cases KRT81 high). The overall survival of patients with HNF1A-positive exocrine-like PDAC was significantly longer than patients with KRT81-positive QM tumors (p < 0.0001, Figure 4g). In the HNF1A-positive cohort, tumors with low levels of uPAR had a significantly better outcome than tumors with high expression and the mortality curve even reached a plateau after 1000 days, indicating long-term survival of some patients. In the KRT81high QM and DN group, there was a trend towards longer survival in patients with tumors with low levels of uPAR that did not reach statistical significance (Figure 4h,i and Supplementary Figure S4), indicating that the prognostic impact of uPAR may vary among different molecular subgroups. PDAC remains one of the human tumors with the highest mortality. uPAR is associated with early invasion, metastasis, and poor prognosis in many solid and hematological tumors [9,10,11]. We have previously shown that PDAC with high-level gene amplifications of uPAR have a particularly poor prognosis [24]. We here show in our cohort of 67 samples and in 168 PDAC samples from the TCGA database that overexpression of uPAR on the mRNA and protein level is also associated with significantly shorter OS. Importantly, although our data suggest that high expression of uPAR is an adverse prognostic factor in all PDAC, its negative impact on survival is more pronounced in some molecular subgroups (especially in exocrine-like tumors) than in others. uPAR has been described to act through its vitronectin-mediated interaction with integrins to transmit mechanical forces across the cell membrane [37,38,39]. The ECM–integrin interaction mediates the intrinsic chemoresistance of cancer cells [40], a phenomenon that has also been called cell-adhesion-mediated drug resistance (CMDR). CMDR has been explained by the strong binding of integrins to the ECM, which activates FAK. Integrin and EGFR signaling activates FAK and influences adhesion, motility, and cell growth [41,42]. FAK has seemingly paradoxical roles in cell migration and metastasis [43]. FAK is a ubiquitously expressed tyrosine kinase that localizes at focal adhesion complexes and transmits adhesion- and growth-factor-dependent signals into the cell [34,43,44]. In contrast to normal cells where FAK is a positive regulator of cell migration and proliferation [45], tumors with constitutive growth factor signaling (such as EGFR) or RAS mutations and consecutive high intrinsic levels of ERK utilize FAK as a negative regulator of cell migration through ERK-dependent dephosphorylation of particular FAK tyrosine residues [43,46]. Constitutive activation of FAK has also been proposed to contribute to the intrinsic chemoresistance against gemcitabine in the pancreatic cancer cell line AsPC-1 [9,33]. We here show that uPAR knockout in AsPC1 cells leads to induction of FAK, Src, CDC42, and p38, as well as chemoresistance towards gemcitabine. Our data further show that this chemoresistance is mediated through p38-induced autophagy. Numerous early clinical trials [47] have shown significant antitumor activity with tolerable toxicity of the autophagy inhibitor chloroquine, in combination with other cytotoxic chemotherapies in a variety of solid cancers, including colorectal and renal cell carcinomas [48]. A randomized clinical phase II trial in 102 PDAC patients treated with gemcitabine and nab-paclitaxel with or without CQ showed no difference in progression-free survival. Still, the authors proposed that preoperative CQ might increase curative resection rates [49]. A total of 90–95% of PDACs harbor activating mutations of KRAS that are thought to occur early in carcinogenesis [16]. Mutated KRAS is a potent oncogenic driver that promotes cell proliferation and migration by activating the downstream MAP kinases ERK1/2 [50]. KRAS has not only been reported to induce uPAR expression by AP-1-dependent transactivation of the uPAR promoter [17], but also mediates FAK dephosphorylation [43]. We here show that a) constitutively active KRAS induces uPAR and b) KRAS and uPAR cooperate in promoting a mesenchymal cell phenotype by activating MEK/ERK signaling and by the suppression of FAK/CDC42/p38 signaling. At the cellular level, this mesenchymal state implies increased cell proliferation and migration as a possible explanation of the poor prognosis of tumors with high levels of uPAR. At the same time, it also implies suppressed cellular dormancy via FAK signaling and p38-mediated autophagy, thus rendering the cells more vulnerable to gemcitabine. These observations highlight a potential therapeutic dilemma that applies both to KRAS and uPAR as emerging targets. Although recent studies propose uPAR as a good candidate for antibody-targeted therapy in cancer [51,52,53,54,55], our results show that these treatments could, at the same time, induce cellular dormancy and render the tumor more resistant to chemotherapy (such as gemcitabine). Tailored strategies should consider this resistance by adding autophagy inhibitors, such as chloroquine, to the regimens. In summary, we have confirmed uPAR as a potent modulating prognostic factor, especially in the large molecular subgroup of exocrine-like tumors. uPAR cooperates with mutated KRAS in the important switch between an active mesenchymal vs. a dormant epithelial cellular phenotype. By keeping tumor cells in the active mesenchymal state, uPAR promotes KRAS-driven proliferation and cell migration as a likely explanation for the poor prognosis of PDAC with high expression of uPAR. At the same time, this active mesenchymal state renders tumor cells more vulnerable to chemotherapy such as gemcitabine. Targeting either uPAR or KRAS could induce cellular dormancy and autophagy, thus leading to relative chemoresistance and limited therapeutic efficacy. Emerging clinical trials should take this possibility into account.

PMC10000459

Ozioma S. Chioma,Elizabeth Mallott,Binal Shah-Gandhi,ZaDarreyal Wiggins,Madison Langford,Andrew William Lancaster,Alexander Gelbard,Hongmei Wu,Joyce E. Johnson,Lisa Lancaster,Erin M. Wilfong,Leslie J. Crofford,Courtney G. Montgomery,Luc Van Kaer,Seth Bordenstein,Dawn C. Newcomb,Wonder Puryear Drake

Low Gut Microbial Diversity Augments Estrogen-Driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease

28-02-2023

estrogen,gut microbiome,lung fibrosis,sarcoidosis,Th17 cells

Although profibrotic cytokines, such as IL-17A and TGF-β1, have been implicated in the pathogenesis of interstitial lung disease (ILD), the interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as the phosphorylation of STAT3, have not been defined. Here, through chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells, we show that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. The genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after the repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. An assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota in relation to lung fibrosis severity. An analysis of female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-β1 levels in CD4+ T cells compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis.

Low Gut Microbial Diversity Augments Estrogen-Driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease Although profibrotic cytokines, such as IL-17A and TGF-β1, have been implicated in the pathogenesis of interstitial lung disease (ILD), the interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as the phosphorylation of STAT3, have not been defined. Here, through chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells, we show that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. The genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after the repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. An assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota in relation to lung fibrosis severity. An analysis of female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-β1 levels in CD4+ T cells compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis. An ever-growing synergy of human and animal investigations supports the important role of sex hormone regulation relating to immunity in the pathophysiology of chronic lung diseases [1,2]. IL-17 signaling has been implicated in numerous chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF), lung cancer and pulmonary sarcoidosis [1,3,4,5]. Moreover, striking clinical disparities according to sex are observed in Th17 cell-mediated diseases. For example, although the incidence of IPF is higher in men, being of the female sex is predictive of better IPF clinical outcomes [6,7,8]. Among patients with pulmonary arterial hypertension, female patients have better survival than males [9,10,11]. These observations support the urgent need to identify relevant sex-specific mechanisms in chronic pulmonary inflammation. Independent reports demonstrate that profibrotic signaling pathways converge on STAT3, an important molecular checkpoint for tissue fibrosis [12,13]. Immune cells, including CD4+ T cells, produce IL-6, which enhances collagen production through the induction of JAK/STAT3/IL-17A or JAK/ERK/TGF-β1 signaling in local and systemic environments [14,15,16,17]. Distinctions in clinical outcomes by sex support an investigation of the interplay of female gonadotrophic hormones with the STAT3-dependent induction of profibrotic cytokine expression. The interactions of the alpha subunit of the estrogen receptor (ERα) and STAT3 protein, both transcription factors, have been reported in breast cancers of epithelial origin, noting enhanced epithelial–mesenchymal transition (EMT) as well as augmented tumor metastasis [18]. However, the immunologic consequences of ERα binding to the STAT3 gene in CD4+ T cells of patients with lung fibrosis remain unexplored. The observed disparate clinical outcomes in chronic lung diseases by sex support the investigation of the impact of gonadotrophic hormones on STAT3 signaling, specifically in the context of the profibrotic cytokines, IL-17A and TGF-β1. Here, we report that human females experiencing a loss of lung function due to progressive fibrosis, as well as female murine models of bleomycin-induced lung fibrosis, demonstrate increased T regulatory cells with TGF-β1 expression (immunosuppressive) in the fibrotic lung microenvironment. Lower estrogen states, such as those found in males and ovariectomized female mice, reveal increased IL-17A expression due to elevated percentages of pulmonary Th17 cells (pro-inflammatory). Moreover, the investigation of this estrogen–adaptive immunity interplay in distinct environments reveals that low gut microbial diversity further increases estrogen-induced lung fibrosis. These data demonstrate a distinct sex-specific role for STAT3 signaling in CD4+ T cells, thus paving the way for developing personalized (e.g., sex-based) immunotherapeutic strategies for chronic lung inflammation. To participate in this study, all of the human subjects signed a written informed consent form, and the patients were enrolled at Vanderbilt University Medical Center. All of the human studies were approved by the appropriate institutional review board (VUMC 040187). For inclusion in this study, the clinical and radiographic criteria used to define sarcoidosis were applied [19]. IPF subjects were defined according to recent American Thoracic Society (ATS) guidelines [20], and systemic sclerosis patients were defined according to the 2013 American College of Rheumatology criteria [21]. Clinical lung progression was defined as previously described [22]. Pulmonary function testing was performed as clinically indicated. FVC decline was defined as a relative reduction of ≥10% in the percent of predicted FVC. There were four human cohorts in this study: 25 healthy controls (7 males and 18 females), 31 sarcoidosis patients (11 males and 20 females), idiopathic pulmonary fibrosis (IPF) patients (36 males and 9 females), and scleroderma patients (5 males and 6 females). Information related to the demographics of the study subjects is provided in Table 1. The Ficoll–Hypaque density gradient centrifugation method was used to isolate peripheral blood mononuclear cells (PBMCs) from the whole blood of all four human cohorts in this study: healthy controls, sarcoidosis, IPF, and scleroderma patients, as previously described [23,24]. The PBMCs were then stored in fetal bovine serum containing 10% dimethyl sulfoxide (DMSO) at a concentration of 10 × 106 cells/mL in a −80 °C freezer before being transferred to liquid nitrogen for prolonged storage or before use. Primary CD4+ T cells were negatively selected using immunomagnetic bead separation (STEMCELL, EasySep #17951). Approximately 1 to 2.5 million total T cells were obtained from 5 to 10 million PBMCs. The T cells were first incubated with 2 mM disuccinimidyl glutarate for 35 min at room temperature; then, formaldehyde was added to a final concentration of 1%, and the cells were incubated for another 10 min at room temperature [25]. The nuclei were isolated using the Covaris truChIP Chromatin Shearing Kit and fragmented by sonication. Immunoprecipitation was performed using an anti-ERα antibody (Cell Signaling #8644) and protein A+G magnetic beads. The chromatins were de-crosslinked and purified using AMPure XP beads. ChIP-seq libraries were prepared according to Illumina protocols and were sequenced using 75 bp paired-end sequencing on an Illumina NextSeq, producing an average of 135,924,844 reads per library. The ChIP-seq reads were examined for technical artifacts using FastQC. No aberrant technical behavior was identified. The reads were trimmed for adapter sequences and decontaminated for sequencing artifacts by using bbduk. The trimming options were set to ktrim = right trimming, mink = 11, hdist = 1, qin = 33, tpe and tbo options enabled. BBDuk’s list of Illumina sequencing adapters was used to perform adapter trimming. Decontamination was performed against phiX adapters and bbduk’s database of sequencing artifacts. The decontaminated reads were aligned to version GRCh38 of the human reference genome using BWA-mem [26], with the following options: -L 100 -k 8 -O 5. Following the alignment, the peaks were called with respect to the input chromatin library using MACS2 [27], with the following options: -nomodel –shift -100 –extsize 200 g hs -q 0.05 -f BAMPE –keep-dup all –broad. All of the murine procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center (protocol #M1700043). For the murine model of bleomycin-induced pulmonary fibrosis, 5- to 8-week-old mice weighing approximately 17–22 g were used. The mice were anesthetized with an intraperitoneal injection of 80 μL of 20 mg/mL Ketamine/1.8 mg/mL Xylazine solution. Then, 75 μL containing 0.04 units of bleomycin (Novaplus Lake Forest IL) in saline or an equal volume of saline (0.9% sodium chloride) (Hospira Inc., Lake Forest IL), used as a control, was administrated intranasally to wild-type or ESR-1-/- mice, as previously described [28]. The lungs were harvested for histology, flow cytometry, or single-cell isolation, as previously described [16]. The mouse strains used are described in Table S1. The degree of fibrosis in the murine lung tissue was assessed using Ashcroft scoring, as previously described [29]. The collagen content was determined using a Sircol Collagen Assay kit (Biocolor, Newtown Abbey, UK), as previously described [30]. Both murine and human flow cytometry experiments were conducted with an LSR-II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the information related to all the antibodies used in this study is listed in Table S2. Live cells were gated based on the forward and side scatter properties, and the surface staining of cells was performed as previously described [31]. Th17 cells were identified by flow cytometry using key transcriptional factors, such as STAT3, as previously described [32]. The cells were gated on singlets, live CD3+ and CD4+ cells. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). A minimum of 50,000 events were acquired per sample. Ovariectomy or sham surgeries were conducted at three weeks of age by the Jackson Laboratory, and the experiments were carried out when the ovariectomized or sham-operated mice were 6 weeks old. At 6 weeks of age, 60-day slow-release pellets (Innovate Research of America, Sarasota, FL, USA) containing 17β- estradiol 0.1 mg (E2), progesterone 25 mg (P4) or a combination of 17β-E2 (0.1 mg) and P4 (25 mg) were surgically placed subcutaneously into ovariectomized C57BL/6J mice, as previously described [33]. As a control, 25.1 mg of vehicle pellets (Innovative Research of America) was surgically placed into the sham-operated females or ovariectomized female mice. Three weeks (21 days) after the pellets were implanted, the mice were challenged with intranasal bleomycin (0.04 Units) and sacrificed 14 days later, as previously described [28]. Studies involving large and independent experimental cohorts of mice were performed at least twice. Fecal pellets were collected from female mice at Day 14 in each housing cohort, and genomic DNA (gDNA) was extracted with the Qiagen DNAeasy extraction kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The gDNA concentration and quality were confirmed using the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). The metagenomic sequencing and analysis of fecal pellets was conducted as previously described [34]. The sequences of gut microbiota have been deposited into BioProject ID PRJNA899808. Wilcoxon Rank Sum tests in R were used to examine differences in Shannon diversity and evenness between the ABSL-1 and ABSL-2 environments. The code for all of the analyses can be found at http://github.com/emallott/PulmonaryFibrosisMicrobiota (accessed on 19 January 2023) [34]. When comparing different experimental groups, we used an unpaired two-tailed Student’s t-test. Multiple-group comparisons were performed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Statistical analysis for all figures was carried out using Prism version 7.02 (GraphPad Software, San Diego, CA, USA). For a result to be considered statistically significant, a p-value of less than 0.05 was used. The estrogen receptor alpha subunit (ERα) is not only a receptor but also serves as a transcription factor. To identify factors that may modulate STAT3 expression during lung fibrosis, we interrogated ChIP-seq datasets in the ENCODE 3 repository [35]. In five human cell lines, including cancers and EBV-transformed B lymphocytes, a significant enrichment of ERα binding was demonstrated within the STAT3 locus. Representative tracks among the technical replicates for each cell line were visualized in the WashU Epigenome Browser [36] (Figure 1A). The numbers of starting reads, decontaminated reads, alignment successes, and enriched peaks are given in Table S3. These findings in the Chip-seq datasets confirmed previous reports indicating that ERα, which is encoded by the ESR1 gene, and STAT3 are important in breast and ovarian cancer [18,37], supporting the hypothesis that the STAT3 gene locus is a frequent target of ERα activity in various cell types. The targeting of ERα to the STAT3 gene locus in T cells has not been previously described. To determine whether ERα interacts with the STAT3 locus in CD4+ T cells through DNA binding activity, we performed genome-wide ChIP-seq for Erα-bound regions. Primary CD4+ T cells were derived from the PBMCs of six healthy individuals with varying demographics (Table S4). Of the six ChIP libraries (four females and two males), sample p1035928-8, which corresponds to a female, identified an over sixfold greater number of ERα-enriched regions relative to any other sample. We used the GREAT algorithm to perform ontology-based functional enrichment analyses on that sample. ERα-enriched sites were statistically significantly enriched in genes related to T-cell function and development (Table S5), suggesting that the peaks obtained from this ChIP capture are specific to CD4+ T-cell function and are not randomly organized across the genome. Finally, we examined the STAT3 locus in detail. We found that sample p1035928-8 contains six ERα-binding regions within or proximal to the STAT3 genomic locus, including two in its promoter region (Figure 1B). Overlaying chromatin accessibility data from the ENCODE project [35], we noted that each of these regions exhibits DNase hypersensitivity in at least one ENCODE cell line. Three of these regions also displayed evidence of estrogen-related receptor alpha (ESRRA) binding in other cell lines (K562, GM12878). Taken together, these results demonstrate that ERα binds the STAT3 locus in CD4+ T cells, specifically at known regions of chromatin accessibility shared with various cell types. Because transcription factor ESR-1 (alpha subunit of ESR) was identified as binding to the STAT3 gene in CD4+ T cells, we investigated the role of the ERα subunit in profibrotic cytokine expression using a murine model of bleomycin-induced lung fibrosis in WT and ESR-1 knockout (ESR-1-/-) mice. Both murine cohorts were challenged intranasally with bleomycin and harvested on day 14. ESR-1-/- mice contain supernormal estrogen levels in their serum due to the loss of the ESR-1 signaling-mediated negative feedback loop [38]. We observed that female ESR-1-/- mice lost significantly less weight and had the same mortality compared to their WT counterparts (Figure 2A,B). Male ESR-1-/- mice also demonstrated reduced weight loss but had significantly increased survival compared to WT males (Supplemental Figure S1). We used flow cytometry to examine profibrotic cytokine expression in pulmonary CD4+ T cells of the murine cohorts. The levels of IL-6 and IL-23R, key mediators of Th17 cell differentiation, were significantly reduced in the lung CD4+ T cells of female ESR-1-/- mice compared to their WT counterparts (Figure 2C,D). Remarkably, the levels of pSTAT3 and IL-17A were increased in ESR-1-/- compared with WT mice (Figure 2E,F). These data demonstrate that ESR-1 has a key role in the induction of IL-6 and IL-23R expression in CD4+ T cells, as well as the repression of pSTAT3 and IL-17A expression in CD4+ T cells during the pulmonary fibrosis of females. To further delineate the contribution of female gonadotrophic hormonal signaling to the progression of proinflammatory cytokine expression in the lung, we used female C57BL/6J mice that were ovariectomized or sham-operated at three weeks of age. Slow-release pellets containing either 17β-estradiol (17β-E2, 0.1 mg), progesterone (P4, 25 mg), the combination of 17β-E2 (0.1 mg) and P4 (25 mg) or a vehicle (25.1 mg) were subcutaneously implanted into adult ovariectomized female C57BL/6J mice at six weeks of age. At nine weeks of age, all groups were challenged with bleomycin, and the lungs were harvested 14 days later. There was no significant difference in weight loss or survival across the hormone treatment groups compared to the ovariectomized mice implanted with placebo pellets (Figure 3A,B). We performed flow cytometric analysis of single-cell lung suspensions (SCLS) to assess alterations of CD4+ T cell populations. TGF-β and IL-17A are profibrotic cytokines that are expressed by regulatory T cells and Th17 cells, respectively. We began by comparing regulatory T and Th17 cell populations in sham-operated, menstruating female mice. We noted a significantly higher population of regulatory T cells compared to Th17 cells in the sham-operated mice (Figure 3C). We then assessed for IL-17A cytokine expression in response to the loss of female hormones. Ovariectomized mice displayed decreased CD4+IL-6+ T cells compared to the sham-operated mice; supplementation with both 17β-E2 and P4 in ovariectomized mice normalized IL-6 expression. Neither hormone individually restored IL-6 expression by CD4+T cells to the same levels as the sham-operated mice (Figure 3D). The same trends held for the IL-6 co-receptor GP130 (Figure 3E). Remarkably, and akin to our observation in ESR-1-/- mice, the levels of pSTAT3 were increased in the CD4+ T cells of ovariectomized mice compared to sham-operated animals, again returning to sham levels in ovariectomized mice by the addition of female hormones (Figure 3F). In accordance with an increase in pSTAT3, we also observed heightened CD4+IL-17A+ T cells in ovariectomized mice compared to sham-operated animals. The addition of 17β-E2, P4 or both to ovariectomized mice decreased IL-17A expression compared to the placebo (Figure 3G). A representative FACS plot is provided (Figure 3H). Overall, these findings reveal that female hormones repress inflammatory profibrotic cytokine expression by inhibiting pSTAT3 signaling and IL-17A expression in murine pulmonary CD4+ T cells following bleomycin administration. To determine the physiologic significance of estrogen signaling for profibrotic cytokine expression, we performed histologic analysis and collagen quantification of the lung using the Sircol assay. Analysis of lung histology using trichrome staining noted significantly less fibrosis in ovariectomized mice without hormone replacement compared to the sham-operated mice or ovariectomized mice given dual estrogen (17β-E2)/progesterone (P4) hormone pellets (Figure 4A). Ashcroft scoring (Figure S2) and the quantification of collagen content (Figure 4B) revealed a nonsignificant decrease in collagen levels in ovariectomized mice compared to mice that underwent sham ovariectomy surgeries. The replacement of female hormones with a combination of estrogen and progesterone pellets increased fibrosis compared to the ovariectomized placebo group (Figure 4B). Similarly, a nonsignificant decrease in pulmonary collagen content was observed in ESR-1-/- mice compared to wild-type mice. The observation of a nonsignificant decline in the pulmonary lung content following the loss of estrogen signaling suggests that additional factors contribute to pathogenesis. We recently reported that the gut microbiota play an important role in lung fibrosis severity. ABSL-1 housing conditions favor gut microbiota diversity, whereas ABSL-2 conditions favor reduced gut microbiota diversity [34]. Using linear discriminant analysis (LDA) to examine species-level differences in the gut microbiota, 10 taxa were overrepresented in ABSL-1 mice, and five taxa were overrepresented in ABSL-2 mice. The overrepresented taxa in ABSL-2 mice included Lachnospiraceae bacterium A2, Lachnospiraceae bacterium 28–4, Firmicutes bacterium ASF500 and Romboutsia ilealis [34]. A higher relative abundance of Firmicutes in the lung microbiota of bleomycin-treated mice with fibrosis has been reported [39]. The species overrepresented in ABSL-1 mice included Staphylococcus nepalensis, Dubosiella newyorkensis, Acetatifactor muris, Lactobacillus animalis, Lactobacillus murinus and Acutalibacter muris [34]. No distinctions in the lung microbiota are present in these mice regarding the housing condition. Specifically, rearing environments that favor low gut microbiota diversity, such as ABSL-2 housing conditions, induce severe lung disease compared to ABSL-1 conditions. To confirm if gut microbiota impact female ILD severity, we began by assessing the lung collagen content in wild-type female mice who received intranasal bleomycin while housed in different environments: germ-free, ABSL-1 or ABSL-2 conditions. We noted significant distinctions in lung collagen content among wild-type females according to the rearing environment, with ABSL-2 female mice demonstrating the most severe disease compared to germ-free or ABSL-1 mice (Figure 4D). To determine the impact of estrogen signaling and gut microbiota on lung fibrosis severity, we assessed the lung collagen content among ovariectomized mice, as well as those ovariectomized with estrogen replacement, while housed under either ABSL-1 or ABSL-2 conditions. Remarkably, we noted that ovariectomized mice housed under ABSL-1 or ABSL-2 conditions did not demonstrate a change in the collagen content (Figure 4E). Equally noteworthy was the observation that a significant increase in lung fibrosis was noted among ovariectomized mice who received estrogen replacement and were housed in ABSL-2 conditions compared to those housed in ABSL-1 conditions. These findings reveal a synergistic relationship between estrogen signaling and gut dysbiosis regarding lung fibrosis severity (Figure 4E). To investigate the hypothesis that the gut microbiota is an important contributor to the differences in fibrosis severity between female mice housed under ABSL-1 and ABSL-2 conditions, we performed metagenomic analysis on fecal pellets from female mice in each housing cohort. We did not detect microorganisms in the stool of female germ-free mice by sequencing and culture, as expected. Shannon alpha diversity, a measure of species richness and evenness, was considerably higher in female ABSL-1 mice compared with female ABSL-2 mice using a Wilcoxon rank sum test (Figure 5A). Additionally, Pielou’s evenness was higher in ABSL-1 compared with ABSL-2 female mice, but species richness did not differ significantly (Shannon diversity: Wilcoxon, W = 108, p = 0.015; Pielou’s evenness: Wilcoxon, W = 106, p = 0.021; Species richness: Wilcoxon, W = 80.5, p = 0.450). The female mice housed under ABSL-1 and ABSL-2 conditions differed significantly in their gut microbiome composition using Jaccard but not Bray–Curtis dissimilarities (PERMANOVA, Bray–Curtis: F1,22 = 2.392, R2 = 0.098, p = 0.079; Jaccard: F1,22 = 8.369, R2 = 0.276, p < 0.001). A similar investigation in male mice revealed that the ABSL-1 and ABSL-2 microbiomes were significantly different using both metrics (PERMANOVA, Bray–Curtis: F1,24 = 4.728, R2 = 0.165, p = 0.004; Jaccard: F1,24 = 6.519, R2 = 0.214, p < 0.001) (Figure 4). Alpha diversity did not differ significantly between floors for male individuals (all p > 0.05). A comparison of female and male gut microbiota diversity according to the housing conditions reveals significantly greater gut diversity among females compared to males under ABSL-1 housing conditions (Figure 5B), whereas only greater species richness was noted among females under ABSL-2 housing conditions (Figure 5C). Beta diversity differences between ABSL-1 and ABSL-2 microbiota compositions also differed significantly when an analysis was conducted using both the Bray–Curtis dissimilarity metric index (Figure 5D) and the Jaccard index (Figure 5E), which account for the presence/absence of taxa and taxon abundance variation, respectively (PERMANOVA, ABSL-1 mice: Bray–Curtis: F1,17 = 4.424, R2 = 0.206, p = 0.014; Jaccard: F1,17 = 2.408, R2 = 0.124, p = 0.053; ABSL-2 mice: Bray–Curtis: F1,29 = 1.952, R2 = 0.063, p = 0.160; Jaccard: F1,29 = 7.944, R2 = 0.215, p < 0.001). These findings support the hypothesis that the female gut microbiome changes according to the rearing environment. Because of the role of female gonadotrophic hormones in reducing the CD4+ T cell-mediated proinflammatory and profibrotic environment in mouse models of lung fibrosis, we probed samples from human patients with fibrotic lung diseases for sex-associated differences. Consistent with the murine model of lung fibrosis, we observed higher levels of STAT3 mRNA and pSTAT3 protein in CD4+ T cells from the male compared to the female sarcoidosis patients (Figure 6A,B). We noted similarly increased mRNA and protein expression of the master transcription factor regulating IL-17A production, RORC, in CD4+ T cells from the male compared to the female sarcoidosis patients (Figure 6C,D). Additionally, among sarcoidosis patients experiencing disease progression, females expressed significantly higher IL-6 levels in their CD4+ T cells compared to males (Figure 6E). We also assessed IL-17A and TGF-β1 production by sex, as CD4+ T cells promote pulmonary fibrosis through the STAT3-medicated production of IL-17A and TGF-β1 [16]. We observed higher IL-17A mRNA and protein expression in CD4+ T cells from male compared to female sarcoidosis patients (Figure 6F,G). CD4+ T cells from female sarcoidosis patients expressed significantly higher free TGF-b1 than males and the healthy female controls (Figure 6H). There were no distinctions in the TGF-b1 precursor protein, latency-associated peptide-TGF-β, among males compared to females (Figure 6I). These findings demonstrate the differential immune modulation of STAT3 signaling pathways in human CD4+ T cells of males (increased) and females (reduced) with fibrotic lung disease. Consequently, CD4+ T cells from males exhibit higher proinflammatory cytokine expression due to enhanced IL-17A production, whereas CD4+ T cells from females exhibit increased immunosuppressive cytokines due to greater TGF-β1 expression. We assessed for a possible contribution of female hormones to other fibrotic diseases, including IPF and Systemic Sclerosis (SSc), by quantifying the serum 17β-E2 levels in age-matched patients and healthy controls. Serum 17β-E2 was greater in male SSc and IPF patients compared to age-matched male healthy controls (Figure 6J). These findings demonstrate the positive interplay of female gonadotrophic hormones in male- and female-predominant fibrotic lung diseases. This original report reveals the “ying–yang” effects of estrogen-induced lung fibrosis in female interstitial lung disease. Estrogen clearly augments the development of lung fibrosis (Figure 4); yet, the binding of ERα to the STAT3 promoter shifts profibrotic cytokine expression away from proinflammatory phenotypes mediated by IL-17A to immunosuppressive phenotypes mediated by TGF-β1 (Figure 2 and Figure 3). Human cytokine expression confirmed reduced pSTAT3 expression in females, leading to increased TGF-β1 production, whereas males display higher IL-17A levels. The beneficial effects of estrogen were apparent. Although ESR-1-/- mice and surgical ovariectomy confirm estrogen’s profibrotic capacity in lung fibrosis, it is worth noting that Th17 cell differentiation is reduced due to the transcription factor ERα‘s ability in relation to the STAT3 promoter (Figure 1, Figure 2 and Figure 3). The loss of STAT3 signaling has been shown to shift the IL-6-JAK2-STAT3 induction of IL-17A to sustained IL-6-ERK-TGF-β1 expression in local and systemic CD4+ T cells [15,16]. This is the most likely explanation for the increased regulatory T cells noted in females and the increased STAT3 signaling and IL-17A production following ovariectomy (Figure 3 and Figure 6). Both ovariectomized and ESR-1-/- mice revealed significantly lower IL-6 and GP130 levels than sham-treated animals but increased pSTAT3 and IL-17A levels in CD4+ T cells (Figure 3). Higher estrogen states augment IL-6 production, but instead of inducing a proinflammatory state supported by increased CD4+ IL-17A levels, estrogen concomitantly inhibits STAT3 signaling. These immune alterations are likely relevant to other IL-17A-mediated diseases in the postmenopausal state, such as myocardial infarctions and osteoporosis [40,41]. Enhanced TGF-β1 expression protects against osteoporosis [42]. The pathologic effects of estrogen were also determined. A prior study noted increased ESR-1 expression in human IPF lung samples and that the chemical inhibition of ESR-1 results in reductions in bleomycin-induced pulmonary fibrosis in male mice [43]. The genetic and surgical ablation of estrogen-dependent signaling resulted in reductions in the pulmonary collagen content, which confirms the profibrotic nature of estrogen in female-predominant ILD (Figure 4). Remarkably, the observed reductions were not statistically significant, suggesting that other factors contribute to lung fibrosis severity in females. The induction of lung fibrosis in females under distinct housing conditions unveiled the role of the gut microbiome in lung fibrosis severity. Wild-type female mice treated with intranasal bleomycin demonstrate the greatest lung severity under ABSL-2 conditions and minimal fibrosis under germ-free conditions, thus confirming the important contribution of gut flora to female lung fibrosis (Figure 4D). Conditions that favor the loss of female gut microbial diversity, such as ABSL-2 housing conditions, lead to greater lung fibrosis compared to ABSL-1 conditions (Figure 4 and Figure 5). Equally noteworthy is the observation that fibrosis is synergistic between estrogen signaling and gut dysbiosis, suggesting that the profibrotic nature of estrogen is heavily influenced by gut microbiota and that the capacity of gut microbiota to induce fibrosis is influenced by the host hormone status. A growing body of literature supports crucial interactions between gut microbiota and estrogens [44,45]. The conjugation of glucuronic acid (GlcA) to a compound, such as estrogen, marks it for elimination via the GI or urinary tract. β-glucuronidase, an enzyme that deconjugates estrogen, mediates estrogen release into the serum in its active form [46,47]. Gut microbiota can inhibit or induce β-glucuronidase activity. In addition, it was previously noted that ABSL-2 stool contains reduced lactobacilli within the microbial community. Lactobacillus spp, which were elevated in ABSL-1 stool, can reduce fecal β-glucuronidase activity [45]; future studies that assess the capacity of lactobacilli to enhance urinary estrogen excretion and lower its serum levels are needed. Future studies defining the mechanisms by which ABSL-2 gut flora augment the estrogen induction of lung fibrosis are also warranted. Considering of the hormone status of the host, as well as defining the gut microbiome, is necessary to explain the clinical observations in females with ILD. TGF-β1 is the master regulator of fibrosis. Figure 6 demonstrates that TGF-β1 is most predominant in female sarcoidosis patients. In Figure 4, we see that gut dysbiosis augments lung fibrosis. When IL-6 induction occurs, downstream signaling can lead to either IL-17A or TGF-B1 expression. IL-17A expression leads to pulmonary inflammation. Estrogen signaling provides protection against proinflammatory fibrosis due to the capacity of the ERα to bind to the STAT3 promoter (Figure 1). This reduction in lung inflammation improves the prognosis. In menopausal females, the gut microbiome continues to drive lung fibrosis, but due to the reduced estrogen state, there is no inhibition of STAT3 expression and Th17 cell development. Lung fibrosis can now be mediated by IL-17A, which likely explains the increased symptoms after menopause. There are some limitations that should be noted. This investigation focused on female ILD; investigations of the role of testosterone in lung fibrosis are needed. There are also reports indicating that estrogen drives Th17 cell differentiation in chronic lung diseases, such as asthma [48,49]. Concomitant immune-gut microbiome investigations of asthma models with ILD models are warranted, including an inquiry into the interplay of gonadal hormones. Additionally, asthma pathogenesis is very distinct from ILD, which may also impact T cell differentiation. Another consideration is that the gut microbiome is influenced by diet. The mice in the murine model had the same diet; future studies assessing the impact of food consumption on the gut microbial community, metabolomic syndromes and inflammation are warranted [50,51,52]. An investigation into the impact of gut dysbiosis on estrogen signaling or of estrogen signaling on gut microbial communities is warranted. Finally, we observed Th17 cell populations increasing following the gavage of ABSL-2 stool into germ-free mice compared to the gavaging of ABSL-1 stool. Future analysis definitively identifying the microorganism(s) responsible for Th17 cell differentiation is warranted, followed by an assessment of their presence in the stool of murine asthma models, as well as asthmatic patients and ILD patients. Taken together, this investigation demonstrates that female gonadotrophic hormones are profibrotic yet, through the ERα binding of the STAT3 locus, reduce the inflammation induced by IL-17A expression in CD4+ T cells. The consequent reduction in inflammation is a likely contributor to the mortality benefit observed in premenopausal females with ILD. This study introduces another key contributor to lung fibrosis severity: gut dysbiosis. The synergistic impact of gut dysbiosis and estrogen on lung fibrosis supports a multi-pronged approach to the treatment of female-predominant lung fibrosis (Figure 7).

PMC10000467

Liang-Wei Lin,Shih-Wei Wang,Wei-Chien Huang,Thanh Kieu Huynh,Chao-Yang Lai,Chih-Yuan Ko,Yi-Chin Fong,Jie-Jen Lee,Shun-Fa Yang,Chih-Hsin Tang

Melatonin Inhibits VEGF-Induced Endothelial Progenitor Cell Angiogenesis in Neovascular Age-Related Macular Degeneration

03-03-2023

melatonin,endothelial progenitor cells,vascular endothelial growth factor (VEGF),platelet-derived growth factor-BB (PDGF-BB),angiogenesis,neovascular age-related macular degeneration

Neovascular age-related macular degeneration (AMD) is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that generates a huge, dark, blind spot in the center of the visual field, causing severe vision loss in over 90% of patients. Bone marrow-derived endothelial progenitor cells (EPCs) contribute to pathologic angiogenesis. Gene expression profiles downloaded from the eyeIntegration v1.0 database for healthy retinas and retinas from patients with neovascular AMD identified significantly higher levels of EPC-specific markers (CD34, CD133) and blood vessel markers (CD31, VEGF) in the neovascular AMD retinas compared with healthy retinas. Melatonin is a hormone that is mainly secreted by the pineal gland, and is also produced in the retina. Whether melatonin affects vascular endothelial growth factor (VEGF)-induced EPC angiogenesis in neovascular AMD is unknown. Our study revealed that melatonin inhibits VEGF-induced stimulation of EPC migration and tube formation. By directly binding with the VEGFR2 extracellular domain, melatonin significantly and dose-dependently inhibited VEGF-induced PDGF-BB expression and angiogenesis in EPCs via c-Src and FAK, NF-κB and AP-1 signaling. The corneal alkali burn model demonstrated that melatonin markedly inhibited EPC angiogenesis and neovascular AMD. Melatonin appears promising for reducing EPC angiogenesis in neovascular AMD.

Melatonin Inhibits VEGF-Induced Endothelial Progenitor Cell Angiogenesis in Neovascular Age-Related Macular Degeneration Neovascular age-related macular degeneration (AMD) is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that generates a huge, dark, blind spot in the center of the visual field, causing severe vision loss in over 90% of patients. Bone marrow-derived endothelial progenitor cells (EPCs) contribute to pathologic angiogenesis. Gene expression profiles downloaded from the eyeIntegration v1.0 database for healthy retinas and retinas from patients with neovascular AMD identified significantly higher levels of EPC-specific markers (CD34, CD133) and blood vessel markers (CD31, VEGF) in the neovascular AMD retinas compared with healthy retinas. Melatonin is a hormone that is mainly secreted by the pineal gland, and is also produced in the retina. Whether melatonin affects vascular endothelial growth factor (VEGF)-induced EPC angiogenesis in neovascular AMD is unknown. Our study revealed that melatonin inhibits VEGF-induced stimulation of EPC migration and tube formation. By directly binding with the VEGFR2 extracellular domain, melatonin significantly and dose-dependently inhibited VEGF-induced PDGF-BB expression and angiogenesis in EPCs via c-Src and FAK, NF-κB and AP-1 signaling. The corneal alkali burn model demonstrated that melatonin markedly inhibited EPC angiogenesis and neovascular AMD. Melatonin appears promising for reducing EPC angiogenesis in neovascular AMD. Age-related macular degeneration (AMD) is an age-related ocular disease that leads to visual impairment, drusen, retinal pigmentary changes, and blood vessel angiogenesis in the retina [1]. Increasing life expectancies and aging populations in most countries worldwide are contributing to a steady increase in the global prevalence of AMD [2]. AMD can be broadly classified as the non-neovascular (dry) or neovascular (wet) type. Neovascular AMD is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that creates a large blind spot in the center of the visual field, causing severe vision loss in more than 90% of patients [3]. Angiogenesis is a complex process that regulates many physiological functions, including wound healing and tissue development, as well as reproduction [4], while it also contributes to pathological processes, such as atherosclerosis [5] and inflammatory diseases [6], as well as neovascular AMD [7]. Angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB) contribute to neovascular AMD [8]. Inhibiting angiogenesis is therefore a critical strategy in neovascular AMD. Currently, anti-VEGF therapy is the only available treatment for neovascular AMD; this therapy targets the vascular endothelial growth factor receptor (VEGFR) [9]. Anti-VEGF/VEGFR2 therapy can effectively inhibit choroidal neovascularization (CNV) and downregulate levels of VEGF mRNA expression in mice with laser-induced CNV [10]. However, although anti-VEGF drugs have provided positive results in clinical trials, these outcomes have not translated into real-world outcomes [3,11]. Thus, other treatment targets besides VEGF and VEGFR are necessary for neovascular AMD. Endothelial progenitor cells (EPCs) are derived from bone marrow-derived endothelial stem cells, and are involved in physiological and pathological angiogenesis [12]. EPCs are recruited in response to angiogenesis, and regulate several cellular functions such as proliferation and migration [13]. Characterized by their surface markers CD34 and CD133, as well as by VEGFR2, EPCs play an important role in the progression of new blood vessel formation [14]. Importantly, VEGF stimulates EPC angiogenesis, including the survival, motility, and tube formation of EPCs [15,16], via the VEGFR2/c-Src/FAK signaling pathway [17] as well as the transcription factors NF-κB [18] and AP-1 [19]; this mobilization of EPCs enables the development of neovascular AMD [20]. EPC-dependent angiogenesis appears to be a valuable treatment target for neovascular AMD, as the anti-VEGF drug ranibizumab significantly reduces the high levels of circulating EPCs that are involved in AMD angiogenesis [21]. The synthesis and release of the neurohormone melatonin enables organisms to respond to circadian and seasonal rhythms [22]. Melatonin is mainly secreted by the pineal gland under the influence of light stimulation of the retina; to a lesser extent, melatonin is also synthesized within the eye, where melatonin uses ocular structures to mediate a variety of diurnal rhythms and physiological processes within the eye [23]. Increasing the concentration of melatonin at night promotes sleep, while decreasing the concentration of melatonin during the day promotes alertness [23]. Melatonin concentrations tend to decrease with age [24]. The effects of melatonin are beneficial for numerous physiological functions, including the promotion of ocular surface wound healing [25], reductions in inflammation and oxidative stress [24], and angiogenesis [24]. The expression and secretion of VEGF is important in retinal physiology [26]. The release of VEGF from retinal pigment epithelial cells helps to protect neuronal cells and the choroid, maintaining a healthy retina [26]. Notably, melatonin reduces retinal levels of VEGF and protects against ocular angiogenesis diseases [26]. However, whether melatonin affects VEGF-induced EPC angiogenesis in neovascular AMD is unclear. This study therefore aimed to determine whether melatonin treatment inhibits VEGF-induced EPC angiogenesis during the development of neovascular AMD. The study also sought to define any other underlying mechanisms, such as signaling pathways, which may mediate this process. Recombinant human VEGF (100-20) was bought from PeproTech (Rocky Hill, NJ, USA). VEGFR2 (07-158), phospho-VEGFR2 (orb106137), CD133 (orb13002), and β-actin (a5441) antibodies, and melatonin (M5250) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospho-FAK (3283S) antibodies was purchased from Cell Signaling (Danvers, MA, USA). FAK (sc-1688), phospho-c-Src (sc-12928-R), c-Src (2105S), phospho-c-Jun (sc-822), c-Jun (sc-74543), phospho-p65 (sc-101752), p65 (sc-8008), CD31 (sc-18916), and CD34 (sc-74499) antibodies, as well as the angiotensin II (FAK activator; sc-363643) and c-Src activator (sc-3052), were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The NF-κB activator (prostratin; ab120880) was purchased from Abcam (Cambridge, MA, USA). PDGF-BB antibody (MBS9404630) was purchased from MyBioSource (San Diego, CA, USA). The VEGFR2 short hairpin RNA (shRNA) plasmid was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). Human primary EPCs were prepared as described in our previous protocols [6]. EPCs were cultured in MV2 complete medium (PromoCell, Heidelberg, Germany) with 20% fetal bovine serum (FBS; HyClone, Logan, UT, USA), and maintained in a humidified incubator at 37 °C, 5% CO2 [27]. Gene expression profile records were downloaded from the eyeIntegration v1.0 database (https://eyeIntegration.nei.nih.gov, accessed on 13 January 2022) for 57 normal retina tissue samples and 448 retina tissue samples from AMD patients, for analysis of CD31, CD34, CD133, VEGF, and PDGF-BB expression [28]. Transwell chambers (8.0 µm pore sizes; Corning, NY, USA) were used for the migration assay. EPCs (8 × 103 in each well) were applied to the upper chambers of 24-well plates in 200 μL of serum-free MV2 complete medium containing 100 ng/mL VEGF. The lower chambers were filled with 300 μL of MV2 complete medium combined with 5% FBS and different concentrations of melatonin (0.1, 0.3, or 1 mM), then incubated for 18 h at 37 °C and 5% CO2. The cells in the lower chambers were fixed with 3.7% formaldehyde and stained with 0.5% crystal violet solution. The numbers of migrated cells were counted by microscope (Nikon, Tokyo, Japan) and analyzed by MacBiophotonics ImageJ software [29]. The 48-well plates were coated with 150 μL of Matrigel (BD Biosciences, MA, USA) and incubated for 30 min at 37 °C. EPCs (2 × 104 cells/well) were seeded onto the gel layer in a 50% MV2 complete medium containing VEGF with melatonin (0.1, 0.3, or 1 mM), then incubated for 6 h at 37 °C. EPC tube formation was evaluated under an inverted phase-contrast microscope (Nikon, Tokyo, Japan). The number of tube branches was quantified by MacBioPhotonics ImageJ software [30]. EPCs (5 × 103) were seeded in a 96-well plate and incubated with melatonin for 24 h or 48 h, before adding 10 μL of CCK-8 reagent (96992, Sigma-Aldrich, St. Louis, MO, USA) and incubating the EPCs at 37 °C for 2–4 h. The samples were quantified by a microplate reader (Bio-Tek, Winooski, VT, USA) at OD 570 nm. The cell lysate was examined with the Human Angiogenesis Protein Array (ARY007, R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol [31]. The results of the protein array were quantified with the ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Chicago, IL, USA). The chromatin immunoprecipitation analysis followed the previously described methodology [32,33]. DNA was immunoprecipitated with an anti-p65 or anti-c-Jun antibody and purified by phenol-chloroform extraction. Immune complexes were collected with protein G-Sepharose beads and eluted from the beads using an elution buffer. The purified DNA pellet was subjected to PCR, and then the PCR products were analyzed on 1.5% agarose gel electrophoresis and visualized by UV light. The primers 5′-CCAAGAGGCTAGATTCACAGTCAC and 3′-TTCAGCTGTTCCGGCCTTT were used to amplify across the PDGF-BB promoter region (−197/−7). The CAM assay was performed according to our previously published method [34]. VEGF and melatonin (0.1, 0.3, or 1 mM) were mixed with Matrigel and dropped onto the developing chicken egg, then incubated at 38 °C in an 80% humidified atmosphere. After 14 days, CAMs were observed by microscopy and photographic documentation. The number of CAM blood vessels was quantified by MacBioPhotonics ImageJ software. The Matrigel plug angiogenesis assay was performed according to our previous research [34,35]. Male nude mice (4 weeks old) were subcutaneously injected with 300 μL Matrigel containing VEGF with the indicated concentrations of melatonin. The plugs were collected after 7 days and processed by immunofluorescence staining for co-staining with CD31, CD34 and CD133 antibodies. All samples were then stained with 40,6-diamidino-2-phenylindole (DAPI) and captured by the TissueFAXS-S-plus imaging system (TissueGnostics, Vienna, Austria). Hemoglobin levels were examined using Drabkin’s reagent and detected at 450 nm with a microplate reader (Bio-Tek, Winooski, VT, USA). The structures of melatonin (PDB code: 5mxb) and VEGFR2 (PDB code: 3V2A) were downloaded from the Protein Data Bank (PDB, https://www.rcsb.org/, accessed on 11 May 2022). The molecular docking analysis between melatonin and the VEGFR2 extracellular domain (ECD) was performed using Discovery Studio software [36]. Male C57BL/6J mice (6–8 weeks old) were anesthetized with an intramuscular injection of Zoletil (0.2 mL/kg). Alkali burn injury was induced by placing 2 mm diameter filter paper (soaked in 1 mol/L NaOH) on the center of the right cornea for 30 s, before gently washing the ocular surface with 30 mL of 1X PBS solution. The mice were then immediately treated with melatonin by intraperitoneal (IP) injection (20 mg/kg or 60 mg/kg), or 1X PBS solution by IP injection, or the VEGF inhibitor bevacizumab (5 mg/μL) by eye drop, continuing with the same treatment regimen every 2 days. The eyeballs were observed by a microscope (Nikon, Tokyo, Japan) on days 0, 3, 5, and 7. On day 7, all mice were sacrificed, and their eyeballs were collected and fixed in Davidson’s fluid for histological analysis [37,38]. All animal procedures were performed according to an approved protocol issued by the Institutional Animal Care and Use Committee of China Medical University (Taichung, Taiwan). All statistical analyses were performed using GraphPad Prism 7.0 software, and all values are expressed as the mean ± standard deviation (SD). Differences between selected pairs from the study groups were analyzed for statistical significance using the Student’s t-test. One-way analysis of variance (ANOVA) followed by post hoc testing was used for statistical analyses of multiple group comparisons. The difference was considered to be significant if the p value was <0.05. Angiogenesis plays an important role in AMD [39]. To confirm the involvement of EPCs in neovascular AMD, we analyzed gene expression profiles from the eyeIntegration v1.0 database. EPC-specific markers CD34 and CD133 and blood vessel markers CD31 and VEGF were all highly expressed in AMD retinas and graded with Minnesota Grading System (MGS) scores of 1 to 4, whereas their expression was low in retinas from healthy individuals (Figure 1), indicating that EPCs contribute to the progression of neovascular AMD. In previous research, melatonin treatment has shown strong antiangiogenic effects by suppressing the proliferation, migration and invasion of endothelial cells [40], and melatonin effectively disrupted the tube formation of human umbilical vein endothelial cells (HUVECs) and dose-dependently reduced the viability of HUVECs [41], although the effects of melatonin are unclear in EPCs. When we examined the apoptotic effects of melatonin in EPCs, MTT assay results showed that incubating EPCs with different concentrations of melatonin (0.1–1 mM) for 24 h or 48 h did not affect cell viability (Figure 2A), so this concentration range was used for further experiments. The incubation of EPCs with VEGF (100 ng/mL) and melatonin (0.1–1 mM) for 24 h showed that VEGF alone promoted the proliferation of EPCs; VEGF plus melatonin significantly reduced this proliferation in a concentration-dependent manner (Figure 2B). Next, the migration assay results showed that melatonin significantly inhibited VEGF-induced cell migration in a dose-dependent manner (Figure 2C), while the tube formation assay showed that VEGF alone stimulated the reorganization and the formation of capillary-like structures in EPCs, whereas the addition of melatonin to VEGF significantly suppressed these effects (Figure 2D). To examine whether melatonin inhibits VEGF-induced angiogenesis in vivo, Matrigel was mixed with VEGF (100 ng/mL) and different concentrations of melatonin as indicated in the CAM assay (Figure 3A). The results showed that melatonin reduced VEGF-induced neoangiogenesis (Figure 3A). Using the Matrigel plug assay to examine the in vivo angiogenesis activity of melatonin, we observed that VEGF promoted microvessel formation and hemoglobin levels over 7 days in the Matrigel plugs, whereas melatonin significantly and dose-dependently inhibited this process (Figure 3B). In the co-immunofluorescent staining results for the blood vessel marker CD31 and EPC-specific markers CD34 and CD133, all markers were significantly and dose-dependently reduced by melatonin (Figure 3C,D). Our findings show that melatonin effectively suppresses VEGF-induced EPC recruitment and angiogenesis in vivo. The results of the angiogenesis protein array show that VEGF treatment increased the amount of angiogenic factors in EPCs and that the addition of melatonin decreased these factors (Figure 4A); in particular, the expression of PDGF-BB was significantly reduced after melatonin treatment (Figure 4B). Furthermore, we found that melatonin decreased VEGF-induced PDGF-BB expression in EPCs in a dose-dependent manner (Figure 4C). Whereas melatonin significantly inhibited VEGF-induced EPC migration and tube formation, these effects were significantly reversed when the EPCs were transfected with PDGF-BB cDNA for 24 h (Figure 4D,E; the tube formation images are shown in Supplementary Figure S1). Moreover, we found that the levels of PDGF-BB gene expression were significantly higher in retinas from patients with neovascular AMD than in retinas from healthy individuals (Figure 4F). These results suggest that melatonin suppresses angiogenesis by inhibiting VEGF-induced PDGF-BB production in EPCs. We examined whether melatonin is capable of blocking the VEGF/VEGFR2 interaction. Using molecular docking software to predict melatonin-VEGFR2 binding affinity, we found that melatonin interacts with the VEGFR2 ECD, with an energy value of −27.121 kCal/mol for the docked complex (Figure 5A), indicating that melatonin inhibits VEGF-induced angiogenesis by directly binding with VEGFR2. Next, we found that melatonin inhibits the VEGF-induced phosphorylation of VEGFR2, c-Src, and FAK in EPCs (Figure 5B–D). We then transfected EPCs with VEGFR2 cDNA or treated the EPCs with FAK or c-Src activators to verify whether VEGF/VEGFR2 signaling regulates EPC angiogenesis. Whereas melatonin treatment significantly inhibited VEGF-induced EPC migration and tube formation, these effects were significantly reversed by transfection with VEGFR2 cDNA, and also by treatment with the FAK and c-Src activators (Figure 5E,F; the tube formation images are shown in Supplementary Figure S2), suggesting that melatonin inhibits the VEGF-induced phosphorylation of VEGFR2, c-Src, and FAK in EPCs via VEGFR2/c-Src/FAK signaling. VEGF-induced phosphorylation of the NF-κB subunit (p65) and AP-1 subunit (c-Jun) was significantly and dose-dependently reduced by melatonin (Figure 6A,B). Next, we tested whether NF-κB or AP-1 are involved in the melatonin-induced inhibition of EPC angiogenesis. Treatment with NF-κB and AP-1 activators significantly promoted EPC angiogenesis, which was significantly reduced by melatonin treatment (Figure 6C,D). We then observed that melatonin significantly reduced NF-κB or AP-1 luciferase activity and their translocation into the nucleus (Figure 6E,F; the tube formation images are shown in Supplementary Figure S3). We also explored whether PDGF-BB is involved in the melatonin-induced inhibition of NF-κB or AP-1 transcriptional activation, using the ChIP assay to assess the in vitro recruitment of NF-κB or AP-1 to the PDGF-BB promoter. As shown in Figure 6G,H, VEGF induced the binding of NF-κB or AP-1 to the PDGF-BB element, and melatonin treatment reduced this binding. These results indicate that melatonin inhibits VEGF-induced PDGF-BB production and angiogenesis in EPCs via the suppression of NF-κB and AP-1 activity. As shown in Figure 7A, corneal neovascularization was increased in the PBS-treated eyes, and decreased after melatonin treatment in a dose-dependent manner. Notably, the effects of melatonin in the highest dose group (60 mg/kg) were similar to those of the bevacizumab treatment group. After the animals were sacrificed on Day 7, the eyeballs were removed to examine corneal epithelial defects. H&E staining showed that corneal swelling was dose-dependently decreased by melatonin (Figure 7B,C). Co-immunofluorescence staining for EPCs and vessel markers showed that melatonin significantly and dose-dependently inhibited EPC angiogenesis and the recruitment of CD31-, CD34- and CD133-positive colonies (Figure 7D). The effects of melatonin 60 mg/kg were similar to those of bevacizumab treatment. Levels of PDGF-BB expression were increased in the control group and significantly reduced after melatonin or bevacizumab treatment (Figure 7E). Thus, melatonin appears to suppress PDGF-BB production, EPC recruitment, and angiogenesis, as well as AMD development in vivo. Neovascular AMD is a serious type of late AMD and an angiogenesis-dependent disease [42]. By triggering the mobilization of EPCs from the bone marrow and their recruitment during pathological states, VEGF supports the differentiation of EPCs into mature endothelial cells at angiogenesis sites [43]. EPCs may serve as a biomarker of neovascular AMD, based on the higher levels of EPC expression in blood from patients with AMD compared with blood from healthy controls [21]. Our analysis of gene expression profiles downloaded from the eyeIntegration v1.0 database confirmed high levels of EPC-specific markers (CD34 and CD133) in the retinal tissue of patients with AMD. Our preclinical findings also indicated that EPCs contribute to angiogenesis and AMD progression. Our results are consistent with pre-existing clinical evidence and emphasize the importance of EPCs in neovascular AMD. VEGF is described as an endothelial cell-specific mitogen [44] that promotes the proliferation of new vessels and increases vascular permeability, and is considered to be a critical angiogenic factor of all angiogenesis processes [45]. Anti-VEGF treatment is an established therapeutic approach for neovascular AMD [46]. However, although the existing anti-VEGF agents, including bevacizumab, effectively inhibit VEGF activation [47] and EPC-derived pathological angiogenesis [48], many patients do not show the expected efficacy of anti-VEGF agents after repeated administration [11]. EPC-targeting therapies may therefore be a promising option to inhibit the angiogenesis process in neovascular AMD. In this study, EPCs were used to investigate the antiangiogenic effects of melatonin. Our findings show that melatonin inhibits VEGF-induced EPC angiogenesis in a concentration-dependent manner, without any evidence of cytotoxicity. Moreover, melatonin significantly suppressed the VEGF-induced stimulation of EPC recruitment and angiogenesis in vivo. Importantly, the corneal alkali burn model indicated that the inhibitory effects of melatonin on EPC-derived pathological angiogenesis are similar to those of bevacizumab, indicating that melatonin has potential against EPC-derived angiogenesis in neovascular AMD. There is much evidence to indicate that the binding of VEGF to VEGFR2 regulates angiogenesis progression in physiological and pathological conditions, and promotes EPC-derived angiogenesis [49,50]. In our study, we found that melatonin inhibits VEGF-induced angiogenesis in EPCs by blocking the VEGF/VEGFR2 signaling pathway. Notably, molecular docking software results identified a good affinity between melatonin and the VEGFR2 ECD. To the best of our knowledge, this study is the first to identify that melatonin has the potential to bind with VEGFR2 and that melatonin may compete with VEGF in this binding process. Our in vitro evidence also showed that melatonin dramatically reduced the phosphorylation of VEGFR2 in EPCs. All of these results reveal that melatonin inhibits EPC-derived angiogenesis by regulating VEGF/VEGFR2 activity. VEGF and PDGF-BB, as well as their receptors, are critical for normal and pathologic angiogenesis [51]. In neovascular AMD, VEGF promotes new vessel growth, while PDGF-BB maintains the interaction between pericytes and endothelial cells in maturing vessels [51]. While VEGF antagonists can inhibit angiogenesis in neovascular AMD, dual VEGF/PDGF inhibitors have proven to be even more beneficial in inhibiting angiogenesis in human endothelial cells and human pericytes in neovascular AMD [51], as well as in suppressing neovascularization in a mouse model of laser-induced CNV [52]. In our study, melatonin significantly reduced VEGF-induced PDGF-BB production in EPCs, as well as EPC migration and tube formation. In addition, our findings from the corneal alkali burn model suggest that melatonin can significantly inhibit corneal levels of PDGF-BB expression and angiogenic activity. Our results suggest that melatonin suppresses angiogenesis in EPCs by inhibiting PDGF-BB production. The promotion or inhibition of angiogenesis is part of the homeostatic balance, with positive and negative effects outside the optimum range. Melatonin influences this balance, with evidence from several clinical research investigations demonstrating that this hormone has antiangiogenic effects in cancer and chronic ocular diseases. For instance, in cancer treatment, adjuvant melatonin appears to be very effective in early-stage disease and helps to reduce the side effect profiles after radiotherapy and chemotherapy [53]. In patients with central serous chorioretinopathy, treatment with oral melatonin (3 mg) three times daily for 1 month resulted in significant improvements from baseline in best-corrected visual acuity (BCVA) and decreases in central macular thickness (CMT), without any adverse effects [54]. The attractive side effects profile and relatively low cost of melatonin suggest that this hormone may be appropriate in a chronic ocular disease such as AMD, although supporting data from large prospective studies are needed before melatonin can be used in the clinic [54]. The FDA-approved the anti-VEGF agents bevacizumab, aflibercept, and ranibizumab, and have shown good therapeutic results in neovascular AMD, but anti-VEGF agents are limited by the necessity for monthly injections in the clinic and the long-term nature of treatment [55]. Moreover, the high cost of anti-VEGF therapy is a heavy burden for patients [56]. Our study evidence suggests that melatonin may overcome new existing therapeutic obstacles with anti-VEGF agents and offer a novel option for treating neovascular AMD. In conclusion, our study indicates that melatonin inhibits VEGF-induced increases in PDGF-BB expression in EPCs by inhibiting the signaling of VEGFR2, c-Src, FAK, NF-κB and AP-1, all of which appear to effectively inhibit EPC angiogenesis (Figure 8). Thus, melatonin shows promising therapeutic potential, alone and in combination with a VEGF inhibitor, for neovascular AMD.

PMC10000468

Sang R. Lee,Moeka Mukae,Kang Joo Jeong,Se Hee Park,Hi Jo Shin,Sang Woon Kim,Young Suk Won,Hyo-Jung Kwun,In-Jeoung Baek,Eui-Ju Hong

PGRMC1 Ablation Protects from Energy-Starved Heart Failure by Promoting Fatty Acid/Pyruvate Oxidation

27-02-2023

Pgrmc1,heart,starvation,ischemia,metabolism

Heart failure (HF) is an emerging epidemic with a high mortality rate. Apart from conventional treatment methods, such as surgery or use of vasodilation drugs, metabolic therapy has been suggested as a new therapeutic strategy. The heart relies on fatty acid oxidation and glucose (pyruvate) oxidation for ATP-mediated contractility; the former meets most of the energy requirement, but the latter is more efficient. Inhibition of fatty acid oxidation leads to the induction of pyruvate oxidation and provides cardioprotection to failing energy-starved hearts. One of the non-canonical types of sex hormone receptors, progesterone receptor membrane component 1 (Pgrmc1), is a non-genomic progesterone receptor associated with reproduction and fertility. Recent studies revealed that Pgrmc1 regulates glucose and fatty acid synthesis. Notably, Pgrmc1 has also been associated with diabetic cardiomyopathy, as it reduces lipid-mediated toxicity and delays cardiac injury. However, the mechanism by which Pgrmc1 influences the energy-starved failing heart remains unknown. In this study, we found that loss of Pgrmc1 inhibited glycolysis and increased fatty acid/pyruvate oxidation, which is directly associated with ATP production, in starved hearts. Loss of Pgrmc1 during starvation activated the phosphorylation of AMP-activated protein kinase, which induced cardiac ATP production. Pgrmc1 loss increased the cellular respiration of cardiomyocytes under low-glucose conditions. In isoproterenol-induced cardiac injury, Pgrmc1 knockout resulted in less fibrosis and low heart failure marker expression. In summary, our results revealed that Pgrmc1 ablation in energy-deficit conditions increases fatty acid/pyruvate oxidation to protect against cardiac damage via energy starvation. Moreover, Pgrmc1 may be a regulator of cardiac metabolism that switches the dominance of glucose-fatty acid usage according to nutritional status and nutrient availability in the heart.

PGRMC1 Ablation Protects from Energy-Starved Heart Failure by Promoting Fatty Acid/Pyruvate Oxidation Heart failure (HF) is an emerging epidemic with a high mortality rate. Apart from conventional treatment methods, such as surgery or use of vasodilation drugs, metabolic therapy has been suggested as a new therapeutic strategy. The heart relies on fatty acid oxidation and glucose (pyruvate) oxidation for ATP-mediated contractility; the former meets most of the energy requirement, but the latter is more efficient. Inhibition of fatty acid oxidation leads to the induction of pyruvate oxidation and provides cardioprotection to failing energy-starved hearts. One of the non-canonical types of sex hormone receptors, progesterone receptor membrane component 1 (Pgrmc1), is a non-genomic progesterone receptor associated with reproduction and fertility. Recent studies revealed that Pgrmc1 regulates glucose and fatty acid synthesis. Notably, Pgrmc1 has also been associated with diabetic cardiomyopathy, as it reduces lipid-mediated toxicity and delays cardiac injury. However, the mechanism by which Pgrmc1 influences the energy-starved failing heart remains unknown. In this study, we found that loss of Pgrmc1 inhibited glycolysis and increased fatty acid/pyruvate oxidation, which is directly associated with ATP production, in starved hearts. Loss of Pgrmc1 during starvation activated the phosphorylation of AMP-activated protein kinase, which induced cardiac ATP production. Pgrmc1 loss increased the cellular respiration of cardiomyocytes under low-glucose conditions. In isoproterenol-induced cardiac injury, Pgrmc1 knockout resulted in less fibrosis and low heart failure marker expression. In summary, our results revealed that Pgrmc1 ablation in energy-deficit conditions increases fatty acid/pyruvate oxidation to protect against cardiac damage via energy starvation. Moreover, Pgrmc1 may be a regulator of cardiac metabolism that switches the dominance of glucose-fatty acid usage according to nutritional status and nutrient availability in the heart. Heart failure is an emerging epidemic, and patients with reduced ejection fraction rates have a mortality rate of >70% [1]. Despite extensive studies on the epidemiology and risk factors, the mortality rate of heart failure remains high [2]. Malnutrition is a known risk factor for myocardial damage [3]. Clinically, individuals are exposed to malnutrition-mediated cardiac risks during surgery, sepsis, and some serious diseases [4]. Currently used drugs for cardiomyopathy, such as angiotensin-converting enzyme inhibitors or beta blockers, reduce vasoconstriction and decrease the risk of death [5]. However, improving the function of the heart itself will provide a more fundamental breakthrough in the treatment of energy-starved heart failure. ATP production is mainly derived from fatty acid oxidation in the heart [6]. Heart failure with hypertension or ischemia is accompanied by decreased cardiac fatty acid oxidation [7]. Similarly, glucose oxidation, another pathway for ATP production, is also suppressed in heart failure [8]. As a failing heart lacks energy due to decreased glucose and fatty acid oxidation, targeting cardiac energy metabolism is the main research focus of many studies [9]. Although subtypes differ between sexes, the overall heart failure risk is comparable between men and women [10]. Some beneficial effects of androgen and estrogen on heart failure have been previously reported [11,12]. While synthetic progestin is considered to have deleterious effects, the influence of progesterone or canonical progesterone receptors in heart failure is neither beneficial nor deleterious [13]. One of the progesterone receptors, progesterone receptor membrane component 1 (Pgrmc1), has been reported to suppress obesity/diabetes-mediated cardiac lipotoxicity [14]. Pgrmc1 is a non-canonical progesterone receptor associated with reproductive functions, such as decidualization [15] and female fertility [16]. Recent studies have revealed the metabolic function of Pgrmc1, beyond the reproductive relationships, in liver [17] and adipose tissue [18], focusing on the anabolism of glucose and lipids. Regulation of insulin, a major anabolic hormone, by Pgrmc1 has also been reported in the pancreas [19]. Although Pgrmc1-related anabolisms have been extensively studied, the mechanism of Pgrmc1-related catabolism remains ambiguous. Furthermore, the regulation of cardiac health by Pgrmc1 has been investigated only in the energy-enriched state in diabetes. In this study, we investigated how Pgrmc1-related catabolism affects cardiac health during energy starvation. Based on previous reports on the apoptosis and necrosis of cardiomyocytes during glucose starvation in vivo and in vitro [20,21], we used glucose starvation mouse models (72 h fasting) to mimic cardiac ischemia under physiological conditions in this study. Additionally, an adrenergic stimulation model using isoproterenol injection was introduced to induce energy starvation in the heart based on previous studies indicating lowered ATP production from ADP in the isoproterenol model [22]. Unlike the overnutrition state, Pgrmc1 loss increased fatty acid and pyruvate oxidation in the heart during malnutrition. Our results indicated that maintenance of the major energy production pathway protected the Pgrmc1-ablated heart from energy starvation-induced injury. Wild-type (WT) and Pgrmc1 global knockout (PKO) littermate mice [23] (8-week-old; C57BL/6 background) were grown in a pathogen-free facility at Chungnam National University under a standard 12:12 h light:dark cycle and fed standard chow diet with water provided ad libitum. The mice were fasted to starvation, and unexpected deaths during the experiment were recorded to assess the survival rate. Isoproterenol (230 mg/kg, subcutaneous) was injected for two weeks to induce adrenergic heart damage. To observe cardiac pumping in WT and PKO mice, fluorescent dye-labeled (DyLight 680 antibody labeling kit, Thermo Scientific, Waltham, MA, USA, 53056) bovine serum albumin (BSA) was intravenously injected into the mice. After 1 h, the mice were anesthetized and placed in an in vivo imaging system (IVIS; FOBI, Vancouver, BC, Canada). A video was recorded to observe cardiac pumping. Images of cardiac contraction/relaxation were also captured. All animal experiments were approved by the Chungnam Facility Animal Care Committee (CNU-00606) and adhered to their ethical guidelines. Public datasets (GEO) were used to determine PGRMC1 transcription levels in patients with cardiomyopathy. GSE29819 and GSE36961 datasets were selected, and all patients were included in the analysis. CLAMS was used to assess the metabolic status of starved mice. Oxygen consumption (VO2) and carbon dioxide production (VCO2) rates were measured using an Oxymax system (Columbus Instruments, Columbus, OH, USA). Mice were placed at least 50 min before experiment for acclimation. The respiratory exchange ratio (RER) and respiratory quotient (RQ) were calculated as the ratio of VCO2 to VO2. The mice were fasted from midway through the light cycle to midway through the dark cycle. RNA pellets were collected from the hearts of mice and H9c2 cells using TRIzol, chloroform, and isopropanol. RNA pellet was washed with ethanol and dissolved in diethyl pyrocarbonate-treated water. RNA concentration was measured, and the same RNA amounts for each sample were used for cDNA synthesis using an Excel RT Reverse transcriptase kit (SG-cDNAS100; Smartgene, Daejeon, Republic of Korea). Real-time PCR was carried out using specific primers (Table 1), Excel Taq Q-PCR Master Mix (SG-SYBR-500; Smartgene), and Stratagene Mx3000P (Agilent Technologies, Santa Clara, CA, USA) in a 96-well optical reaction plate. Negative controls containing water instead of the sample cDNA were used in each plate. Protein samples were resolved on 8–12% sodium dodecyl sulfate (SDS) polyacrylamide gels (running buffer: 25 mM Tris, 192 mM Glycine, 0.1% SDS, and D.W.). After electrophoresis, the gels were blotted onto a polyvinylidene difluoride membrane (IPVH 00010; Millipore, Burlington, MA, USA) at 350 mA for 1–2 h with the transfer buffer (25 mM Tris, 192 mM Glycine, and 20% (v/v) methanol). Membranes were blocked in 3% BSA and incubated with primary antibodies overnight at 4 °C. Membranes were washed thrice with TBS-T to remove the excess antibodies and incubated overnight at 4 °C with the following secondary antibodies: goat anti-rabbit IgG horseradish peroxidase (HRP) (Catalog #31460) and goat anti-mouse IgG HRP (Catalog #31430; Thermo Fisher Scientific, Waltham, MA, USA) antibodies. After washing thrice with TBS-T, immunoreactive proteins were observed with ECL solution (Eta C Ultra 2.0; Cyanagen, Bologna, Italy) using a ChemiDoc system (Fusion Solo, Vilber Lourmat, Eberhardzell, Germany). The following primary antibodies were used: PGRMC1 (13856; Cell Signaling Technology, Danvers, MA, USA), ribosomal protein lateral stalk subunit P0 (RPLP0; A13633; Abclonal, Woburn, MA, USA), poly(ADP ribose) polymerase (PARP; 9532; Cell Signaling Technology), C/EBP homologous protein (CHOP; #MA1-250; Invitrogen, Waltham, MA, USA), β-actin (sc-47778; Santa Cruz, Dallas, TX, USA), glycolysis antibody sampler kit (8337; Cell Signaling Technology), pAMPK, tAMPK (9957; Cell Signaling Technology), LC3B (L7543, Sigma-Aldrich, St. Louis, MO, USA), and α-tubulin (66031-1-Ig; Proteintech, Rosemont, IL, USA). For blood glucose measurement, the tail was snipped, and the blood glucose levels were measured using an Accu-Chek Active kit (Roche, Basel, Switzerland). During necropsy, blood was collected from the IVC. Plasma samples were analyzed to determine the levels of free fatty acids (FFAs; BM-FFA100, Biomax, Planegg, Germany), triglycerides (TGs; TG-1650, Fuji Film, Tokyo, Japan), and total cholesterol (TCHO; TCHO-1450). All the cell culture reagents were purchased from Welgene (Gyeongsan, Republic of Korea). H9c2 rat cardiomyocytes were maintained in Dulbecco’s modified Eagle’s medium (LM001-05; Welgene) supplemented with 5% (v/v) fetal bovine serum (FBS, Punjab, Pakistan), penicillin (100 U/mol), and streptomycin (100 μg/mL). To reflect the plasma profile of mice, cells were incubated with a low-glucose/fatty acid medium (500 mg/L glucose, 110 µM palmitic acid, 220 μM oleic acid) for 24 h. For Pgrmc1 knockdown/overexpression experiments, cells were incubated with Opti-MEM (31985070; Gibco; without FBS) for 0.5 h and treated with the siRNA/plasmid and lipofectamine 2000 (11668027; Thermo Fisher Scientific). The siRNA sequence used was: 5′-CAGUUCACUUUCAAGUAUCA-U-3′. Medium containing FBS was later added after 6 h. Tissues were fixed with neutral-buffered formalin, and trimmed tissues were washed with tap water. Tissues were subjected to serial dehydration and embedded in paraffin. The paraffin block was cut (5 μm) using a microtome, and the cut sections were attached to a silane-coated slide. Slides were immersed in xylene overnight and processed using a commercial kit (MST-100T; Biognost, Zagreb, Croatia), according to the manufacturer’s protocol, for Masson’s Trichrome staining. Regions of interest were observed under a light microscope. Frozen tissues were embedded in an optimal cutting temperature compound and cut (8 μm) using a cryostat. Slides were dried overnight and washed with TBS-T. TUNEL assay (11684795910; Roche, Basel, Switzerland) was performed according to the manufacturer’s protocol. After 4′,6-diamidino-2-phenylindole staining, the region of interest was observed under a fluorescence microscope. For immunostaining, frozen tissue slides were dried overnight and heated in oven (65 °C) for 10 min. Slides were immersed in distilled water and subsequently TBS-T. After blocking with 3% BSA, slides were incubated with primary antibody (CD31, ab56299; Abcam, Cambridge, UK) overnight at 4 °C. The next day, slides were washed with TBS-T and incubated with secondary antibody (A21202, Life Technologies, Carlsbad, CA, USA) for 4 h at room temperature. The region of interest was observed under a fluorescence microscope. Data are reported as the mean ± standard deviation. Differences between means were analyzed via Student’s t-test and one-way analysis of variance followed by Tukey’s multiple comparison test using the Graph Pad Software (GraphPad Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05. Using public clinical datasets, we collected data to investigate the relationship between PGRMC1 expression and cardiomyopathy. In GSE29819, both ventricles from patients with dilated cardiomyopathy showed lower PGRMC1 expression levels than those from non-failing donor hearts (Figure 1A). In GSE36961, the hearts of patients with dilated cardiomyopathy with left ventricular systolic dysfunction showed decreased PGRMC1 expression levels compared to those of normal individuals (Figure 1A). Interestingly, the expression levels of key enzymes involved in fatty acid oxidation and glycolysis were lower in the hearts of patients with dilated cardiomyopathy (Figure 1A). Through several in vitro and in vivo experiments, we attempted to delineate the effects of energy starvation on cardiomyocyte health. We induced energy starvation in H9C2 cardiomyocytes and mice via glucose starvation (glucose 0 mg/L, FBS 1%) and fasting (72 h), respectively. As shown in Figure 1B, cells under glucose starvation were predisposed to apoptotic cell death. Furthermore, hearts from mice under starvation (72 h) showed increased protein levels of apoptotic markers (cleaved PARP) and endoplasmic reticulum stress markers (CHOP) compared to those under resting conditions (Con) (Figure 1C). PGRMC1 protein expression was markedly suppressed by fasting (Figure 1C). These results indicate that PGRMC1 levels are closely related to energy starvation-induced cardiomyocyte injury. Since there is no information on the physiological profile of PKO mice under starvation, we used CLAMS for comprehensive assessments. In CLAMS, VO2 levels were markedly reduced from 14 h fasting and reached baseline after 20 h fasting in WT mice. In contrast, VO2 levels were generally maintained at high levels in PKO mice during fasting. VCO2 levels showed a similar pattern as the VO2 levels. Levels of VCO2 markedly decreased after 14 h of fasting and reached baseline after 20 h of fasting in WT mice. In contrast, PKO mice maintained high VCO2 levels during fasting (Figure 2A). Additionally, the RER (VO2/VCO2) ratios were lower in PKO mice than in WT mice during prolonged fasting (Figure 2B). RQ calculation revealed that PKO mice are more likely to consume fat than glucose during prolonged fasting (Figure 2C). The heat production of PKO mice was highly maintained during fasting, notably from 14 h fasting, compared to that of WT mice (Figure 2D). The physical activity of PKO mice was also maintained during the prolonged fasting period, while that of WT mice was substantially diminished during the same period (Figure 2E). When mice were starved for a long period, some died unexpectedly due to an energy deficit. PKO mice were resistant to starvation-induced death compared to WT mice (Figure 2F). These results indicate that PKO mice are physiologically resistant to energy starvation. To investigate how Pgrmc1 will affect the heart under starvation, WT and PKO mice were starved for 72 h and exposed to cardiac malnutrition. Blood glucose levels were at baseline in both starved WT and PKO mice, showing no difference between the two groups (Figure 3A). Plasma lipid profiles increased in starved PKO mice. Notably, plasma FFA and TG levels were significantly higher in starved PKO mice than in starved WT mice (Figure 3A). Heart weight (HW) decreased in starved PKO mice, while the ratio of HW per body weight (BW) was similar (Figure 3B). Western blotting showed that starved PKO hearts had decreased cleaved PARP levels, which is an apoptotic marker, compared to starved WT hearts (Figure 3C). Concordantly, PKO hearts showed seemingly increased cardiac contractions in the IVIS using fluorescence (Figure S1). Most hearts with hypertrophy or failure undergo metabolic alterations characterized by decreased fatty acid oxidation [24]. Fatty acid oxidation accounts for almost 70% of cardiac energy production [25]. PKO hearts under starvation conditions showed significantly increased expression levels of mitochondrial fatty acid oxidation enzymes (carnitine palmitoyltransferase 2 (Cpt2) and very long-chain acyl-CoA dehydrogenase (Vlcad)) and peroxisomal fatty acid oxidation enzyme (acyl-CoA oxidase 1 (Acox1)) compared to WT hearts under starvation conditions (Figure 3D). Glycolysis is a rapidly induced cardiac metabolism process associated with heart failure [26]. PKO hearts under starvation had markedly decreased protein levels related to glycolysis (hexokinase (HK)-1, HK2, and pyruvate kinase M2 (PKM2)) (Figure 3E). Glucose oxidation accelerates cardiac function recovery following myocardial injury [27]. Likewise, dichloroacetate, a pyruvate dehydrogenase (PDH) activator, increases myocardial efficiency [28]. Cardiac PDH was higher in PKO than in WT plants under starvation conditions (Figure 3E). These results indicate that starved PKO hearts increase their main energy production and fatty acid/pyruvate oxidation and do not need to be exposed to metabolic alterations. As plasma FFA levels were highly maintained in PKO mice, it should be tested whether these metabolic alterations are influenced by the levels of physiologically induced substrates. To limit the influential factors in vivo, we introduced H9c2 rat cardiomyocytes and knocked down Pgrmc1 by siRNA. The cells were exposed to low glucose (500 mg/L) and fatty acids (palmitic acid (110 µM)/oleic acid (220 µM)). PGRMC1 protein levels were lower in the PK (Pgrmc1 knockdown) group than in the CK (control knockdown) group (Figure 4A). Cleaved PARP levels were lowered in PK group (Figure 4A). Metabolic alterations followed in vivo results. The mRNA expression levels of Cpt2, Vlcad, and Acox1 were higher in the PK group than in the CK group (Figure 4B). The protein levels of HK1 and HK2 decreased in the PK group (Figure 4C). PDH levels increased in the PK group (Figure 4C). Collectively, in vitro Pgrmc1 knockdown in low-energy cardiomyocytes induced fatty acid/pyruvate oxidation and decreased cellular injury. To investigate whether metabolic alterations in the PK group increased energy production compared to that in the CK group under energy deficit, we introduced a seahorse flux analyzer system to measure cellular respiration. H9c2 cells were knocked down and starved in a medium containing low glucose (500 mg/L) and fatty acids (palmitic acid (110 µM)/oleic acid (220 µM)). In the mitochondrial stress test, the PK group had a higher maximal respiration rate than that of the CK group (Figure 4D). We also measured the mitochondrial fusion/fission gene expression levels to assess the mitochondrial balance [29]. PKO hearts had a mildly increased fission gene (dynamin-related protein 1; Drp1) expression level compared to WT hearts (Figure S2A). These results confirm that fatty acid/pyruvate oxidation by PK increases energy production even under reduced glycolysis. We investigated the possible mechanism of metabolic alterations induced by Pgrmc1. AMPK is a multi-functional protein kinase involved in the oxidation and uptake of metabolites [30]. Western blotting revealed that starved PKO hearts had increased phosphorylated AMPK (pAMPK) levels and decreased total AMPK (tAMPK) levels. Starved PKO hearts showed a higher p/t AMPK ratio than WT hearts (Figure 5A). In H9c2 cells, PK cells showed higher pAMPK and lower tAMPK levels than CK cells. Concordantly, PK cells showed an increased p/t AMPK ratio compared to that in CK cells (Figure 5A). Metabolic effects of AMPK activation and inactivation in cardiomyocytes were assessed. PGRMC1 levels were not directly regulated by AMPK activation because treatments with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; AMPK activator) and compound C (Com C; AMPK inactivator) suppressed PGRMC1 expression. AMPK phosphorylation was increased by AICAR and decreased by Com C treatment (Figure 5B). HK1 levels were lowered by AICAR, whereas HK2 and PKM2 levels were increased by Com C. PDH levels were decreased by Com C (Figure 5B). In contrast, the expression levels of fatty acid oxidation enzymes were markedly increased by AICAR treatment (Figure 5C). Com C treatment decreased Cpt2 and Vlcad expression levels (Figure 5C). In summary, AMPK activation was related to the induction of fatty acid/pyruvate oxidation and decreased glycolysis. As Pgrmc1 loss increased AMPK activation and showed similar metabolic alterations to AMPK-activated cells, AMPK may be linked to metabolic modulation by PGRMC1 in starved hearts. We introduced isoproterenol cardiac injury model according to previous studies [31,32]. Mice were injected with isoproterenol (five times, total 230 mg/kg, 14 days) and sacrificed (Figure 6A). Masson’s trichrome staining revealed that isoproterenol-WT hearts showed large positive areas with fibrosis (Figure 6B). In contrast, isoproterenol-PKO hearts showed decreased fibrotic areas compared with WT hearts (Figure 6B). Transforming growth factor-beta mRNA expression levels decreased in isoproterenol-PKO hearts (Figure 6C). As heart failure markers, mRNA expression levels of actin alpha 1 and brain natriuretic peptide were decreased in isoproterenol-PKO hearts compared to those in WT hearts (Figure 6D). In metabolic assessments, isoproterenol-PKO hearts showed higher levels of fatty acid oxidation enzymes (Cpt2) than isoproterenol-WT hearts (Figure 6E). Furthermore, isoproterenol-PKO hearts had decreased glycolysis enzyme levels and increased PDH levels. Additionally, isoproterenol-PKO hearts showed an increased p/t ratio of AMPK (Figure 6F). Hence, isoproterenol-PKO hearts had altered cardiac metabolism, such as fasting-PKO cardiac metabolism, increased fatty acid/pyruvate oxidation and AMPK phosphorylation, and decreased glycolysis. Maintenance of the ATP-producing pathway, i.e., fatty acid/pyruvate oxidation, may provide cardioprotection under ischemic injury. Ischemic heart failure is prevalent worldwide [33]. Beyond traditional surgery, various methods using protein, cell, and gene therapeutics have been suggested for treatment [34]. Notably, several regulators of cardiac metabolism have been identified [35]. The heart relies heavily on long-chain fatty acids and utilizes glucose low-proportionally for energy production in the normal state [36]. Both fatty acid oxidation and glucose oxidation produce acetyl-CoA, which directly participates in the tricarboxylic acid cycle and electron transport chain and accounts for 95% of myocardial ATP production [7]. In failing hearts, fatty acid availability substantially affects the myocardial function and efficiency [37]. Additionally, pyruvate oxidation, leading to the production of acetyl-CoA from glucose-derived pyruvate, is limited in heart failure, resulting in impaired ATP production [7]. Thus, failing hearts are etiologically or resultantly associated with impaired energy production via fatty acid/pyruvate oxidation. During cellular stress, AMPK phosphorylation downregulates fatty acid synthesis but upregulates fatty acid oxidation [38]. Although fatty acid oxidation itself can suppress pyruvate oxidation, AMPK activation increases glycolysis and pyruvate oxidation. Due to its diverse effects, whether AMPK improves or deteriorates the cardiac health may differ according to the physiological state of the patient [39]. AMPK has been reported to increase overall ATP production to respond to the energy demand and provide tolerance against cardiac ischemia [40]. When the hearts were exposed to fasting or isoproterenol-induced energy starvation, PKO increased AMPK phosphorylation. Catabolic activation by PKO differed according to metabolic pathways; fatty acid and pyruvate oxidation increased, but glycolysis decreased. Fatty acid oxidation takes place predominantly in the mitochondria and peroxisomes in less magnitude [41]. Mitochondrial fatty acid oxidation enzymes [42], namely Cpt2 and Vlcad, and the peroxisomal fatty acid oxidation enzyme [43] Acox1 increased in PKO hearts. The high availability of plasma fatty acids in PKO may influence catabolic processes. However, exposure to the same amount of fatty acids in in vitro experiment also increased fatty acid oxidation in PK cells. Conversely, Pgrmc1-overexpressing (POE) cells exhibited decreased fatty acid oxidation (Figure S3). Hence, an increase in the fatty acid oxidation pathway affects cardiac energy metabolism in PKO. Paradoxically, PKO hearts have decreased levels of glycolytic enzymes, hexokinases, and pyruvate kinase but increased PDH [44]. When cells are exposed to the same amounts of glucose and fatty acids, PK cells still increase pyruvate oxidation but suppress glycolysis. Similarly, POE cells showed a mild increase in glycolysis (Figure S3). We speculated that the lactate source must be induced to increase pyruvate substrate and pyruvate dehydrogenase in limited sources from glycolytic products. Our results (data not shown) also showed the induction of lactate dehydrogenase in starved PKO hearts. Further studies on the regulation of lactate metabolism by Pgrmc1 should be performed. Glycolysis only accounts for <10% [45], while the oxidation of fatty acids (50–70%) [46] and pyruvate (20–40%) [7] comprises the majority of cardiac ATP production. Hence, starved PKO hearts may have increased overall ATP production. Mechanistically, PKO hearts showed increased AMPK phosphorylation, and AMPK inhibitor (Com C) treatment resulted in the opposite cardiac metabolism pattern compared to that of PKO. In line with this, AMPK activator (AICAR) treatment showed a cardiac metabolism pattern similar to that of PKO. Concordantly, PKO-altered cardiac energy metabolism may be linked to AMPK phosphorylation during cardiac injury. We also measured the cardiac autophagy, as AMPK is an autophagy promoter [47], but observed significantly down-regulated LC3B levels in PKO hearts. As Pgrmc1 is an autophagy promoter [48], cardiac autophagy was mainly affected by Pgrmc1 compared to AMPK. This is in accordance with our results, as autophagy is up-regulated in ATP-depleted and ischemic hearts [49]. We insist on the interpretation of conflicting metabolic alterations and functions of PKO hearts in light of a previous study. In our previous study, PKO hearts in diabetic conditions showed increased TG and fatty acyl-CoA accumulation [14], leading to lipotoxicity. However, TG deposits play an ATP-providing role [50], and fatty acyl CoA is directly related to oxidative phosphorylation in the heart [51,52]. In contrast to overnutrition hearts, the large pool of lipids in PKO can be the ATP pool for energy-deficient hearts. Additionally, in our previous study, cardiac glycolysis was induced only in overnutrition PKO and slightly decreased in normal PKO hearts [14]. In the energy-deficient state, glycolysis was significantly decreased in PKO hearts. In contrast, fatty acid oxidation was decreased in normal and overnutrition PKO hearts [14] but increased in malnutrition PKO hearts. We concluded that cardiac metabolic alteration by Pgrmc1 depends on glucose availability. In re-fed and diabetic mice, blood glucose levels were approximately 200 mg/dL [14], which were higher than those in starved mice (approximately 60 mg/dL). Pgrmc1 may be a physiological switch that regulates the preference of cardiac substrates for ATP production depending on the body’s nutrition. In energy-deficit conditions, Pgrmc1 reduces oxidation of fatty acids/pyruvates, thereby limiting ATP production in the heart. The failing heart possesses a nearly 30% ATP volume [53] and reduces the ATP-supplementing flux from the reserve (creatine kinase) by 50% compared to the normal heart [54]. ATP depletion in the failing heart directly leads to contractile dysfunction because continuous ATP production/turnover is necessary for cardiac function [24]. Fatty acid oxidation is the major cardiac ATP-producing pathway, but it suppresses glucose oxidation, as per the Randle cycle [55]. Since glucose oxidation is a much more efficient ATP-production and less-oxygen-consuming pathway than fatty acid oxidation [28], its activation is therapeutically effective in a failing heart [56]. The fatty acid oxidation inhibitor etomoxir has been reported to exert cardioprotective effects by switching from energy metabolism to glucose oxidation [57,58]. However, adverse effects of fatty acid oxidation inhibition can also be observed in experimental/clinical reports [59,60]. Based on our results, Pgrmc1 inhibition increases both fatty acid and pyruvate oxidation and improves overall ATP production during energy starvation. Therefore, improvement in ATP-production via a Pgrmc1 inhibitor can be used as a novel therapeutic approach for energy-starved failing hearts. Additionally, PKO hearts reduced CD31 abundance in immunostaining (Figure S2C). This result is of clinical importance, as CD31 levels are markedly observed in the necrotic myocardium of deceased patients under ischemic heart disease [61]. Furthermore, CD31 blockade reduces damage in ischemia/reperfusion heart injury [62]. As Pgrmc1 promotes cellular processes of microvascular endothelial cells of the brain [63], further study is expected regarding Pgrmc1 and the cardiovascular system.

PMC10000476

36899881

Kalee N. Holloway,Marisa R. Pinson,James C. Douglas,Tonya M. Rafferty,Cynthia J. M. Kane,Rajesh C. Miranda,Paul D. Drew

Cerebellar Transcriptomic Analysis in a Chronic plus Binge Mouse Model of Alcohol Use Disorder Demonstrates Ethanol-Induced Neuroinflammation and Altered Glial Gene Expression

25-02-2023

AUD,astrocyte,microglia,oligodendrocyte,neuroinflammation,transcriptomics

Alcohol use disorder (AUD) is one of the most common preventable mental health disorders and can result in pathology within the CNS, including the cerebellum. Cerebellar alcohol exposure during adulthood has been associated with disruptions in proper cerebellar function. However, the mechanisms regulating ethanol-induced cerebellar neuropathology are not well understood. High-throughput next generation sequencing was performed to compare control versus ethanol-treated adult C57BL/6J mice in a chronic plus binge model of AUD. Mice were euthanized, cerebella were microdissected, and RNA was isolated and submitted for RNA-sequencing. Down-stream transcriptomic analyses revealed significant changes in gene expression and global biological pathways in control versus ethanol-treated mice that included pathogen-influenced signaling pathways and cellular immune response pathways. Microglial-associated genes showed a decrease in homeostasis-associated transcripts and an increase in transcripts associated with chronic neurodegenerative diseases, while astrocyte-associated genes showed an increase in transcripts associated with acute injury. Oligodendrocyte lineage cell genes showed a decrease in transcripts associated with both immature progenitors as well as myelinating oligodendrocytes. These data provide new insight into the mechanisms by which ethanol induces cerebellar neuropathology and alterations to the immune response in AUD.

Cerebellar Transcriptomic Analysis in a Chronic plus Binge Mouse Model of Alcohol Use Disorder Demonstrates Ethanol-Induced Neuroinflammation and Altered Glial Gene Expression Alcohol use disorder (AUD) is one of the most common preventable mental health disorders and can result in pathology within the CNS, including the cerebellum. Cerebellar alcohol exposure during adulthood has been associated with disruptions in proper cerebellar function. However, the mechanisms regulating ethanol-induced cerebellar neuropathology are not well understood. High-throughput next generation sequencing was performed to compare control versus ethanol-treated adult C57BL/6J mice in a chronic plus binge model of AUD. Mice were euthanized, cerebella were microdissected, and RNA was isolated and submitted for RNA-sequencing. Down-stream transcriptomic analyses revealed significant changes in gene expression and global biological pathways in control versus ethanol-treated mice that included pathogen-influenced signaling pathways and cellular immune response pathways. Microglial-associated genes showed a decrease in homeostasis-associated transcripts and an increase in transcripts associated with chronic neurodegenerative diseases, while astrocyte-associated genes showed an increase in transcripts associated with acute injury. Oligodendrocyte lineage cell genes showed a decrease in transcripts associated with both immature progenitors as well as myelinating oligodendrocytes. These data provide new insight into the mechanisms by which ethanol induces cerebellar neuropathology and alterations to the immune response in AUD. Excessive alcohol consumption in adolescents and adults has significant societal impacts, with an estimated economic cost of $249 billion in the U.S. alone [1]. Studies have shown that alcohol misuse can lead to low academic achievement, an increased risk of suicide, and a lifetime struggle with addiction [2,3,4]. Furthermore, alcohol use disorder (AUD) is one of the most prevalent mental health disorders, with 15.7 million Americans aged 12 and older diagnosed [5,6], and is associated with many physical and psychiatric comorbidities [7,8]. Despite the known consequences of excess alcohol consumption, 29.7% of men and 22.2% of women were diagnosed with an AUD in 2019 [9]. AUD is associated with pathology to organ systems including the central nervous system (CNS). Animal models of AUD have been developed which simulate the behavioral abnormalities and neuropathologies associated with human AUD, thus allowing researchers to investigate the biological mechanisms associated with AUD [10]. Within the CNS, the cerebellum is responsible for coordinating motor movements, cognitive processing, and sensory discrimination. In individuals with AUD, these cerebellar functions are often disrupted, which may persist following abstinence from alcohol [11,12]. Alcohol can induce an immune response in the CNS termed neuroinflammation, which may result in neurodegeneration [13] and an increased risk of developing an AUD [14]. In adult rodents, the extent of alcohol-induced neuroinflammation can depend on the experimental paradigm of ethanol exposure utilized [15,16,17,18,19]. In the current study, we evaluated the effects of ethanol on the transcriptomic profile of adult mouse cerebella, utilizing a chronic plus binge ethanol exposure paradigm adapted from an alcoholic liver disease model developed by the Gao laboratory, in which liver injury and systemic inflammation were reported [20,21]. Using a top-down approach, we analyzed the effects of ethanol on global gene expression in the cerebellum. Our studies indicated that ethanol altered the expression of immune-related transcripts and pathways in the adult cerebellum, and may alter the function and phenotype of CNS glial cells. Thus, the current studies aid in advancing our understanding of the neuroinflammatory transcriptomic changes induced in AUD, unraveling potential targets for therapeutic strategies. All animal use protocols were reviewed and approved by the University of Arkansas for Medical Sciences (UAMS), Institutional Animal Care and Use Committee (IACUC). Adult C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; stock #000664) and were housed in the UAMS Division of Laboratory Animal Medicine, where a breeding colony was established to produce experimental animals. Adult male mice aged 10–14 weeks and weighing ≥20 g were housed individually and were randomly separated into 2 experimental groups, ethanol (E) or vehicle control (C), (n = 5 mice per group). Solid food was removed from cages, while water was provided ad libitum for the duration of the study. On study days 1–5, both experimental groups of mice were allowed to acclimate to the Bio-Serv Rodent Liquid Diet, control formulation (Flemington, NJ, USA; #F1259SP) provided freely in a fresh tube each day just before the start of the dark cycle. Following acclimation, the ethanol group underwent ethanol ramping, in which mice received successive increases of the Bio-Serv ethanol formulation (#F1258SP) with either 1% (day 6), 2% (day 7), or 3% ethanol (day 8) diluted using 95% v/v ethanol (Acros, a part of Thermo Fisher Scientific, Waltham, MA, USA; #AC615110010). On study day 9, chronic ethanol administration began, in which the ethanol-treated mice received 4% ethanol for 10 days, followed by 5% ethanol for 7 days. Pair-feeding for the control group began on study day 10 (the second day of 4% ethanol administration), in which the control group was fed an equivalent volume of control diet to match the mean ethanol group consumption volume from the previous day. On the morning of study day 26, immediately following the start of the light cycle, the ethanol group underwent an acute binge administration of 5 g/kg of 31.5% ethanol (v/v) diluted from 95% v/v ethanol delivered in water via gavage. The control group received 45% (w/v) Maltose Dextrin (10 DE Food Grade #3585) diluted in water and delivered via gavage. At this time, the liquid diet was removed from all cages and standard food pellets were provided. 24 h following the ethanol binge administration, mice were euthanized and transcardially perfused with 1X PBS containing 5 U/mL heparin. Brains were removed and cerebella were micro-dissected into two halves along the midline and snap frozen in liquid nitrogen. Blood ethanol concentrations (BECs) from a separate set of animals were determined to be 230 (±59.7) mg/dL following 4% administration, 311.7 (±49.8) following 5% administration, and 718 (±6.9) mg/dL following bolus administration, as reported previously when using this model [22]. BECs were not measured at the time of tissue collection, though we suspect BECs were at or near 0 based upon preliminary studies using this model. One whole cerebellar hemisphere from each experimental animal was homogenized using a B2X24B Bullet Blender and 0.5 mm glass beads, as described by the manufacturer (Next Advance, Troy, NY, USA). RNA was isolated using the RNeasy Lipid Tissue Mini Kit with on-column Dnase digestion using the Rnase-free Dnase Set (Qiagen, Valencia, CA, USA, Cat #74804 and #79254), as described previously [23]. RNA quantity was assessed using the Qubit 3.0 fluorometer with the Qubit Broad-Range RNA Assay Kit (Thermo Fisher Scientific), and an Agilent Fragment Analyzer with the Standard Sensitivity RNA Gel Kit (Agilent Technologies, Santa Clara, CA, USA) was used to ensure RNA quality. RNA-seq libraries were prepared using an Illumina TruSeq mRNA Library Prep Kit with TruSeq Unique Dual Indexed adapters (Illumina, San Diego, CA, USA), and were quantified with Qubit 1X dsDNA High-Sensitivity NGS Gel Kit (Thermo Fisher Scientific). KAPA Library Quantification (Roche, Basel, Switzerland) was used for further library characterization, and an Agilent Fragment Analyzer with the High-sensitivity NGS Gel Kit (Agilent) was used for determining fragment size. Library molarities were calculated followed by dilution and denaturation according to manufacturer’s specification for clustering. The control and ethanol-exposed animals were clustered on a high-output NextSeq 500 flow cell and paired-end sequenced with 150-cycle SBS kit for 2X75 reads (Illumina). To identify significant differences in mRNA gene expression and global biological pathways associated with alterations of cerebellar genes between the control and ethanol treatment groups, raw RNA-sequence data (NCBI GEO accession GSE222445) were analyzed. RNA-seq reads were quality-checked, trimmed, and aligned to the GRCm39 reference genome (accession: GCA_000001635.9) using the Nextflow RNAseq pipeline, nf-core/rnaseq (version 3.4), available at DOI 10.5281/zenodo.1400710. The resulting gene counts were transformed to Log2 counts per million (CPM) [24]. Lowly expressed genes were filtered out, and libraries were normalized by trimmed means of M-values [25]. The Limma R package was used to calculate differential expression among genes [26]. Log2 fold change values were calculated for ethanol compared to control, and genes with an adjusted (adj.) p ≤ 0.05 were considered statistically significant. Heat map and principal component analysis (PCA) plots were created from the processed differential gene expression data using R statistical software. The R-based EnhancedVolcano package was used to make the volcano plots [27]. Pathway and network analysis were conducted using the QIAGEN Ingenuity Pathway Analysis (IPA) software (QIAGEN Inc., Valencia, CA, USA, https://digitalinsights.qiagen.com/IPA, accessed on 22 July 2022 ) using the “Core Expression Analysis”. IPA analysis parameters were set with the “species” parameter as “mouse”, and the “tissues and cell lines” parameter as “cerebellum”, with gene cut offs of an adj. p ≤ 0.05 and Log2 fold change ≥0.5 or ≤−0.5. To obtain a better understanding of the specific cellular processes and cell types of the cerebellum that are most sensitive to ethanol exposure, we extracted cell type-specific gene lists from publicly available single-cell RNA-seq (scRNA-seq) resources, which have been used previously to deduce the cell composition of bulk RNA-seq tissue [28]. Using this approach, we identified a total of 822 microglia-associated genes from scRNA-seq resources [29,30,31,32,33] (Supplemental Table S1A). We compared this list of microglia-associated genes to the list of genes significantly differentially regulated by ethanol (adj. p ≤ 0.05) in our dataset, which identified 151 microglia-associated genes whose expression was altered by ethanol (Table 1). We were able to characterize 23 of the 151 genes as being either homeostatic or neurodegenerative (Table 2), as defined in previous studies [33,34,35,36,37] (Supplemental Table S1B,C). To further evaluate the effects of ethanol on homeostatic versus neurodegenerative microglial phenotypes, we computed mean z-scores to compare control versus ethanol for the transcripts associated with these phenotypes. Since the goal was to determine relative gene expression changes in our dataset, i.e., to determine whether the genes are up- or down-regulated due to ethanol, the average z-score was computed. We calculated the average z-score across individual genes in our extracted microglia homeostatic and neurodegenerative-associated gene lists, and then averaged these individual gene z-scores within each sample. The average z-score of each sample in the homeostatic and neurodegenerative group was then evaluated using a two-tailed Student’s t test, with p ≤ 0.05 being considered statistically significant. R statistical software was used to conduct the Student’s t-test as well as construct the average z-score graphs. Similar to microglia, we utilized scRNA-seq data to compose a list of 309 astrocyte-associated genes (Supplemental Table S2) [37]. From this list we identified 56 astrocyte-associated genes that were differentially expressed in response to ethanol in our current study. We then characterized these transcripts as being associated with an astrocyte phenotype common to acute injury, chronic neurodegenerative diseases, or pan-injury (Table 3), the last of which includes genes associated with both acute injury and chronic neurodegenerative disease phenotypes [37]. To test for statistical significance, the average z-scores of each gene in our extracted acute, chronic, and pan-injury astrocyte-associated gene lists were generated, and these individual gene z-scores were then averaged within each sample in a manner consistent with the microglia described above. The Student’s t-test and average z-score graphs were constructed using R statistical software. Due to the small number of chronic neurodegenerative disease astrocyte-associated genes (n = 3), no z-score graph was generated for this group. For oligodendrocyte lineage-associated genes, we extracted gene lists for oligodendrocyte precursor cells (OPCs) (381 genes), committed oligodendrocyte precursor cells (COPs) (55 genes), newly formed oligodendrocytes (NFOL) (9 genes), myelin-forming oligodendrocytes (MFOL) (347 genes), and mature oligodendrocytes (MOL) (7 genes) from publicly available scRNA-seq studies [29,30,39] (Supplemental Table S3), in a manner consistent with microglia and astrocytes described above, to determine which genes were significantly differentially regulated by ethanol. From these lists, we identified 71 differentially expressed genes associated with OPCs, 12 genes associated with COPs, 2 genes associated with NFOL, 2 genes associated with MOL, and 108 genes associated with MFOL within our significantly differentially regulated dataset (Table 4). We performed statistical analyses in a manner similar to the microglia and astrocytes above. Briefly, the average z-scores of each gene in our OPC, COP, and MFOL-associated gene lists were generated, and the individual gene z-scores were then averaged between each sample. The Student’s t-test and average z-score graphs were constructed using R statistical software. Due to the small number of NFOL and MOL-associated genes differentially regulated by ethanol, z-score graphs were not generated for these groups. A principal component analysis (PCA) was performed to provide an overview of the transcriptomic changes that occurred in response to ethanol. PCA analysis demonstrated that gene transcripts correlating and anticorrelating to the first and second principal components could differentiate control animals from those exposed to ethanol. (Figure 1A). Hierarchical clustering analysis of significant genes was conducted using Pearson’s correlation, while controlling for false discovery rate adj. p ≤ 0.05 (Figure 1B). RNA-seq analysis identified 732 genes that were significantly differentially regulated (adj. p ≤ 0.05 and log2FC 0.5). Of these 732 genes, 269 were upregulated genes (36.75%) and 463 were downregulated genes (63.25%), (Figure 1C). IPA analysis was performed to determine the specific pathways altered by ethanol in the cerebella of adult mice. The results of the top canonical pathways altered by ethanol exposure included those related to the generation of precursor metabolites and energy, pathogen-influenced signaling, cellular immune response, degradation/utilization/assimilation, cellular stress and injury, biosynthesis, disease-specific pathways, cardiovascular signaling, nuclear receptor signaling, and ingenuity toxicity list pathways (Figure 2A). A description of the pathway names, p-values, and molecules associated with each significantly altered pathway category is shown in Table 5. The top disease and biological function categories altered by ethanol exposure included nervous system development and function, tissue/cell morphology, cell-to-cell signaling and interaction, cell death and survival, cellular compromise, immune cell trafficking, and inflammatory response [−log(p.val) range = 5.5–2.1] (Figure 2B). The diseases and biological function annotations that correlate to the diseases and biological functions categories, as shown in Figure 2B, are myelination (p.val = 2.88 × 10−6 ) or demyelination (p.val = 0.0053) of the cerebellum; quantity (p.val = 0.000125) or coupling (p.val = 0.000556) of oligodendrocytes; thickness of myelin sheath (p.val = 0.000556); quantity of cells (p.val = 0.00783); activation of microglia (p.val = 0.00783); permeability of blood–brain barrier (p.val = 0.0236); and astrocytosis of cerebella (p.val = 0.0467), (Table 6). These results suggest that in the cerebellum, ethanol alters biological functions that pertain to alterations in the formation of myelin, along with possible microglia and astrocyte phenotypic changes. Alcohol has been demonstrated to induce neuroinflammation in both humans and rodents which may include microglial activation, characterized by shortening and thickening of processes, along with the secretion of proinflammatory cytokines and chemokines that may contribute to neuropathology [19,40,41]. We performed hierarchical clustering analysis on homeostatic and neurodegenerative disease microglia-associated genes that were differentially expressed (adj. p ≤ 0.05) in response to ethanol (Figure 3A). A Student’s t-test comparing the average z-scores across all relevant genes indicated that ethanol caused an overall significant downregulation of microglia homeostatic genes (p.val = 3.191 × 10−6) (Figure 3B, Table 2) and an overall significant upregulation of microglia genes associated with neurodegenerative diseases (p.val = 7.786 × 10−5) (Figure 3C, Table 2). Collectively, these data suggest that ethanol may alter the microglial phenotype from a homeostatic and protective phenotype to a more activated phenotype observed in neurodegenerative diseases. Astrocytes are one of the most abundant cell types in the CNS and play a critical role in regulating CNS functions in health and disease by maintaining homeostasis, providing energy to neurons, regulating synapse development and plasticity, modulating blood-brain-barrier integrity, and controlling neurological function and behavior [42,43,44,45,46]. Similarly to microglia, astrocytes play a role in CNS inflammation [47,48], and ethanol has been demonstrated to trigger an immune response in astrocytes [49,50]. In the current study, we performed hierarchical clustering analysis on acute injury, chronic neurodegenerative, and pan-injury astrocyte-associated genes that were differentially expressed (adj. p ≤ 0.05) in response to ethanol (Figure 4A). A Student’s t-test comparing the average z-scores across all relevant genes indicated that ethanol caused an overall significant increase in astrocyte genes related to acute injury (p.val = 7.085 × 10−5) (Figure 4B, Table 3) and an almost even number of up- and down-regulated genes (12 up vs. 13 down) pertaining to pan-injury (p.val = 0.6266) (Figure 4C, Table 3). Ethanol only altered the expression of three genes associated with the chronic neurodegenerative disease category (Table 3), thus the effect of ethanol on this small number of genes was not statistically evaluated. These data suggest that alcohol-induced transcriptomic changes in astrocytes are consistent with an acute injury phenotype. Ethanol has been demonstrated to alter myelination in adult humans and rodents [51,52]. We performed hierarchical clustering analysis on genes associated with distinct oligodendrocyte lineages (immature and myelinating) whose expression was altered by ethanol (Figure 5A,B). Evaluation of the effects of ethanol on immature oligodendrocyte lineages indicated that ethanol significantly decreased the expression of genes associated with COPs (p.val = 0.0006784) (Figure 5C, Table 4), and that ethanol skewed toward decreasing the expression of genes associated with OPCs (p.val = 0.1702) (Figure 5D, Table 4). For the myelinating oligodendrocyte lineage cells, ethanol significantly decreased the expression of genes associated with MFOLs (p.val = 2.905 × 10−05) (Figure 5E, Table 4). NFOL and MOL groups only contained two differentially expressed genes; therefore, statistical significance was not evaluated for these categories (Table 4). These results suggest that ethanol effects both immature and myelinating oligodendrocyte lineage cells, which could potentially lead to altered myelination. Pathway analysis indicated that ethanol had significant effects on immune processes in the cerebella of adult mice. In addition, these analyses suggested that ethanol may alter the phenotype and function of glial cells including microglia, astrocytes, and oligodendrocyte lineage cells. We and others have previously demonstrated that ethanol induces neuroinflammation in adult rodents. However, the amount of neuroinflammation varies depending on the ethanol administration paradigm. For example, acute 4-day ethanol exposure did not alter the expression of pro-inflammatory molecules, although microglial activation was observed [17,53]. Following 10-day ethanol exposure, increased expression of pro-inflammatory molecules was observed, although it was somewhat modest [16,18,19]. Chronic ethanol exposure over a period of 3–5 months resulted in more robust neuroinflammation [15,49,54,55]. Using a variation of the same model as the current study, in which gene expression in both male and female mice was evaluated in control, ethanol, and ethanol + pioglitazone experimental groups, we have previously demonstrated robust neuroinflammation following chronic plus binge exposure to ethanol in less than one month [22]. This model is similar to an alcoholic liver disease model used previously by the Gao laboratory, in which they showed systemic inflammation and liver injury [20,21]. At this point, it is unclear in our studies whether ethanol induces CNS inflammation directly or indirectly through ethanol induced inflammation outside of the CNS. In order to begin to understand the possible mechanisms by which ethanol induces neuroinflammation in this chronic plus binge model of AUD, we have treated a unique set of male mice for the purpose of RNAseq analysis in the current study. We acknowledge that the use of only male mice is a limitation of the current study. Furthermore, some of the pathways identified in the current study only contain 1 or 2 genes, and some genes are represented in multiple pathways. Thus, we have exercised caution to not overinterpret the results. We evaluated the transcriptomic data to identify immune-regulated genes whose expression was most strongly induced by ethanol, which included FOSB, CCL2, CCL7, C5AR1, SPP1, CD68, SOCS3, C3AR1, and KLF4. The most highly upregulated gene is FOSB, which encodes a transcription factor that dimerizes with Jun protein to form AP-1 and plays a critical role in alcohol and drug addiction [56]. Alcohol increases the expression of FOSB in the mesocorticolimbic system, which is believed to contribute to alcohol use disorder [57,58]. Furthermore, ethanol was demonstrated to alter synaptic plasticity and epigenetic alterations in the FOSB promoter, resulting in increased FOSB expression in the medial prefrontal cortex in wild-type but not TLR4 deficient mice. Since ethanol is believed to activate TLR4, resulting in downstream immune signaling [59], a role of ethanol-induced neuroinflammation is suggested in these processes. FOSB has also been demonstrated to contribute to excitotoxic microglial activation through regulation of complement C5a receptors in these cells [60]. Interestingly ethanol strongly increased the expression of complement C5AR1 and C3AR1 in our RNA-Seq studies. C5AR1 expression is increased in the liver of patients with alcoholic hepatitis [61], and is believed to contribute to alcohol-induced inflammation and liver injury [62,63]. Additionally, ethanol induces the expression of complement receptors including C3AR1 expression in microglia, resulting in altered phagocytosis [64]. We previously demonstrated that ethanol induces the expression of the chemokine CCL2 or MCP-1 following acute ethanol exposure in adult rodents [65], as well as in animal models of fetal alcohol spectrum disorders (FASD) [66]. It is interesting that in the current study, ethanol induced the expression of CCL2 as well the related chemokine CCL7 or MCP-3 in this chronic plus binge model. It should also be noted that transcriptomic changes were only evaluated at one timepoint, 24 h after the final ethanol exposure. Future studies may wish to evaluate transcriptomic changes at different times following the final ethanol exposure. It is also noteworthy that the other immune-related molecules we identified previously in this model were not indicated in the current study; this may be due to less sensitivity and smaller “n”, both of which are limitations that come with RNAseq when compared to quantitative real-time PCR [22]. Microglia are capable of responding to signals, resulting in activation and an altered phenotype. Our IPA analysis indicated that ethanol treatment resulted in microgliosis or microglial activation in the cerebellum. Upon activation, microglia have traditionally been hypothesized to undergo classical activation, resulting in a M1 pro-inflammatory phenotype, or alternative activation, resulting in an M2 anti-inflammatory or protective phenotype [67,68]. However, more recently it has become clear that microglial phenotypes are complex, and cannot be defined or categorized effectively using this simple binary system [69]. One recent nomenclature to distinguish microglial phenotype focuses on homeostatic versus neurodegenerative disease phenotypes. Under homeostatic conditions, microglia have a homeostatic phenotype, described by playing a role in synaptic plasticity and synaptogenesis, trophic support, chemotaxis and immune cell recruitment, and neurogenesis [37]. During insult to the CNS, microglia commonly lose their homeostatic signature and assume a chronic inflammatory signature [70,71,72]. Evaluation of the phenotype of microglia in a variety of neurodegenerative diseases have resulted in the identification of a common neurodegenerative disease-associated microglia phenotype [34,37,71,73]. In the current study, ethanol induced a microglia phenotypic switch in the cerebellum. This phenotypic switch was similar to that observed in neurodegenerative diseases, with a downregulation of homeostatic signature genes and an upregulation of neurodegenerative signature genes. Astrocytes, like microglia, are capable of functioning in the innate immune response in the CNS. Once astrocytes are activated, commonly referred to as astrogliosis/astrocytosis, they produce cytokines and chemokines, nitric oxide, and other reactive oxygen species as part of an inflammatory response [74], Our IPA analysis indicated that ethanol treatment resulted in “astrocytosis”. Astrocytes were classically defined to respond to various stimuli to become reactive A1 astrocytes (neurotoxic or reactive A2 astrocytes) which are protective and neurotrophic [75,76]. However, as with microglia, this binary system of classifying reactive astrocytes appears inadequate to fully define and distinguish astrocyte phenotypes. More recently, Serrano-Pozo and colleagues performed a meta-analysis of mouse transcriptomic studies which resulted in a nomenclature that classified reactive astrocytes as being consistent with acute injury, chronic neurodegeneration, or pan-injury reactive astrocytes which exhibited characteristics of both acute injury and chronic neurodegenerative phenotypes [38]. In the current study, we determined that ethanol induced changes consistent with an acute injury astrocyte phenotype. Interestingly, LPS was previously shown to trigger an acute injury astrocyte phenotype [38]. ethanol has also been shown to activate TLR4 receptors, suggesting that ethanol-mediated neuroinflammation could occur in response to recruitment of TLR4 during alcohol use/abuse [77,78,79]. Therefore, we speculate that in this model of AUD, in the cerebellum, ethanol induces an acute injury astrocytic phenotype through the activation of TLR4, subsequently inducing an immune response. Oligodendrocytes are responsible for forming a myelin sheath around axons of neurons in the CNS, facilitating the efficient propagation of action potentials [80]. OPCs are produced during embryogenesis, and migrate to their functional location wherein they differentiate into mature myelinating oligodendrocytes. Most myelination occurs at later stages of CNS development but can occur throughout life [81]. Ethanol has profound effects on the developing CNS and is believed to significantly contribute to the pathology associated with FASD, at least in part by altering myelination [82]. Ethanol also alters myelination in adults with AUD [83,84]. Ethanol is highly toxic to oligodendrocyte lineage cells, with OPCs being particularly susceptible [85,86]. Alcohol exposure is known to disrupt OPC differentiation and survival by decreasing the expression of platelet-derived growth factor receptor α (PDGFRα), a molecule crucial for differentiation of OPCs into mature oligodendrocytes [87]. In the current study, we found that adult chronic plus binge-like alcohol exposure depletes the expression of genes associated with both immature oligodendrocyte precursor cells as well as myelinating oligodendrocytes. Future studies are needed to determine the mechanism by which ethanol effects oligodendrocyte lineage cells and myelination in AUD. The current study demonstrates that ethanol alters the transcriptomic profile in the adult cerebellum in a chronic plus binge model of AUD. The pathways altered by ethanol included those involved in immune response. Ethanol caused a shift in the expression of microglial-associated genes, with a decrease in homeostatic and an increase in chronic neurodegenerative-associated transcripts. Ethanol also increased the expression of astrocyte-associated genes common to acute injury. Finally, ethanol decreased the expression of genes associated with immature oligodendrocyte progenitor cells, as well as myelinating oligodendrocytes. These results provide clues about the mechanisms by which ethanol induces neuroinflammation and altered glial function in AUD.

PMC10000477

Natasa Nikolic,Branka Basica,Aljosa Mandic,Nela Surla,Vera Gusman,Deana Medic,Tamas Petrovic,Mirjana Strbac,Vladimir Petrovic

E6/E7 mRNA Expression of the Most Prevalent High-Risk HPV Genotypes in Cervical Samples from Serbian Women

28-02-2023

E6,E7,HPV,cervical intraepithelial lesion,biomarker,Serbia

Cervical cancer caused by persistent infection with HR HPV genotypes is the second leading cause of death in women aged 15 to 44 in Serbia. The expression of the E6 and E7 HPV oncogenes is considered as a promising biomarker in diagnosing high-grade squamous intraepithelial lesions (HSIL). This study aimed to evaluate HPV mRNA and DNA tests, compare the results according to the severity of the lesions, and assess the predictive potential for the diagnosis of HSIL. Cervical specimens were obtained at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia, during 2017–2021. The 365 samples were collected using the ThinPrep Pap test. The cytology slides were evaluated according to the Bethesda 2014 System. Using a real-time PCR test, HPV DNA was detected and genotyped, while the RT-PCR proved the presence of E6 and E7 mRNA. The most common genotypes in Serbian women are HPV 16, 31, 33, and 51. Oncogenic activity was demonstrated in 67% of HPV-positive women. A comparison of the HPV DNA and mRNA tests to assess the progression of cervical intraepithelial lesions indicated that higher specificity (89.1%) and positive predictive value (69.8–78.7%) were expressed by the E6/E7 mRNA test, while higher sensitivity was recorded when using the HPV DNA test (67.6–88%). The results determine the higher probability of detecting HPV infection by 7% provided by the mRNA test. The detected E6/E7 mRNA HR HPVs have a predictive potential in assessing the diagnosis of HSIL. The oncogenic activity of HPV 16 and age were the risk factors with the strongest predictive values for the development of HSIL.

E6/E7 mRNA Expression of the Most Prevalent High-Risk HPV Genotypes in Cervical Samples from Serbian Women Cervical cancer caused by persistent infection with HR HPV genotypes is the second leading cause of death in women aged 15 to 44 in Serbia. The expression of the E6 and E7 HPV oncogenes is considered as a promising biomarker in diagnosing high-grade squamous intraepithelial lesions (HSIL). This study aimed to evaluate HPV mRNA and DNA tests, compare the results according to the severity of the lesions, and assess the predictive potential for the diagnosis of HSIL. Cervical specimens were obtained at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia, during 2017–2021. The 365 samples were collected using the ThinPrep Pap test. The cytology slides were evaluated according to the Bethesda 2014 System. Using a real-time PCR test, HPV DNA was detected and genotyped, while the RT-PCR proved the presence of E6 and E7 mRNA. The most common genotypes in Serbian women are HPV 16, 31, 33, and 51. Oncogenic activity was demonstrated in 67% of HPV-positive women. A comparison of the HPV DNA and mRNA tests to assess the progression of cervical intraepithelial lesions indicated that higher specificity (89.1%) and positive predictive value (69.8–78.7%) were expressed by the E6/E7 mRNA test, while higher sensitivity was recorded when using the HPV DNA test (67.6–88%). The results determine the higher probability of detecting HPV infection by 7% provided by the mRNA test. The detected E6/E7 mRNA HR HPVs have a predictive potential in assessing the diagnosis of HSIL. The oncogenic activity of HPV 16 and age were the risk factors with the strongest predictive values for the development of HSIL. It is estimated that approximately every fourth malignancy can be linked to an infectious agent, that is, its contribution to various stages of cancer development (reviewed in [1]). About a third of this contribution is related to the human papillomavirus (HPV) (reviewed in [2]). Today, significant evidence confirms the association of high-risk (HR) HPV as a carcinogen or promoter in developing malignant diseases in different locations: the cervix, vulva, vagina, penis, anus, and certain head and neck regions. In first place are neoplasias of the lower genital tract, such as cervical cancer [3]. According to estimates by the World Health Organization (WHO), that is, by the International Agency for Research on Cancer (IARC), 604,000 new cases and 342,000 deaths were registered around the world in 2020, which makes cervical cancer the fourth most frequently diagnosed cancer in women [4]. In Serbia, organized cervical cancer screening has been conducted since 2012, using the PAP test, based on the cytomorphological examination of cervical samples. Screening is mandatory for women aged 25 to 69. However, despite organized screening, cervical cancer remains one of the most common cancers among women in Serbia [5]. The incidence of cervical cancer in Serbia is still among the highest and is approximately twice the average in Europe (10.7 to 100,000) [6,7]. It is necessary to emphasize that data on the HPV prevalence and genotype distribution among women with normal cervical cytology, precancerous cervical lesions, and cervical cancer are missing in the updated IARC Human Papillomavirus and Related Diseases Report for Serbia [6]. HPV vaccination is a crucial prevention tool against HPV infection and HPV-related precancers and cancers [8]. If vaccination against HPV is carried out before initial sexual activities, it is one of the most effective ways to prevent cervical cancer [9]. Still, in Serbia, vaccination against HPV infection is not part of the mandatory national immunization program, but it is recommended for children aged 9 to 19 years [5]. Strong evidence for HPV as a causative aetiology of cancers of various locations was provided by the IARC, which classified HPV according to its potential to cause malignant cell alteration as follows [10]: Group 1 (carcinogenic to humans, HR) includes HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59; Group 2A (probably carcinogenic) includes HPV genotype 68; Group 2B (potentially carcinogenic) includes HPV genotypes: 26, 53, 66, 67, 70, 73, 82, 30, 34, 69, 85 and 97; Group 3 (low risk, LR) includes HPV genotypes 6 and 11. Persistent HPV infection is the most critical risk factor for the development of cervical cancer, which is confirmed by the presence of HR HPV in over 99% of cervical cancer samples. Concerning the oncogenic potential, infection with a particular HR HPV genotype carries a specific risk for cellular transformation and malignancy (reviewed in [3]). Namely, one of the most critical determinants of the degree of pathogenicity of different HPV genotypes is the functional differences between their oncoproteins, E6 and E7 [11]. During viral genome integration into the host cell genome, E1 or E2 are usually disrupted [12]. This gene disruption leads to uncontrolled transcription of E6 and E7 genes as the E2 repression on these oncogenes disappears [13,14]. Their protein products lead to unregulated cell proliferation, differentiation, and loss of the reparative abilities of the host cell, wherefore they are considered the main actors of virus-induced oncogenesis of cervical cancer [15,16]. Thanks to the use of cervical cancer screening tests, this cancer is classified as one of the most preventable malignancies. The most common test for this purpose is the cytological abnormality test, the Papanicolaou (PAP) test. However, considering the etiological role of HR HPV in developing cervical cancer, DNA tests have been incorporated into the primary screening of developed countries. Still, this test is characterized by high sensitivity and low specificity, which indicates the necessity of improving the test’s characteristics concerning specificity [17]. In this context, the results of numerous studies state that using the HR HPV mRNA test as a basic or additional test in primary screening would improve these characteristics [18]. Given the above, this research aimed to determine the oncogenic activity of the most commonly diagnosed HR HPVs in cervical smear samples using the mRNA test and compare the results according to the severity of the cervical intraepithelial lesion. Furthermore, it aimed to examine the clinical characteristics and predictive potential in assessing the diagnosis of high-grade cervical intraepithelial lesions of HPV DNA and mRNA tests. From 2017 to 2021, cervical smears were obtained from a sample of 365 female patients (age 20–74 years) with normal and abnormal results of cervical cytology who were undergoing gynecological exams at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia. All of the women in the study did not receive any prior treatment for cervical dysplasia or cancer, and all were unvaccinated against HPV infection. The samples were collected using the ThinPrep Pap test (Hologic Inc.) according to the manufacturer’s instructions and sent for further analyses to the Center of Virology, Institute of Public Health of Vojvodina, Novi Sad, Republic of Serbia. The classification of cytological findings was performed according to the criteria of the Bethesda System 2014. It was categorized into negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL). All of the women enrolled in the study were informed about the research objective and signed an informed written consent form. The study protocol was reviewed and approved by the Medical Ethical Committee of the Institute of Public Health of Vojvodina, Novi Sad, Serbia (approval number: 01-252/3). The ThinPrep cervical smear samples were stored at 4–8 °C for up to 3 days from the sampling day. The 2 mL of collected samples were transferred to nuclease-free tubes and centrifugated at 8000× g for 5 min. The formed pellet was dissolved in 200 µL of nuclease-free water and used for nucleic acid extraction. According to the manufacturer’s instructions, DNA extraction was carried out using the SaMag STD DNA Extraction Kit (Sacace Biotechnologies, Como, Italy). The extracted DNA was eluted in 100 μL elution buffer. The detection and genotyping of 12 HR HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), marked as the HPV DNA test, were performed using the High Risk Typing Real-TM Kit (Sacace Biotechnologies, Como, Italy) following manufacturer’s instructions. The E7 gene of specific HPV genotypes was amplified using primers and TaqMan probes in the multiplex reaction performed in a total of 13 μL. The β globin gene is used as an internal control. Real-time PCR was performed on the SaCycler-96 (Sacace Biotechnologies, Como, Italy). After the initial activation of the DNA polymerase at 95 °C for 15 min, five cycles of amplification were performed under the following conditions: 95 °C/5 s, 60 °C/20 s, and 72 °C/15 s, and 40 amplifications were performed under the following conditions: 95 °C/5 s, 60 °C/30 s (fluorescence detection), and 72 °C/15 s. The kinetics of the detected fluorescence signals were monitored using the SaCycler-96 software package (Sacace Biotechnologies, Como, Italy). E6/E7 mRNA of the most prevalent HPVs was tested in the cervical samples positive for the most prevalent HR HPVs DNA and HR HPV DNA negative samples. The HR-HPV-negative samples were included in E6/E7 mRNA testing because the study aimed to determine the mRNA test’s clinical characteristics by evaluating and comparing it with the HPV DNA test. Total RNA was extracted from the prepared sample using the miRNeasy Mini Kit and QIAcube robotic workstation (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The total RNA was eluted in 50 µL ultrapure water free from nucleases. Following the manufacturer’s recommendations, potentially present contaminants were removed using the TURBO DNA-free Kit (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA). The routine procedure for removing contaminants using the kit above included the addition of 5 µL of 10× TURBO DNase Buffer and 1 µL of TURBO DNase enzyme into each sample of extracted total RNA, with incubation for 30 min at a temperature of 37 °C. After the action of the enzyme, 5 µL of inactivation reagent (Dnase Inactivation Reagent) was added, with incubation for 5 min, at room temperature and occasional vortexing. After that, centrifugation was performed (90 s, 10,000× g). The supernatant was carefully transferred to a nuclease-free tube. The real-time reverse transcription PCR (RT-PCR) analysis, marked as the HPV mRNA test, was performed using specific primers and TaqMan probes to detect the E6/E7 mRNA of individual HPV genotypes. The sequences for the primers and probes (Table 1) were adopted from Lindh et al. (2007) [19] and purchased from Life Technologies (Carlsbad, CA, USA). The AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Waltham, MA, USA) was used for the real-time RT-PCR. A separate reaction mixture was prepared for each set of primers and TaqMan probes. The reaction was prepared to a final volume of 25 μL containing: 12.5 μL 2× RT-PCR Buffer, 1 µL of 25× RT-PCR Enzyme Mix, primers to a final concentration of 300 nM, the probe to a final concentration of 200 nM, 1 µL of RNase Inhibitor reagent (Applied Biosystems, Waltham, MA, USA), 5 μL of isolated total RNA, and DEPC-treated nuclease-free water (Invitrogen, Waltham, MA, USA). Real-time PCR was performed on the Applied Biosystems 7500 Real-Time PCR Systems (ThermoFisher Scientific, Waltham, MA, USA). After the reverse transcription reaction at 48 °C for 30 min, the inactivation of reverse transcriptase and the activation of Taq polymerase were performed at 95 °C for 10 min. After that, 45 cycles of PCR amplification were carried out with denaturation at 95 °C for 15 s and annealing and elongation at 58 °C for 1 min. The data were analyzed with the Applied Biosystems Software v2.0.6 (ThermoFisher Scientific, Waltham, MA, USA) and the GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). All of the statistical analyses were performed using SPSS statistics software Version 21.0 (Chicago, IL, USA). Testing the difference in frequencies of attributive features was performed using the Chi-square (χ2) test of independence and quality of the match. The Student’s t-test was used to compare values between the two age groups, a numerical characteristic. A one-way analysis of variance (ANOVA) and the Bonferroni post-hoc test were applied to compare values between three or more data groups. Frequencies were used to present the analysis of the oncogenic activity of multiple HR HPV infections. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and their 95% confidence intervals (CIs) of HR HPVs DNA and E6/E7 mRNA HPVs detection and cytology test were calculated. To quantify the diagnostic capabilities of the selected test and evaluate its significance, the receiver operating characteristics (ROC) curve was used, which enables testing the significance of differences in the discriminating potential of different variables for the same binary outcome. It is based on a graphical presentation of pairs of sensitivity and specificity that can be obtained by estimating the threshold value for all values of discontinuous variables of the sample. Univariate and multivariate logistic regression were used to analyze the connection between two or more features, generating adequate statistical models. Multivariate logistic regression analysis was applied to all of the analyzed factors to construct a predictive model and named the most relevant predictors for the development of HSIL. A p-value of less than 0.05 defined as statistically significance. A total of 365 specimens obtained from women in the north part of the Republic of Serbia (Vojvodina) were classified based on the Bethesda System 2014 into four categories by cytological criteria. The cervical samples were analyzed for 12 HR HPVs, where 246 out of 365 (67.4%) had HPV-DNA-positive results, which indicates that the overall prevalence of HPV in the study population was 67.4%. All of the HPV genotypes covered by the HPV DNA test were identified (n = 274) in the study population (246 HR-HPV-positive cervical samples). The most prevalent HPV genotype is HPV 16 which makes up 38.3% (105/274) of the total HPV-detected genotypes in 42.7% (105/246) of HP- DNA-positive samples. HPV 31 takes second place with 17.2% (47/274) of total HPV-detected genotypes in 19.1% (47/246) of HPV-DNA-positive samples. Equally represented are HPV 33 and HPV 51, each with 8.8% (24/274) of total HPV-detected genotypes in 9.8% (24/246) of HPV-DNA-positive samples (Figure 1). The results show that those HR HPVs make up 73% (200/274) of the detected genotypes, including multiple infections, which determined those cervical samples (n = 172) for further examination of oncogenic activity, according to the study’s aim. Multiple HPV infections were found in 15.7%. The most common co-infections were those with HPV 16 and 31, found in 7.6% (13/172) of cases with multiple infections (Table 2). Molecular data were compared with the cytological results and age categories of patients. The distribution of cytological groups was analyzed within the most prevalent HR-HPV-DNA-positive samples (172 cervical samples), including multiple infections. A minority of women, 16.9% (n = 29), had normal results, whereas 83.1% (n = 143) showed different abnormalities. A total of 26.7% (n = 46) of the examined women had ASCUS; in 25.6% (n = 44), LSILs were found, whereas HSILs were detected in 30.8% (n = 53). The mean age of the patients was 36.7 years. Among the specimens, the number of Serbian women who were ≤30 years, 31–44 years, and ≥45 years old accounted for 36.5% (n = 68), 36.0% (n = 62), and 24.4% (n = 42) of the samples, respectively (Table 3). The distribution of the most frequently detected HPVs concerning cytology is shown in Table 4. The prevalence rates of HR HPV 16 ranged from 44.8% in the group of NILM cytology to 75.5% in the HSIL group. Contrarily, the prevalence of HR HPV 31 is similar in the groups of NILM (37.9%), ASCUS (34.8%), and LSIL (31.8%), while it is lower in the group of HSIL (11.3%). HPV genotypes 33 and 51 are present in all of the cytological groups in less than 21%. The statistically significant difference in the prevalence between the number of positive findings of HPV 16 (χ2 test; p = 0.035) and HPV 31 (χ2 test; p = 0.017) was determined, depending on the degree of severity of the cytological findings, which was not determined for HPV 33 and 51 (χ2 test; p = 0.706, p = 0.790, respectively) (Table 4). A statistically significant difference was found in the number of female patients concerning the cytological findings and the age of the patients (χ2 test; χ2 = 29.500; p = 0.000) (Table 5). The statistically significant difference was determined regarding the cytological findings and the age of the patients, where the female patients diagnosed with HSIL were significantly older compared to the other groups (ANOVA; F = 9.321; p < 0.001). The Bonferroni post-hoc test determined that the female patients with HSIL are statistically significantly older than those with ASCUS (p < 0.001), NILM (p < 0.001), and LSIL (p = 0.012) (Figure 2, Table 5). Female patients with confirmed HPV 31 are statistically significantly younger (33 years) than the other HR-HPV-DNA-positive patients (t = 2.317; p = 0.022). The average age of the female patients with confirmed HPV DNA 16 was 36.9 years; with HPV DNA 33, it was 34.1 years, while the average age of patients with HPV DNA 51 was 40.5 years. The statistical analyses show that the proportion of HPV 16 positivity maintained at the same level as age. Conversely, the proportion of HPV 31 positivity decreases with age. The detection of HR HPV 33 genotypes decreases with age, while HR HPV 51 increases (Table 5). Figure 3 shows the study design with sample processing to analyze the expression of the E6/E7 mRNA of the most prevalent HR HPV in cervical samples from Serbian women. A total of 291 cervical samples, which include HPV 16-, 31-, 33- and 51-positive (n = 172) and HR-HPV-negative samples (n = 119), were tested by the HPV mRNA test. The E6/E7 mRNA HPV was detected exclusively in HR-HPV-DNA-positive samples (Table 6). E6 and E7 transcripts of the four most frequent HR HPVs were detected in 57.5% (115/200) of the HR HPV DNA confirmed genotypes. Accordingly, the distribution of E6/E7 mRNA HR HPV 16, 31, 33, and 51 are shown in Table 6. The E6/E7 mRNA HR HPV 16 was the most abundant, which accounted for 25.5% (51/200) of HR HPV genotypes. Next in frequency was the E6/E7 mRNA HR HPV 31 in 16.5% (33/200), while the E6/E7 mRNAs HR HPV 33 and 51 were equally represented in 8% (16/200) and 7.5% (15/200), respectively. Almost every second HPV 16 genotype is oncogenically expressed (48.6%; 51/105), and it was detected in 29.7% (51/172) HPV-DNA-positive samples. The oncogenic activity of HPV 31 was detected in approximately every fifth (19.2%; 33/172) HPV-DNA-positive sample. Regarding the oncogenic activity of the remaining tested genotypes, HPV 33 and HPV 51 were detected in roughly every tenth HPV-DNA-positive sample (Table 6). The results of expressing E6 and E7 HR HPV oncogenes were expressed through the dispersion of the obtained Ct values. The oncogenic activity of HPV 16 is detected by the lowest registered value (Ct = 16) (Supplementary Materials Figure S1). The expression of the E6 and E7 genes as indicators of the oncogenic activity of HR HPV 16, 31, 33, and 51 was analyzed concerning cytological findings. The oncogenic activity of the tested genotypes increases with the severity of the cervical intraepithelial lesion. A statistically significant difference in E6/E7 mRNA HPV expression among the various cytological groups was observed (χ2 test; χ2 = 108.623; p < 0.001). E6/E7 mRNA HPVs are the most prevalent in patients with HSIL cytological findings (88.9%). In the group of patients with LSIL cytological findings, it was demonstrated in a lower percentage (60%). A two-fold lower prevalence is observed in patients with ASCUS (29.4%). Oncogene activity in women with normal cytological findings is present in 10.9% of samples (Table 7). Subsequently, the E6/E7 mRNA distribution of HR-HPV-DNA-positive samples according to genotype and cytological groups was analyzed. E6/E7 mRNA HR HPV 16 is the most represented in patients with HSIL cytological findings (64.2%), while in the other groups of cytological findings, it was demonstrated in a lower percentage (3.4–22.7%). A statistically significant difference was found in the number of positive findings of E6/E7 mRNA HR HPV 16 concerning the cytological status (χ2 test; χ2 = 46.881; p < 0.001). This result singled out the HR HPV 16 genotype for further analyses. The presence of E6/E7 mRNA HR HPV 31 was the least detected in HSIL (7.5%) compared to other cytological groups (22.7–26.1%). The distribution of the oncogenic activity of the remaining genotypes (HR HPV 33 and 51) is approximately the same across different cytological groups and remains at a low level (2.2–11.4%) (Table 7). The analyses of the oncogenic activity of multiple HR HPV infections are presented by frequencies. The overall oncogenic activity, including both single and multiple HR HPV infections detected using the E6/E7 mRNA HR HPV test, increases with the degree of cervical lesion severity (60–100%). The oncogenic activity detected in single-genotype infections (20.0–85.7%) is higher compared to the oncogenic activity of multiple genotypes (0–40%) (Table 8). The results were categorized according to age categories (≤30, 31–44, ≥45 years) to analyze the prevalence of E6/E7 mRNA HPV in the specific age groups. The lowest percent of E6/E7 mRNA HR HPV 16 was detected in the younger group and the highest percent was detected in patients over 44 years. A statistically significant difference was observed in patients with E6/E7 mRNA HR HPV 16 expression (χ2 test; χ2 = 7.331; p = 0.026), wherein individuals with positive E6/E7 mRNA HPV results were older than the others. The detection of E6/E7 mRNA HR HPV 31 and 33 decreases with the increasing age of the patient, while E6/E7 mRNA HR HPV 51 increases (Table 9). The comparison of tests for the detection of the most prevalent HR HPV genotypes and their oncogenic activity revealed that the presence of the HR HPV genotypes is higher than the presence of the oncogenic activity of the genotypes in younger women (≤30 years), similar in middle-aged women (31–44 years), and lower in women 45 years and older. The prevalence of oncogenic activity of the HPV genotypes increases with the severity of the cervical intraepithelial lesion. Compared to the prevalence of the examined HR HPV genotypes, the same parameter is lower in women with normal and undefined cytological findings, approximately the same in low-grade lesions, and significantly higher in high-grade lesions (Figure 4). The calculated clinical characteristics of the DNA and E6/E7 mRNA HR HPV tests are shown in Table 10. The sensitivity and NPV of both tests were increased with the severity of the cervical intraepithelial lesion, with the HR HPV DNA test showing a statistically significantly higher level (67.6–98.2%). The specificity of the E6/E7 mRNA HR HPV test (89.1%) is statistically significantly higher than the HR HPV DNA test (75.6%). The PPV of the HR HPV DNA test is approximately the same for all types of cytological findings (60.3– 64.6%), while for the mRNA HR HPV test, it increases with the degree of the cervical intraepithelial lesion (69.8– 78.7%) and it is statistically significantly higher (Table 10). To quantify the diagnostic capabilities of the E6/E7 mRNA HPV test and evaluate its significance, an ROC curve was used to assess the assays for detecting HSIL. The area determined by the ROC curve (AUC) of E6/E7 mRNA HR HPV is 0.812 (CI (95%): 0.752–0.871), while the area under the ROC curve formed by the parameters of the HR HPV DNA test is 0.740 (CI (95%): 0.680–0.799) (Table 11, Figure 5). The relationships between the oncogenic activity of all of the tested HPVs and previously singled out HPV 16 vs. the cytological results were analyzed using Spearman’s (ρ) correlation (Figure 6). There is a statistically significant positive moderate correlation between the presence of HPV 16 oncogenic activity and cytological status (Spearman’s correlation; ρ = 0.494; p < 0.001) (Figure 6A). The oncogenic activity of the tested HPV genotypes is associated with a strong statistically significant positive correlation with the degree of cervical intraepithelial lesion severity (Spearman’s correlation; ρ = 0.594; p < 0.001) (Figure 6B). Univariate multinomial logistic regression examined the individual factors (the HR HPV DNA 16, the oncogenic activity of all of the tested HR HPVs, the oncogenic activity of the HR HPV 16 genotype, and the age category) that indicated an increased probability of developing HSIL (Supplementary Materials Tables S1–S4). For the influence of HR HPV 16 on the probability of HSIL, a statistically significant predictive value was determined in the NILM and LSIL cytological groups. Patients with the confirmed presence of the HPV 16 genotype through a DNA test have a higher probability of being diagnosed with HSIL than NILM (3.8-fold), while they will have a 2.6-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with LSIL (Supplementary Materials Table S1). The influence of the oncogenic activity of all tested of the HR HPV genotypes (E6/E7 mRNA HR HPVs) on diagnosing high-grade lesions of the cervical epithelium has a statistically significant prediction found in all cytological groups, NILM, ASCUS, and LSIL. If patients have confirmed indicators of oncogenic activity, they will have an almost seven-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with NILM. The same patients have a 19-fold higher probability of being diagnosed with HSIL compared to the probability of detecting ASCUS and a 5-fold higher probability of detecting HSIL compared to LSIL (Supplementary Materials Table S2). E6/E7 mRNA HR HPV 16 is an indicator for diagnosing HSIL, and a statistically significant predictive value was determined for all types of cytological findings, NILM, ASCUS, and LSIL. In a patient with confirmed HPV 16 oncogenic transcripts, the probability of diagnosing HSIL is 50-fold higher than the probability of diagnosing NILM. Patients with a positive result of E6/E7 mRNAs HR HPV 16 are 12-fold more likely to be diagnosed with HSIL compared to the detection of ASCUS, while in patients with the same result, the probability of detection of HSIL is 6-fold higher than the probability of detection of LSIL (Supplementary Materials Table S3). A statistically significant predictive value for the age category was determined in all cytological groups, NILM, ASCUS, and LSIL. The probability of detecting HSIL concerning normal results in HR-HPV-positive patients is 6.3-fold higher if they are ≥ 45 years of age than women under 30. A similar prediction for the detection of HSIL was shown concerning ASCUS (6.5-fold). Patients from the oldest age category have a 3.4-fold higher probability of detecting HSIL concerning LSIL than the youngest (Supplementary Materials Table S4). The predictive model contains four independent variables: HR HPV DNA 16, E6/E7 mRNA HR HPVs, E6/E7 mRNA HR HPV 16, and age category. The HSIL lesion, as a representative of a high degree of cervical atypia, represented a dependent variable concerning all of the analyzed relevant factors. Multivariate logistic regression analysis was applied to all of the analyzed factors to construct a predictive model and named the most relevant predictors for the development of HSIL (Table 12). Analyzing the mutual influence of the examined relevant factors for diagnosing HSIL compared to the probability of diagnosing a normal cytological finding, the strongest statistically significant predictor was determined to be the oncogenic activity of the HPV 16 genotype. If it is detected, the probability of diagnosing HSIL concerning the probability of diagnosing normal results increases 19-fold (OR = 19.10; CI (95%): 1.54–236.98; p = 0.022). A statistically significant but almost three-fold weaker predictor for the detection of HSIL compared to NILM is the patient belonging to the oldest age category (OR = 6.65; CI (95%): 1.66–26.60; p = 0.007), while female patients from the age category 31–44 years have a slightly lower statistically significant probability (OR = 5.38; CI (95%): 1.36–21.30; p = 0.016) for the detection of the same lesion. The age category was determined as the strongest statistically significant predictor by analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detecting ASCUS. HR HPV DNA-positive patients belonging to the oldest age category (≥45 years) have an 8.7-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with ASCUS compared to patients belonging to the younger age category (OR = 8.74; CI (95%): 2.15–35.57; p = 0.002). Female patients with the proven oncogenic activity of HPV 16 have a probability of detecting HSIL 6.4-fold higher compared to the probability of being diagnosed with ASCUS (OR = 6.38; CI (95%): 1.22–33.54; p = 0.029). By analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detection of LSIL, the strongest statistically significant predictor was determined to be the oncogenic activity of HPV 16 (OR = 5.10; CI (95%): 1.09–23.83; p = 0.038), while weaker statistically significant predictability is shown by the patient’s belonging to a specific age category. Female patients belonging to the oldest age category (≥45 years) have a 3.7-fold greater probability of detecting an HSIL change compared to the detection of LSIL compared to the younger age category (≤30 years) (OR = 3.72; CI (95 %): 1.16–11.92; p = 0.027) (Table 12). Persistent infection caused by HR HPV is the leading risk factor for developing cervical intraepithelial lesions and cervical cancer (reviewed in [20]). Many countries have introduced screening programs based on the cytomorphological examination of cervical samples using the PAP test during the last 60 years to reduce the morbidity and mortality caused by cervical cancer. However, this procedure has shown less than optimal sensitivity (50%) and high inter- and intra-individual variability [21,22]. HPV DNA detection and genotyping provide an efficient screening method and enable risk stratification. However, just proving the presence of the HPV genome in a cervical epithelium does not provide insight into the type of infection. It does not answer whether there is a transient or persistent infection in which the virus is actively multiplying and in which there is a high risk of cancer developing (reviewed in [23]). It was established that cervical carcinogenesis is strongly associated with the HPV-caused infection in which the transcription of E6 and E7 HR HPV oncogenes occurs, with the consequent increase of their mRNA and protein levels. For this reason, the detection of E6 and E7 mRNA of HR HPVs can serve as a promising biomarker of their persistence and oncogenic activity (reviewed in [23]), which could enable a better assessment of the progression to high-grade cervical intraepithelial lesions and, in this regard, significantly influence the algorithm for monitoring patients (reviewed in [18,23,24]). In our study, 365 female patient samples from Serbia were tested for the twelve HR HPV genotypes. It should be emphasized that the study has limitations. Used HPV tests are non-validated according to European Guidelines for use in primary screening (Meijer HPV test criteria) or according to the VALidation of HPV GENoyping Tests (VALGENT) protocols [25]. The commercial molecular HPV test was chosen for research according to Serbia’s general requirements for molecular diagnostics and the limited research expenses. From the total number of tested samples, 246 (67.4%) samples were positive for at least one of the 12 tested HR genotypes. These results agree with previous studies in the same area and European countries, where a high prevalence of HPV was registered. Studies in Serbia have reported that the presence of HR HPV infections range from 50% to 79% of women [26,27]. In the countries of our region, such as Croatia, a similar prevalence was shown (59%) [28] and in Bulgaria (61%) [29], while a slightly lower prevalence was registered in Italy (53%) [30]. Contrarily, a low prevalence of HPV infection was registered in Western and North European countries. Some of them are Great Britain (20.6%), Sweden (9.7%), and the Netherlands (3.8%) [7]. HPV genotype 16 (38%) emerged as the most frequently detected genotype in the study group, in concordance with previous reports [26,27]. The presence of HPV 16 is more often present (76%) in high-grade lesions compared to lower-grade lesions of the cervical epithelium. Studies on the HPV 16 genotype’s prevalence concerning cytological findings confirm these results (reviewed in [6]). The following frequencies also represented the Alpha-9 genotype, HPV 31 (17%) and HPV 33 (9%). A meta-analysis study on five continents shows that HPV 31 is especially frequent in Europe [31]. The results of this research indicate that the frequency of the HPV 31 genotype statistically significantly decreases with the progression of the intraepithelial lesion. The decreasing trend of the same genotype’s presence according to the degree of severity of the cytological lesion, more precisely, a lower prevalence in cancers compared to precancerous lesions, is observed in research from other studies [32,33,34]. Like HPV 31, a prevalence decrease of HPV 33 in HSIL compared to normal cytology was also registered in this research. Globally, HPV 33 ranks fourth in frequency and is responsible for 4.2% of all registered cervical cancers [6]. The surprising fact is a markedly high prevalence (9%) of the Alpha-5 genotype HPV 51 in our research. In the context of the causative agents of cervical cancer, HPV 51 is not included in the top ten most frequent HPV genotypes registered worldwide [6]. However, the detection of this genotype within this research, as well as previous studies from our region [26] and certain European countries [35,36,37,38,39,40,41], places it among the first four most prevalent genotypes detected in precancerous lesions of the cervix. Since HPV vaccines do not protect against all oncogenic HPVs, such as HPV 51, a complete understanding of its oncogenic activity is particularly significant [42]. The remaining tested genotypes (HR HPV 52, 56, 45, 18, 59, 58, 39, and 35) were present in a low percentage which is in concordance with previous reports [26]. Although HPV 18 is considered to be responsible for 15% of invasive cervical cancers, it is essential to note that its prevalence is similar to some studies from neighboring countries [29,43,44]; we found that in our area, HPV 18 was present at a lower percentage. Its frequency (3.6%) was 10-fold lower than that of HPV 16 (Figure 1). The HPV vaccine was introduced in over half of the WHO member countries in 2020 [5]. There is scientific data from numerous countries that have implemented HPV vaccines in their routine immunization programs on decreasing the burden of cervical HPV infections and precancers [45]. According to our data, using the nine-valent vaccine could prevent more than 80% of the cervical precancerous lesions identified in this study. The presence of HR HPVs determined further examination of their oncogenic potential. In our study, the expression of E6/E7 mRNA HR HPV was identified in 67% of the HPV-positive samples (Table 6). The percentage of expression of the E6 and E7 HPV-examined HR HPVs is proportional to the degree of severity of the cervical lesion (Table 7). It can be observed that approximately every tenth HPV-infected woman (11%) with normal cytological findings is infected with an oncogenically expressed HPV. At the same time, in HSIL, this relationship is the opposite. Namely, the absence of indicators of HPV oncogenic activity is detected in approximately every tenth woman with HSIL status (11.1%). An undoubted trend in E6/E7 mRNA HR HPV positivity with increasing cytology severity has been observed in the data of studies conducted in different regions of the world [46,47,48,49,50]. In agreement with the report of Argyri et al. (2013) and according to the mRNA test, E6/E7 expression was prevalent in 9.1% of women with normal cytology, similar to our study [51]. However, other previously published data indicated the prevalence of those transcripts in a lower proportion of women with normal cytology findings than our study (0%) [52]. In our study, HPV 16 constituted 29.7%, followed by genotype 31 (19.2%), genotype 33 (9.3%), and genotype 51 (8.7%). The results showed that approximately every second HPV 16 genotype is oncogenically expressed (48.6%). Our data agree with the results reported in other studies. Rossi et al. (2017) observed a positivity rate for E6/E7 mRNA HR HPV, ranging from 58% to 77% in HPV-DNA-positive women [50]. A study by Tüney et al. (2017) in Turkey registered 55.6% E6/E7 mRNA HR HPVs, where HPV genotype 16 constituted 57.8% [24]; similar to that, Bruno et al. (2018) found that the HPV 16 genotype was the most oncogenetically active [46]. As confirmed in this research, it is also stated by several other authors that the transcription product HPV 16 is most often detected, which indicates that the tendency of expression of the essential oncogenes of this genotype is significantly higher compared to the other examined genotypes [24,53,54], which gives it the status of the HPV with the most carcinogenic potential [55]. The oncogenic activity of HPV 31 was detected in approximately every fifth (19.2%) HPV-DNA-positive sample. Contrary to HPV 16, the opposite trend is observed with the degree of cervical lesion severity. It can be assumed that the negative transcriptional status of E6 and E7 oncogenes is more prevalent due to the presence of an episomal form of the virus or an established transcriptional control that enables the spontaneous elimination of infection. Within this research, the results of E6/E7 mRNA HPV 51 detection are approximately the same as those of HPV 33. The distribution of oncogenic activity of these two genotypes is approximately the same across cytological groups and remains at a low level (2.2–11.4%) (Table 7). Other studies have registered different percentage ranges of transcriptional detection of oncogenes of a particular genotype (HPV 33), from its absence [52] to complete expression of 100% [55]. The reason for those differences can be explained by the variations in their number in the total sample [55]. Furthermore, the total oncogenic activity increases with the degree of cervical lesion severity (60–100%). The oncogenic activity of individual genotypes (20–86%) is higher than that of multiple genotypes (0–40%) in the same type of lesion and increases with the degree of its severity (Table 8). These data are supported by the literature. Each of the detected genotypes is considered to have an independent mechanism of action in oncogenesis [56,57,58,59], which supports the hypothesis that different cells being infected with different viral genotypes rather than their intracellular coexistence is possible [60]. To analyze the oncogenic activity of the four most frequently diagnosed HR HPVs related to the age categories, a statistically significantly higher prevalence of positive E6/E7 mRNA HPV 16 findings was observed in the older age groups (Table 9). In agreement with this result, studies have shown that E6 and E7 mRNA HR HPV detection is significantly higher after 30 years [48,61]. The same result is supported by population-based cohort studies, which state that the majority of young women have an asymptomatic HPV infection which, thanks to the immune response, acquires transitory status [49,62]. The growing interest in molecular diagnostics methods has led various authors to compare the characteristics of the HPV DNA test with the mRNA test, evaluating the diagnostic accuracy in identifying high-grade cervical lesions. Our data showed that the specificity (89%) and PPV (70–79%) of the mRNA test are statistically significantly higher, while the same test has a statistically significantly lower sensitivity than the HPV DNA test. These results agree with the statements of different authors, who emphasized slightly lower values of the sensitivity of the mRNA test compared to the sensitivity of the HPV DNA test. At the same time, the specificity is expressed at a higher level [48,49] (reviewed in [63]). In contrast, others suggest that the sensitivity is similar and that its application would significantly improve the assay’s specificity characteristics [49]. Studies evaluating the reliability of the mRNA assay showed heterogeneous findings (reviewed in [32]). The meta-analysis results indicated that the obtained sensitivity value ranged from 41% to 95%, while the registered specificity was 42–97% (reviewed in [64]). The observed difference is explained by the heterogeneous participation of cervical pathology [64] and methodological quality in different studies, which highlights the limitations in the general interpretation of these test characteristics [32]. Although the results are presented broadly, they maintain a unique trend and suggest that the mRNA test over the HPV DNA test improves specificity [64]. By comparing the clinical characteristics of the test, it can be concluded that the detection of E6 and E7 mRNA HR HPV compared to HPV DNA represents a much better marker for more accurate screening of high-grade cellular atypia of the cervix (reviewed in [32]), which make it an appropriate tool for the secondary screening of cervical cancer [47,64]. In our study, the results of the ROC curve analysis indicated that the probability of detecting HPV infection with the mRNA test (81%, AUC = 0.812) in patients with high-grade lesions is higher than the possibility of diagnosing them with the HPV DNA test (74%, AUC = 0.740). The obtained results are in line with the other studies emphasizing the potential usefulness of this test. Sun et al. (2021) also found higher AUC values for the mRNA assay compared to the DNA HPV (0.929 vs. 0.833) [65], while according to Yao et al. (2017), the clinically relevant portion of the AUC of mRNA was 0.721 [66]. According to the previously established degree of influence on diagnosing HSIL, relevant factors were selected in our research. Factors that represent an increased risk for more severe cervical changes were gradually examined (the HPV 16 genotype, the total oncogenic activity of all of the tested HPV genotypes, the HPV 16 oncogenic activity, and the age category of the patient). Firstly, the HPV-DNA-16-positive results have a statistically significant predictive value for diagnosing HSIL (Table S1). In the same context, the study of Bruno et al. (2018) stated that the presence of the HPV 16 genotype was associated with a five-fold higher risk of developing a high-grade lesion compared to women with the presence of another HPV genotype [46]. Similarly, the data from a recent study indicate that type-specific HPV persistence predicted high-grade lesions, with HPV 16 being the most common type [67]. Secondly, the oncogenic activity of all of the tested HR HPVs has a statistically significant predictive value for diagnosing HSIL (Table S2). The obtained results support the statements related to the research on the importance of oncogenic activity, which show that the detection of E6 and E7 mRNA HR HPV could have a prognostic value in monitoring the development of carcinogenesis [68,69]. The results of the study by Fontecha et al. (2016) [55] showed that in the highest percentage of E6 and E7 mRNA HPV-positive women, the progression of the lesion is diagnosed over time (53%), followed by the persistence of abnormal cytological findings (42%), while regression was recorded in 10-fold lower cases (4%). Furthermore, in patients with positive results of indicators of the oncogenic activity of HPV 16, a statistically significantly higher probability of diagnosing a high-grade lesion was determined in all types of cytological groups (Table S3). According to the previous insights into the published statements, one of the few prospective follow-up studies that dealt with the predictive value of E6 and E7 mRNA HPV 16 indicated that through the detection of this biomarker, it is possible to identify 87.5% of the HPV infections that progressed. In this case, the risk of progression of negative cytology and low-grade cervical lesions was 10-fold higher than that detected in mRNA-negative women during a follow-up period of 35 months [70]. This is supported by the results of Johansson et al. (2015), which indicated that the absence of E6/E7 mRNA HPV demonstrated a high negative predictive value for the future development of high-grade lesions of the cervix among HR-HPV-DNA-positive women with ASCUS and LSIL [68]. Within the results of this research, a statistically significant final model with all of the predictors was constructed, which shows the strength of the potential of the examined factors, that is, biomarkers for diagnosing precancerous lesions in women with HR HPV infection. Looking at each cytological group individually, two independent variables made a statistically significant contribution to the model and were thus named as the strongest predictors. These are the oncogenic activities of HR HPV 16 and the age category (≥45 years). It is known that the presence of HPV infection is confirmed in all age categories. However, belonging to a particular age group is a determinant significantly associated with the risk of acquiring this infection, depicting the peak in prevalence, which generally takes place around 20–25 years of age. For lesions to progress to more severe forms of cervical disease, a period of 5–14 years is necessary, during which the infection persists and the process of oncogenesis takes place [71]; the results of this research confirm the stated findings. In the examined women, the age category (≥45 years) is a statistically significant prognostic factor for diagnosing HSIL in all of the cytological groups (Table 12 and Table S4). Loopik et al. (2020) [58] indicated that the risk of progression of an existing high-grade lesion increases with age, i.e., in women over 50, the risk of developing cervical cancer increases by seven fold. In addition, the risk of developing cervical abnormalities and the need to use an mRNA test in the diagnostic protocol of HPV-DNA-positive postmenopausal women with normal cytology is emphasized by Asciutto et al. (2020) [72]. In summary, this study describes the detection rates of the most common HR HPVs (16, 31, 33, and 51) and E6/E7 mRNA HR HPV expression in 365 Serbian women who showed normal and abnormal cytological findings. Those HR HPV genotypes are oncogenically active in more than half of the examined cases, and the detected oncogenic activity has predictive potential in diagnosing high-grade cervical intraepithelial lesions. According to the constructed predictive model, the oncogenic activity of HPV 16 and age are risk factors with the strongest predictive values for developing those lesions. Thus, our data indicate that mRNA testing may be more relevant than HPV DNA for assessing lesion grade and diagnosing and monitoring women at risk of progressive cervical disease. This way, the mRNA test as a tool for better risk stratification of HPV infection could overcome unnecessary examinations, increased costs, and patient anxiety. However, further follow-up studies are needed to determine the clinical utility of the mRNA HR HPV test.

PMC10000481

Kristin E. Zorn,Ashley M. Cunningham,Alison E. Meyer,Karen Sue Carlson,Sridhar Rao

Pediatric Myeloid Sarcoma, More than Just a Chloroma: A Review of Clinical Presentations, Significance, and Biology

24-02-2023

myeloid sarcoma,chloroma,acute myeloid leukemia,pediatric

Simple Summary Childhood acute myeloid leukemia (AML) remains a cancer with poor overall outcomes. Myeloid sarcomas (MS) are extramedullary masses of leukemia cells that can develop in patients with AML. In children, MS occurs more frequently than described in adults. Their clinical significance in both pediatric and adult patients with AML is unclear. In this review, we aim to summarize the current knowledge of MS in children and its underlying biology in the hopes of sparking future studies and ultimately improving treatment options for children with AML. Abstract Myeloid sarcomas (MS), commonly referred to as chloromas, are extramedullary tumors of acute myeloid leukemia (AML) with varying incidence and influence on outcomes. Pediatric MS has both a higher incidence and unique clinical presentation, cytogenetic profile, and set of risk factors compared to adult patients. Optimal treatment remains undefined, yet allogeneic hematopoietic stem cell transplantation (allo-HSCT) and epigenetic reprogramming in children are potential therapies. Importantly, the biology of MS development is poorly understood; however, cell-cell interactions, epigenetic dysregulation, cytokine signaling, and angiogenesis all appear to play key roles. This review describes pediatric-specific MS literature and the current state of knowledge about the biological determinants that drive MS development. While the significance of MS remains controversial, the pediatric experience provides an opportunity to investigate mechanisms of disease development to improve patient outcomes. This brings the hope of better understanding MS as a distinct disease entity deserving directed therapeutic approaches.

Pediatric Myeloid Sarcoma, More than Just a Chloroma: A Review of Clinical Presentations, Significance, and Biology Childhood acute myeloid leukemia (AML) remains a cancer with poor overall outcomes. Myeloid sarcomas (MS) are extramedullary masses of leukemia cells that can develop in patients with AML. In children, MS occurs more frequently than described in adults. Their clinical significance in both pediatric and adult patients with AML is unclear. In this review, we aim to summarize the current knowledge of MS in children and its underlying biology in the hopes of sparking future studies and ultimately improving treatment options for children with AML. Myeloid sarcomas (MS), commonly referred to as chloromas, are extramedullary tumors of acute myeloid leukemia (AML) with varying incidence and influence on outcomes. Pediatric MS has both a higher incidence and unique clinical presentation, cytogenetic profile, and set of risk factors compared to adult patients. Optimal treatment remains undefined, yet allogeneic hematopoietic stem cell transplantation (allo-HSCT) and epigenetic reprogramming in children are potential therapies. Importantly, the biology of MS development is poorly understood; however, cell-cell interactions, epigenetic dysregulation, cytokine signaling, and angiogenesis all appear to play key roles. This review describes pediatric-specific MS literature and the current state of knowledge about the biological determinants that drive MS development. While the significance of MS remains controversial, the pediatric experience provides an opportunity to investigate mechanisms of disease development to improve patient outcomes. This brings the hope of better understanding MS as a distinct disease entity deserving directed therapeutic approaches. Myeloid sarcomas (MS) are extramedullary tumors of myeloid blasts forming masses disrupting normal tissue architecture in patients with acute myeloid leukemia (AML) [1,2,3]. They are also known as myeloblastomas, granulocytic sarcomas, chloroleukemia, and chloromas given the historically green appearance of the tumors resulting from myeloperoxidase exposure to air. Importantly, there is no clearly accepted definition of what qualifies as MS. Most agree that discrete tumor masses of myeloid blasts are MS; however, whether gingival infiltration and masses within lymph nodes, the liver, or spleen should also be considered MS is debated given their propensity for generalized infiltration. Central nervous system (CNS) leukemia is also a challenge with categorization including both cerebral spinal fluid (CSF)-positive disease and CNS infiltrates/masses on imaging. This has made clear, consistent reporting of clinical presentation and outcome data difficult given the lack of consensus within the literature. Given this limitation, the following terms will be used for this review: extramedullary disease and MS. Extramedullary disease will more broadly refer to leukemic disease outside the bone marrow/peripheral blood, while MS will be specific to myeloid blast tumors. These terms are not used interchangeably and are used as defined to more accurately portray the referenced literature, with extramedullary disease as an umbrella term that also includes MS. MS most frequently presents with a mass in subcutaneous/soft tissue, bone, and skin (also known as leukemia cutis). Case reports include masses and infiltrative involvement in nearly every conceivable tissue including the GI tract, reproductive organs, CNS, heart, lungs, kidneys, and breast [4]. Interestingly, MS, while most often seen concurrently with intramedullary AML, can occur in isolation in the absence of bone marrow disease. MS can also occur in the setting of a preceding hematologic disease such as myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN). Finally, MS can develop as a relapse following a hematopoietic malignancy, including after allogeneic hematopoietic stem cell transplantation (allo-HSCT) [1,2]. Although AML is seen primarily in older adults with a median age at diagnosis of 68 years, AML accounts for 10 to 15% of acute leukemias in children [5,6]. Pediatric AML differs from AML in adults in terms of clinical course, outcomes, and genomic landscape [7,8,9]. MS in pediatrics represent an inadequately understood aspect of AML. MS presentations offer another distinction between pediatric and adult AML with opportunities for improvement in diagnosis, management, and further investigation into the biological mechanisms of development and treatment resistance. This review will discuss the clinical presentations and reported outcomes of pediatric patients with MS including post-allo-HSCT, imaging approaches to diagnosis, and finally, the biology of MS will be addressed. While this review focuses on pediatric MS, important comparisons with adults will also be discussed. Although generally considered a rare presentation, MS and extramedullary AML are common in children with AML. Numerous cooperative groups and large institution studies have reported both characteristics and outcomes associated with extramedullary disease (Table 1). The inconsistent terminology surrounding MS and extramedullary disease prevents direct comparison across these studies. Despite this limitation, these studies provide helpful data about the clinical features and associations seen as well as insight into outcomes. The incidence of MS varies widely, particularly when comparing adults and children. This is predominantly related to the lack of consistency in MS evaluation and reporting. There is no standard recommendation for patients with AML to undergo screening evaluation for MS and the true incidence is likely higher than that reported given the potential for asymptomatic occult tumors. Pediatric studies describe an incidence of MS ranging from 5.7% to as high as 40% with the expansive definition of extramedullary disease, although most commonly it is between 10 and 25% [10,13]. The distribution of common anatomic sites in children is illustrated in Figure 1. A lower incidence of MS is generally reported in adults (4–9%) with newly diagnosed AML, although this is likely an underestimate considering a recent prospective study [24]. It is unclear why such differences exist between children and adults, but this may be related to differences in diagnostic evaluations performed or inherent differences in the leukemias that children develop compared to adults in terms of mutational spectrum [7,8,9]. In children, MS is typically associated with a younger age at diagnosis, particularly in infants, and more frequently in males, with 55–75% of patients with MS being male [19,21,22]. Higher WBC counts and hepatosplenomegaly are often seen in the presence of MS, although less consistently. Additionally, the FAB M4 (acute myelomonocytic) and M5 (acute monocytic) subtypes are most commonly associated with MS. Cytogenetically, the most frequently described associations are inv(16), t(8;21), and chromosome 11 abnormalities, namely 11q23, with both deletions and rearrangements [12,15,17,18,19,26]. By contrast, in adults, a recent study of 1,583 patients identified increased odds of extramedullary disease in patients with PTPN11, NPM1, and FLT3-ITD mutations with no association with inv(16) or t(8;21) and decreased odds of extramedullary disease with IDH2 and CEBPA mutations [27]. Pediatric AML has distinct mutational profiles in comparison to adult AML, and similarly, the AML genetic profile associated with pediatric MS appears distinct from that of adults. This suggests different biologic drivers of pediatric AML and MS or extramedullary disease development, and potential significance for treatment and outcomes. There is no consensus on the influence of MS on prognosis in children with AML. Table 1 summarizes many of the larger studies, with notably conflicting results. Although typically considered a presentation of advanced disease, event free survival (EFS) and relapse free survival (RFS) do not consistently demonstrate worse outcomes for MS. Reports from Children’s Oncology Group (COG) demonstrate an improved EFS in subsets of MS patients with non-skin MS, later defined as orbital MS and CNS MS, with otherwise similar outcomes to non-MS patients in those with other sites of MS disease including skin [12,15]. A single center report from India similarly describes improved EFS and overall survival (OS) in patients with MS excluding CSF-only disease [18]. By contrast, other studies describe no significant association between MS or more general extramedullary disease and EFS [11,16,17]. A Turkish single center study found significant effects on outcomes for patients with MS only when less intensive treatment was given [13]. The Japanese childhood AML cooperative study group also only found inferior EFS with extramedullary disease in the setting of WBC count >100 × 109/L [14]. By contrast, other reports describe significantly worse EFS and OS for children with AML and MS compared to those without MS [10,19,25]. Collectively, this indicates that whether MS is a critical driver of outcomes remains an important unanswered question within the field. Favorable cytogenetics, including core binding factor mutations, are common in patients with MS. In evaluation of children with low risk AML (e.g., inv(16), t(8;21), NPM1 mutated without FLT3-ITD mutation, and CEBPA mutation), reports demonstrated worse RFS and EFS in patients with MS present compared to those without MS present [20,21,22]. This suggests that even in otherwise favorable AML, the presence of MS could be relevant for both prognosis and potentially risk stratification. Although controversy remains in broadly assigning prognostic impact to the presence or absence of MS in pediatric leukemia, the significance should not be simply ignored. Particularly in patients with t(8;21), inv(16), or chromosome 11/KMT2A abnormalities, evaluating for the presence of MS may be significant in considering therapeutic approaches. Additional studies are needed to prospectively identify patients with MS, as defined by clear criteria, to determine the impact on prognosis, the potential need for altering risk stratification, and defining remission status. This will be important to identify which patients may benefit from specific or intensified therapy regimens to improve outcomes. Relapse of AML remains the predominant cause of treatment failure and death with allo-HSCT as the only curative option for many patients. The role of allo-HSCT in the setting of MS is a moving target in children. However, new data are emerging to address this important point because anecdotal evidence suggests isolated MS relapse, in the absence of bone marrow relapse, is a common occurrence post-allo-HSCT. The Japan Society for Hematopoietic Cell Transplantation (JSHCT) used their national database to identify pediatric AML patients that underwent allo-HSCT and found that the presence of extramedullary disease (both CNS disease and MS) had no impact on OS or leukemia-free survival (LFS) after transplant. However, the patients with extramedullary disease prior to transplant were more likely to have extramedullary relapses after transplant, with 41% of relapses being extramedullary. In comparison, those without prior extramedullary disease had only 6% extramedullary relapse, although the overall rates of recurrence were the same between the two groups [28]. Relapse with isolated MS was not separated out as a group and was therefore difficult to directly assess. The Turkish Pediatric Bone Marrow Transplantation Registry recently reported on their experience with isolated extramedullary relapse (iEMR) in children following allo-HSCT, although they included both acute lymphoblastic leukemia (ALL) and AML. They found different risk factors for medullary relapse post-allo-HSCT versus iEMR. Transplant in CR2 or later or active disease at time of transplant and matched sibling donor transplants were independently associated with increased risk of medullary relapse as well as iEMR. The presence of chronic graft versus host disease (cGVHD) was conversely associated with decreased risk of medullary relapse with no impact on the risk of iEMR. iEMR rates were, however, independently higher in those with prior extramedullary disease [29]. A higher rate of second iEMR was also seen following a first iEMR at 58.8% versus after a first medullary relapse at 13% [29]. Local radiotherapy of extramedullary disease sites prior to transplantation and the presence of cGVHD had no impact on post-allo-HSCT iEMR, while cGVHD was protective in preventing medullary relapse [29]. This suggests that although a graft versus leukemia effect is helpful in preventing medullary relapse, this immune-mediated mechanism is not effective against MS masses and extramedullary sites of leukemia. A single site report from the University of Michigan found that children were also three times more likely than adults to experience an extramedullary relapse with an associated higher pretransplant extramedullary disease incidence [30]. The significance of extramedullary relapse, particularly in these settings, provides insight into mechanisms of disease resistance specific to the MS phenotype following allo-HSCT. Despite these concerns, allo-HSCT remains the best disease management for patients with high-risk AML. Extramedullary disease prior to transplant is consistently associated with increased risk of extramedullary relapse after HSCT in both children and adults [30,31]. The presence of prior extramedullary disease (including CNS disease and MS) in adults with AML was not found to be an independent risk factor for post-allo-HSCT relapse, DFS, or OS in both a large CIBMTR analysis and a Canadian report [32,33]. This confirms the anecdotal clinical concern that allo-HSCT is more effective for medullary versus extramedullary disease. Adults with iEMR post-allo-HSCT are more likely to have had prior extramedullary disease and GVHD present compared to those with medullary relapse [30,31,34,35]. Additionally, extramedullary relapse has a higher incidence following allo-HSCT than intensive chemotherapy alone [36]. These iEMRs may represent sanctuary sites in which immune-based therapies may be less effective and may result from a different mechanism of pathogenesis compared to medullary relapse. There is currently no treatment consensus for iEMR post-allo-HSCT, with a range of treatment approaches taken including local radiotherapy and systemic chemotherapy [37,38,39,40]. The significance of iEMR on outcomes compared to medullary relapse is more controversial, with conflicting studies limited by inclusion of both ALL and AML patients with known differences in the efficacy of graft versus leukemia effect between the two diseases [31,34]. One retrospective adult study, however, did report that allo-HSCT was an effective treatment for patients with MS compared to chemotherapy alone, although lack of complete MS remission prior to transplant had independently worse OS and PFS [41]. In summary, the presence of known or occult MS prior to allo-HSCT may be clinically important for a subset of patients, although additional studies are needed to define which groups may benefit. Furthermore, identifying the increased risk for iEMR following allo-HSCT can inform evaluations and management of patients during their post-transplant course. This emphasizes the importance of identifying and following MS in patients with AML prior to allo-HSCT and remaining vigilant to the possibility of iEMR. The use of imaging to identify occult MS as well as re-evaluation of disease presence has remained inconsistent, both in frequency and modality, particularly in children. Ultrasounds of MS lesions typically show homogenously hypoechoic lesions with hypervascularity [42]. Computed tomography (CT) scans identify MS as isodense lesions compared to muscle with moderate enhancement with IV contrast media. Enhancement is more commonly homogenous (65%) versus inhomogenous (35%) [43]. MRI scans demonstrate predominantly T2 hyperintense (82%) or isointense (18%) lesions compared to muscle and T1 isointense (61%) or hypointense (39%) lesions with homogenous contrast enhancement and a mean apparent diffusion coefficient (ADC) on diffusion weighted imaging (DWI) of 0.57 × 10−3 mm2/s [43]. Fluorodeoxyglucose (FDG)-positron emission tomography (PET) scans are increasingly being used for diagnosing extramedullary disease, with MS lesions displaying moderate uptake of FDG [44]. A retrospective study including pediatric patients showed a sensitivity of 93% and a specificity of 71.4% limited by difficulty differentiating extramedullary leukemia disease from infectious/inflammatory entities [45]. The recent PETAML trial however prospectively evaluated adult patients with AML prior to therapy initiation with total body 18FDG PET/CT scans to determine prevalence of extramedullary disease. This showed a prevalence of 22% with a sensitivity of 77% and specificity of 97% [24]. Interestingly, leukemia cutis and CNS meningeal involvement were not necessarily 18FDG-PET-avid [24,45]. In addition, there were four patients who remained with residual 18FDG-PET-positive lesions despite complete marrow remission. Three of those four subsequently relapsed, suggesting there may be a specific role for 18FDG-PET imaging for remission evaluation of patients with AML [24]. Consideration should be given to prospectively evaluating patients with AML to identify MS lesions requiring focused follow-up and possible treatment modifications and may be of value in designing de novo AML clinical trials, particularly in pediatrics with a high incidence of MS. MS are infiltrative tumor masses of myeloid blasts that efface or disrupt the normal architecture of the involved organ. The leukemic blasts found in MS have heterogeneous morphology; however, monocytic differentiation is common where the blasts will show either myelomonocytic or monoblastic morphology [46]. An example of the histology of MS is shown in Figure 2. Immunophenotypic profiling by flow cytometry or immunohistochemical stains is often necessary for a definitive diagnosis, as many of these tumor masses may resemble carcinoma. Evaluation of markers of immaturity, including CD34 and CD117, are helpful in addition to other markers which are variably expressed on myeloid blasts including CD13, CD33, CD68 (KP1), CD45, and myeloperoxidase (MPO). In the setting of monocytic differentiation, expression of monocytic markers such as CD68, CD163, CD14, and/or non-specific esterase (NSE) can be seen. Several translational studies have begun to investigate the genetic landscape of MS lesions in isolation as well as in comparison to their paired intramedullary leukemia counterparts. One recent study evaluated 7 adult trios of AML, MS, and normal tissue using capture-based next generation sequencing (NGS) of 479 cancer genes. Genes recurrently altered in these patients included KMT2A, FLT3, NRAS, CEBPA, TP53, WT1, and NPM1, with 84% of variants found in the AML also present in the MS [47]. Three of the seven patients had additional variants detected in the MS compared to the AML including additional FLT3, SETD2, and NF1 mutations in the MS, while two had additional variants of U2AF1 and RAD21 in the AML but not the MS [47]. In the relapsed MS samples, there were increased single nucleotide variants (SNV) in the MS [47]. Another study evaluated 6 isolated MS tumors (without concurrent AML) and performed a 21 gene targeted panel of AML and MDS associated genes. They found recurrent variants in the genes for FLT3 (50%), NPM1 (33%), and KIT (67%) and additional variants in WT1, SF3B1, EZH2, ASXL1, and TET2 in one MS each [48]. The genomic reports of patient-derived MS are all limited by targeted NGS sequencing without exploration of novel gene variants that may be specific to MS pathogenesis. Additionally, RNA transcriptome analysis of MS is lacking in the literature and provides an opportunity for investigation of transcriptome-based changes that may contribute to MS development outside of genetic mutations. Such studies may also facilitate the identification of cryptic translocations, which are common in pediatric AML [7]. Although, typically, the genomic profile of MS is in concordance with the AML and marrow, this is not always true. Particularly in cases of isolated MS, NGS and molecular evaluation may inform targeted treatment options and should be included in diagnostic evaluation of patients. The biology underlying development of MS remains poorly defined with no clear molecular determinants. Biological features such as cytogenetic changes, molecular abnormalities, and cell surface marker expression are not consistent across studies. Much of the work on MS development surrounds the invasiveness of AML as studied using in vitro transwell assays and infiltration in the spleen and liver. The simple infiltration of hematopoietic organs, however, appears distinct from MS development in non-hematopoietic sites with no clear biological explanation. The development of MS appears to require leukemia mobilization/release from the marrow environment, tissue invasion, and further changes leading to a tumor/mass phenotype, as illustrated in Figure 3. These steps will be further discussed below. CXCR4 (CXC chemokine receptor 4, CD184) is the receptor for the chemokine matrix cell derivative-1 (SDF-1/CXCL12) and is expressed by most tissues as well as hematopoietic stem cells and leukemic blasts wherein it facilitates the retention of hematopoietic stem cells in the bone marrow niche [49,50]. The CXCR4/SDF-1 axis may contribute to chemoresistance through downstream signaling cascade dysregulation within leukemia cells [49,51]. Higher CXCR4 expression has been seen in AML patients with extramedullary infiltration at diagnosis and extramedullary infiltration in childhood ALL [49,52]. The proposed mechanism of extramedullary involvement in acute leukemias is altered bone marrow homing and increased peripheral blood dissemination via a chemotactic gradient of SDF-1 with increased CXCR4 expression on the leukemia cells [53]. CXCR4/SDF-1 can promote the retention of AML cells within the skin of children with AML; however, CXCR4 expression by peripheral blood blasts was no different in patients with or without skin involvement [54]. Furthermore, a lack of association between SDF-1 polymorphisms and MS implies that small variants do not contribute to extramedullary disease development, although these have been previously of interest and described [55]. More common in adults, NPM1-mutated AML is associated with extramedullary disease and is associated with downregulation of CXCL12 and CXCR4 gene pathways [50]. While CXCR4 expression and signaling may be a contributing factor to extramedullary disease, its impact appears limited to initial release and migration of leukemia cells from the marrow and is not specific to MS development. Further work is needed to better characterize this mechanism and whether or how CXCR4 is contributing to discrete MS formation. CD56 (also known as neural cell adhesion molecule-1 or NCAM1) is normally expressed by natural killer (NK) cells and other immune cell subtypes and is housed on chromosome 11q23.1. It is frequently described as part of the immunophenotype of AML with MS [1,56,57]. Expression patterns of CD56 are not consistently described, however, and in a population of adult t(8;21) AML patients, there was no association between CD56 expression and presence of extramedullary disease [58]. AML in adults with CD56 positivity is more commonly associated with worse 5-year EFS and OS; however, a report in low-risk patients shows no association with outcome [21,25,59]. Additionally, post-allo-HSCT CD56 positivity is not associated with extramedullary relapse [30]. Despite the frequent CD56+ immunophenotype, there is no described mechanism or in vitro data to suggest the significance of this finding. Additional experimental studies are required to determine if CD56 is simply a biomarker of MS or is required for MS development. An AML-extracellular matrix interaction is likely critical to the development of MS. This is illustrated in transcriptome analysis of adult patient-derived AMLs demonstrating enrichment of cell surface gene sets in those AMLs with concomitant MS [60]. This includes integrin-α7 (ITGA7), which showed a higher expression in AML with associated MS in addition to high expression in MS samples [60]. Laminin 211 is a specific ligand of integrin-α7 that signals through the ERK signaling cascade [60]. While there is much described about the role of integrins and selectins in migration and homing of hematopoietic stem cells, there remains no clear mechanism by which these molecules facilitate MS formation [61,62]. Further study is needed to better evaluate the role of cell adhesion molecules in MS development and whether targeting these cell interactions may provide therapeutic benefit for patients with MS. Angiogenesis plays a notable role in acute leukemia with increased microvascular density in AML and adult MS [63]. VEGFR2, the major mediator of the mitogenic, angiogenic, and permeability effects of VEGF, may contribute to the development of MS [64]. VEGF signaling via the PI3K/Akt pathway in the setting of hERG1 expression was necessary for an in vitro migratory phenotype in AML cells [65]. In adults, the small molecule VEGFR2 tyrosine kinase inhibitor apatinib (also known as TN968D1) demonstrated enhanced antileukemic effects in ex vivo cytotoxicity studies from patient-derived AML samples with associated extramedullary disease [66]. Angiogenesis is well-described in the pathogenesis of other malignancies and it is reasonable to think that a unique perturbation may play a role in the migration or tumor formation of MS. In vitro studies have described the role of MMP secretion (MMP-2 and MMP-9) by leukemia cells contributing to invasion capacity, most notably of the blood-brain barrier, with upstream regulation by mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3-K)/AKT pathways [67,68]. Additionally, TIMP-2 (tissue inhibitor of metalloproteinase 2) upregulation has been seen with increased leukemia cell line (i.e., SHI-1) invasion both in vitro and in vivo with more extensive and severe extramedullary infiltration through both MMP-2-dependent and independent activities [69,70]. Other in vitro studies propose a role for the β2 integrin-proMMP-9 complex in the extramedullary phenotype of AML [71]. Type IV collagenase secretion enhanced by TNFα and TGFβ from a patient-derived MS cell line increased in vitro cell invasion with collagenase secretion demonstrated in the MS AML cell line but not other leukemia cell lines [72]. While tissue invasion by leukemia cells is likely required for MS development, not all of the critical players have been identified. Epigenetic dysregulation has been reported in the context of extramedullary disease and infiltration in AML. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), which deposits Histone 3 Lysine 27 trimethylation (H3K27me3). High EZH2 expression is correlated with higher peripheral blood blast percentages as well as extramedullary infiltration in patients with AML with numerous well-established biological roles. In vitro studies suggest that migration of AML cells appears to be regulated by EZH2/p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways [73]. EZH2 is a frequently mutated gene in AML; however, EZH2 has a variety of biologic influences and a unique role in MS formation remains undefined [7,9]. Altered DNA methylation is another described mechanism in the development of extramedullary disease with key enzymes frequently mutated in AML. DNA methyltransferase 3A (DNMT3A) mutations contribute to altered DNA methylation, subsequently resulting in increased expression of a subset of genes with specific roles in myeloproliferation and extramedullary hematopoiesis [74]. DNMT3A mutation appears to contribute to extramedullary CNS infiltration mediated by overexpression of TWIST1, a key epithelial mesenchymal transition transcription factor, which is not otherwise well described in AML [75]. Furthermore, TET2 is a member of the ten-eleven translocation (TET) gene family and is a key enzyme for DNA demethylation and a critical regulator for hematopoietic stem cell homeostasis. Models using TET2-deficient mice demonstrated not only high incidence of MS development but also transplant ability of the MS cells as well as an in vivo response to azacitidine treatment [76]. Decreased TET2 expression was also seen in patient-derived MS samples with further suggestion of methylation changes impacting MS development [77]. AML has many examples of mutations in epigenetic pathways that are enriched in AML more than many other disease entities and may not be directly related to their involvement in MS [9]. The role of epigenetic dysregulation in leukemia migration and invasion with described MS phenotypes is intriguing yet requires further study. Additional research may uncover future targetable pathways for MS treatment, and as noted below, case reports have demonstrated the safety and efficacy of hypomethylating agent use for patients with MS. Mesothelin (MSLN) is a cell surface protein hypothesized to be involved in cell adhesion and is overexpressed in a subset of AML patients. MSLN overexpression was strongly associated with KMT2A-R, t(8;21), and inv(16) as well as the presence of extramedullary disease in children and young adults with AML. Methylation profiling further demonstrated an inverse association between MSLN promoter methylation and MSLN expression, suggesting another impact of epigenetic dysregulation [78]. Versican (VCAN) overexpression in the setting of NPM1-mutated AML is associated with an invasive phenotype and higher expression levels in patients with skin infiltration [79]. Lysyl oxidase (LOX), which has roles in pediatric acute megakaryoblastic leukemia and in the creation of a growth permissive fibrotic microenvironment, was associated with increased extramedullary disease in adults with AML and high plasma LOX activity [80]. WT1 overexpression has also been described in MS cases as well as in extramedullary relapsed disease [81,82]. ERG transcription factor overexpression, similar to that of Ewing sarcoma, has been seen in patient-derived MS samples [83]. Multiple studies describe other associations observed in AML and extramedullary disease, including increased expression of amyloid precursor protein (APP) in AML1/ETO leukemia cells perhaps mediating the p-ERK/c-Myc/MMP-2 pathway, expression of miR-29c&b2, circular RNA expression patterns, and expression of CD25 and CD117 [84,85,86,87,88,89,90]. Polo-like kinase 1 (PLK1), which is involved in cell cycle control, was effectively inhibited in vivo using a patient-derived leukemia in mice with improvement in extramedullary disease [91]. PD-1 and PD-L1 have been investigated given the described efficacy of checkpoint inhibitors; however, there was no difference in expression of PD-1/PD-L1 in MS tested, and they may instead have more impact in the surrounding tumor microenvironment [92,93]. Using mouse models, others observe a maturation plasticity of leukemia cells, with potential implications for chemotherapy resistance as a mechanism for extramedullary relapse [94]. A PIM2/MYC co-expressed mouse model demonstrated consistent and lethal in vivo MS development with MYC expression likely contributing to the phenotype [95]. Mouse models have also demonstrated cooperation between MLL/AF10 and activating KRAS mutations, with increased cell adhesion properties contributing to in vivo MS formation via Adgra3 and Hoxa11 [96,97]. While many different mechanisms have been suggested in the development of MS, there remains no clear understanding of the pathogenesis. As such, it is hard to definitively identify the potential molecular determinants causing MS formation is some AML patients but not others. While the pathogenesis remains to be fully elucidated, prior studies suggest that there are likely multiple steps leading to MS development, including release from the bone marrow (which may be represented by higher WBC counts associated with MS), tissue invasion, and discrete mass formation, with the latter being the most consequential with regards to leukemia and the least described. Investigating how these different steps may cooperate and ultimately how the leukemia cells aggregate and sustain an aggregated phenotype requires dedicated study. Furthermore, the immune evasive or immunosuppressive microenvironment of MS illustrated in the post-allo-HSCT setting highlights that there is much more to learn about the pathogenesis of MS and its uniqueness with respect to its systemic/intramedullary AML counterpart. There is no consensus on the best treatment approach for management of MS, particularly isolated extramedullary disease. In pediatrics, systemic chemotherapy has been favored with consideration of allo-HSCT independent of the presence of MS. The general approach to management in pediatric patients has evolved over the last three decades. In prior large cooperative group study treatment protocols (e.g., CCG 2961), children with MS would receive radiation therapy to the affected sites following initial induction chemotherapy, given MS responsiveness to irradiation. Although part of protocol therapy, many patients were not irradiated and outcomes demonstrated no difference in 5-year EFS, similar to smaller cohorts [12,98]. In adults, radiation therapy is more commonly used to treat isolated relapses, but the effects are not typically sustained and both localized and medullary relapse following radiotherapy are common [30,99]. Although prior COG studies included radiotherapy for treatment of MS sarcomas, this is no longer standard of care in the US; however, it continues to be recommended for MS in Berlin–Frankfurt–Munster (BFM) studies [17,100]. Given the epigenetic basis of AML development, inclusion of novel epigenetic approaches in treatment are increasing in utility for AML [9,101,102]. The hypomethylating agents azacitidine and decitabine have demonstrated efficacy in management of extramedullary disease in AML, including in pediatric patients [103]. Case reports in pediatrics have demonstrated complete response to monotherapy with azacitidine in MDS patients who received allo-HSCT as consolidation [103]. In the setting of post-allo-HSCT relapses of MS, multiple case reports in adults demonstrate efficacy of azacitidine or decitabine including complete response [104,105]. Hypomethylating agents in adults with AML and extramedullary disease showed improvement after one–two cycles and complete or near complete resolution of MS following four–five cycles [106,107,108,109]. Venetoclax has also shown activity against MS [109,110,111]. The utility of venetoclax and hypomethylating agents suggests a role for epigenetic reprogramming as a means for MS treatment, although DNA methylation-based mutations including DNMT3A, IDH1, IDH2, and TET2 are far less common in children than in adults and translation of utility is more challenging [9,112]. While no consensus exists regarding optimal treatment of MS, particularly in pediatrics, radiation therapy is unlikely to contribute to durable remission of disease and expanding chemotherapeutic options to include hypomethylating agents and venetoclax in both initial chemotherapy regimens or as maintenance therapy following allo-HSCT should be considered and deserves further investigation. Children with AML and MS are distinct from adults. Given these differences, it is necessary to further study MS in the context of the driver lesions specific to children. MS remains a known clinical presentation with unclear impact on prognosis, risk stratification, and potential consequences in the setting of allo-HSCT. The advancement of imaging techniques and data for MS provides the opportunity for more directed and prospective evaluation in children. Collectively, this highlights the need for further large-scale cooperative group studies with clear criteria for the identification of MS in children. While there is no specific treatment approach for children with MS, the use of intensive systemic chemotherapy remains at the forefront. However, additional studies are required to determine if epigenetic or immuno-oncology therapies may be beneficial. The role of allo-HSCT continues to be important as a curative option for many patients with high-risk AML; although, considering its potential lack of efficacy in the setting of extramedullary disease, the role of an immunosuppressive microenvironment in MS requires additional study. Further investigation into the potentially immunosuppressive MS microenvironment will be crucial to improving efficacy of allo-HSCT and managing isolated extramedullary relapses post-HSCT. Although many different biological associations exist, there is an ongoing lack of clarity as to how leukemic blasts can not just invade tissues but form discrete tumors. Experiments delineating the potential epigenetic and transcriptomic differences between medullary AML disease and MS are required to identify the underlying molecular mechanisms that promote MS development. Understanding how leukemic blasts transform into and sustain an MS phenotype is critical to identifying specific targetable mechanisms. Understanding and combatting chemotherapy resistance and immune escape will ultimately improve survival in patients with AML and MS.

PMC10000483

MHD Ouis Al Khatib,Giulia Pinton,Laura Moro,Chiara Porta

Benefits and Challenges of Inhibiting EZH2 in Malignant Pleural Mesothelioma

28-02-2023

EZH2,malignant pleural mesothelioma,epigenetic,tumor microenvironment,macrophages,immune infiltrate,immunotherapy,targeted therapy

Simple Summary Malignant pleural mesothelioma (MPM) is an aggressive cancer linked to asbestos exposure with an extremely poor outcome. Despite the recent approval of immune checkpoint blockade-based therapies, MPM still remains a fatal cancer that challenges physicians and scientists. Enhancer of zeste homolog 2 (EZH2) has emerged as a promising therapeutic target. In addition to being an oncogenic driver, EZH2-dependent epigenetic reprogramming modulates tumor-immune infiltrate. Therefore, we argue that a better understanding of the molecular mechanisms that sensitize cancer cells to EZH2 inhibition and modulate tumor microenvironment will likely provide important insights for new treatment options for MPM. Abstract Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer that is mainly associated with prior exposure to asbestos fibers. Despite being a rare cancer, its global rate is increasing and the prognosis remains extremely poor. Over the last two decades, despite the constant research of new therapeutic options, the combination chemotherapy with cisplatin and pemetrexed has remained the only first-line therapy for MPM. The recent approval of immune checkpoint blockade (ICB)-based immunotherapy has opened new promising avenues of research. However, MPM is still a fatal cancer with no effective treatments. Enhancer of zeste homolog 2 (EZH2) is a histone methyl transferase that exerts pro-oncogenic and immunomodulatory activities in a variety of tumors. Accordingly, a growing number of studies indicate that EZH2 is also an oncogenic driver in MPM, but its effects on tumor microenvironments are still largely unexplored. This review describes the state-of-the-art of EZH2 in MPM biology and discusses its potential use both as a diagnostic and therapeutic target. We highlight current gaps of knowledge, the filling of which will likely favor the entry of EZH2 inhibitors within the treatment options for MPM patients.

Benefits and Challenges of Inhibiting EZH2 in Malignant Pleural Mesothelioma Malignant pleural mesothelioma (MPM) is an aggressive cancer linked to asbestos exposure with an extremely poor outcome. Despite the recent approval of immune checkpoint blockade-based therapies, MPM still remains a fatal cancer that challenges physicians and scientists. Enhancer of zeste homolog 2 (EZH2) has emerged as a promising therapeutic target. In addition to being an oncogenic driver, EZH2-dependent epigenetic reprogramming modulates tumor-immune infiltrate. Therefore, we argue that a better understanding of the molecular mechanisms that sensitize cancer cells to EZH2 inhibition and modulate tumor microenvironment will likely provide important insights for new treatment options for MPM. Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer that is mainly associated with prior exposure to asbestos fibers. Despite being a rare cancer, its global rate is increasing and the prognosis remains extremely poor. Over the last two decades, despite the constant research of new therapeutic options, the combination chemotherapy with cisplatin and pemetrexed has remained the only first-line therapy for MPM. The recent approval of immune checkpoint blockade (ICB)-based immunotherapy has opened new promising avenues of research. However, MPM is still a fatal cancer with no effective treatments. Enhancer of zeste homolog 2 (EZH2) is a histone methyl transferase that exerts pro-oncogenic and immunomodulatory activities in a variety of tumors. Accordingly, a growing number of studies indicate that EZH2 is also an oncogenic driver in MPM, but its effects on tumor microenvironments are still largely unexplored. This review describes the state-of-the-art of EZH2 in MPM biology and discusses its potential use both as a diagnostic and therapeutic target. We highlight current gaps of knowledge, the filling of which will likely favor the entry of EZH2 inhibitors within the treatment options for MPM patients. Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer that derives from the mesothelial cells of the pleura and is mainly associated with prior exposure to asbestos fibers [1]. Even though it is a rare cancer, the global rate of MPM is increasing because of the constant use of asbestos in some countries and the difficulty in its removing from the environment, even in countries that banned its use in the 1990s [2]. MPM has long been classified in three main histological subtypes, which are characterized by different frequencies and prognoses [3]. Specifically, epithelioid MPM represents the most common (50–70%) and the less aggressive subtype; sarcomatoid MPM is the rarest (10–20%), most aggressive and chemo-resistant subtype; and biphasic MPM is characterized by epithelial and mesenchymal components and is the subtype whose frequency and outcome are in between the previous ones. In addition to histology, both their stromal and molecular features are increasingly recognized as important prognostic determinants and are included in the updated classification of pleural tumors published by the World Health Organization (WHO) in 2021 [4]. Due to the long latency of tumor development—which usually takes approximately 40 years—and the poor specificity of the clinical symptoms, MPM is usually diagnosed in old individuals at an advanced stage, when malignant cells have already spread to all the pleural layers [2]. Therefore, MPM clinical management is challenging, and the high resistance of malignant cells to treatments further worsens the patient’s outcome. Overall, this results in a dismal prognosis and a-5-year survival rate of approximately 10%. Despite its poor effectiveness, the combination chemotherapy with cisplatin and pemetrexed has remained the only first-line therapy for MPM for almost two decades [5]. The addition of bevacizumab to combination chemotherapy showed a two-month-survival improvement, but it didn’t receive any approval because of the increased frequency of severe adverse events [6]. In contrast, the combination of Tumor Treating Fields (TTFields)—which is a non-invasive approach based on the transcutaneous delivery of low-intensity alternating electric fields—with gold standard chemotherapy showed promising results in terms of safety and efficacy in a single-arm phase-II multicentric study. As a result, it was approved in 2019 by the Food and Drug Administration (FDA) as a front-line therapy for unresectable, locally advanced or metastatic MPM. Nevertheless, the lack of randomized evidence limits its entry into the clinical guidelines. Meanwhile, the success of immune checkpoint blockade (ICB)-based immunotherapy for the treatment of melanoma [7] has fostered its evaluation for other deadly cancers, such as MPM. Despite the disappointing results of the first trials, in 2021 the publication of the Checkmate 743 trial, which was a large randomized open-label phase III study, showed that the combination of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) in a frontline setting is more effective than standard chemotherapy. As a result, both the FDA and the European Medicines Agency (EMA) rapidly approved the combined ICB as a new first-line treatment option for unresectable MPM. Although this undoubtedly represents a breakthrough for MPM, many patients are still refractory or relapse after a few months of therapy. Thus, MPM is still a fatal cancer that urgently needs new treatment options. The identification of reliable biomarkers that enable researchers to anticipate the diagnosis at a “pre-invasive” stage is obviously a key step towards better clinical management. Equally important are new therapeutic strategies along with predictive biomarkers to guide clinical decision making toward the best treatment for each patient, which are very active fields of research that challenge physicians and scientists. Enhancer of zeste homolog 2 (EZH2) is a well-known oncogenic driver in different malignancies, wherein it regulates gene expression in a PRC2-dependent and -independent manner [8]. Although EZH2 alone is enzymatically inactive, biochemical and structural studies have shown that in association with EED, SUZ12 and RbAp46/48, it becomes the catalytic subunit of polycomb repressive complex 2 (PRC2), which represses gene expression by the trimethylation of histone H3 on lysine K27 (H3K27me3) [8]. Although this epigenetic repression of genes plays a key role during tissue development and stem cell fate decision, its dysregulation can bring about the silencing of tumor suppressor genes and the promotion of carcinogenesis. Additionally, emerging studies have pointed out that EZH2 can promote the activation of key oncogenic programs through its direct interaction with transcription factors, such as NF-κB, estrogen and androgen receptors [9]. Collectively, the overexpression or gain-of-function mutations of EZH2 have been reported in a variety of solid and hematological cancers [8]. Accordingly, EZH2 has been recently introduced by the WHO as a diagnostic marker that enables the distinction of MPM from benign mesothelial proliferation [10]. Because of the association between its overexpression and a worse outcome [11,12,13], its inhibition has also been evaluated for new therapeutic perspectives. In models of MPM overexpressing EZH2 due to BRCA-1-associated protein 1 (BAP1) loss, pharmacological EZH2 inhibition showed significant anti-tumor activity [14]. However, treatment with tazemetostat, an EZH2 inhibitor that has recently been entered into the clinical treatment for epithelioid sarcoma [15], showed only a modest response rate in patients with relapsed or refractory BAP1-inactivated mutations [16]. Therefore, there is a need to better understand the mechanisms that sensitize cancer cells to EZH2 inhibitors, along with their effects on the tumor microenvironment (TME). Indeed, EZH2-dependent epigenetic reprograming has emerged as a crucial modulator of tumor-infiltrating immune cells in different types of malignancies, but it has never been fully explored in MPM. As a result, combining EZH2 inhibitors with other treatment approaches, including immunotherapy, is currently a hot topic of research in solid tumors [17] and might represent the next key challenge for the clinical management of MPM. Based on these premises, this manuscript reviews the current state-of-the-art of EZH2 in MPM pathogenesis, diagnosis and therapy. Specifically, we provide a comprehensive narrative synthesis of the evidence regarding the identification of EZH2 in the context of MPM, we critically describe its value as a diagnostic biomarker, and we discuss the pre-clinical and clinical studies that identify EZH2 as a new promising therapeutic target. We also highlight current gaps of knowledge and argue about the putative therapeutic perspectives of EZH2 inhibitors in combination with ICBs for MPM. The mutational landscape that has emerged over the previous years has highlighted extensive genetic variation and gene expression deregulation both between and within MPM patients [18,19,20,21]. This molecular heterogeneity suggests the existence of a continuum of MPM clinical phenotypes whose understanding will remarkably improve MPM classification and prognostication. In addition to the mutated genes that characterized each tumor, most MPMs also harbor loss-of-function mutations or the genetic loss of a few tumor suppressor genes (CDKN2A/2B, BAP1, NF2, TP53, LATS2 and SETD2), which likely plays a key role in neoplastic transformation [18,19]. The inactivation of such tumor driver genes is mainly due to chromosomal instability rather than point mutations. As a result of chromoplexy or chromothripsis, multiple chromosomal rearrangements and deletions are commonly observed in MPM cells [22,23]. In addition, tumor suppressor genes can be silenced by epigenetic modifications. Since 2009, it has been known that the expression of up to 11% of the genes in MPM cells are repressed by histone and DNA methy24 is nowlation [24]. With the exception of some overlaps, the majority of the genes enriched with H3K27me3 have no detectable level of DNA hypermethylation on the CpG promoters, while most of the DNA hypermethylated genes have no H3K27me3 marks. Thus, it appears that H3K27me3 and DNA hypermethylation may contribute to MPM development through the silencing of specific target genes. Two years later, Kemp C.D. et al. provided the first evidence of the aberrant expression of the polycomb group (PcG) proteins in MPM and proposed its targeting as a new potential treatment for this malignancy [12]. They revealed that the majority of MPM cell lines and primary MPM cells express higher levels of EZH2—a core component of PRC-2—than normal mesothelial cells. More importantly, the immunohistochemical (IHC) analysis of the MPM specimens demonstrated that EZH2 overexpression was associated with aggressiveness and the advanced stage of disease, and it decreased patient survival. Albeit poorly studied in the context of pleural mesothelial cells [25], it is widely recognized that the balanced activity of EZH2 methyltransferase with KDM6A (UTX) and KDM6B (JMJD3) demethylase controls the physiological levels of H3K27me3, which drives proper cell differentiation during development. Accordingly, an accumulating amount of evidence has indicated that the dysregulated activity of these proteins is linked with cancer cell features (e.g., proliferation, survival, stemness, migration, epithelial-mesenchymal transition) in different tumor types [26]. However, the interplay between EZH2 methyltransferase and KDM6A (UTX) and KDM6B (JMJD3) demethylase has yet to be explored in MPM. The analysis of surgical samples from MPM patients showed that both KDM6A and KDM6B transcript levels were increased in malignant tumors [27]. However, their pharmacological inhibition resulted in stronger anti-proliferative effects in normal mesothelial compared to MPM-derived cell lines, reducing the interest in KDM proteins as therapeutic targets [27]. In contrast, the upregulation of EZH2 observed in tumor tissue biopsies were retained in the MPM-derived cell lines, suggesting that EZH2 expression is under the control of tumor-specific factors. Specifically, the expression of a number of PcG genes, including EZH2, is transcriptionally regulated by E2F1. Nevertheless, this control can be dysregulated in MPM due to frequent CDKN2A deletions or epigenetic modulation [28]. Loss of BAP-1, which is another common oncogenic driver in MPM, was also found to be associated with EZH2 upregulation in human MPM cell lines [14]. Additionally, epigenetic regulators such as microRNA (miR)-101 and miR-26a, which are down-regulated in primary MPM, negatively affect the expression of EZH2 [12]. Recently, we have demonstrated that the silencing or inhibition of SIRT1 in MPM cells induces EZH2 protein acetylation and stability, as well as augmented H3K27me3 levels [28]. Over the last years, the analysis of TCGA data has confirmed that EZH2 mRNA is highly expressed in MPM and is significantly associated with decreased survival [11]. Along the same line, the analysis of transcriptomic datasets of MPM by bioinformatic tools, which allows for the prediction of protein–protein interaction networks (PPIs), has recently identified EZH2 as well as Hyaluronan Mediated Motility Receptor (HMMR) as “core” genes of MPM development, progression and outcome [29]. In agreement with in silico analysis, the role of PRC2-dependent gene expression in MPM pathogenesis has been strengthened by different in vitro studies [11]. Having corroborated the observation that a subset of genes repressed in MPM exhibits H3K27me3 without DNA hypermethylation, McLoughlin K.C. and co-workers used microarray, qRT-PCR, immunoblot and immunofluorescence techniques to examine PcG gene/protein expression in a panel of MPM cell lines and normal mesothelial cells. The results demonstrated that the overexpression of EZH2 and, to a lesser extent, EED and SUZ12 is associated with the increase of H3K27me3 in approximately 80% of primary MPMs. EZH2 or EED knock-down by shRNA decreased global H3K27me3 levels and significantly inhibited the proliferation, migration, clonogenicity and tumorigenicity of MPM cells [11]. BAP1 loss has been found functionally linked with EZH2 overexpression. Data obtained by LaFave L.M. et al. suggested that BAP1 interacted and co-occupied the EZH2 promoter with L3MBTL2, a protein that binds E-box motifs and maintains H4K20me1. BAP1 loss led to reduced L3MBTL2 stability and increased EZH2 transcription. Therefore, the silencing or pharmacological inhibition of EZH2 has been reported to induce apoptosis in BAP1-mutant MPM cell lines and reduce their growth when subcutaneously injected in mice [14]. Recently, we have reported that low SIRT1 sensitized MPM cells to EZH2 inhibition, which significantly reduced MPM cell proliferation in vitro by arresting cells in the G0/G1 phase and inducing a senescent phenotype [28]. Collectively, these studies indicate that despite the existence of different mechanisms leading to EZH2 overexpression, this epigenetic regulator is a central orchestrator of MPM pathogenesis. Therefore, EZH2 might represent both a reliable diagnostic marker of malignancy and a novel target for the development of new therapeutic interventions. Currently, MPM is primarily diagnosed with imaging procedures, followed by the immunophenotyping of paraffin-embedded sections from thoracoscopic biopsies or, in some cases, of cells recovered from pleural effusion samples [30]. In addition to cytological/histological analyses, molecular markers are essential to differentiate MPM from either metastatic adenocarcinoma [31] or reactive mesothelial hyperplasia (RMH) [32]. Despite being a benign process, RMH cytologically resembles epithelioid MPM, which is the most common and diverse subtype in terms of cytological and architectural complexity [33]. Moreover, in the attempt to advance both the diagnosis and prognosis of MPM, a growing number of researchers have focused their attention on the identification of a reliable panel of biomarkers for distinguishing mesothelial tumors at the “pre-invasive” stage from those that have already infiltrated the pleural layers. These studies will likely pave the way for earlier therapeutic interventions, which might also be more effective. According to recent International Mesothelioma Interest Group (IMIG) guidelines [34], the homozygous deletion of the 9p21 locus detected by fluorescence in situ hybridization (FISH) and/or BAP1 loss detected by IHC [34,35,36,37] are the most accurate biomarkers for distinguishing malignant from benign mesothelial proliferations. Nevertheless, there are some concerns regarding their clinical use. Regarding FISH analysis of the 9p21 locus, it is hard to define an appropriate cutoff to differentiate homozygous from hemizygous deletions. Additionally, FISH is an expensive and time-consuming technique that cannot be performed in every facility. Interestingly, Girolami I. et al. [38] have recently reported high concordance between 9p21 homozygous deletion by FISH and methylthioadenosine phosphorylase (MTAP) loss by IHC. Thus, the latter could represent a reliable option for detecting 9p21 deletion in a low-resource setting. MTAP might also be useful in combination with BAP1 to improve MPM diagnosis. Although the number of studies was insufficient to perform a pooled analysis, [39,40,41] it seems that a lack of MTAP and BAP1 has a higher sensitivity than BAP1 loss only. To distinguish MPM from RMH, additional IHC markers such as desmin, epithelial membrane antigen (EMA), insulin-like growth factor mRNA binding protein 3 (IMP3), glucose transporter-1 (GLUT-1) and CD146 have also been evaluated [42,43]. Yoshimura et al. reported that GLUT1 (up to 89%) and IMP3 (up to 94%) have the highest sensitivity, while Sheffield et al. found that EMA with p53 (64%) and BAP1 with 9p21 locus (100%) are the most sensitive and specific combinations, respectively. A recent systematic literature review confirmed that, unless they are used in combination, biomarkers such as GLUT1 and IMP3 have an unsatisfactory diagnostic performance [38]. Given that different studies have reported that EZH2 is overexpressed in a remarkable number of MPM cases (44.4–57%) but not RMH cases, EZH2 has emerged as an interesting diagnostic marker [42,44,45]. Indeed, EZH2, which is known to be upregulated in different solid cancers, is not a tissue-specific marker of malignancy. Therefore, high EZH2 expression can be exploited to distinguish MPM from benign mesothelial proliferations, but not from other lung malignancies. In contrast, there is evidence that BAP1 is a specific and useful marker for distinguishing non-mesothelial malignancies from epithelioid and biphasic but not sarcomatoid MPM in the thoracic or abdominal cavities. The latter rarely harbors BAP1 loss and is usually well-diagnosed on the bases of its histological features only. Even though enhanced EZH2 expression can be functionally associated with BAP1 loss in MPM cell lines [14], different studies have demonstrated that BAP1 loss is not statistically associated with EZH2 expression in human MPM biopsies [45], indicating that the mechanisms underlying EZH2 overexpression and BAP1 loss may be distinct. Thus, the combination of BAP1 and EZH2 detection by IHC could be a highly sensitive (90.0%) and specific (100%) approach for MPM diagnosis. Additionally, the lack of correlation among BAP1 or MTAP loss and EZH2 overexpression (p = 0.973, p = 0.284) suggests that the combination of the three different markers might further increase the accuracy of MPM diagnosis [42]. Recently, EZH2 has been evaluated in combination with Survivin, whose expression was detected in 67.9% of MPM cases, but not in RMH cases [44]. With the exception of some variations in terms of the prevalence of Survivin-positive MPMs across different cohorts of patients, [46,47], this study confirmed the diagnostic value of Survivin. Along the same line, the authors corroborated a highly significant direct association between BAP1 loss and Survivin expression [32], but also revealed an inverse association between high EZH2 expression and either BAP1 loss or Survivin expression. Therefore, the combinations of EZH2high and/or BAP1 loss with Survivin+ might be exploited to gain sensitivity in the differential diagnosis between epithelioid MPM and RMH. It is worth noting that BAP1 and EZH2 are the only markers that are localized in the nuclei of tumor cells, whereases the IHC analysis of the other markers results in a cytoplasmic staining wherein variable intensity can challenge the detection of a positive signal from the background. Therefore, the inclusion of BAP1 and EZH2 in the panel of markers for the IHC analysis of tissue biopsies could greatly improve the accuracy of MPM diagnosis. Previous systematic reviews have failed to define a reliable panel of diagnostic biomarkers for MPM. The variations in marker expression reported across the different studies may be reasonably assumed to be due to the differences in terms of sample sizes, antibodies used, staining and quantification techniques. Therefore, the standardization of IHC procedures will likely allow for the determination of the appropriate combination of markers that, together with histologic analysis and clinical evaluation, might anticipate the diagnosis of MPM. A growing number of studies have indicated the therapeutic potential of EZH2 targeting (Figure 1). The first evidence dates back to 2012, when 3-deazaneplanocin A (DZNep) demonstrated a significant cytotoxic effect against MPM cells [12]. DZNep is a S-adenosylhomocysteine hydrolase (SAH) inhibitor that indirectly inhibits EZH2 by interfering with S-adenosyl-methionine (SAM) and SAH metabolism. However, H3K27 demethylation observed upon DZNep treatment is due to the proteolytic degradation of EZH2 and other PRC2 components, rather than specific EZH2 catalytic inhibition [12]. In vitro, DZNep triggers the expression of several tumor suppressor genes, which inhibits MPM cell proliferation and induces cell senescence but not apoptosis [12]. These data are in accordance with p21cip upregulation and the delay of the G2/M transition, which have been respectively observed in melanoma and breast cancer cells upon EZH2 knockdown [48,49]. Additionally, the effect of DZNep was evaluated on MPM xenografts. The results demonstrated a significant reduction of tumor size after each cycle of treatment and an approximately 50% decrease in tumor mass at the end of the treatment course, along with no signs of systemic toxicity. Therefore, the authors claimed that DZNep recapitulated, in vitro and in vivo, the effects of EZH2 or EED depletion in MPM cells. Successively, LaFave L.M. et al. [14] proved that human BAP1-mutant MPM cell lines were sensitive to the selective EZH2 inhibitor EPZ011989. Accordingly, EPZ011989 significantly reduced the growth of sub-cutaneous transplanted BAP1-mutant MPM cells and abrogated pulmonary metastasis when mice were injected with a BAP1-mutant MPM cell line with metastatic potential. Because the wild-type tumors were less responsive to EZH2 inhibition, they concluded that BAP1 mutations, which typically result in increased EZH2 expression, render MPM cells addicted to PRC-2. Despite the strong association between BAP1 mutations and repression of PRC-2 targets [51], it seems that BAP1 mutant MPMs harbor different clinical phenotypes, since different studies have reported an overexpression of EZH2 in BAP1 wild-type MPM biopsies [28,42,45]. Recently, we have demonstrated that in low SIRT1 conditions, EZH2 inhibition significantly reduced the proliferation of BAP1 wild-type MPM cells [28]. Interestingly, we have observed that EZH2 inhibition induced cell senescence by promoting CDKN2A/p16ink4a expression, whereas CDKN2A null cells underwent apoptosis upon treatment with the EZH2 inhibitor EPZ6438 [28]. These findings indicate that patients carrying homozygous deletion or loss-of-function mutations of CDKN2A should be more responsive to EZH2 inhibition. Therefore, in a translational perspective, studies are warranted to evaluate CDKN2A status as a marker for patients’ stratification and/or potentiation of EZH2 inhibition efficacy. A high-throughput screening (HTS) campaign followed by hit triaging led to the discovery of the EPZ005687 compound by the company Epizyme. This EZH2 inhibitor has a greater than 500-fold selectivity against 15 other protein methyltransferases and a 50-fold selectivity against the closely related enzyme EZH1 [52]. The EPZ005687 has a similar affinity for wild-type and Y641 mutant EZH2, but a greater affinity for the A677G mutant. In spite of the remarkable reduction of H3K27me3 in both EZH2 wild-type and mutant lymphoma cell lines, similar to other EZH2 inhibitors, EPZ005687 significantly inhibited the proliferation of mutant EZH2 cells only. A further-improved version of EPZ005687 is EPZ-6438 (tazemetostat), which is a potent and selective SAM competitive small molecule that retains the cellular activity and selectivity of EPZ005687 but gains better oral bioavailability and pharmacokinetic properties [53]. In addition to hematological malignancy, the inhibition of EZH2 can be beneficial for the treatment of solid cancers. Firstly, EPZ-6438 has demonstrated significant anti-tumor activity against malignant rhabdoid tumors (MRTs), provided that SMARCB1 is deleted. Indeed, EZH2 inhibition by EPZ-6438 induced apoptosis in SMARCB1-mutant MRT cells and dose-dependent tumor regression in xenograft-bearing mice [54]. Subsequently, accumulating preclinical studies have substantiated the therapeutic potential of EPZ-6438 for a variety of solid tumors [55], leading to the initiation of clinical trials worldwide. In 2020, tazemetostat (TasverikTM) was approved by the FDA for the treatment of adults with locally advanced or metastatic epithelioid sarcoma not eligible for complete resection [15]. Along the same line, a phase-1 study recently conducted in Japan has reported that tazemetostat has a favorable safety profile and promising anti-tumor activity in patients with relapsed, refractory or advanced B-cell non-Hodgkin lymphoma [56]. However, some B-cell malignancies are resistant to EZH2 inhibitors [57], and in many solid cancers, despite the overexpression of EZH2, its inhibition alone doesn’t achieve a sufficient level of efficacy [58]. Consistent with that, the results of a recent multicenter single-arm open-label phase-II study with tazemetostat in BAP1-inactivated relapsed or refractory MPM patients provide the first evidence of safety along with a moderate anti-tumor activity [16]. The study enrolled patients with a more indolent disease after initial systemic therapy and included a substantial proportion of patients who had a surgical resection that did not reflect the real average of patients usually eligible for surgery. BAP1 mutation was determined by DNA sequencing, while loss of protein expression was done by IHC. The primary end-point of the study was the disease control at 12 weeks. Indeed, meta-analyses of trials conducted in MPM patients indicates that this parameter is a reliable positive predictor of survival. The end-point was reached in about a half of the patients, and the drug showed a favorable safety and tolerability profile. Two patients had a partial response, with a 30-week median duration of response. Noteworthy, a preliminary exploration of the TME composition before and after treatment with tazemetostat highlighted a significant reduction of intra-tumoral and stromal B-cells. That effect on immune cells warrants future studies to gather its role on clinical response. Altogether, these findings indicate that tazemetostat is a promising therapeutic option, whose efficacy might likely be improved by a better definition of predictive biomarkers for the stratification of MPM patients, as well as by novel combination strategies of EZH2 inhibitors with therapies such as chemo-, immuno- and targeted therapy. Indeed, many preclinical studies have demonstrated the efficacy of EZH2 inhibition in combination with cisplatin in different tumor types, such as lung, ovarian, and breast cancers [59]. EZH2 inhibition can rescue cisplatin resistance and mitigate the adverse effects [59]. Given that cisplatin-based chemotherapy is the standard-of-care for MPM, future studies are warranted to evaluate the putative beneficial effects of the combination of EZH2 inhibitor with cisplatin. In addition to cancer cells, the TME, including immune and non-immune cells, the extracellular matrix and the soluble mediators released by the different cells, plays a key role in MPM development, growth, progression and response to therapy [60,61,62]. Here, we focus on immune cells only (Figure 2), among which macrophages emerge as key orchestrators of both early tumor-promoting inflammation in response to asbestos fibers and immunosuppression at the advanced stage of MPM. Alveolar macrophages, which efficiently eliminate dust particles and environmental pollutants [63], struggle to clear fibers longer than 5 μm, which consequently remain in the lungs—triggering the neoplastic transformation of mesothelial cells. Although the underlying mechanisms have not yet been fully understood, it is widely recognized that ‘’frustrated phagocytosis’’ promotes a chronic inflammatory microenvironment that supports the carcinogenesis, survival and proliferation of neoplastic cells through the production of reactive oxygen and nitrogen species (ROS and NOS), as well as cytokines, such as IL-1β [64,65] and TNFα [66]. Additionally, High Mobility Group Box1 Protein (HMGB1), a damage-associated molecular pattern released by both mesothelial cells and macrophages, plays a key role in tumor development and progression by enhancing both macrophage-driven inflammation and mesothelial/neoplastic cell survival, proliferation, autophagy and epithelial-mesenchymal transition (EMT) [67,68]. Accordingly, HMGB1 dramatically increased in the blood of asbestos-exposed individuals, and its high levels in MPM patients are associated with a worse outcome [69,70]. Tumor-associated macrophages (TAMs), which are the most abundant population of immune cells in human MPM [61], largely stems from monocytes recruited by chemotactic factors like CCL2, which is produced abundantly by mesothelial cells exposed to asbestos [60,71]. As a result, CCL2 levels increased significantly both in the pleural effusion (PE) and in the blood of MPM patients, in particular at the advanced stage, supporting the central role of macrophages across all stages of MPM development [72]. Accordingly, the number of TAMs defined by the pan-macrophage marker CD68 was associated with worse outcomes in non-epithelioid MPM [71]. Similar to other tumor types, TAMs upregulate M2 markers like CD163 and CD206, indicating a shift of polarized activation toward the alternative (M2) immunosuppressive program. In agreement, a positive correlation between stromal CD68+ macrophages and immunosuppressive Tregs was observed in MPM specimens [73]. Additionally, pleural effusion is enriched in molecules, such as macrophage colony stimulating factor (M-CSF) [74], transforming growth factor β (TGF-β) [75] and prostaglandin E2 (PGE2) [76], which are released by tumor cells and drive immunosuppressive macrophage differentiation in vitro. In line with human evidence, the accumulation of immunosuppressive and tumor-promoting TAMs was also confirmed in different pre-clinical models of MPM [77,78], where their depletion and/or M1-reprograming rescued anti-tumor immunity [78], in particular in combination with anti-PD1/PD-L1 blockades [79]. Although these studies overall support the therapeutic value of the approaches that target macrophages, the increasing evidence of the intra- and inter-tumor heterogeneity of human MPM [20,80,81] points out the need for a better understanding of TME and its cross-talk with cancer cells. Even though they account for less than 10% of immune infiltrate, both polymorphonuclear (PMN) and monocytic (M-) myeloid-derived suppressor cells (MDSCs) exert different tumor-promoting activities that negatively affect MPM outcome [82]. Both subsets exert important immunosuppressive activities, as demonstrated by the inhibition of proliferation and cytotoxic activity of autologous human T lymphocytes [82]. Further supporting the therapeutic potential of targeting MDSCs, the neutralization of GM-CSF in a preclinical model of MPM inhibits the accumulation of tumor-infiltrating PMN-MDSC, boosting anti-tumor immunity [83]. Dendritic cells (DC), which play a key role in inducing an antigen-specific immune response, are not only reduced in number but also in migratory and antigen presentation capability. Although these cells maintain expression of IL-12, they also tend to produce higher amounts of anti-inflammatory and pro-angiogenic factors such as IL-10 and vascular endothelial growth factor (VEGF) [84]. So far, cytotoxic immune cell populations like NK and NKT cells have been poorly studied in human MPM. Different evidence indicates that, despite playing a relevant role in anti-tumor immunity, NK frequency in MPM is not associated with a better outcome [60]. A reasonable explanation is that the immunosuppressive microenvironment of MPM hampers their effector functions [85]. According to this hypothesis, in the PE of MPM patients, NK cells express high levels of the checkpoint inhibitors T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) [60]. Additionally, a reduced expression of activating receptor-like NKp46 and an enrichment of a CD56Bright NK subset have been reported in the blood of MPM patients [86]. Interestingly, anti-CTLA-4-based immunotherapy seems to enhance the cytotoxic activity of NK cells since an increase of CD56Dim/CD56Bright NK ratio has been observed in the blood of tremelimumab-treated patients [86]. NKT cells, whose activation by alpha-galactosylceramide in combination with cisplatin has demonstrated a relevant anti-tumoral activity in mouse models of MPM [87,88], represent an additional population of cytotoxic immune cells that warrants more study in MPM patients. Beyond the impact of each immune cell population, understanding the cross-talk among the stromal, immune and cancer cells is a key challenge for improving patients’ stratification and clinical management. Indeed, different studies based on the IHC analysis of immune infiltrate have observed that the combination of different immune cells has a better prognostic value than the frequency of single immune cell subsets. For example, although a high frequency of either T (CD3+, CD8+, or CD4+ cells) or B (CD20+ cells) lymphocytes has been reported as favorable prognostic markers [89,90,91,92,93] in epithelioid MPM, CD20+B cellshigh CD163+ TAMlow and CD8+ T cellslow CD163+ TAMhigh combinations showed a superior accuracy in predicting better and worse outcomes, respectively [92]. Additionally, in a cohort of patients with non-epithelioid MPM, it has been observed that despite the presence of a high number of anti-tumoral CD8+ T lymphocytes, when a significant level of CD68+ macrophages and PD-L1+ tumor cells are present as well, the response to chemotherapy and the outcome are poor [93]. In contrast, a higher number of B lymphocytes, along with the presence of tertiary lymphoid structures (TLS) consisting of B and T lymphocytes, have been associated with a response to chemotherapy and a longer survival for patients with epithelioid MPM [94]. These studies highlight the importance of TME composition not only as prognostic marker, but also as a predictor of response to therapy. Accordingly, a recent study performed on a small cohort of patients showed that a high number of CD8+ T cells is an independent factor associated with better survival in epithelioid MPMs treated with hypo-fractionated radiation therapy [95]. Besides chemo- and radiotherapy, the immune contexture obviously holds great promise as a predictor of response to immunotherapy. To overcome the limits of IHC, the development of innovative multiplex immunophenotyping techniques has marked a milestone for a more comprehensive characterization of the TME. Nevertheless, only Lee H. S. and colleagues have hitherto analyzed the MPM immune infiltrate by mass-cytometry [96]. As a result, MPM patients were stratified in two groups characterized by a distinct immunogenic immune signature, which was associated with favorable outcomes and a response to checkpoint blockade [96]. Although the multiplex immunophenotyping technique allows for the analysis of intratumor heterogeneity at the single-cell resolution level, transcriptional profiling is an easier approach that has become widespread over the last years. As a result, an underestimated level of cancer cell heterogeneity beyond histological subtypes has emerged. Additionally, due to the consistent increase of publicly available datasets, different algorithms have been generated to unravel the MPM microenvironment and determine the immune signatures to predict outcomes and response to treatments. For example, the application of the ESTIMATE algorithm has indicated a prognostic signature consisting of 14 stromal/immune-related genes, which could also be useful to predict response to ICB [97]. Recently, using non-negative matrix factorization (NMF) and nearest template prediction (NPT) algorithms, Yang and co-workers developed an in silico classification system that stratifies MPM in different immune subtypes that are associated with different prognoses [98]. In addition, because of the high lymphocyte infiltration, TCR and BCR diversity, and IFNγ signature, the “immune activated” subtype has a favorable response to ICB, while the “immune suppressed” subtype, which is characterized by a huge number of immunosuppressive Treg and myeloid cells (TAM, MDSC) along with a TGF-β signature, is resistant to ICB, but it could benefit from drug targeting macrophages such as CSF1/CSF1R antibody. Therefore, improving our understanding of the TME contexture prior to therapy could be crucial to guide clinical decision making, whereases gathering the effects of treatments on TME would provide a more comprehensive knowledge of their efficacy and might open new strategies to enhance their therapeutic effects. It has long been known that, besides the cancer cell-autonomous effect, the anti-cancer activity of drugs targeting epigenetic modulators is due to the promotion of anti-tumor immunity [99,100,101,102]. Although poorly studied in MPM, EZH2-dependent epigenetic reprograming can modulate tumor cell immunogenicity and TME composition, and it can directly regulate immune cell differentiation and functional activation (Figure 3). Specifically, in different types of both hematological (e.g., diffuse large B-cell lymphoma) and solid cancers (e.g., neuroblastoma, melanoma, breast, prostate and lung cancer), gain-of-function mutations or the overexpression of EZH2 increases H3K27me3, which represses genes encoding tumor-specific antigens and MHC molecules [103,104,105,106]. Therefore, EZH2 inhibitors can enhance tumor cell immunogenicity by reshaping the epigenetic landscape of cancer cells and favoring the expression of genes associated with both the presentation of new antigens and the recruitment of anti-tumor immune cells. Consistently, in preclinical models of ovarian cancer and melanoma, epigenetic reprogramming due to EZH2 knock down or pharmacological inhibition enhanced the expression of Th1-recruiting chemokines (e.g., CXCL9, CXCL10), increased tumor-infiltrated CD8+ T cells, and improved the efficacy of ICB-based immunotherapy [106,107]. Additionally, in a poorly immunogenic melanoma model, the inhibition of EZH2 triggered the expression of STING and consequently sensitized cancer cells to STING agonists. As a result, a combination of a EZH2 inhibitor and a STING agonist synergistically reduced tumor growth in association with an increased CD8+ T-cell infiltration [108]. Although the mechanism is different, the activation of STING upon treatment with EZH2 inhibitors has been also reported in prostate cancer. Indeed, in prostate cancer cells, EZH2 inhibitors can rescue the expression of endogenous retrovirus (LTR/ERV), which results in a “viral mimicry” state. Specifically, dsRNA molecules activate STING receptors, which triggers the expression of interferon-stimulated genes (ISGs). This brings about an increase of antigen presentation, cytotoxic CD8+ T cell recruitment and anti-PD1 responsiveness [109]. In line with these studies, using a MPM multicellular spheroid model (MCS), we have found that treatment with the EZH2 inhibitor tazemetostat lead to the upregulation of chemokines specific for the recruitment of cytotoxic immune cells such as CXCL9 and CXCL10 [50]. However, we have also found an increased expression of different monocyte chemoattractants (e.g., CCL2, M-CSF, CCL5, CXCL12, VEGF) in association with a significantly higher recruitment of tumor-promoting monocytes in the MCS [50]. This was the first study that had evaluated the effect of EZH2 on MPM TME composition, specifically on human monocytes and their impact on cancer cell responsiveness to tazemetostat. Subsequently, a functional association between EZH2 and TAM infiltration has been also reported in other types of tumors, such as breast and colorectal cancer (CRC) [110,111]. Recently, the effect of EZH2 on the composition of human MPM immune infiltrate has been explored using bioinformatic analysis on TCGA datasets. Interestingly, the results showed that high EZH2 expression, which is significantly associated with a worse outcome, negatively correlated with the number of tumor-infiltrating mast, NK and Th17 cells [13,60]. Overall, these studies provide the proof-of-concept that EZH2 modulates the composition of both innate and adaptive immune infiltrate in MPM. Besides recruitment, EZH2 affects anti-tumor immunity by modulating the differentiation and functional activation of the immune cells [112]. Concerning T cells, EZH2 promotes the lineage-specification, identity, maintenance and survival of differentiated antigen-specific CD4+ T helper cells, whereas effector CD8+ T cell differentiation is restrained by EZH2, which favors the formation of precursor and mature memory CD8+ T cells [113]. Additionally, Treg differentiation and suppressive activity require the EZH2-dependent deposition of H3K27me3 marks [114,115]. Indeed, mice carrying Treg-specific Ezh2 deficiency showed a reduced growth of different types of tumors (e.g., CRC, melanoma, prostate cancer) in association with the reprograming of tumor-infiltrating Tregs in anti-tumor effector cells (e.g., IL-2, IFNγ, and TNF) [116]. Regarding innate lymphoid cells, EZH2 inhibits invariant natural killer T (iNKT) cell differentiation and function, as well as the maturation, activation, survival and cytotoxicity of NK cells [117]. Accordingly, in hepatic cancer, the inhibition of EZH2 in tumor cells enhanced NK recruitment via CXCL10 [118], and it enhanced their activation through the expression of NKG2D ligands [119]. EZH2 also modulates the differentiation of MDSC. In murine models of either CRC or Lewis lung cancer (LLC), blocking EZH2 with GSK126 in immunocompetent mice impaired anti-tumor immunity by boosting systemic MDSCs expansion and accumulation in TME. Depleting MDSCs by anti-GR1 neutralizing antibodies or low doses of gemcitabine/5-Fluorouracil rescued GSK126 efficacy by recovering the anti-tumor effector T-cell activity [120]. Divergent effects of EZH2 on TAM functional activation have been reported in different settings. In a murine model of MPM, it has been observed that the treatment of murine RAW264.7 macrophages with a EZH2 inhibitor led to the upregulation of the phagocytosis inhibitory checkpoint PD-1 and, consequently, impaired their cytotoxic activity toward the MPM cells in vitro and in vivo [121]. Accordingly, by using an MCS model consisting of human MPM cells and monocytes, we have demonstrated that tazemetostat enhances both the recruitment and M2-polarized activation of monocytes, blocking the anti-proliferative effects of EZH2 inhibition in cancer cells [50]. Therefore, combining EZH2 inhibition with TAM-targeted therapy, such as anti-CSF1R [122], might synergistically improve the anti-tumoral efficacy. Along the same line, the treatment of breast cancer cells with EZH2 inhibitors promotes recruitment and favors M2 polarized macrophage activation by inducing CCL2 upregulation [110]. In contrast, EZH2 depletion caused an miR-124-3p-dependent inhibition of CCL2 expression in the tumor cells, leading to the inhibition of M2 polarized activation [110]. This highlighted an additional level of complexity in EZH2 activity, whose non-enzymatic modulatory functions are still poorly characterized. Moreover, cancer cell intrinsic and TME signals may account for the distinct effects of EZH2 inhibitor in different tumor types. Indeed, in a murine colorectal cancer (CRC) model, tazemetostat induced the accumulation of anti-tumor macrophages [111]. Accordingly, in glioblastoma multiforme, EZH2 inhibition by DZNep favored macrophage M1 polarization, as demonstrated by the upregulation of pro-inflammatory cytokines and the downregulation of anti-inflammatory ones, and it enhanced phagocytic capability [123]. These divergent results suggest that cancer cell intrinsic and TME signals may account for the distinct effects of EZH2 inhibitor in different tumor types. After decades of failed trials, the approval of immunotherapy based on the combination of ipilimumab and nivolumab has marked a milestone for MPM, particularly the sarcomatoid subtype, which is more aggressive and resistant to chemotherapy. However, MPM remains a deadly cancer with an unacceptably poor survival rate after diagnosis. Besides histology, the increasing advancements in MPM classification by molecular markers represent a key step towards better clinical management. Indeed, if we were able to bring the diagnosis toward the “pre-invasive stage” and to improve prediction of outcome, we would increase the chances of effective treatment regimens. In this context, EZH2 has emerged as a valuable diagnostic marker with a prognostic potential. Similar to many other solid cancers, its overexpression in MPM is recognized as an oncogenic driver. Consequently, inhibitors of EZH2 such as tazemetostat, which has recently entered into clinical use for epithelioid sarcoma, has attracted a lot of interest and has recently demonstrated some promising results of efficacy in preliminary clinical trials. Along with a better understanding of reliable biomarkers to identify the patients who most likely benefit from EZH2 inhibition, combinations of EZH2 inhibitors with different therapeutic modalities holds promise for enhancing efficacy. Being an epigenetic modulator, EZH2 has a profound effect not only on cancer cells, but also on TME. Given that EZH2 inhibitors can modulate both anti-tumor and pro-tumor immune cell populations, a better understanding of the effect of EZH2 inhibitors on the MPM immune infiltrate will likely help physicians determine the most effective combination approaches. Notably, the growing number of pre-clinical studies looking at different models of solid cancers indicate that EZH2 inhibitor synergizes with ICB-based immunotherapy thanks to the increased expression of PD-L1, immunogenic antigen and chemokine-recruiting cytotoxic T cells [124,125,126,127]. On the other hand, it is well-recognized that the efficacy of ICB-based immunotherapy could benefit by combination therapeutic strategies. So far, clinical trials conducted with MPM patients have evaluated ICBs with chemotherapy, targeted therapy like bevacizumab, and stereotactic body radiation therapy [128]. Epigenetic modulators, such as EZH2 inhibitors, which have been demonstrated to have a favorable safety profile along with a promising immunogenic potential, could represent a new potential therapeutic approach that warrants evaluation in combination with immunotherapy.

PMC10000491

Lei Wu,Liming Gao,Xiang Jin,Zhikang Chen,Xutong Qiao,Xiting Cui,Jianhua Gao,Liwei Zhang

Ethanol Extract of Mao Jian Green Tea Attenuates Gastrointestinal Symptoms in a Rat Model of Irritable Bowel Syndrome with Constipation via the 5-hydroxytryptamine Signaling Pathway

04-03-2023

Mao Jian Green Tea,ethanol extract,gastrointestinal motility,5-hydroxytryptamine,gut microbiota,flavonoids

In a previous study, we demonstrated that the hydro extract of Mao Jian Green Tea (MJGT) promotes gastrointestinal motility. In this study, the effect of MJGT ethanol extract (MJGT_EE) in treating irritable bowel syndrome with constipation (IBS-C) in a rat model constructed via maternal separation combined with an ice water stimulation was investigated. First, a successful model construction was confirmed through the determination of the fecal water content (FWC) and the smallest colorectal distension (CRD) volume. Then, the overall regulatory effects of MJGT_EE on the gastrointestinal tract were preliminarily evaluated through gastric emptying and small intestinal propulsion tests. Our findings indicated that MJGT_EE significantly increased FWC (p < 0.01) and the smallest CRD volume (p < 0.05) and promoted gastric emptying and small intestinal propulsion (p < 0.01). Furthermore, mechanistically, MJGT_EE reduced intestinal sensitivity by regulating the expression of proteins related to the serotonin (5-hydroxytryptamine; 5-HT) pathway. More specifically, it decreased tryptophan hydroxylase (TPH) expression (p < 0.05) and increased serotonin transporter (SERT) expression (p < 0.05), thereby decreasing 5-HT secretion (p < 0.01), activating the calmodulin (CaM)/myosin light chain kinase (MLCK) pathway, and increasing 5-HT4 receptor (5-HT4R) expression (p < 0.05). Moreover, MJGT_EE enhanced the diversity of gut microbiota, increased the proportion of beneficial bacteria, and regulated the number of 5-HT-related bacteria. Flavonoids may play the role of being active ingredients in MJGT_EE. These findings suggest that MJGT_EE could serve as a potential therapeutic pathway for IBS-C.

Ethanol Extract of Mao Jian Green Tea Attenuates Gastrointestinal Symptoms in a Rat Model of Irritable Bowel Syndrome with Constipation via the 5-hydroxytryptamine Signaling Pathway In a previous study, we demonstrated that the hydro extract of Mao Jian Green Tea (MJGT) promotes gastrointestinal motility. In this study, the effect of MJGT ethanol extract (MJGT_EE) in treating irritable bowel syndrome with constipation (IBS-C) in a rat model constructed via maternal separation combined with an ice water stimulation was investigated. First, a successful model construction was confirmed through the determination of the fecal water content (FWC) and the smallest colorectal distension (CRD) volume. Then, the overall regulatory effects of MJGT_EE on the gastrointestinal tract were preliminarily evaluated through gastric emptying and small intestinal propulsion tests. Our findings indicated that MJGT_EE significantly increased FWC (p < 0.01) and the smallest CRD volume (p < 0.05) and promoted gastric emptying and small intestinal propulsion (p < 0.01). Furthermore, mechanistically, MJGT_EE reduced intestinal sensitivity by regulating the expression of proteins related to the serotonin (5-hydroxytryptamine; 5-HT) pathway. More specifically, it decreased tryptophan hydroxylase (TPH) expression (p < 0.05) and increased serotonin transporter (SERT) expression (p < 0.05), thereby decreasing 5-HT secretion (p < 0.01), activating the calmodulin (CaM)/myosin light chain kinase (MLCK) pathway, and increasing 5-HT4 receptor (5-HT4R) expression (p < 0.05). Moreover, MJGT_EE enhanced the diversity of gut microbiota, increased the proportion of beneficial bacteria, and regulated the number of 5-HT-related bacteria. Flavonoids may play the role of being active ingredients in MJGT_EE. These findings suggest that MJGT_EE could serve as a potential therapeutic pathway for IBS-C. Irritable bowel syndrome (IBS) is a common gastrointestinal disease associated with changes in gastrointestinal motility, secretion, and visceral sensation. It manifests clinically mainly as abdominal pain accompanied by intermittent or persistent irregular bowel movements, as well as abnormalities in stool texture and shape [1,2,3]. Several basic and clinical studies have investigated the etiology of IBS from different perspectives, including the effects of genetic factors, low-grade mucosal inflammation and immune activation following severe gastrointestinal infection, increased intestinal mucosal permeability, changes in gut microbiota, abnormalities in bile salt metabolism, allergies to certain dietary components, abnormalities in neurotransmitter pathways, and changes in brain function [4,5,6,7,8]. Although the pathogenesis of IBS has not yet been fully elucidated, researchers speculate that hypersensitivity and alteration in visceral perception as well as gastrointestinal dysmotility form the main pathophysiological basis of the disorder [9]. IBS can be divided into four subtypes according to the Rome IV criteria: IBS with constipation (IBS-C), IBS with predominant diarrhea (IBS-D), IBS with mixed bowel habits (IBS-M), and IBS unclassified (IBS-U) [10,11]. Approximately one third of all IBS cases are of the IBS-C subtype [12]. Medical treatments for IBS often yield unsatisfactory results, thereby imposing a heavy disease burden on patients [13]. For IBS-D, most clinicians recommend the use of serotonin type 3 receptor (5-HT3R) antagonists to block the excessive action of 5-HT on 5HT3R and reduce intestinal motility. Common medications used for IBS-C treatment include the serotonin type 4 receptor (5-HT4R) agonists, prucalopride and tegaserod [14], the type 2 chloride channel activator, lubiprostone [15], and the guanylate cyclase C agonists, linaclotide [16,17] and plecanatide [18], which can promote intestinal peristalsis. In effect, tegaserod has already been approved for use by the U.S. Food and Drug Administration for the treatment of IBS-C. However, there are age-related limitations and contraindications to the use of lubiprostone and tegaserod, with the latter only being approved for use in a limited IBS-C patient population (women aged < 65 years without cardiovascular disease risk-related contraindications) [19]. Only a few studies have investigated the effects of lubiprostone in Asian patients; therefore, its use in the treatment of IBS-C has not been recommended in South Korea and Japan [20,21]. Given the scarcity of IBS-specific drugs, the treatment of the disease often requires the introduction of other adjuvant strategies, such as good lifestyle habits (eating regular meals and increasing dietary fiber intake), traditional Chinese medications or acupuncture [22,23,24], or acupoint catgut embedding [25] that contribute to improving the condition of the patient. During history, herbal medicines have also been developed by several countries and regions with specialties to deal with various diseases. Some of them can treat or relieve gastrointestinal disorders. For example, the herbal therapy STW-5 (Iberogast®) including angelica roots (Angelicae radix), chamomile flowers (Matricariae flos), caraway fruit (Carvi fructus), St. Mary’s thistle fruit (Cardui mariae fructus), balsam leaves (Melissae folium), peppermint leaves (Menthae x piperitae), greater celandine (Chelidonii herba), and licorice root (Liquiritiae radix) has been in clinical use in German-speaking countries for decades and is sold as an over-the-counter medicine in Europe. It acts on 5-HT4, 5-HT3, muscarinic M3, and opioid receptors to relieve intestinal spasm and reduce gastric acid secretion [26]. There is much evidence that peppermint oil reduces visceral pain and modulates gastrointestinal motility via TRPM8 and/or TRPA1 receptors [27]. Curcumin, contained in turmeric (Curcuma longa), treats abdominal pain as well as other gastrointestinal symptoms present in IBS [28]. Atractylodes lanceolata oil was able to ameliorate the rat IBS-D by inhibiting the SCF/c-kit pathway, thereby reducing inflammation and protecting the intestinal barrier from damage via the MLCK/MLC2 pathway [29]. Dracocephalum rupestre Hance, called Mao Jian Cao (MJC) in Chinese, is a perennial herb of the Dracocephalum genus and the Lamiaceae family that is native to the Northern Shanxi Province of China. MJC is a traditional Chinese medicine, with the effect of relieving headaches, soothing sore throats, subsiding coughs and preventing icterohepatitis [30]. MJC is rich in flavonoids, among which dihydroflavonoids and their corresponding glycosides such as luteolin, luteolin-7-O-β-D glucoside, eriodictyol, and eriodictyol-7-O-β-D glucoside, and terpenoids such as β-sitosterol, betulinol, and betulinic acid, are representative components [31]. Interestingly, the herbal tea made from its leaves as a daily drink to aid digestion is a more popular way. This is because people in these regions often consume the slower-digesting coarse grains. From June ending to September each year, fresh MJC leaves are harvested by locals for making Mao Jian tea (MJT), including MJGT (green tee) or MJBT (black tea). The making method could lead to the production of different metabolites [32]. For example, 130, 136, and 95 compounds were detected in the MJC, MJGT, and MJBT, respectively. There were 28 differential metabolites in MJGT compared to MJC; MJBT had 29. The MJGT-making method led to the significant intensity of some flavonoids such as apigenin, eriodictyol, luteolin, and naringenin, whereas the corresponding glycosides of eriodictyol and luteolin decreased. Interestingly, a similar trend was observed in MJBT, except that the content of some flavonoid glycosides decreased sharply, including the 7-O-glucoside of the four flavonoids mentioned above. Common green tea is made from the steamed and dried leaves of the Camella sinesis plant. The main active ingredients include tea polyphenols and tea polysaccharides, which have anticancer, antioxidant, neuroprotective, and hypoglycemic pharmacological activities [33]. Green tea contains caffeine (~3%) [34], which enhances the autonomic activity of the vagus nerve, releases acetylcholine, and promotes gastrointestinal motility [35], but such ingredients can also cause euphoria and insomnia after consumption. The caffeine content of MJC is very low at 0.495%, so it does not affect sleep after consumption [36]. In addition, caffeine is one of the main components that form the bitter taste of the tea [37]; therefore, MJC has a lighter taste compared to other common green teas. Flavonoids have multiple effects on the gastrointestinal tract, including (1) protecting the intestinal epithelium from drug damage and food toxins; (2) regulating the activity of enzymes involved in lipid and carbohydrate absorption; (3) maintaining the intestines of the intestinal barrier; (4) regulating the secretion of intestinal hormones; (5) modulating the gastrointestinal immune system; (6) exerting potential anti-colorectal cancer activity; and (7) shaping the composition of the bacterial flora [38]. For example, quercetin inhibits gastrointestinal toxicity induced by diclofenac and aggravated by ranitidine, improves gastrointestinal bleeding, intestinal permeability, and restores intraluminal pH in rats [39]. The ability of polyphenols in oranges and apples to alter the microbiota of systemic lupus erythematosus (SLE) patients, with their flavonoids increasing the levels of lactobacilli and dihydroflavonols increasing the levels of bifidobacteria, suggests the possibility of correcting the ecological dysbiosis associated with SLE by altering the flavonoid diet [40]. Apigenin showed a dose-dependent relaxation effect on acetylcholine (ACh)-induced muscle strips in the concentration range of 0.1 to 100 μmol/L. Luteolin and quercetin showed a similar performance to apigenin with the exception that, at low doses (0.001–0.1 μmol/L), they were able to further increase the induction effect of ACh [41]. Previously, we demonstrated that MJGT promotes small intestinal propulsion and gastric emptying in normal rats and improves gastrointestinal motility by increasing the abundance of beneficial bacteria [42]. Flavonoids constituted the main active ingredients in MJGT. Considering the easier concentration and higher efficiency for the extraction of flavonoids without the concerns of impacts on toxicity and biodegradability [43], we chose ethanol as the extraction solvent in the present study. A rat model of IBS-C was treated with the ethanolic extract of MJGT (MJGT_EE), and the response of the key enzymes and downstream targets of the serotonin (5-hydroxytryptamine; 5-HT) biosynthesis pathway, which is an essential signaling pathway in the gastrointestinal tract, was investigated. MJGT was purchased from Jiufeng Cooperative (Ningwu County, Shanxi Province, China) in December 2018, and samples were conserved at the Chinese Medicine Resources and Sciences Laboratory of Shanxi Agricultural University (JF18001-2). MJGT (200 g) was subjected to heat reflux extraction in 4 L of 70% ethanol for 60 min at 70 °C followed by vacuum concentration for ethanol recovery; then, the ethanol extract obtained was dried and stored at 4 °C until use. Six pregnant specific-pathogen-free (SPF) rats were purchased from Si Pei Fu Biotechnology Co., Ltd. (Beijing, China). The rats were provided clean water, fed daily, and reared under a 12-h light–dark cycle. At 7 days of age, 24 offspring rats were randomly selected and subjected to 3 h of maternal separation from 9 am to 12 noon daily for 14 days. Subsequently, the rats were assigned to three groups: the model (MG), positive drug (MSP) (1 mg/kg of mosapride), and 70% MJGT ethanol extract (MJGT_EE) (17 mg/mL, determined based on the daily dose for humans, with 0.1% dimethyl sulfoxide added in each group for solubilization) groups. Eight rats that were not subjected to maternal separation were assigned to the negative control (NC) group (saline). With the exception of animals in the NC group, animals in all the other groups were administered ice water at 0–4 °C (1.5 mL/rat) via gavage for 14 days. A fecal water content (FWC) measurement and intestinal sensitivity testing were performed to confirm successful IBS-C modeling. Subsequently, drug administration was performed in the different groups for 30 days via gavage. Next, rats were selected from each group and sacrificed. Then, two 5 × 5-mm proximal colonic tissue specimens were collected from each sacrificed rat and washed thrice with saline for intestinal content removal. One specimen was fixed in 4% paraformaldehyde for immunohistochemical analysis, and the other was stored at −80 °C for western blot analysis. In addition, the cecal contents of the sacrificed rats were collected in 1 mL sterile centrifuge tubes and immediately stored at −80 °C. All specimens were transported on dry ice prior to testing. All animal experiments were approved by the laboratory animal ethics committee of Shanxi Agricultural University (Taigu, China) (Approval No.: SXAU-EAW-2018R.0406001) and performed in accordance with the regulations and guidance of this committee. On days 14 and 28 of the IBS-C model construction and drug administration periods, respectively, the rats were separated with each rat reared individually, and the amount of feces passed out within 24 h by the rats in each group was recorded for FWC calculation. Then, the wet weight of the feces was measured using an electronic balance; next, the feces was dried at 105 °C to a constant weight and the dry weight of the feces was recorded. FWC was calculated using Equation (1). The smallest threshold colorectal distention (CRD) volume was also measured on days 14 and 28 of the IBS-C model construction and drug administration periods, respectively. Each rat was anesthetized using a small amount of diethyl ether and placed in a rat holder. Then, a glycerol-lubricated urinary catheter with an attached balloon was inserted into the colorectum of each rat and taped to the base of the tail, with the end of the balloon positioned approximately 1 cm away from the anus. When the rats regained consciousness and were fully acclimatized to the environment for 30 min, normal saline at 26–28 °C was injected into the balloon, and the smallest injection volume that induced CRD in the rats was recorded. This process was repeated thrice at 15-min intervals, and the smallest threshold volume was calculated by taking the average of the three volumes. After continuous gavage for 29 days, the rats were starved for 24 h but allowed access to water. A semi-solid paste was prepared following a slightly modified version of the method described by [44]; first, 10 g of sodium carboxymethyl cellulose was dissolved in 250 mL of distilled water. Then, 16 g of milk powder, 8 g of glucose, 8 g of starch, and 2 g of activated charcoal powder were added to this solution, and the resulting mixture was uniformly mixed to obtain 300 mL of a black semi-solid paste. This paste was stored in a refrigerator and warmed to a temperature of 20 °C before use. At the end of the drug administration period (day 30), 2 mL of the semi-solid paste was measured (Mp: mass of paste) and administered to each rat by gavage. Forty minutes following administration, the animals were anesthetized using pentobarbital sodium (40 mg/kg) and sacrificed. Then, the total mass of the stomach (Mfs), the net mass of the stomach (Mns), the total distance from the pylorus to the ileocecal junction (Lt), and the distance from the pylorus to the front edge of the black semi-solid paste (Lc) were measured. The gastric emptying and small intestinal propulsion rates were calculated using Equations (2) and (3), respectively, as represented below: For H&E staining, colonic tissues were fixed in 10% neutral buffered formalin solution, sectioned, deparaffinized at 65 °C, rehydrated, and stained using an H&E staining kit (Solarbio, G1121). For immunohistochemical analyses, colonic tissue specimens were sectioned, deparaffinized, and rehydrated. High-temperature antigen retrieval was performed on the tissue specimens using trisodium citrate, and endogenous peroxidase activity and non-specific binding sites in the specimens were blocked with H2O2 and goat serum, respectively (Zhongshan Jinqiao ZLI-9022). Anti-5-HT antibodies (primary antibodies; 1:1000) were added to the tissue sections and incubated at 4 °C for 12 h. Next, goat anti-rabbit IgGs (secondary antibodies; 1:100) and the peroxidase-antiperoxidase complex (PAP; 1:100) were successively added to the samples and incubated for 1 h at 37 °C each time; staining was performed using diaminobenzidine (DAB)/H2O2. The sections were adequately rinsed with 0.01 mol/L PBS (Na2HPO4 8 mM, NaCl 136 mM, KH2PO4 2 mM, KCL 2.6 mM, pH: 7.4) in between the steps described above. After staining, the sections were mounted onto gel-coated slides, dehydrated using graded alcohol, cleared in xylene, mounted, and observed under an optical microscope (Olympus BX-51 biological microscope, white light, Japan, Olympus). Five high-power (200×) fields of view were randomly selected for the calculation of the average optical density (AOD, IOD/Area) using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). For each rat, 50 mg of colonic tissue was precisely weighed and placed in an Eppendorf tube. First, an appropriate amount of protease inhibitor-containing cell lysis buffer was added to the tissue and the mixture was homogenized using an electric homogenizer. Next, the sample loading buffer was added to the homogenate, and the mixture was boiled for 5 min; then, the resulting mixture was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skimmed milk powder. Second, primary antibodies against 5-hydroxytryptamine receptor 3 (5-HT3R; bs-2126R), 5-hydroxytryptamine 4 receptor (5-HT4R; bs-12054R), the serotonin transporter (SERT, bs-1893R), tryptophan hydroxylase 1 (TPH1; bs-1215R), tryptophan hydroxylase 2 (TPH2; bs-8729R), calmodulin (CaM; bs-3666R), or myosin light chain kinase (MLCK) (Bioss Biotechnology Co., Ltd., Beijing, China) were added (1:1000) to the membrane and incubated at 4 °C for 12 h. Next, the membrane was washed thrice with TBST buffer (T1081, Solarbio Science & Technology Co., Ltd., Beijing, China); then, secondary antibodies (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) or HRP-conjugated Affinipure Goat Anti-Rabbit IgGs (H+L); 1:5000) were added to the membrane and incubated at room temperature for 1 h. Finally, at the end of the incubation, the membrane was again washed thrice with the TBST buffer, developed, exposed in a dark room, and imaged using a ChemiDoc MP Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA). GAPDH or α-tubulin was used as the housekeeping protein. DNA extraction: Total microbiota DNA was extracted from rat fecal samples using the E.Z.N.A.® soil DNA kit (Omega Bio-Tek, Norcross, GA, USA) following the instructions of the manufacturer. The quality of the extracted DNA was evaluated via 1% agarose gel electrophoresis, and DNA concentration and purity were measured using the NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA) device. 16S rRNA gene amplification and sequencing through the polymerase chain reaction (PCR): The V3–V4 variable regions of the 16S rRNA gene were subjected to PCR (ABI GeneAmp® 9700, ABI, Los Angeles, CA, USA) using the 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′GGACTACHVGGGTWTCTAAT-3′) primer sequences under the following cycling conditions: initial denaturation at 95 °C for 3 min, 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, stable extension at 72 °C for 10 min, and storage at 4 °C. The PCR reaction system was constituted of 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and ddH2O to make up a final volume of 20 μL. PCR was performed in triplicate for each sample. Illumina Miseq sequencing: DNA fragments were recovered from a 2% agarose gel after mixing, purified using the AxyPrep DNA Gel Extraction Kit (Axygen, San Francisco, CA, USA), detected using 2% agarose gel electrophoresis, and quantified using a Quantus™ Fluorometer (Promega, Madison, WI, USA). Sequencing libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA) according to the following steps: (1) adaptor ligation; (2) magnetic bead screening for the removal of self-ligated adaptors; (3) library template enrichment via PCR amplification; (4) magnetic bead recovery of PCR products to obtain the final sequencing libraries. Sequencing was ultimately performed on the Illumina MiSeq PE300 platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China), and raw sequencing data were uploaded to the NCBI Sequence Read Archive (SRA) (Accession No.: PRJNA906308). Data processing: Reads were filtered by removing bases with tail quality values of less than 20 using a 50-bp window. If the average quality value within the window was less than 20, bases were trimmed from the back end starting from the window. Reads with lengths < 50 bp after quality control were filtered and those containing N bases were removed. Based on the extent of overlap between the paired-end reads, read pairs were merged into a sequence with a minimum overlap length of 10 bp. Sequences that exceeded the maximum overlap region mismatch ratio allowed (0.2) were removed. Samples were distinguished based on the barcodes at the head and tail ends of sequences and primers, and sequence directions were adjusted based on the number of permitted mismatches (allowable barcode mismatches: 0; maximum allowable primer mismatches: 2). Using the UPARSE software (http://drive5.com/uparse/, version 7.1, accessed on 20 June 2021) package, operational taxonomic unit (OTU) clustering was performed based on a 97% similarity threshold, and chimeric sequences were removed. Each sequence was subjected to species classification and annotation using the RDP classifier and compared against the SILVA 16S rRNA database (version 138), with the confidence threshold set at 70%. Chromatographic conditions: Chromatographic system: Agilent Technologies; chromatographic column: Agilent 5 TC-C18(2), 250 mm × 4.6 mm, 5 μm; flow rate: 1 mL/min; column temperature: 25 °C; injection volume: 10 μL; mobile phase: 0.3% acetic acid (A)-methanol (B) (0–22 min: 32% B, 22–23 min: 32–37% B, 23–36 min: 37% B, 36–37 min: 37–45% B, 37–46 min: 45% B, 46–47 min: 45–60% B, 47–60 min: 60–80% B); UV absorption of eriodictyol and eriodicty-7-O-glucoside was monitored at 284 nm, while monitoring of luteolin and luteolin-7-O-glucoside was at 350 nm.. Preparation of mixed control solution (S1): A mixed solution containing eriodictyol-7-O-glucoside (0.348 mg/mL), eriodictyol (0.200 mg/mL), luteolin-7-O glucoside (0.270 mg/mL), and luteolin (0.156 mg/mL) in methanol was prepared as the control. Preparation of test solution (S2): Dried MJGT was pulverized and passed through a 40-mesh sieve. A total of 1.7 g of MJGT was precisely weighed and placed in a 250-mL distillation flask. Then, 70% ethanol was added to the flask, which was securely stoppered and weighed. The contents of the flask were subjected to heat reflux extraction for 60 min and cooled to room temperature. Then, 70% ethanol was added to the flask to make up for the lost weight, and the contents of the flask were filtered through a 0.22-μm organic filtration membrane to obtain the test solution. All data are expressed as the mean ± standard error of the mean (SEM). SPSS version 26.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical comparison of data via one-way analysis of variance (ANOVA). Differences were considered statistically significant when p was less than 0.05. Lowercase and uppercase letters were used to denote the results of comparisons at significance levels of 0.05 and 0.01, respectively. All graphs were plotted using GraphPad Prism version 7.0 (GraphPad software, Inc., La Jolla, CA, USA). Figure 1A shows the process flow diagram for model construction and administration. There was a significant decrease in FWC in the MG group (before administration; p < 0.01; Figure 1B). However, rats administered MJGT_EE or MSP exhibited a significant recovery in FWC, with the recovered FWC in the MSP group being similar to that in the NC group (after administration). Visceral sensitivity changes constitute some of the most important pathophysiological characteristics of IBS patients. In this study, the smallest threshold CRD volume was adopted as a measure of visceral sensitivity in the different groups. A significant decrease in the smallest threshold CRD volume in the MG group was observed (before administration; p < 0.05; Figure 1C), indicating a significant increase in rat visceral sensitivity. After drug administration, the smallest threshold CRD volume was restored to normal levels in the MJGT_EE and MSP groups as compared to the MG group (after administration; Figure 1C). Previous studies have demonstrated that IBS-C patients exhibit delayed gastric emptying and small intestinal transit [45,46]. Therefore, these two indicators were selected and evaluated in our in vivo experiments. As compared to rats in the NC group, those in the MG group exhibited a significant decrease in gastric emptying and small intestinal propulsion rates (p < 0.01). MJGT_EE administration restored both gastric emptying and small intestinal propulsion rates to levels similar to those in the NC group (p < 0.01); the treatment effects of MJGT_EE were in line with those of MSP (Figure 2A,B). Rat colonic tissues were subjected to H&E staining for morphological evaluation. Clear structures were observed in the various colonic tissue layers. Mucosal epithelial cells exhibited a simple columnar structure, with cells and glands arranged in an orderly manner; in addition, normal colonic crypts were present. No significant inflammatory cell infiltration or pathological damage, interstitial hyperemia or edema, ulcerations, or organic lesions were observed. These findings indicated that the method used for model construction did not induce organic lesion development (Figure 3A,B), and that both the MJGT_EE (Figure 3C) and MSP (Figure 3D) treatments had no effects on rat tissue morphology. To determine whether MJGT_EE affects 5-HT expression, 5-HT accumulation in rat colonic tissues was evaluated with the immunohistochemical method. As shown in Figure 4, 5-HT was mainly distributed in the submucosal and muscular layers of the colon. Calculated AOD values indicated that colonic 5-HT secretion significantly increased in the MG group as compared to the NC group (p < 0.01; Figure 4A,B), suggesting that the model construction method induced an increase in colonic 5-HT levels. After MJGT_EE or MSP intervention, colonic 5-HT secretion significantly decreased in these groups as compared to the MG group (p < 0.01, Figure 4C,D). Therefore, MJGT_EE and MSP exhibited similar regulatory effects on 5-HT, and this is consistent with the results described above (Figure 4E). To further evaluate the cause of the decrease in 5-HT synthesis observed, western blotting was performed to quantitatively determine changes in the levels of tryptophan hydroxylases (TPHs), which are key enzymes of the 5-HT metabolic pathway. A significant increase in the expression levels of TPH1 and TPH2 in the colons of IBS-C rats was identified (MG group) (p < 0.01), suggesting that changes in 5-HT expression levels in the model were significantly associated with TPH synthesis (Figure 5A,B). Following treatment with MJGT_EE, the expression levels of both TPH1 and TPH2 decreased (p < 0.05, Figure 5A,B), and its effects were similar to those of the positive drug, MSP. Treatment with both MSP and MJGT_EE restored TPH expression to levels similar to those of the NC group (Figure 5A,B). 5-HT synthesized in the intestines can be transported by the SERT from the interstitial spaces of the lamina propria to the mucosal epithelial cells and presynaptic neurons. This process is known as 5-HT reuptake [47]. Thus, the SERT content is also a major factor that affects 5-HT expression. IBS-C negatively affected colonic SERT expression in rats in the MG group as compared to rats in the NC group (p < 0.01). Following drug administration (MJGT_EE and MSP), SERT expression levels were restored to levels similar to those observed in rats in the NC group at the 0.01 level (Figure 5C). Among the seven confirmed 5-HT receptor subtypes, 5-HT3R and 5-HT4R are significantly related to gastrointestinal motility and pain sensitivity. Therefore, these two indicators were evaluated to determine the effects of the drug. As compared to rats in the NC group, those in the MG group exhibited a significant decrease in 5-HT3R and 5-HT4R expression (p < 0.01). Both MSP and MJGT_EE restored 5-HT4R expression levels (p < 0.05, Figure 5D,E), with the expression levels in rats in the MSP group being higher than those in rats in the NC group (p < 0.01). However, a similar trend was not observed with 5-HT3R expression. The CaM-MLCK pathway is a classical downstream pathway of 5-HT4R that regulates smooth muscle contraction. As compared to rats in the NC group, those in the MG group exhibited significantly lower CaM (p < 0.05) and MLCK (p < 0.01) expression levels. Following treatment with MJGT_EE and MSP, both CaM and MLCK expression levels were restored to levels similar to those in rats in the NC group (Figure 5F,G). IBS-related pathophysiological changes involve alterations in gut microbiota composition or microbiota dysbiosis [48,49,50]. Therefore, 16S rDNAs of the gut microbiota in rats in the different treatment groups were analyzed. β diversity analysis revealed the existence of significant differences in the microbiota between rats in the MG and NC groups (Figure 6A). The number of OTUs decreased from 882 (NC) to 834 (MG) following model construction but increased following drug administration (MJGT_EE and MSP). Notably, the number of OTUs was highest following treatment with MJGT_EE (926) (Figure 6B). The ACE, Chao, Sobs, and Shannon α diversity indices also showed that the diversity of the gut microbiota in rats with IBS-C was lower than that in rats in the NC group; however, this diversity was restored following treatment with MJGT_EE or MSP (p > 0.05) (Figure S1). At the phylum level, bacteria of the phyla Firmicutes and Bacteroidota accounted for the greatest proportion of microbes, with their proportions in the NC, MG, MJGT_EE, and MSP groups being 68.95% and 13.49%, 78.57% and 7.94%, 76.59% and 11.00%, and 74.27% and 15.00%, respectively (Figure 7A). The Bacteroidetes/Firmicutes ratio in the MG group (0.10) decreased onefold as compared to that in the NC group (0.20). This is consistent with the previously reported significant increase in Firmicutes bacterial strain counts [51] and the decrease in the Bacteroidetes/Firmicutes ratio in IBS-C patients [52]. Following drug administration, the Bacteroidetes/Firmicutes ratio increased in the MJGT_EE group (0.14) but was still lower than that in the MSP group (0.20). At the family level, changes in the abundance of bacteria of the Prevotellaceae family in the different groups (0.80% and 0.43% in the NG and MG groups, respectively) followed a similar trend to those of bacteria of the Bacteroidota phylum, i.e., the restoration of bacterial abundance was more significant in the MSP group (1.75%) than in the MJGT_EE group (0.69%). The proportion of Clostridia_UCG-014 strains in the MG group (1.59%) decreased by 58.6% as compared to that in the NC group (3.75%); following drug administration, in the MJGT_EE group, this proportion (7.08%) was 4.45 and 2.79 times that in the MG and MSP groups, respectively. Linear discriminant effect size analysis (LFEse) revealed Clostridia_UCG-014 to be the only characteristic bacterium enriched in the MJGT_EE group (LDA > 4, Figure S2). There was an increase in the abundance of some bacterial families in IBS-C rats. For instance, the abundance of bacteria of the Lachnospiraceae family in the MG group (28.94%) was 1.64 times that in the NC group (17.61%); however, this increase was reversed following treatment with MJGT_EE (14.94%) and MSP (14.37%). The abundance of bacteria of the Corynebacteriaceae family ranged from 1.68% in the NC group to 2.16% in the MG group, but decreased to 0.92% following treatment with MJGT_EE. The proportions of some bacteria remained unchanged before and after model construction, but changed following drug treatment. For instance, bacteria of the Ruminococcaceae family accounted for 4.54% and 4.68% of the microbiota in rats in the NC and MG groups, respectively. Their abundance increased to 6.67% following treatment with MJGT_EE but decreased to 3.86% following treatment with MSP (Figure 7B). At the genus level, the proportion of Lactobacillus sp. decreased by approximately 76.08% in the MG group (1.11%) as compared to the NC group (4.64%). Their abundance was restored following the administration of MJGT_EE (6.19%) and MSP (4.04%), with MJGT_EE inducing a 0.33-fold increase in their abundance as compared to that in the NC group (Figure 7C). MJGT_EE was analyzed using high-performance liquid chromatography (HPLC). Mixed control (S1) and test (S2) solutions were prepared as described in Section 2.10 and analyzed under appropriate chromatographic conditions. Through a comparative analysis of retention time and ultraviolet (UV) spectra, four main chromatographic peaks, which represented luteolin-7-O-glucoside (Figure S3(1)), luteolin (Figure S3(2)), eriodictyol-7-O-glucoside (Figure S3(3)), and eriodictyol (Figure S3(4)), were identified in MJGT_EE. In IBS patients, gastrointestinal motility disorders are often accompanied by visceral hypersensitivity responses, which are exaggerated sensational responses to environmental stimuli, possibly induced by alterations in the processing of afferent signals from visceral neurons [53]. The IBS-C rat model can be constructed via water limitation, ice water stimulation, and the maternal separation plus ice water stimulation. The first two methods ignore the psychological factors in the pathogenesis of IBS-C, so the last one that is more closely related to the patient’s pathogenesis was chosen in the study. The chronic stress in newborn rats was applicated using maternal separation to induce stable changes in the central nervous system through the hypothalamic–pituitary–adrenal axis (HPA), as well as cognitive and emotional functions. This led to the gradual development of a disease state characterized by increased visceral sensitivity at the level of the large intestines after the maturation of the animals. Subsequently, to establish the IBS-C model, ice water at 4 °C was administered daily via gavage to the rats to induce symptoms of constipation. A comparison of the FWC and smallest threshold CRD volume between the MG and drug administration groups showed that drug administration not only reversed the significant decrease in FWC, but also decreased intestinal sensitivity in rats in the MG group. Therefore, MJGT_EE is able to reverse the decrease in FWC, as well as the increase in intestinal sensitivity, exhibited by IBS-C rats. Another characteristic symptom of IBS is the chronic disruption of normal gastrointestinal peristaltic activity [54], which mainly manifests as delayed gastric emptying and small intestinal transport [55]. Our experimental results showed that rats in the MG group exhibited delayed gastric emptying and decreased small intestinal propulsion functions, and these were significantly improved via treatment with MJGT_EE. This provides preliminary evidence of the effects of MJGT_EE in promoting gastrointestinal motility in IBS-C patients. Notably, this method used to construct IBS-C in the present study can only represent one of the psychological factors that cause the onset of IBS. Studies on the gastrointestinal motility-promoting mechanism of MJGT_EE have shown that it elicits this effect by inhibiting the secretion of 5-HT, which is a typical indicator of alterations in IBS patients. In addition, 5-HT plays a key role in the regulation of gastrointestinal motility, secretion, and sensation [56,57]. In this study, colonic tissue H&E staining revealed no inflammatory exudates nor pathomorphological changes. This is consistent with the findings of previous studies, which reported that IBS patients typically do not manifest organic lesions [58] and that IBS-C rarely induces inflammatory responses. To further evaluate the causes of alterations in the 5-HT signaling system, we investigated rate-limiting enzymes that directly affect 5-HT synthesis, i.e., TPH (TPH1 and TPH2) [59,60], as tryptophan (Trp) is a precursor of 5-HT [61] that is first converted to 5-hydroxytryptophan (5-HTP) under the action of TPH and is subsequently converted to 5-HT under the action of 5-HTP decarboxylase (5-HTPDC). TPH plays a vital role in the conversion of Trp to 5-HTP [62,63] and directly affects 5-HT secretion. Synthesized 5-HT, which is stored in enterochromaffin (EC) cells, is released into the lamina propria in response to luminal pressure, as well as chemical or mechanical stimuli, where it interacts with nerve endings and immune cells [64]. As with 5-HT release, its deactivation is of equal importance in maintaining dynamic equilibrium. The SERT plays an indispensable role in 5-HT deactivation as it transports 5-HT from the interstitial spaces of the lamina propria to the intestinal mucosal cells and presynaptic neurons and is subsequently involved in 5-HT degradation. Insufficient SERT synthesis leads to 5-HT accumulation [65]. This induces high contractility in the digestive tract smooth muscles, as well as high gland sensitivity and increased endocrine secretion, which result in diarrhea and pain. A significant increase in 5-HT levels in the MG group was observed as compared to the NC groups, and this was the possible cause of the abnormalities in gastrointestinal motility; however, treatment with MJGT_EE exerted a significant downregulatory effect on 5-HT levels. These findings are consistent with the trend observed in the changes in TPH expression in the different groups, demonstrating that MJGT_EE reduced 5-HT secretion by decreasing TPH synthesis. The decrease in SERT expression was identified in the MG group; however, this expression was upregulated following treatment with MJGT_EE, indicating that drug administration inhibited excessive 5-HT accumulation. Therefore, MJGT_EE evidently restores abnormally increased 5-HT levels by regulating TPH and SERT expression. MJGT_EE regulates gastrointestinal responses by regulating 5HT4R. 5-HT released from EC cells can regulate gastrointestinal motility by effectively activating 5-HT3R and 5-HT4R at the vagal afferent nerve endings of the intestinal mucosa. Consequently, the activation of these receptors enhances gastrointestinal transport [66,67]. Our findings indicated that the expression levels of both 5-HT3R and 5-HT4R were downregulated in IBS-C rats, and that their expression levels were negatively correlated with visceral sensitivity, and this is consistent with findings reported in the literature [68,69]. Interestingly, in a similar way as MSP, a selective 5-HT4R agonist, MJGT_EE only exerted restorative effects on 5-HT4R expression but did not affect 5-HT3R expression. This phenomenon could be explained by the fact that 5-HT released by EC cells can mediate digestive functions through the activation of endogenous or exogenous sensory nerve endings at high concentrations [70,71] and activate 5-HT4 or 5-HT1P receptors at a low concentrations, thereby regulating gastrointestinal motility [72,73]. MJGT_EE reduced 5-HT synthesis, inducing lower 5-HT luminal concentrations in the colon; this may explain the lack of effect of MJGT_EE on 5-HT3R secretion. The CaM-MLCK pathway is a core pathway through which 5-HT4R regulates downstream smooth muscle contraction. Following the activation of Ca2+ ion channels, free Ca2+ concentrations within cells rapidly increase, leading to the formation of Ca2+–CaM complexes. Consequently, MLCK is activated and induces the phosphorylation of the 19th serine residue on myosin light chain 20 (MLc20). This in turn activates myosin ATPase, which hydrolyzes ATP and coverts the chemical energy it contains to mechanical energy that enables myosin to slide past actin filaments and achieve smooth muscle contraction, ultimately resulting in the acceleration of intestinal peristalsis [74]. The findings of our study also indicate that changes in CaM and MLCK expression in the MG group before and after drug administration were positively correlated with 5-HT4R expression, further corroborating the effects of MJGT_EE. Intestinal microbes would also be involved in the gastrointestinal motility impacted by MJGT_EE. Several clinical studies indicated the variation of gut microbiota composition in IBS patients [75,76,77], and this might be a possible etiology for the disorder. In this study, we found that treatment with MJGT_EE led to the following. (1) Increase in gut microbiota diversity. The gut microbiota in IBS patients is significantly different from that in healthy individuals and is characterized by lower bacterial diversity [75,78]. We found that the number of OTUs in rats with IBS-C was 5.4% lower than that in normal rats (NC group), however, increased by 11% and 4.8% following treatment with MJGT_EE and MSP, respectively. This indicates that not only was MJGT_EE beneficial in increasing microbiota diversity, but it also elicited effects superior to those of MSP. (2) Increase in the number of bacteria with known benefit. MJGT_EE increased the counts of some beneficial bacteria such as those belonging to the Lactobacillus genus and the Prevotellaceae and Ruminococcaceae families. Lactobacillus sp. strains alleviate gastrointestinal diseases [79,80], reduce allergic symptoms [81], and are considered as potential antimicrobial probiotic strains against human pathogens [3,82] through various mechanisms. Bacteria of the Ruminococcaceae family can generate short-chain fatty acids (SCFA) [83], which are generally believed to elicit beneficial effects in the human body, such as improving intestinal health and protecting the intestinal mucosal barrier [84]. This increase in beneficial bacterial counts may be related to the MJGT_EE-induced decrease in 5-HT secretion, as 5-HT directly inhibits the growth of beneficial bacteria [52]. (3) Regulation of gut microbes involved in 5-HT synthesis. Studies have shown that Corynebacterium sp. strains promote 5-HT synthesis in tissues [85,86]; we observed an increase in the proportion of Corynebacterium sp. strains in rats in the MG group as compared with that in the rats in the NC group; in the MG group, this proportion decreased to approximately half the initial level following treatment with MJGT_EE. The proportions of bacteria of the Lachnospiraceae family, which induce 5-HT biosynthesis and release by EC cells [87], were also restored to levels similar to those in rats in the NC group following treatment with MJGT_EE and MSP. Notably, Clostridia_UCG-014 strains, which are beneficial bacteria associated with tryptophan metabolism and that regulate intestinal homeostasis, were only enriched in rats in the MJGT_EE treatment group [88]. Similar effects were observed for MSP, persuading us to speculate that Clostridia_UCG-014 strains may be key bacterial species that affected gastrointestinal motility in rats in the MJGT_EE group. Generally, an organism has a core native microbiota that remains relatively stable during adulthood. However, each individual has a unique gut flora profile due to a variety of factors such as gut type, body mass index (BMI) level, frequency of exercise, lifestyle, culture, and diet. Therefore, there is no one best gut microbiota composition [89]. For example, gut microbiota are not only able to respond to the various physiological activities of flavonoids, but also to metabolize them and produce new active products [90]. Through an HPLC analysis, four major MJBT_EE components were identified: eriodictyol-7-O-glucoside, luteolin-7-O-glucoside, eriodictyol, and luteolin. In the previous trial, we found that these four components are the active ingredients in hydro extracts of MJGT that affect gastrointestinal motility [42]. The regulation effect of luteolin and other flavonoids, such as apigenin and quercetin, on muscle tissue contraction in cows were also identified [41]. Thus, we believe that they should also be important components of MJBT_EE to alleviate the symptoms of IBS-C. Notably, whether there are other active ingredients needs to be further investigated. MJGT_EE promoted gastrointestinal motility and reduced intestinal sensitivity in IBS-C model rats established via maternal separation. These effects were mainly related to a decrease in 5-HT secretion and an upregulation in 5-HT4R expression and were not related to 5-HT3R expression. The possible mechanism underlying the effects of MJGT_EE on 5-HT secretion may involve the decrease and increase in TPH and SERT secretion, respectively, while underlying that its effects on 5-HT4R expression involve the upregulation of the CaM-MLCK pathway. MJGT_EE also increased microbiota diversity and beneficial bacterial counts, while restoring gut microbiota composition disturbed by IBS-C. Since the flavonoids are important active ingredients in MJGT_EE, the final observations would result from the complicated interactions among flavonoids and gut microbiota bioactivity and a wide range of their metabolites, which are worthy of further investigation.

PMC10000492

Ching-Hu Wu,Chien-Wei Feng,Chiu-Lin Wang,Zhi-Hong Wen,Cheng-Yu Long,Feng-Hsiang Tang

Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell

03-03-2023

zinc finger protein 90,ovarian cancer,cisplatin sensitivity,apoptosis,nuclear factor E2-related factor 2

Simple Summary Our data suggested that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic proteins expression (p-P38, p-ERK, p-Akt, Bcl-2, Bax, active caspase-3). The anti-oxidative signal (Nrf2, HO-1, SOD) was also activated, which could inhibit cell migration (MMP-2, MMP-9, E-cadherin). The intervention of Zfp90 could significantly enhance the apoptosis pathway and inhibit the migrative pathway to regulate the cisplatin sensitivity in ovarian cancer (OC) cells. Abstract Our study discussed the role of Zfp90 in ovarian cancer (OC) cell lines’ sensitivity to cisplatin. We used two OC cell lines, SK-OV-3 and ES-2, to evaluate their role in cisplatin sensitization. The protein levels of p-Akt, ERK, caspase 3, Bcl-2, Bax, E-cadherin, MMP-2, MMP-9 and other drug resistance-related molecules, including Nrf2/HO-1, were discovered in the SK-OV-3 and ES-2 cells. We also used a human ovarian surface epithelial cell to compare the effect of Zfp90. Our outcomes indicated that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic protein expression. The anti-oxidative signal was also stimulated, which could hinder cell migration. The intervention of Zfp90 could greatly improve the apoptosis pathway and block the migrative pathway to regulate the cisplatin sensitivity in the OC cells. This study implies that the loss of function of Zfp90 might promote cisplatin sensitization in OC cells via regulating the Nrf2/HO-1 pathway to enhance cell apoptosis and inhibit the migrative effect in both SK-OV-3 and ES-2 cells.

Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell Our data suggested that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic proteins expression (p-P38, p-ERK, p-Akt, Bcl-2, Bax, active caspase-3). The anti-oxidative signal (Nrf2, HO-1, SOD) was also activated, which could inhibit cell migration (MMP-2, MMP-9, E-cadherin). The intervention of Zfp90 could significantly enhance the apoptosis pathway and inhibit the migrative pathway to regulate the cisplatin sensitivity in ovarian cancer (OC) cells. Our study discussed the role of Zfp90 in ovarian cancer (OC) cell lines’ sensitivity to cisplatin. We used two OC cell lines, SK-OV-3 and ES-2, to evaluate their role in cisplatin sensitization. The protein levels of p-Akt, ERK, caspase 3, Bcl-2, Bax, E-cadherin, MMP-2, MMP-9 and other drug resistance-related molecules, including Nrf2/HO-1, were discovered in the SK-OV-3 and ES-2 cells. We also used a human ovarian surface epithelial cell to compare the effect of Zfp90. Our outcomes indicated that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic protein expression. The anti-oxidative signal was also stimulated, which could hinder cell migration. The intervention of Zfp90 could greatly improve the apoptosis pathway and block the migrative pathway to regulate the cisplatin sensitivity in the OC cells. This study implies that the loss of function of Zfp90 might promote cisplatin sensitization in OC cells via regulating the Nrf2/HO-1 pathway to enhance cell apoptosis and inhibit the migrative effect in both SK-OV-3 and ES-2 cells. Ovarian cancer (OC) is one of the most malignant gynecological tumors [1]. According to the Taiwan Ministry of Health and Welfare data, OC became eighth place in the occurrence of cancer in Taiwan in 2018. Its five year death rate of between 60 and 70% renders it one of the highest in all gynecologic cancers [2,3]. In addition, the ovary is found in the abdominal cavity’s depths, which is more inconducive for direct sampling pathological slices and presently lacks effective screening for the early detection of OC. Overall, 70% of OC patients have been detected with stage III or stage IV in recent years [4]. Debulking surgery followed by postoperative chemotherapy and radiotherapy is the standard golden treatment for OC. Cisplatin-based therapy is the first-line treatment for OC patients [5]. However, drug resistance has become a big concern for OC treatment [6]. Drug resistance and toxicities are often the main problems for OC chemotherapy [7]. The mechanisms implicated in anti-cancer drug resistance are often complex. However, some anti-oxidant cascade-induced chemoresistance have been frequently discussed [8,9]. Studies about inhibiting such resistance to cancer medication could offer more beneficial information about improving chemotherapy [10]. Prior studies have depicted that the anti-cancer effect influenced by cisplatin is through generating reactive oxygen species (ROS) or DNA double-strand breaks (DSBs) [11,12]. Thus, some studies have revealed that the mechanism of chemo-resistance from cancer cells influences the NF-E2-related factor 2 (Nrf2)/anti-oxidant response elements (ARE) pathway [13,14]. Some literature has demonstrated that the activation of the Nrf2/ARE pathway could protect cancer cells from cisplatin damage [15,16,17]. Thus, some research has also sought to inhibit this pathway to modulate the chemo-resistance effect [18,19]. To enhance the treatment rate of OC, there has been numerous research to develop a proper way to deal with cisplatin chemoresistance [20]. Some of these studies have aimed at realizing the pathobiology to expand the currently available treatments and develop tailored therapies. Some studies used genome-wide association studies (GWAS) to determine the possible genetic polymorphisms as predictors of OC clinical outcome [21,22,23]. There are more than 30 variants found by GWAS linked to OC susceptibility [24,25,26], such as BRCA1 and BRCA2, which are the most well-known pathogenic variants. Pathogenic variations in the BRCA1 and BRCA2 responsible for most OC syndromes have been reported in numerous ethnic groups [27]. Prior studies have also illustrated that women with harmful BRCA1 (39–44%) or BRCA2 (11–17%) variants will develop OC easily at 70–80 years old [28,29]. To further discover more possible candidate pathogenic variants, many studies have performed post-GWAS analysis [30,31]. Among them, numerous GWAS studies have highlighted that p-value < 5 × 10–8 acted as a significant index for identifying whether the variant has an impact or not. However, Chen et al., 2021 indicated that studies with sample sizes over 20,000 to 120,000 could relax the p-value threshold to 5 × 10−7 and increase the discovery of potential candidates [32], such as TREM2 variant rs75932628 in Alzheimer’s disease. The analysis of participants indicated a strong, highly substantial association with Alzheimer’s disease (p = 1.4 × 10−7; OR = 4.59; CI = 2.49–8.46) [33]. Other studies also further confirmed the feasibility of the strategies [34,35,36]. We therefore assessed the GWAS data and discovered Zinc finger protein 90 (Zfp90) (rs137866923) (p = 3 × 10 −7; OR = 3.82; CI = 2.29–6.36) as a potential target. Zinc finger proteins (Zfp) are a broad family of proteins characterized by the coordination of one or more zinc ions to regulate the fold. It performs various biological functions, including the development and differentiation of several tissues. A prior study demonstrated that Zfp90 regulated cardiac development [37]. In addition, some studies have also disclosed that Zfp90 played a critical role in initiating colitis-associated colorectal cancer through modulating the nuclear factor of stimulated T-cells and the cytoplasmic 2 (NFATC-2)/bone morphogenic protein-4 (BMP-4) pathway [38]. The team also expressed that Zfp90 facilitated the development of colitis-associated colorectal cancer via a microbiota-dependent strategy [39]. However, the role of Zfp90 in OC remains largely equivocal. Due to the data expressed in the GWAS, our study sought to evaluate the role of Zfp90 in OC and the impact of Zfp90 on cisplatin sensitization. We assessed Nrf-2-related molecules, including p-Nrf2 and HO-1; the SOD activity; the apoptotic pathway, including p-P38, p-Akt, p-ERK, Bcl-2, Bax and active caspase-3; and the migration pathway, including MMP-2 and MMP-9 to assert its role in OC treatment. The SK-OV-3 cell line was acquired from the American Type Culture Collection (No. HTB-77TM). SK-OV-3 and ES-2 were maintained with McCoy’s 5A medium (No. CC120-0500, GeneDireX Inc., Zhunan Township, Taiwan) (5% CO2; 37 °C). The cell numbers for each assay were presented as follows: cell viability assay: 2 × 103/well for 96-well microplates; 2 × 105/dish in a 6 cm culture dish containing coverslips (24 × 24 mm) for the transferase dUTP nick end labeling (TUNEL) staining and wound healing assay; 1 × 106/dish in a 10 cm culture dish for the Western blot analysis, SOD activity and Enzyme-linked immunosorbent assay (ELISA). The ES-2 cell line was obtained from the Bioresource Collection and Research Center (No. 60067). The cells were maintained with McCoy’s 5A medium (No. CC120-0500, GeneDireX Inc, Taiwan) (5% CO2; 37 °C). The cell numbers for each assay were presented as follows: cell viability assay: 2 × 103/well for 96-well microplates; 2 × 105/dish in a 6 cm culture dish for the TUNEL staining (with coverslips (24 × 24 mm)) and wound healing assay (without coverslips); 1 × 106/dish in a 10 cm culture dish for the Western blot analysis, SOD activity and ELISA. The normal cell line used in this study was human ovarian surface epithelial cells (HOSE), which were purchased from the ScienCell Research Laboratories (No. 7310, San Diego, CA, USA). The HOSE was maintained in an ovarian epithelial cell medium (OEpiCM) (No. 7311, San Diego, CA, USA). 2 × 104/well for 96-well microplates The cells were seeded in 10 cm dishes for the transfection of Zfp90 siRNA. The SK-OV-3 cells and ES-2 cells were transfected with Zfp90 siRNA (SASI_Hs01_0036-3148/ZFP90, SASI_Hs01_0017-3518/ZFP90, SASI_Hs01_0036-3519, Sigma, St. Louis, MO, USA), The normal control group were treated with the siRNA negative control (No. SIC001, Sigma, MI, USA). The transfections of siRNAs and the negative control were executed with DharmaFECT 1 Transfection Reagent (No. T-2001-03, Horizon Discovery, Cambridge, UK). The cultured cells were washed once with phosphor buffer saline (PBS) and replaced with normal McCoy’s 5A medium after transfection for 48 h. The cultured cells (si-Ctrl or si-Zfp90) were seeded in 96-well microplates and treated with 1, 10, 15 or 20 μM cisplatin (No. 15663-27-1, Sigma, MI, USA) for 24 or 48 h. Then, 10 μL alamarBlueTM (No. DAL1025, Invitrogen, Carlsbad, CA, USA) in each well was included and calculated by the ELISA reader (595 nM). The cell viability was calculated as 100 × [(optical density (OD) of treated cells − OD of blank-treated cells)/(OD of control cells − OD of blank-treated cells)]. The cells (si-Ctrl or si-Zfp90) in 6 cm dishes with coverslips were treated with 20 μM cisplatin for 24 h. The cells were washed with PBS and also fixed with 4% paraformaldehyde. Then, 3% H2O2 in methanol was used to inhibit and was stained with a TUNEL reaction mixture (No. 11684795910, Roche Diagnostics, Mannheim, Germany) for 1 h. The cells on the coverslips were washed by ddH2O and mounted by the mounting medium with DAPI (No. ab104139, abcam, Cambridge, UK). The apoptotic signal was detected by fluorescence microscopy (IX51, Olympus, Tokyo, Japan). The lysed cell extracts were controlled to an adequate concentration with a lysis buffer. Then, the mixture of sample buffer and cell extracts was loaded in a 10% SDS-polyacrylamide gel and the electrophoresis was run for 90 min at 100 volts. Then, a PVDF membrane was employed in the following transfer at 125 mA for overnight at 4 °C. Next, 5% non-fat milk in TTBS was blocked for 40 min at room temperature (RT), corresponding to the primary antibody for 24 h at 4 °C. After washing thrice in TTBS, the membrane was incubated with a secondary antibody for one hour at RT. Images were collected using the UVP BioChemi Imaging System, and the LabWorks 4.0 software (UVP) was employed to quantify the relative densitometry. The primary antibodies used in this study are listed as followed: ZFP90 (zinc finger protein 90; dilution 1:1000) (No. 26120-1-AP, Thermofisher, Waltham, MA, USA); β-actin (loading control; dilution1:1000) (No. A5441, Sigma, MI, USA); p-ERK (extracellular signal-related kinases; dilution 1:1500) (No. 9190, Cell Signaling Technology, Danvers, MA, USA); ERK (extracellular signal-related kinases; dilution 1:1000) (No. 9102, Cell Signaling Technology, USA); p-AKT (dilution 1:1000) (No. 9271, Cell Signaling Technology, USA); AKT (dilution 1:1000) (No. 4685, Cell Signaling Technology, USA); p-P38 (dilution 1:1000) (No. 9211, Cell Signaling Technology, USA); P38 (dilution 1:1000) (No. 9212, Cell Signaling Technology, USA); active caspase-3 (dilution 1:1000) (No. MA5-32015, Invitrogen, USA); Bcl-2 (dilution 1:2000) (No. ab59348, abcam, UK); Bax (dilution 1:2000) (No. ab32503, abcam, UK); MMP-2 (dilution 1:1000) (No. ab13132, abcam, UK); MMP-9 (dilution 1:1000) (No. AB19016, Sigma, MI, USA); E-cadherin (dilution 1:1000) (No. GTX100443, GeneTex, CA, USA); p-Nrf2 (dilution 1:1000) (No. PA5-67520, Invitrogen, USA); HO-1 (dilution 1:1000) (No. ab13248, abcam, UK) The SK-OV-3 cells (2 × 105/dish) or ES-2 cells (2 × 105/dish) were seeded in 6 cm dishes for 24 hr. We then utilized a 200mL pipette tip to inscribe a scratch wound. The cells (si-Ctrl or si-Zfp90) were treated with 20 μM cisplatin for 24 hr. Images of the scratched wounds were collected after treatment. The closing of the scratched wounds was regarded as the completion of the migration process. The migrated areas were assessed and identified using the ImageJ software. The SK-OV-3 cells or ES-2 cells (Si-Ctrl or Si-Zfp90) were held in 6-well microplates for 24 hr. Then, the cells were treated with or without 20 μM cisplatin for another 24 h in a serum-free medium. The matrix metalloproteinases (MMPs) and bone morphogenic protein-7 (BMP-7) excreted in the culture medium were quantified using the MMPs activity and BMP-7 ELISA kit following the manufacturer’s instructions (No. ab112146; ab99985, abcam, UK). The data were presented as mean ± SEM. The data were evaluated using one way analysis of variance (ANOVA), followed by Tukey’s test. A p value less than 0.05 was deemed statistically significant. The intensity of each band was indicated as the relative integrated density segmented by the average integrated density values from all of the internal controls in western blotting. To analyze the role Zfp90 played in cisplatin treatment, we transfected si-Zfp90 to suppress its expression in SK-OV-3 cells and ES-2 cells. The cytotoxic impact of multiple concentrations (1, 10, 15 and 20 μM) of cisplatin in the SK-OV-3 and ES-2 cells with or without si-Zfp90 were identified by an alamarBlueTM assay in 24 or 48 h treatment. Our data indicated that 10, 15 and 20 μM cisplatin could significantly inhibit SK-OV-3 cell viability (*, p < 0.05, versus si-Ctrl group) and the knockdown of Zfp90 significantly enhanced cisplatin-induced cytotoxicity in both the 24 h treatment (1, 10, 15 and 20 μM) (Figure 1A) and the 48 h treatment (1, 10, 15 and 20 μM) (Figure 1B) (#, p < 0.05, versus same concentration cisplatin in si-Ctrl group individually). We also conducted a TUNEL stain to determine the cell apoptosis. The apoptotic impact of 20 μM cisplatin in the SK-OV-3 and ES-2 cells with or without si-Zfp90 was executed after 24 h treatment. The data revealed that the treatment of 20 μM cisplatin could significantly up-regulate the TUNEL signal (from 1.0 ± 0.5 to 16.4 ± 1.2) (*, p < 0.05, versus si-Ctrl group) and si-Zfp90 alone did not influence the SK-OV-3 cell death. In addition, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the SK-OV-3 sensitivity to cisplatin (from 16.4 ± 1.2 to 26.7 ± 2.2) (Figure 1C) (#, p < 0.05, versus 20 μM cisplatin group). Furthermore, we also employed an ES-2 cell line to validate the result. Our data demonstrated that the knockdown of Zfp90 significantly enhanced the cisplatin-induced cytotoxicity in both the 24 h treatment (1, 10, 15 and 20 μM) (Figure 1D) and 48 h treatment (10, 15 and 20 μM) (Figure 1E) (#, p < 0.05, versus same concentration cisplatin in si-Ctrl group individually). We also revealed that the treatment of 20 μM cisplatin could significantly up-regulate the TUNEL signal (from 2.5 ± 1.4 to 17.2 ± 3.5) (*, p < 0.05, versus si-Ctrl group) and si-Zfp90 alone did not affect the ES-2 cell death. In addition, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the ES-2 sensitivity to cisplatin-induced damage (from 17.2 ± 3.5 to 32.3 ± 4.6) (Figure 1F) (#, p < 0.05, versus cisplatin group). We used the human ovarian surface epithelial cells (HOSE) as normal cells to compare with the two OC cell lines. Zfp90 protein expression was performed in the HOSE, SK-OV-3 and ES-2 cells (Figure 1G), original blot shown in Figure S1. The cytotoxic effect of the different concentrations (1, 10, 15 and 20 μM) of cisplatin in the HOSE cell with or without si-Zfp90 were performed for 24 h (Figure 1H). The data showed that the inhibition of Zfp90 did not affect the cisplatin-induced damage to the HOSE cell. We then evaluated the phosphorylation of p-P38, p-ERK, p-Akt and downstream apoptotic related protein expression to further determine the correlation between Zfp90 and cisplatin sensitization. The protein expression of 20 μM cisplatin in the SK-OV-3 and ES-2 cells with or without si-Zfp90 was identified by western blotting after 1 h treatment. Our data depicted that 20 μM cisplatin significantly increased the p-P38 protein expression and decreased the p-ERK and p-Akt expression (*, p < 0.05, versus si-Ctrl group). The knockdown of Zfp90 alone did not affect the p-P38, p-ERK and p-Akt protein expression (Figure 2A). Furthermore, the co-treatment of 20 μM cisplatin and si-Zfp90 did not affect the cisplatin-induced up-regulation of p-P38 (Figure 2B) and significantly inhibited the cisplatin-induced down-regulation of p-ERK (Figure 2C) and p-Akt (Figure 2D) (#, p < 0.05, versus cisplatin group), original blot shown Figure S2. In the ES-2 cells, the data showed some trend. The outcome indicated that 20 μM cisplatin significantly decreased the p-Akt protein expression and increased the p-P38 protein expression. The knockdown of Zfp90 decreased the p-Akt protein expression (Figure 2E). Moreover, the co-treatment of 20 μM cisplatin and si-Zfp90 only significantly enhanced the cisplatin-induced up-regulation of p-P38 (Figure 2F) (*, p < 0.05, versus si-Ctrl group) and did not affect the p-ERK (Figure 2G) and p-Akt expression (Figure 2D), original blot shown in Figure S3. Apoptotic proteins, such as active caspase-3, Bcl-2 or Bax, were then conducted. The apoptotic protein of 20 μM cisplatin in the SK-OV-3 and ES-2 cells with or without si-Zfp90 were determined by western blotting after 24 h treatment. The data highlighted that 20 μM cisplatin significantly increased the active-caspase-3 and Bax, and decreased the Bcl-2 protein expression (*, p < 0.05, versus si-Ctrl group). However, the knockdown of Zfp90 alone did not affect the active-caspase-3, Bax and Bcl-2 protein expression (Figure 3A). Furthermore, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the cisplatin-induced up-regulation of active-caspase-3 (Figure 3B) and Bax (Figure 3C), and inhibited the cisplatin-induced down-regulation of Bcl-2 protein expression (Figure 3D) (#, p < 0.05, versus cisplatin group), original blot shown Figure S4. In the ES-2 cell, the data also expressed the same trend. The outcomes implied that 20 μM cisplatin significantly elevated the active-caspase-3 and Bax, and decreased the Bcl-2 protein expression (*, p < 0.05, versus si-Ctrl group). The knockdown of Zfp90 did not affect the active-caspase-3, Bax and Bcl-2 protein expression (Figure 3E). Similarly, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the cisplatin-induced up-regulation active-caspase-3 (Figure 3F) and Bax (Figure 3G), and did not affect the Bcl-2 protein expression (Figure 3H) (#, p < 0.05, versus cisplatin group), original blot shown in Figure S5. Cell migration was assessed to determine the role of Zfp90 in OC. The anti-migrative effect of si-Zfp90 in the SK-OV-3 and ES-2 cells with or without 20 μM cisplatin were identified by a wound healing assay after 24 h treatment (Figure 4A). The quantitative outcome indicated that 20 μM cisplatin significantly decreased the wound recovery area, more than the si-Ctrl group (*, p < 0.05, versus si-Ctrl group), and the knockdown of Zfp90 alone did not impact the wound recovery area. However, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the cisplatin-induced anti-migrative effect (Figure 4B) (#, p < 0.05, versus cisplatin group). We also conducted matrix metalloproteinases (MMPs) activity and migrative-related protein expression to confirm the anti-migrative effect of inhibiting Zfp90 in the molecule level. The MMPs activity and protein expression of si-Zfp90 in the SK-OV-3 and ES-2 cells with or without 20 μM cisplatin were observed after 24 h treatment. Our data showed that 20 μM cisplatin significantly decreased the MMPs activity (from 100.6 ± 4.5 to 57.0 ± 5.6) (*, p < 0.05, versus si-Ctrl group) and the knockdown of Zfp90 alone did not affect the MMPs activity. Furthermore, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced down-regulation of the MMPs activity (from 57.0 ± 5.6 to 33.8 ± 6.6) (Figure 4C) (#, p < 0.05, versus cisplatin group). We also determined the migrative-related proteins, including MMP-2, MMP-9 and E-cadherin expression. Our data demonstrated that 20 μM cisplatin significantly decreased the MMP-2, MMP-9 and E-cadherin protein expression (*, p < 0.05, versus si-Ctrl group) and the knockdown of Zfp90 only affected the E-cadherin protein expression (Figure 4D) (*, p < 0.05, versus si-Ctrl group). The co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced down-regulation of MMP-2 (Figure 4E) and MMP-9 (Figure 4F), but did not affect the E-cadherin (Figure 4G) protein expression (#, p < 0.05, versus cisplatin group), original blot shown in Figure S6. In the ES-2 cells, we also detected the wound healing assay (Figure 4H). The quantitative result revealed that 20 μM cisplatin and the knockdown of Zfp90 mitigated the wound recovery area significantly more than the si-Ctrl group individually (*, p < 0.05, versus si-Ctrl group). The co-treatment of 20 μM cisplatin and si-Zfp90 significantly enhanced the cisplatin-induced anti-migrative effect (Figure 4I). We also performed MMPs activity and protein expression in the ES-2 cells. Our data showed that 20 μM cisplatin significantly decreased the MMPs activity (from 100.6 ± 4.5 to 65.2 ± 4.5) (*, p < 0.05, versus control group) and the knockdown of Zfp90 did not affect the MMPs activity. In addition, the co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced down-regulation of the MMPs activity (from 65.2 ± 4.5 to 40.5 ± 5.3) (Figure 4J) (#, p < 0.05, versus cisplatin group). In terms of protein expression, our data showed that 20 μM cisplatin significantly decreased the MMP-2, MMP-9 and E-cadherin protein expression, and the knockdown of Zfp90 affected the MMP-2 and E-cadherin protein expression (Figure 4K) (*, p < 0.05, versus si-Ctrl group). The co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced down-regulation of MMP-2 (Figure 4L), but did not affect the MMP-9 (Figure 4M) and E-cadherin (Figure 4N) protein expression. (#, p < 0.05, versus cisplatin group), original blot shown in Figure S7. The anti-oxidative stress pathway nuclear factor erythroid 2–related factor 2 (Nrf2)/heme-oxygenase-1 (HO-1) cascade has often been highlighted as being involved in the mechanism of drug resistance. We then tested the associated protein, such as p-Nrf2, HO-1 and bone morphogenic protein-7 (BMP-7). The anti-oxidative effect of si-Zfp90 in the SK-OV-3 and ES-2 cells with or without 20 μM cisplatin were determined by western blotting and ELISA after 24 h treatment. Our data indicated that 20 μM cisplatin significantly increased the p-Nrf2 and HO-1 protein expression (Figure 5A) (*, p < 0.05, versus si-Ctrl group) and the knockdown of Zfp90 alone did not affect the protein expression mentioned above. The co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced up-regulation of the p-Nrf2 (Figure 5B) and HO-1 (Figure 5C) protein expression. The cisplatin-induced up-regulation of the BMP-7 concentration was also inhibited (Figure 5D) (#, p < 0.05, versus cisplatin group), original blot shown in Figure S8. In the ES-2 cells, the data showed that 20 μM cisplatin significantly increased the p-Nrf2 and HO-1 protein expression (Figure 5E) (*, p < 0.05, versus si-Ctrl group) and the knockdown of Zfp90 alone did not affect the protein expression mentioned above. The co-treatment of 20 μM cisplatin and si-Zfp90 significantly inhibited the cisplatin-induced up-regulation of p-Nrf2 (Figure 5F) and HO-1 (Figure 5G) protein expression. The cisplatin-induced up-regulation of the BMP-7 concentration was also inhibited (Figure 5H) (#, p < 0.05, versus cisplatin group), original blot shown in Figure S9. We utilized the HO-1 inducer, carnosol, to revalidate the mechanism of action of Zfp90 in the cell viability and SOD activity. The cell viability of 12.5 and 25 μM carnosol in the SK-OV-3 and ES-2 cells with or without si-Zfp90 or cisplatin were determined by an alamarBlueTM assay in a 24 h treatment. Our data expressed that 12.5 and 25 μM carnosol both significantly reversed the si-Zfp90-induced down-regulation of the cell viability in the SK-OV-3 cells (Figure 6A) (and, p < 0.05, versus si-Zfp90 plus cisplatin group). In terms of the SOD activity, 12.5 and 25μM carnosol both significantly reversed the si-Zfp90-induced down-regulation of the SOD activity in the SK-OV-3 cells (Figure 6B) (and, p < 0.05, versus si-Zfp90 plus cisplatin group). In the ES-2 cells, the same trend was shown. The outcome implied that 12.5 and 25 μM carnosol both significantly reversed the si-Zfp90-induced down-regulation of the cell viability in the ES-2 cells (Figure 6C) (and, p < 0.05, versus si-Zfp90 plus cisplatin group). In addition, 12.5 and 25μM carnosol also significantly reversed the si-Zfp90-induced down-regulation of the SOD activity in the ES-2 cells (Figure 6D) (and, p < 0.05, versus si-Zfp90 plus cisplatin group). OC is an extensive gynecology disease in women and the major limitation of OC therapy is cisplatin resistance [40,41,42]. The statistical findings in the GWAS revealed that Zfp90 may play a crucial role in the modulation of drug resistance or tumorigenesis. To the best of our best knowledge, we are the first to investigate the role of Zfp90 in OC chemoresistance. This protein was originally discovered via the screening of zinc-finger-encoding genes [43]. It is known to exhibit inhibitory activity and possesses a zinc finger domain. Later, the literature indicated that Zfp90 contributed to obesity [44]. Hata et al., 2011 also demonstrated that Zfp90 inhibited the neuron-restrictive silencer factor (NRSF)-mediated transcriptional repression of fetal cardiac genes by inhibiting the NRSF binding to the neuron-restrictive silencer element (NRSE) [37]. The original study of the correlation between Zfp90 and cancer was conducted by Yim et al., 2006 [45]. The study indicated that the protein expression of Zfp90 significantly increased when 10μM cisplatin was treated in cervical carcinoma cells (HeLa). The same tendency was also found in the GWAS-related studies in OC [46,47]. However, no study has explored the mechanism of the Zfp90 effect in OC development or drug resistance. To the best of our best knowledge, we are the first to investigate the role of Zfp90 in OC chemoresistance. In the current study, we presented the enhanced effect of Zfp90 inhibition on cisplatin sensitization. We initially confirmed that the inhibition of Zfp90 substantially increases the sensitization of the SK-OV-3 and ES-2 OC cells to cisplatin, which was validated by the decreasing IC50 of cisplatin. Prior studies also validated that the knockout of Zfp90 could help modulate cancer development. Yu et al., 2020, indicated that Zfp90 was regulated by NFATC2, and the knockout of Zfp90 could significantly impact the colorectal cancer (CRC) malignant phenotype, including the CRC sphere formation and tumor formation potential. They also discovered the smaller tumor size and the decrease in the tumor number in Zfp90−/− mice. Furthermore, the modulation of Zfp90 impacted the BMP4 and some oncogenic related pathways in CRC mouse models and patients [38]. The same research team also discovered that Zfp90 might have played an important role in colitis-associated colorectal cancer (CAC) through the systemic analyses of the GWAS. They disclosed that gut microbiota depletion abolished the tumorigenic effect of Zfp90 in the CAC mouse model. The mechanistic studies indicated that Zfp90 elevated CAC development via the TLR4-PI3K-Akt-NF-κB pathway. This cascade facilitated an oncogenic environment and an innovative target for CAC prevention and treatment [39]. Our studies showed a similar trend in that the knockdown of Zfp90 could improve the sensitization of two OC cell lines, SK-OV-3 and ES-2, to cisplatin damage in terms of cell viability and the TUNEL stain (Figure 1). In addition, we also confirmed that the effect of the knockdown of Zfp90 in normal human ovarian surface epithelial (HOSE) cells did not cause a significant difference in the cell viability (Figure 1H). The HOSE cells were used as normal cells compared to the OC cell line in previous studies [48,49]. Xie et al., 2016, used human normal ovarian surface epithelial cells to compare the expression of MUS81 with the SOC tissues at both the transcript and protein levels, and the expression level of the MUS81 protein in ovarian cancer cell lines was also higher than that in human normal ovarian surface epithelial cell lines. The above result showed a similar trend as us. The low expression of Zfp90 might also explain the minimal effect the knockdown of Zfp90 had on the cell viability. Many studies have highlighted that one mechanism of chemo-resistance is alterations of the apoptotic signal, which facilitates cell death not only in OC, but also in different kinds of cancer [50,51,52]. Chowdhury et al., 2017, revealed that the binding of lectin with the receptors attributed to the phosphorylation of the Akt and ERK pathways, which also influence the downstream apoptosis signal, including Bcl-2 and Bax, is linked to the cytochrome c release and the generation of ROS in mitochondria that influence cell death [52]. In addition, Wang et al., 2017, revealed that pterostilbene, an analog of resveratrol, could prevent Akt-modulated cytoskeleton assembly and lung cancer cell metastasis. The apoptotic pathway was also implicated in the inhibition of treating lung metastasis in the review [53]. Furthermore, Yu et al., 2018, indicated that the knockdown of long non-coding (lnc) RNA HOTAIR could aid OC cells’ sensitization to cisplatin via the stimulation of the autophagy. They observed that the transfection of si-Atg7 substantially enhanced the cisplatin-induced cytotoxic signal, including caspase-3 and Bax, and inhibited the anti-apoptosis molecule Bcl-2 in OC cells [54]. Our analysis also revealed a similar trend in Zfp90. The inhibition of Zfp90 alone did not impact the apoptosis-related protein, such as p-P38, p-Akt and p-ERK. However, it enhanced the cisplatin-induced up-regulation of p-P38 and the down-regulation of p-Akt and p-ERK in the SK-OV-3 and ES-2 OC cells (Figure 2), which induced the downstream proteins Bcl-2, Bax and active caspase-3. The elevation of Bax and active caspase-3 and of the decrease in Bcl-2 induced by cisplatin were also enhanced by the knockdown of Zfp90 (Figure 3). Except in case of the apoptosis cascade, the migrative effect was extensively investigated. Almost 90% of cancer deaths are attributed to cancer metastasis and damage of secondary tumors [55]. To treat OC more effectively, some analyses have focused on the inhibition of metastatic carcinoma cells and took metastasis as being crucial for cell proliferation. Qian et al., 2021, demonstrated that the kinesin family member 18A (KIF18A) was overexpressed in esophageal cancer (EC) patients, and the modulation of KIF18A could impact cancer cell migration and invasion in the EC cell lines. They also knocked-down si-KIF18A and activated the Insulin-like growth factor-II mRNA binding protein 3 (IGF2BP3) to reconfirm the role of the KIF18A function in cell movement [56]. In addition, Zhang et al., 2019, also indicated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) demethylase (KDM1A) as metastasis promoters in papillary thyroid cancer. They revealed that KDM1A could increase the MMP-9 expression and activity via binding to the active site of the tissue inhibitors of metalloproteinases-1 (TIMP-1). These effects influenced the decrease in the migrative effect in the wound healing assay and invasion assay [57]. Furthermore, Si et al., 2020, expressed that the knockdown of cell adhesion molecules-1 (CADM1) influenced the growth, migration and invasion of OC cells. They also overexpressed CADM1 in OC cells and observed an increase in the cell growth and movements via a wound healing assay. The mechanism of action in CADM1 passed through the PI3K/Akt signaling pathway, which is comparable to our results [58]. We conducted a wound healing assay in SK-OV-3 and ES-2 cells to evaluate the effect of Zfp90 knockdown on cell migration. The data indicated that the inhibition of Zfp90 could further decrease the cisplatin-induced down-regulation of the migrative effect in both cells. The MMPs activity also expressed the same trend. The inhibition of Zfp90 could substantially reduce the cisplatin-induced down-regulation of the MMPs activity (Figure 4). Some related protein expressions, including MMP-2, MMP-9 and E-cadherin, were also conducted. The cisplatin-induced down-regulation of MMP-2, MMP-9 and E-cadherin were greatly suppressed by the knockdown of Zfp90 (Figure 4). Although cisplatin played a significant role in the OC treatment, the issue of drug resistance in OC still requires attention. Drug resistance is always a critical issue in OC treatment [59,60]. Among all the related pathways, the Nrf2/HO-1 signaling pathway has been explored significantly in cisplatin sensitization [61,62,63]. Deng et al., 2020, revealed that the Nrf2/HO-1 pathway was determined as a drug resistance mechanism in SK-OV-3 cells, and the peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is implicated in the regulation of Nrf2 via increasing the p-GSK3β and p-GSK3β, which would conversely modulate the transcriptional activity of PGC1α. They demonstrated that the intervention of Nrf2 or PGC1α led to the enhancement of cisplatin sensitization in SK-OV-3 and A2780 cell lines [62]. Li et al., 2021, outlined some factors that react to Nrf2 and cause resistance to cisplatin, including P62, CD99, ABCF2 and ATF2 [17]. Xia et al., 2020, indicated that the overexpression of p62 in SK-OV-3 could shield the cell against vitamin K3-induced damage via an increase in the anti-oxidant genes, such as Nrf2, and the downstream factors, including HO-1 and NQO-1 [64]. Bao et al., 2017, revealed that ABCF2 was impacted by the low expression of Nrf2, which influenced the upregulation of the cisplatin sensitization of A2780 by regulating the drug efflux pump [65]. In our studies, we propose that Zfp90-regulated cisplatin sensitization in SK-OV-3 and ES-2 cells might occur through the Nrf2/HO-1 pathway. The up-regulation of Nrf2 and HO-1 protein expression was significantly mitigated by the knockdown of Zfp90. The upstream factor BMP-7 was also conducted by an ELISA, and the up-regulation of the BMP-7 concentration was greatly inhibited by the knockdown of Zfp90 (Figure 5). Conversely, we validate the association between Zfp90 and the Nrf2/HO-1 pathway via treating carnosol, a facilitator of HO-1. The data expressed that the treatment of carnosol could greatly abolish the si-Zfp90-induced enhancement of the cytotoxic effect. The SOD activity was also evaluated, and its trend is comparable to the cell viability (Figure 6). According to the results of our study, Zfp90 was shown to be a potential target to deal with cisplatin chemoresistance in OC via oxidative stress and the apoptotic pathway. However, further in vivo tests should be conducted to examine its role in OC chemoresistance. The data from the GWAS also showed a great difference between OC patients and normal people in terms of Zfp90 expression. We hope the above findings could help the development of target strategies employing Zfp90 siRNAs to complement the conventional chemotherapies for advanced OC. In conclusion, our data highlight that cisplatin treatment produces ROS that modulate apoptotic proteins expression (p-P38, p-ERK, p-Akt, Bcl-2, Bax and active caspase-3). The anti-oxidative signal (Nrf2, HO-1 and SOD) was also stimulated, which could inhibit cell migration (MMP-2, MMP-9, E-cadherin). The intervention of Zfp90 could substantially enhance the apoptosis pathway and inhibit the migrative pathway to regulate the cisplatin sensitivity in OC cells.

PMC10000495

Praewa Suthapot,Wararat Chiangjong,Parunya Chaiyawat,Pongsakorn Choochuen,Dumnoensun Pruksakorn,Surasak Sangkhathat,Suradej Hongeng,Usanarat Anurathapan,Somchai Chutipongtanate

Genomics-Driven Precision Medicine in Pediatric Solid Tumors

23-02-2023

precision medicine,pediatric solid tumor,actionable mutations

Simple Summary The detection of genomic aberrations in cancers has yielded a wealth of information to discover oncogenic drivers or pathogenic variants that are relevant for the development of precise treatment strategies. Recent studies have shown promising outcomes in adult cancer patients with well characterized cancer genetic biomarkers. However, the development of precise treatments for pediatric cancers is difficult due to the limited number of accessible samples and the fact that well-defined target genetic aberrations are limited. Here, we review the current landscape of pediatric precision oncology compared to adults and highlight the examples of single-arm and multiple-arm designs of pediatric precision treatments. Abstract Over the past decades, several study programs have conducted genetic testing in cancer patients to identify potential genetic targets for the development of precision therapeutic strategies. These biomarker-driven trials have demonstrated improved clinical outcomes and progression-free survival rates in various types of cancers, especially for adult malignancies. However, similar progress in pediatric cancers has been slow due to their distinguished mutation profiles compared to adults and the low frequency of recurrent genomic alterations. Recently, increased efforts to develop precision medicine for childhood malignancies have led to the identification of genomic alterations and transcriptomic profiles of pediatric patients which presents promising opportunities to study rare and difficult-to-access neoplasms. This review summarizes the current state of known and potential genetic markers for pediatric solid tumors and provides perspectives on precise therapeutic strategies that warrant further investigations.

Genomics-Driven Precision Medicine in Pediatric Solid Tumors The detection of genomic aberrations in cancers has yielded a wealth of information to discover oncogenic drivers or pathogenic variants that are relevant for the development of precise treatment strategies. Recent studies have shown promising outcomes in adult cancer patients with well characterized cancer genetic biomarkers. However, the development of precise treatments for pediatric cancers is difficult due to the limited number of accessible samples and the fact that well-defined target genetic aberrations are limited. Here, we review the current landscape of pediatric precision oncology compared to adults and highlight the examples of single-arm and multiple-arm designs of pediatric precision treatments. Over the past decades, several study programs have conducted genetic testing in cancer patients to identify potential genetic targets for the development of precision therapeutic strategies. These biomarker-driven trials have demonstrated improved clinical outcomes and progression-free survival rates in various types of cancers, especially for adult malignancies. However, similar progress in pediatric cancers has been slow due to their distinguished mutation profiles compared to adults and the low frequency of recurrent genomic alterations. Recently, increased efforts to develop precision medicine for childhood malignancies have led to the identification of genomic alterations and transcriptomic profiles of pediatric patients which presents promising opportunities to study rare and difficult-to-access neoplasms. This review summarizes the current state of known and potential genetic markers for pediatric solid tumors and provides perspectives on precise therapeutic strategies that warrant further investigations. Cancer occurrence before the age of 20 years is rare, but it is one of the leading causes of disease-related mortality in children and adolescents globally [1,2]. Approximately 300,000 children aged 0–19 years old worldwide are diagnosed with cancer each year [1], and 80% of these patients live in low- and middle-income countries (LMCs). Hematologic malignancies are more common among pediatric cancers, comprising about half of all cases. Solid malignancies are rarer and heterogenous as following an age-specific pattern. In early childhood, embryonal-type solid tumors are common, such as neuroblastoma, retinoblastoma, medulloblastoma, hepatoblastoma, and Wilms tumor [3]. The prognosis for childhood cancer has improved dramatically over the past four decades, particularly for hematologic malignancies [2]. Nonetheless, treatment outcomes for childhood solid malignancies remain unsatisfactory, especially in LMCs [4,5]. Genetic sequencing studies have led to the identification of somatic gene alterations as cancer hallmarks and germline predisposition and targeted the molecular abnormalities for the development of precise treatment [6,7,8]. Dramatic differences in the genetic repertoire between normal and cancer cells provide advantages of molecular targeted therapies over traditional strategies based on the target selectivity [9,10,11]. Several components in cellular signaling pathways, i.e., tyrosine receptor kinase (TRK), mitogen-activating protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K)-mammalian target of rapamycin (mTOR), have been commonly identified as actionable mutations that would recommend appropriately targeted therapies [12,13]. These generic biomarker-driven precise treatments have been investigated in several pre-clinical and clinical trials since the early 2000s [14]. Progress in designing treatments targeting molecular alterations specific to pediatric cancers is considerably slow due to the rare and unique genetic alterations in children compared to adults [15]. A report from the European Union (E.U.) revealed that up to 26 anticancer drugs approved for adults might be also effective in pediatric malignancies; however, only four of these drugs have been approved for childhood cancers [16]. Nishiwaki S. and Ando Y. reported that only 3 out of 66 drugs with adult indications have been approved for pediatrics in the E.U., United States, and Japan [17]. Thus far, larotrectinib and entrectinib have been two of the most successful molecularly targeted therapies for children with solid tumors and have shown their promising responses in patients with NTRK-fusion [9]. In 2018, larotrectinib became the first drug to receive FDA approval to treat NTRK fusion-positive solid tumors in children and adults [18]. Similarly, entrectinib, a multi-kinase inhibitor, also received approval for the treatment of TRK fusion solid tumors in patients aged ≥ 12 years [19]. Combinatorial treatment of dabrafenib and trametinib has been recently approved by FDA (June 2022) for use in adult and pediatric patients > 6 years of age with unresectable or metastatic solid tumors with BRAF V600E mutation [New Drug Application (NDA): 202806 and 204114]. Note that abnormalities in NRAS, ABL1, JAK2, KIT, ALK and BRAF were among the group of common genetic variants found in adult and childhood cancers. In this review, we summarize the progress in the identification of actionable mutations in pediatric malignancies, FDA-approval status for pediatric and childhood treatment, and the recent update from clinical studies to explore the feasibility and utility of genomics-driven precision medicine. Cancers are driven by changes in cellular DNA which further promote the transition of genetic landscape, especially in cell survival programs, leading to unstoppable cell growth with abnormal cellular characteristics [20]. In contrast to normal tissues, cancer cells can dysregulate their own signaling cascades autonomously, thus controlling their own cell fate [21]. Besides their proficiency in cancer hallmarks in evading growth suppressors, resisting cell death, reprogramming cellular mechanisms, and avoiding immune destruction, cancer cells can also acquire the capability to sustain proliferative signaling in several alternative ways [22,23]. Cancer cells may send signals to activate normal cells within the tumor parenchyma, which reciprocally communicate to supply cancer cells with various growth-promoting factors [24,25]. Furthermore, common downstream components in distinct signaling cascades also allowed cancer cells to control cell fate in a growth factor-independent manner by triggering the downstream molecules directly, negating the need for ligand-mediated receptor activation [23,26]. Hence, the vast majority of different cancers are coordinately modulated by canonical oncogenic drivers, including KRAS, MYC, NOTCH, and TP53. This factors highlights the need to fully elucidate their regulatory networks for further therapeutic development [27]. Cancer gene mutations can be either inherited or acquired. Hereditary or germline mutations refer to the genomic changes that occur in germ cells and can be detected in all cells of the offspring and are passed inter-generationally [28,29]. Genetic predisposition has been described by certain characteristics, including [30]; Familial history of the same or related cancers; Occurrence of bilateral or multifocal cancers; Earlier age at disease onset; Physical suggestive of a predisposition syndrome; Appearance of specific tumor types corresponding to the genetic predisposition. Several studies have described germline mutations in cancer including BRCA1/2, TP53, ATM, CHEK2, MSH2 and PALB2 [31,32,33]. Cancer cells harboring these germline predispositions are prone to increase cancer susceptibility, developing cancers at younger ages than usual. Using the 565 cancer-predisposing gene (CPG) panel for germline mutation analysis in children and adolescents with pan-cancer (n = 1120), Zhang et al. [31] reported that 95 pathogenic variants were detected in 21 of the 60 autosomal dominant CPGs in 94/1120 patients. Interestingly, the prevalence of germline mutation was greatest among patients with non-CNS solid tumors (16.7%), followed by brain tumors (8.6%) and leukemia (4.4%) [31]. Genetic predisposition syndromes associated with rare cancers of pediatric solid malignancies are provided in Table 1 [34,35,36]. Cancer predisposition syndrome such as Li–Fraumeni syndrome (LFS) with TP53 mutation generally promotes the onset of various benign and malignant neoplasms, such as neuroblastoma (NB), osteosarcoma (OS), soft tissue sarcomas (STS), and brain tumors [37]. Mutations in NF1 are associated with neurofibromatosis (NF), low- and high-grade gliomas (L/HGGs), and malignant peripheral nerve sheath tumors. Mutations in SUFU or PTCH1 in Nevoid basal cell carcinoma are relevant to the development of the sonic hedgehog (SHH) subgroup-medulloblastoma (MB) [38]. Somatic mutations are de novo genetic alterations that spontaneously develop in an individual cell over time and play a vital role in cancer development and progression [51]. Studies have shown that the number of genetic abnormalities identified in each cancer patient may increase over time, leading to tumor survival against the selective pressure of drug actions, thereby acquiring resistance and causing disease progression [13,52]. Commonly identified somatic mutations include those involved in RTK signaling (PDFGRA, ERBB2 and EGFR), MAPK signaling (NF1, KRAS, and MAP2K1), PI3K-mTOR signaling (PIK3CA, MTORC1/2 and PTEN), cell cycle (CDKN2A/B, RB1 and ATM), DNA maintenance (TP53), transcriptional regulators (MYC and MYCN), and epigenetic modifiers (SMARCB1 and ATRX) [12,53]. Cancers usually involve a different spectrum of mutation which are strongly associated with pathogenesis and disease prognosis. A pan-cancer analysis reported by Grobner et al. [33] showed that 93% of adult cancer patients harbor at least one significantly mutated gene, while only 47% presented such mutations in pediatric tumors. However, approximately 30% of recurrent hot-spot mutations in pediatrics overlapped with adult cancers, highlighting some potential druggable targets based on finding from adult cancers. Hence, advances in identifying and understanding oncogenic drivers and actionable mutations would further improve the current therapeutic strategies for the development of precision medicine in cancers. In the context of defining mutational actionability, the relevant effects of genomic aberration participating in cancer phenotypes are considered. DNA aberrations include missense, nonsense, frameshift mutations, and chromosome rearrangements, with some changes affecting only a single DNA base that may or may not alter the protein’s property and some point mutations completely abrogating protein expression. A wide variety of gene alterations have been detected such as activating point mutation in BRAF, ALK, EGFR and FGFR1 genes, high copy number gains in PDGFRA and ERBB2, loss-of-function mutation affecting PTEN, PTPN11, PIK3R1, and MTORC1, CDKN2A/2B deletions, or in-frame expression of large indels (NOTCH1 and FOXA1) [12]. Other changes involving larger stretches of DNA may include rearrangements, deletions, or duplications of long stretches of DNA [54]. For example, exon skipping on MET exon 14 proto-oncogenes resulting from intronic mutation increases the protein lifespan and promotes MET activation in lung carcinogenesis [55]. The significance of genetic variants may vary depending upon their potential effects on cellular functions. An “actionable” mutation is defined as a genetic aberration that is potentially responsive to targeted therapy, while a “driver” mutation refers to variants that confer a growth advantage to cancer cells but may not be targetable with a specific treatment yet. Passenger mutation is used to designate cancer-neutral variations and is unlikely to be under selective pressure during the evolution of the cancerous cells [56,57]. The “passenger” mutation has the lowest tendency to impact protein function, most of which are synonymous substitutions; however, these mutations occur more frequently than driver or actionable mutations. Unraveling the passenger mutational paradigm has otherwise revealed the existence of pre-existing latent driver mutations in which certain combinations of the passenger mutations could indeed be functional drivers. One example is the non-hotspot, passenger mutation of the Akt1 gene at position L52R, C77F, and Q79K, which promotes its membrane localization similarly to the E17K driver. In contrast, the co-existence of D32Y, K39N, and P42T passenger mutations can lead to Akt conformational inactivation, suggesting that treatment decisions based only on genetics may overlook crucial actionable components [56,58]. In addition, silent mutations occurring near the donor splice junction could contrarily affect exon splicing. For example, T125T mutation in TP53 is a recurrent mutation that is generally considered a non-functional passenger event; however, its existence at the −1 donor site of exon 4 raises the possibility that this mutation affects splicing. Further integration with RNA-seq data demonstrated that T125T mutation resulted in the retention of intron 4 and introduced a premature stop codon such as nonsense-mediated decay [59]. Thus, aberrant splicing caused by silent mutations should be carefully evaluated during interpretation of the sequencing results. The accumulated data of genetic composition data from the tumors of patients has become a growing compendium of molecular biomarkers for precise treatment with FDA-approved drugs. Figure 1 summarizes the actionable mutations currently approved by FDA consortium for targeted therapy in adult cancers and pediatric solid tumors. Common actionable genetic aberrations associated with the National Comprehensive Cancer Network (NCCN) guidelines or FDA-approved targeted therapies are extensively summarized in Table 2. The data were predominantly gathered from the OncoKB database and the representative cancer types, and levels of evidence were included [60]. Pediatric cancers reflect a heterogeneous group of disorders distinct from adult cancers in terms of cellular origins, genetic complexity, and specific driver alterations [62,63]. Pediatric malignancies typically occur in developing mesoderm rather than adult epithelia (ectoderm) and are often induced by inherited or sporadic errors during development [33]. Studies have quantified the mutation burden in many pediatric cancers, identifying approximately 5 to 10 protein-coding variants identified across multiple tumor types except in osteosarcoma, which showed an average of 25 protein-affecting mutations. In contrast, the average number of mutations in adult cancers ranges between 33 to 66 in pancreatic, colon, breast, and brain cancers while mutagen-caused adult tumors (such as melanoma and lung cancers) can include up to 200 protein-coding variants [64,65,66]. At diagnosis, patients with pediatric cancers tend to have less complexity on mutational spectra than those in adult cancers; however, with treatment-refractory tumors and recurrence—the mutation rates in pediatric tumors have increased to be comparable to adult tumors [67,68]. Moreover, the rare occurrence of pediatric cancers and the low frequency of recurrent genomic alterations have a great impact on the investigations and the availability of targeted agents. Thus, there is an urgent need to accelerate the pace of genomic data acquisition and clinical trials in children to design more effective strategies for pediatric precision oncology. Single nucleotide variations (SNVs) and small indels are the usual mutations identified in adult cancers. In contrast, childhood cancers show a relatively high prevalence of copy number aberrations (CNAs) and specific structural variations (SVs). Note that insertion and deletion lead to adding and removing at least one nucleotide to the gene, respectively, which can affect protein functions and contribute to carcinogenesis. Current data suggest that approximately 10% of pediatric cancers are caused by genetic predisposition [32]. Zhang et al. [31] revealed that 95 out of 1120 (8.5%) patients younger than 20 years of age harbor germline mutations in cancer-predisposing genes. Diets et al. [69] performed trio-based whole-exome sequencing on the germline DNA of 40 selected children with cancer and their parents. Of these, germline pathogenic mutations were identified in 20% (8/40) of children with cancer [69]. Similarly, Grobner et al. [33] reported that most germline variants were related to DNA repair genes from mismatch (MSH2, MSH6, PMS2) and double-stranded break (TP53, BRCA2, CHEK2) repair. Using combined somatic and germline sequencing for children with solid tumors, Parsons et al. [32] identified actionable mutations in up to 40% (47/121) of pediatric solid tumor tissues. Likewise, Wong et al. [12] performed the combination of tumor and germline sequencing (WGS) and RNA sequencing (RNA-seq) to identify 968 reportable molecular aberrations (39.9% in both WGS and RNA-seq; 35.1% in WGS only and 25.0% in RNA-seq only) in 247 high-risk pediatric cancer patients with 252 tumor tissues. Interestingly, 93.7% of these patients had at least one germline or somatic aberration, 71.4% had therapeutic targets, and 5.2% had a change in diagnosis [12]. These cohort studies emphasized that comprehensive molecular profiling could resolve molecular aberration in high-risk pediatric cancer and provide clinical benefits in a significant number of patients. In the era of next-generation sequencing, publicly genomic data access is considered one of the keys to accelerate research. The St. Jude Cloud is one of the most promising data-sharing ecosystems, with genomic data from >10,000 pediatric patients with cancer and long-term survivors. When exploring the mutational profile of pediatric solid tumors, the resource has revealed common genetic alterations among the different cancer types, as shown in Table 3. This integrative view of genomic data could be further used to expedite studies of pediatric cancer-associated risk factors and initiate novel therapeutic investigations for improving treatment outcomes. The reports of actionable mutations identified in various studies have ranged from 27% to 100%, depending on the study design [6]. Several methods have been adopted for comprehensive molecular analysis to discover the actionable mutations that result in the targeting of cancer-associated elements. Table 4 contains a comprehensive, up-to-date summary of genomic aberrations found in pediatric solid tumors, together with potential targeted treatments, based on several public databases [60,70,71,72,73]. We systemically reviewed genomic alterations with high prevalence in pediatric cancers using comprehensive WES and RNA-seq data via the St. Jude Cloud (www.stjude.cloud; accessed on 26 September 2022) [70]. Importantly, the genomic point mutations and gene fusions reported by this public domain are unique and different from those variants identified in the OncoKB database (the mutational collection of adult cancers) [60]. In addition, the potential druggable targets of these significant genomic alterations required further testing in pediatric solid tumor patients. A significant number of studies [60,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85] were reported by the Clinical Interpretation of Variants in Cancer (CIViC) database (https://civicdb.org; accessed on 18 September 2022) [71] which matched genomic alteration and molecularly targeted therapies tested in pediatric patients. These treatment designs were translated from the clinical care of adults across different tumor types but harboring the same genetic dysregulation, which gave satisfactory clinical outcomes. For pediatric solid tumors with no clinical evident support or undruggable genomic alterations, we listed the potential targeted therapies based on the knowledge from adult cancers as suggested by cBioPortal (www.cbioportal.org; accessed on 30 April 2022) [72,73] and OncoKB (https://www.oncokb.org; accessed on 17 April 2022) [60] that should be considered for further investigation and optimization for pediatric treatments. As of now, fewer number of patients could hinder the availability of molecular characterization and statistically meaningful preclinical/clinical outcomes. However, this challenge can be overcome by the initiation of multi-institutional cooperation and international data sharing, which would enable clinicians to effectively explore optimized therapeutic interventions toward pediatric precision oncology. Genomic precision medicine has demonstrated preferential outcomes among ongoing genomic-driven clinical trials in adult cancers. Yet, clinical investigations based on pediatric tumor genetics are still lacking. Based on the patient genetic profile screening, scattered reports on molecularly defined pediatric patients are showing prominent responses to some targeted therapies. For example, targeting ALK has shown success in treatments of ALK(+) non-small cell lung cancers and also in childhood anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor using the ALK inhibitor crizotinib [92]. While ALK mutation is the most common somatic mutation in neuroblastoma, crizotinib was compromised due to the interference by common ALK mutation F1174 [93]. Since then, ceritinib, alectinib, brigatinib, and lorlatinib have been approved against advanced ALK+ NSCLC [94,95,96,97]. Intriguingly, the third-generation TKI that targets both ALK and ROS1, lorlatinib, has recently shown promise in patients with ALK mutated neuroblastoma, but most of the studies are still at phase I clinical trial. [98]. Nonetheless, repotrectinib, a next-generation ROS1/TRK inhibitor with >90-fold potency against ROS1 than crizotinib in NSCLC patients is also being tested for dose escalation in phase II clinical trial with patients aged ≥ 12 years [99]. Another promising example is the targeted therapy against Ras-Raf-MEK-ERK signaling cascade which include somatic BRAF alterations (BRAF V600E and BRAF fusions). The prototype for targeting BRAF V600E/K is cutaneous melanoma, where 40–60% of patients with these mutations are eligible for the FDA-approved BRAF-inhibitor, vemurafenib [100]. Low-grade-gliomas have been identified to contain multiple alterations in Ras-Raf-MEK-ERK pathway, and a single treatment of vemurafenib in malignant glioma resulted in tumor regression [85,101]. Recently, Jain et al. [102] reported that a combination of BRAF-inhibitor dabrafenib and MEK-inhibitor trametinib enhanced treatment efficacies in pediatric low-grade-glioma carrying KIAA1549-BRAF fusion. Additionally, several studies have utilized the combination of molecularly targeted agents and traditional chemotherapy or radiation to reduce the severe side effects caused by an intensive dose of chemo/radiotherapy while minimizing acquired drug resistance due to selective pressure (Table 5). The following large-scale pediatric and young-adult precision oncology programs have been launched with multiple-arm trials for patients with matched molecular profiles: TAPUR (ClinicalTrials.gov identifier NCT02693535), NCI-COG Pediatric MATCH (NCT03155620), the Tumor-Agnostic Precision Immuno-Oncology and Somatic Targeting Rational for You (TAPISTRY) (NCT04589845). These global, multicenter, open-label, multi-cohort studies are now at phase II, and the treatment assignment has relied on the basis of relevant onco-genotypes as identified by a Clinical Laboratory Improvement Amendments (CLIA)-certified or a validated next-generation sequencing (NGS) assay. While the eligible criteria of TAPUR are open for patients aged 12 years old or older, most of the patients enrolled are reported to have adult cancer phenotypes [103,104,105]. In contrast, the NCI-COG Pediatric MATCH aims to evaluate the molecular-targeted therapies with selected biomarkers of childhood and young adult patients with a reported detection rate of actionable alterations of 31.5% from the first 1000 tumors screened. Assignments to treatment arms were made for 28% of patients screened and 13% of patients enrolled in the treatment trial [106]. In the TAPISTRY study, nine targeted treatments are being examined, and eleven non-randomized treatment arms are available for participants of all ages with locally advanced/metastatic solid tumors. The purpose of this study is to evaluate the safety and efficacy of different targeted therapies and immunotherapies in patients as single agents, but the results of the study are still to be released. Overall, the advancements in high-throughput sequencing technology have closed the gap between the current treatment paradigm and precision medicine, markedly improving rates of response, progression-free survival (PFS), and overall survival (OS) compared to traditional randomized trials. Moreover, the multicenter, open-label, multi-arm treatment designs can further benefit treatment strategies by yielding efficacy and toxicity data in a timely manner with cost-effectiveness. Therefore, in the future, international coordination will be crucial to generate a database to inform rational trial design and to evaluate the combination of treatments/interventions that ensure more favorable outcomes. The current applications of precision study designs for pediatric cancers (summarized from clinicaltrials.gov; accessed on 17 August 2022) are shown as single-arm and multiple-arm designs in Table 5 and Table 6, respectively. Large-scale cancer sequencing studies such as the 1000 Genomes Project [107], The Cancer Genome Atlas (TCGA) [108], and the International Cancer Genome Consortium (ICGC) [109] provide an extensive landscape of tumor genomic profiles which substantially facilitate the predication of recurrent hot-spot mutations on the selected type of cancers. Other large databases aim to collect the profile of childhood cancers include St. Jude/Washington University Pediatric Cancer Genome Project (PCGP) [110] and NCI’s Therapeutically Applicable Research to Generate Effective Treatments (TARGET) [53] which are accessible via the St. Jude Cloud (https://www.stjude.cloud, accessed on 26 September 2022) public data repository. These large-scale studies have confirmed that the spectra of genomic alterations and their relevant mechanisms differ in childhood tumors from those predominantly occurring in adult cancer—at least by half. Thus, the actionability of pediatric-driven mutations needs to be carefully interpreted before translating into a targeted treatment option. Several challenges need to be addressed when researchers launch the study/trial for pediatric cancer treatment. Many pediatric cancers are rare, and finding the right patient population for the drugs is challenging. In fact, a small patient population and a prolonged trial duration are not uncommon issues in the settings of rare diseases and low-incidence pediatric cancers [111,112,113,114]. Optimal statistical designs for less stringent comparisons, for example, by relaxing type I error (higher than 5%) or power (lower than 80%) can still provide meaningful results from small but faster trials [111,112,113,114]. Implementing multi-arm multi-stage trial design would allow patients with poor prognosis to be stratified into multiple phase II arms; receiving the window-of-opportunity/experimental therapies and restaging by serial biopsies and molecular characterizations to inform ongoing treatment choices [113,114]. These approaches remain useful to increase the overall feasibility for rare disease trials, i.e., keeping the sample size as small as possible while maintaining the power and ability to address the trial objectives. Only 45% of pediatric cancer driver genes are shared with adult cancers, suggesting that novel therapeutic agents are required for pediatric cancer. Additionally, pediatric cancers are often driven by structural variants that can be challenging to identify and target. Nonetheless, children with cancers have accumulated fewer genetic mutations, thus making genomic targeting simpler than adults [113]. In a broad view, cancer intrinsic targets (e.g., mutated oncogene, tumor suppressor, epigenetics, synthetic lethal, and DNA damage) play crucial roles in cancer pathogenesis and thus could serve as the key stones for drug development against childhood cancers [115]. Another approach in drug development strategy is a mechanisms-of-action (MoA)-driven approach which successfully exemplified the efficiency of nivolumab and larotrectinib as targeted anticancer drugs against programmed cell death protein-1 (PD1) and TRK receptors, respectively [116]. Nonetheless, lessons learned from adult cancers have warned us that many pediatric cancers would have failed to express mutated kinase targets, and resistance to targeted therapies would rapidly occur. Recently, newly emerging cancer targets have been discovered upon multidimensional complexity of the dynamic oncogenic states, for example, tumor archetypes, master regulators, cancer-associated protein–protein interactions, and metabolic vulnerabilities [115,117,118,119,120]. The development of drugs against the emerging classes of cancer targets may deliver adjunct/complementary agents for combination with targeted therapeutic regimens [115]. The emergence of gene editing technologies such as transcription activator-like effector nucleases (TALENS) and clustered regularly interspaced palindromic repeats (CRISPR) paired with the CRISPR-associated endonuclease 9 (CRISPR-CAS9) offer the powerful customizable therapeutic options to precisely edit the targeted genes [121,122,123], thus providing hope to all pediatric cancers to be benefited from genomic-driven precision medicine approach. Comprehensive molecular profiling of the genetic variants/mutations, gene expression at both transcripts and protein levels, and perhaps information on post-translational modifications and metabolites are coordinately utilized to improve the accuracy of molecularly targeted agents. Challenges in this grand scheme, besides big data sharing and multi-omics integration, are interpreting complex high-dimensional data in the biological sense, prioritizing findings into actionable targets/pathways, and achieving the candidate compounds/drugs for precise treatment. Aberrant expression of messenger RNA associated with genomic changes could contribute to the biology of tumor progression. In most cases, RNA-seq analysis can increase the coverage number of variant curations, especially the comprehensive gene fusion discovery and tumor expression subgroup analysis, when compared to WGS alone [124]. A novel molecularly guided approach, so-called transcriptomic connectivity analysis, utilizes the power of RNA-seq to detect aberrant gene expression and employs transcriptomic reversal of cancer cells/tissues for repurposing FDA-approved drugs [125,126,127]. This molecularly guided therapeutic approach could be an asset for prioritizing the approved drugs for off-label use in childhood cancer trials. Despite the promising demonstration of ongoing genomic-driven clinical trials of targeted anticancer small molecules, cancer immunotherapies have become significant advances for pediatric solid tumors [128,129]. Ganglioside GD2 is a sialic acid-containing glycosphingolipid that highly expressed on the surface of multiple pediatric solid tumors, i.e., neuroblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, and brain tumors including diffuse intrinsic pontine glioma (DIPG) and medulloblastoma [128,129]. Thus, GD2 is recognized as one of the most promising targets for pediatric cancer immunotherapy. Dinutuximab, anti-GD2 monoclonal antibody, has been approved as the first-line therapy for high-risk pediatric neuroblastoma [128,129,130], while GD2-specific chimeric antigen receptor (CAR) T cell therapy is under investigation in the early phase trials for children with neuroblastoma, osteosarcoma, and brain tumors (ClinicalTrials.gov identifier NCT03721068, NCT04539366, NCT04099797, NCT04196413). Besides GD2, newly emerging targets for pediatric cancer immunotherapy, including PD1/PD-L1 (NCT04544995, NCT04796012), B7-H3 (CD276; NCT04864821, NCT04743661), HER2 (NCT00902044, NCT04616560) and CD47 (NCT04525014, NCT04751383), have been actively investigated for pediatric sarcomas and brain tumors. Last but not least, it should be noted that new therapeutics often lack dosage guidelines for children [12]. Acknowledging children have different drug responses and tolerance profiles compared to adults, it is crucial to define the optimal dosages of new drugs/biologics (and the off-label use of FDA-approved medications) to achieve preferred therapeutic outcomes. Recent innovations in study designs (i.e., phase I dose-finding design for pediatric population, the potential inclusion of children in adult trials, cooperative group trials) [131,132,133,134], together with the regulatory initiatives in the United States (US) and the E.U. which encourage the development of novel anticancer therapies in children [134,135], provide guidance to address this challenge while accelerating the pace of genomic-driven precision medicine in pediatric oncology. Essential questions that need to be addressed in applications of precision therapeutic program include the applicability of the genetic testing, the significance of the mutation variant, and the existence of an approved targeted therapy. Although targeted agents are approved for a set of tumors harboring specific mutations, future development of clinical guidelines may recommend these agents to be used off-label in different tumor types with the same mutations. Identifying the mutational signatures of pediatric solid tumors will open opportunities for new targeted therapeutic strategies since their malignant origin manifests differently from the adults. Similar genomic-driven precision medicine approaches have been launched by several institutes, while the long-term effects of many of those novel agents are just beginning to be evaluated. These treatments could improve survival and reduce toxicity in pediatric patients and maximize therapeutic advantages when incorporated into standard care.

PMC10000504

Chengcheng Liu,Yuying Huo,Yansong Zhang,Fumei Yin,Taoyu Chen,Zhenyi Wang,Juntao Gao,Peng Jin,Xiangyu Li,Minglei Shi,Michael Q. Zhang

Development and Experimental Validation of a Novel Prognostic Signature for Gastric Cancer

05-03-2023

gastric cancer,prognostic signature,tumor microenvironment,oncogenic mutation,clinical outcomes,machine learning

Simple Summary Gastric cancer (GC) accounts for a considerable amount of morbidity and mortality worldwide. This study developed and experimentally validated a prognostic risk gene signature (PRGS). This is a stable and robust signature for assessing the prognosis of gastric cancer. We performed multiple analyses through consensus clustering and binary classification to assess the robustness of the PRGS in other independent datasets. Additionally, this PRGS exhibited a superior accuracy compared to most traditional clinical markers, including molecular features, and other published signatures. Besides, we also detected the tumor purity, immune cell infiltration, and oncogenic mutation status of high- and low-PRGS groups. Abstract Background: Gastric cancer is a malignant tumor with high morbidity and mortality. Therefore, the accurate recognition of prognostic molecular markers is the key to improving treatment efficacy and prognosis. Methods: In this study, we developed a stable and robust signature through a series of processes using machine-learning approaches. This PRGS was further experimentally validated in clinical samples and a gastric cancer cell line. Results: The PRGS is an independent risk factor for overall survival that performs reliably and has a robust utility. Notably, PRGS proteins promote cancer cell proliferation by regulating the cell cycle. Besides, the high-risk group displayed a lower tumor purity, higher immune cell infiltration, and lower oncogenic mutation than the low-PRGS group. Conclusions: This PRGS could be a powerful and robust tool to improve clinical outcomes for individual gastric cancer patients.

Development and Experimental Validation of a Novel Prognostic Signature for Gastric Cancer Gastric cancer (GC) accounts for a considerable amount of morbidity and mortality worldwide. This study developed and experimentally validated a prognostic risk gene signature (PRGS). This is a stable and robust signature for assessing the prognosis of gastric cancer. We performed multiple analyses through consensus clustering and binary classification to assess the robustness of the PRGS in other independent datasets. Additionally, this PRGS exhibited a superior accuracy compared to most traditional clinical markers, including molecular features, and other published signatures. Besides, we also detected the tumor purity, immune cell infiltration, and oncogenic mutation status of high- and low-PRGS groups. Background: Gastric cancer is a malignant tumor with high morbidity and mortality. Therefore, the accurate recognition of prognostic molecular markers is the key to improving treatment efficacy and prognosis. Methods: In this study, we developed a stable and robust signature through a series of processes using machine-learning approaches. This PRGS was further experimentally validated in clinical samples and a gastric cancer cell line. Results: The PRGS is an independent risk factor for overall survival that performs reliably and has a robust utility. Notably, PRGS proteins promote cancer cell proliferation by regulating the cell cycle. Besides, the high-risk group displayed a lower tumor purity, higher immune cell infiltration, and lower oncogenic mutation than the low-PRGS group. Conclusions: This PRGS could be a powerful and robust tool to improve clinical outcomes for individual gastric cancer patients. Gastric cancer (GC) is a leading cause of cancer morbidity and mortality worldwide [1]. According to GLOBOCAN 2020, there were about 1,089,103 new cases of gastric cancer patients (5.6% of the total cancer burden) [1]. Disease progression and a lack of effective treatment cause most of the mortality [2]. Therefore, preventing “high-risk” GC is the key to improving clinical outcomes. The tumor, node, metastasis (TNM) classification and the American Joint Committee on Cancer (AJCC) classification [3] are commonly used methods to assess the risk and treatment demand for patients in the clinical setting. Nevertheless, due to the limitation of the current grading system, it cannot provide the best clinical treatment for patients. For example, in the clinic, the decision of adjuvant chemotherapy (ACT) is mainly dependent on the clinical–pathological stage rather than molecular biological characteristics [4]. This approach is insufficient and may result in latent overtreatment or undertreatment. Hence, in the era of individualized treatment, it is imperative to identify effective biomarkers to optimize the prognosis of GC. The ideal biomarker should have a consistent expression within and between tumor tissues to perform stably among all patients. Hence, a multigene signature may be an effective approach for addressing this heterogeneity. To date, there are three categories of clinically important GC markers, CEA, CA19-9, and CA72-4, with positive rates of 21.1%, 27.8%, and 30.0%, respectively [5]. However, they are present in a limited number of patients with GC, and the sensitivity and specificity of these biomarkers are not sufficient. With the development of bioinformatics technology, many prognostic biomarkers have been published [6,7,8]. Unfortunately, most of the identified biomarkers failed in the validation. GC is a heterogeneous malignant disease. Histologically, the human gastric mucosa can be divided into three zones, i.e., the cardiac zone, the fundus/corpus zone, and the pyloric zone [9,10]. These zones differ vastly in their histology, regeneration rates, and profiles [11]. In addition, there are different morbidity and mortality rates among GC in these three zones. It is believed that GC patients with the GC localized in the cardiac zone have the worst prognosis. As the patient’s age progresses, the location of the gastric cancer moves upward, and the incidence of it occurring in the cardiac zone increases [12]. The composition of cell types within them is also discrepant. For instance, the quantities of gastric parietal cells are considerably different in these three zones. Namely, parietal cells account for 25%, 50–100%, and 0–1% of all cells in the cardiac zone, the fundus/corpus zone, and the pyloric zone, respectively [12]. Of note, most researchers who investigate gastric cancer biomarkers have ignored the differences between these three zones and analyzed them as if all gastric cancers are of one single area. Hence, it is essential to construct gene co-expression networks and select prognostic-related genes separately for GC data in these three zones. In this work, we attempted to computationally develop and experimentally validate a prognostic risk gene signature with 1226 GC patients from three independent public datasets (Tables S1–S4), a gastric cancer cell line, and several clinical samples to assess the prognosis, tumor growth, and molecular characterization of GC. A multi-step procedure of machine-learning approaches was performed to develop and cross-validate the prognostic risk gene signature (PRGS) model based on the co-expression networks of GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone. This PRGS may help optimize precision treatment and further improve the clinical outcomes of GC patients. In this study, we used three independent public datasets, including 1226 GC patients obtained from the UCSC Toil Recompute Compendium of The Cancer Genome Atlas TARGET and Genotype Tissue Expression project datasets (TCGA target GTEx, primary_site = stomach) [13] and the Gene Expression Omnibus (GEO) (Tables S1–S4). These datasets (TCGA target GTEx, GSE66229, and GSE15459), which encompass complete overall survival (OS) information, were used. Among these, we converted the RNA-seq raw read count from the TCGA target GTEx database to transcripts per kilobase million (TPM) and then log2-transformed. And the data has been removed batch effects among these patients [14]. We retrieved GSE15459 and GSE66229 from the Affymetrix® GPL570 platform (Human Genome U133 Plus 2.0 Array). We selected the most highly expressed probe for each gene to ensure reliable results in consensus clustering and we reserved all probes of each gene to ensure accurate results in binary classification. The ATAC-seq and somatic variation data were obtained from the database of the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/, accessed on 1 February 2022). In this study, the collection of gastric tissues was approved by the Department of Gastroenterology, Seventh Medical Center of Chinese PLA General Hospital, Beijing, China, on 1 June 2022. Overall, one advanced GC sample, an early GC sample, and a normal gastric sample were collected. All patients provided written informed consent, and the ethics committee of the PLA General Hospital also approved our research. The DEGs between cancer and normal samples were detected by edgeR [15]. The genes with an absolute log2 (fold change) ≥ 1.5 were considered to be significant differentially expressed genes (DEGs) between tumor and normal tissues. The volcano plots of upregulated or downregulated genes were generated by the ggplot2 R 4.0 package. The Venn diagram was plotted by the VennDiagram R 4.0 package. The co-expression gene networks were constructed by the WGCNA package [16]. To recognize modules of significantly correlated clusters in the cardiac zone, the fundus/corpus zone, and the pyloric zone, the module that displayed the highest correlation was selected. The brown, purple, and salmon modules of the result of WGCNA were used for the edges, signifying the correlations in the cardiac zone, the fundus/corpus zone, and the pyloric zone, respectively. The filter criterion of a weight value was set to greater than 0.02. A total of 25,350, 1819, and 706 edges and 308, 69, and 54 nodes correlated with these zones were separately obtained and processed in Cytoscape 3.8.1 [17]. To construct the network and select the hub genes, the Cytoscape software was used with the CytoHubba method [18]. LASSO is a regularization and dimensionality reduction technique combined with Cox models, which can be applied in biomarker screening [19]. To identify the hub genes, the top 25 genes of the co-expression network from the cardiac zone, the fundus/corpus zone, and the pyloric zone by Cytoscape were inputted into the LASSO–Cox regression [19]. To ensure the stability of the gene and the model, this procedure was repeated 1000 times. Then, the regression coefficients of each gene were calculated (Table S5). Ultimately, genes with positive regression coefficients were selected. Four genes (APOB, VCAN, ABCA6, and CTSF) whose p-values in the Kaplan–Meier analysis were also < 0.05 were identified to generate the PRGS model. The PRGS risk score of GC patients was calculated by the formula: PRGS score = βk × RNAk. In this formula, k means the genes in the PRGS—we used k = 1, 2, 3, 4 to index the genes in the PRGS—the β value was the multivariate Cox regression coefficient; and RNAk means the expression level of gene k of each patient. The peak regions on chromosomes were shown by the R package chromosome locator. Using the R-packaged ChIPseeker, the alignment can be mapped to peaks in the TSS region to build a signature matrix. Peak position annotation and motif enrichment analyses were performed using HOMER (V4.10). In the range of ±1 kb around the TSS, the peak overlapping the gene initiator was considered as the peak of gene regulation. This process was accomplished through the ConsensusClusterPlus package [20]. We subsampled 80% of the samples, and then used the k-means algorithm to divide each subsample into k (k = 9) according to the Euclidean distance. This procedure was repeated 1000 times. Finally, using the PAC with the smallest k value, the optimal cluster (k = 2) was derived. (1) We pre-processed the GSE66229 data and selected the top 5 genes from the three generated co-expression networks of the three zones’ expression spectrum matrices, then divided the data into normal and tumor samples. We also generated the GSE66229 data and generated PRGS, CEA [21], and GCscore [6] expression spectrum matrices. (2) We performed 5-fold cross validation using the logistic regression classifier (LR) and the random forest classifier (RF) on the GSE66229 and computed the ROC curve for each fold. We then calculated the means of every fitting curve to generate the plot. We then divided the data into five subsets based on the sample tags “Tumor/Normal”, “Stage 1/Normal”, “Stage 2/Normal”, “Stage 3/Normal”, and “Stage 4/Normal”. Details of the data are shown in Table S4. The parameters were set as follows: RF, max_depth = 5, n_estimators = 5, random_state = 123; LR, solver = “liblinear”, penalty = “l2”, C = 1.0. The rest were set as default. We trained and validated each fold and calculated the FPR, TPR, and AUC. We fitted each result into the ROC curve using np.interp and took the means of all fitting curves to craft the figure. For HE staining, a 4% paraformaldehyde solution was used to fix GC tissues. After 24 h, the GC tissues were dewaxed in xylene, dehydrated in ethanol, and subjected to hematoxylin staining (5 min), then dehydrated in eosin solution (10 s), dehydrated in graded alcohol, removed in xylene, and sealed with neutral glue. They were observed and photographed with a microscope (Olympus, Tokyo, Japan). For IHC, 5% bovine serum was used to incubate sections. Then, they were mixed with primary antibodies (anti-APOB, 1:100; anti-VCAN, 1:100; anti-ABCA6, 1:200; and anti-CTSF, 1:200, Abcam, UK) and a secondary antibody (1:800, Abcam, Cambridge, UK). Then, they were stained with a DAB kit and photographed with an optical microscope (Olympus, Japan). GES-1 gastric cancer cells (a gift from Prof. Jun Qin) were cultured with 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, Waltham, MA, USA, C11995500BT) with 20% FBS and 100 mg/mL penicillin/streptomycin. For the siRNA treatment, Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher, L3000001) was used to transfect cells using a standard procedure. For immunostaining, poly-l-lysine-coated coverslips were used to culture cells for 72 h to perform the siRNA transfection; 4% paraformaldehyde was used to fix the cells for 10 min, and then the cells were washed with PBSTr buffer (PBS plus 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA T8787). The cells were incubated with the anti-phosphorylated histone 3 (pH3) antibody overnight at 4 °C. The cells were incubated with a secondary antibody at room temperature and washed with PBSTr buffer. Then, 0.01 mg/mL DAPI and the Vectashield Antifade Mounting Medium (Vector Laboratories, San Francisco, CA, USA H-1200) were used to incubate and mount the cells, respectively. For flow cytometric analysis of the cell cycle with propidium iodide (PI) staining, a standard procedure was used [22]. The Eastep Super Total RNA Extraction Kit (Promega, Madison, WI, USA LS1040) was used to extract the total RNA of siRNA-treated GES-1 cells. The Eastep RT Master Mix Kit (Promega, LS2050) was used to synthesize the cDNA. An Applied Biosystems 7500 real-time PCR system (Thermo Fisher) was used to perform real-time PCRs with the PowerUp SYBR Green Master Mix (Thermo Fisher, A25776). The comparative CT method and Graphpad Prism 8 (GraphPad Software, La Jolla, CA, USA) were used to analyze the data. All experiments were repeated three times. ESTIMATE [23] and CIBERSORT [24] were used to evaluate immune infiltrates. The immune scores, stromal scores, and tumor purity were calculated by the ESTIMATE algorithm. CIBERSORT [24] was used to analyze the levels of infiltrating immune and stromal cells. Maftools was used to calculate significantly mutated genes (p < 0.05) between the low- and high-PRGS groups [25]. A one-sided z-test and two-sided Chi-square test were used to calculate the statistical test for the proportion of mutations (p < 0.05). A functional enrichment analysis was performed on DEGs and peak-related genes. The possible peak-related genes of GO/KEGG enrichment were used to analyze the ClusterProfiler package in R [26]. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to annotate the tumor-related pathways. The gene set variation (GSVA) method [27] was used to enrich the pathways. We used the enrichment score of the GSVA to obtain the expression pathway of the PRGS. The workflow of this work is shown in Figure 1. First, we identified the differentially expressed genes (DEGs) in the TCGA target GTEx with the edgeR package independently to screen DEGs in normal samples and GC samples from the cardiac zone, the fundus/corpus zone, and the pyloric zone. For GC in the cardiac zone, 1498 upregulated and 883 downregulated DEGs were identified. A total of 1661 upregulated and 826 downregulated DEGs were found between normal and GC samples from the fundus/corpus zone. There were 1542 upregulated and 889 downregulated DEGs for GC in the pyloric zone compared with normal samples (Table S5). The distributions of DEGs are shown by volcano plots (Figure 2A). There were 1553 DEGs shared among the GC samples from the three zones (Figure 2B). We adopted GO and KEGG enrichment analysis methods to investigate the annotation of the DEGs. The DEGs of GC samples from the cardiac zone were mainly enriched in sensory perception (GO:0050907), the collagen-containing extracellular matrix (GO:0062023), the sarcomere (GO:0030017), and olfactory transduction. Sensory perception (GO:0050907), the ion channel complex (GO:0034702), olfactory receptor activity (GO:0004984), and olfactory transduction were detected in GC samples from the fundus/corpus zone. In addition, the GC samples from the pyloric zone were highly associated with digestion (GO:0007586), the collagen-containing extracellular matrix (GO:0062023), receptor–ligand activity (GO:0048018), and neuroactive ligand–receptor interactions (Figure S1A,B). To further identify the gene modules related to GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone, the WGCNA method [16] was applied. We assured a scale-free network (soft threshold = 3) with a high scale independence and a low mean connectivity (near 0) (Figure S1C). DEGs in the GC samples from the cardiac zone, the fundus/corpus zone, and the pyloric zone were respectively divided into 22 modules by a cluster analysis (Figure 2C). The brown module related to GC in the pyloric zone was the most significant (cor = 0.76, p = 2 × 10−112). For GC in the cardiac zone and the fundus/corpus zone, the purple and salmon modules were chosen according to the correlation (cor = 0.22, 0.33; p = 3 × 10−8, 1 × 10−16). We selected the brown, purple, and salmon modules for the edges, representing the correlations in GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone, respectively, by the WGCNA algorithm [16]. The Cytoscape software [16] was used to visualize the gene co-expression networks [17], and Cytohubba was used to select hub genes [18] (Figure 2D and Table S6). We also performed a GO and KEGG enrichment analysis of the purple (correlated with the cardiac zone), salmon (correlated with the pyloric zone), and brown (correlated with the fundus/corpus zone) modules (Figure S2A–F). We performed a survival analysis of the genes from the co-expression networks of the three zones on GC patients in the TCGA target GTEx and GSE66229 datasets (GSE15459 did not have information on the three zones). The results indicated that the top five genes (APOA4, MS4A10, SLC28A1, AQP10, and APOB) from the cardiac zone co-expression network correlated most significantly with the outcomes of GC patients in the cardiac zone compared to GC patients of the other two zones (Figure S3A,B). The same was true for patients with GC in the fundus/corpus zone. The top five genes (VCAN, COL1A2, FAP, PODNL1, and SULF1) from the fundus/corpus zone co-expression network also displayed the vastest correlation with the fundus/corpus zone GC patients compared to GC patients of the other two zones (Figure S3C,D). Meanwhile, we a performed binary classification using the logistic regression classifier (LR) and random forest classifier (RF) with the hub genes (APOA4, MS4A10, SLC28A1, AQP10, and APOB) of GC in the cardiac zone as the feature genes to predict whether patients have GC, and we achieved the highest AUC in patients with GC of the cardiac zone compared to the other two zones in the GSE66229 datasets (Figure S3E). The same was true for the hub genes (VCAN, COL1A2, FAP, PODNL1, and SULF1) of GC patients in the fundus/corpus zone (Figure S3F). These results further indicated that discriminating among different zones of GC is of great importance. To further determine the prognostic genes related to the three zones of GC, we continued to generate predictive genes using the TCGA target GTEx and cross-validated these genes with two independent datasets (GSE66229 and GSE15459). Based on the expression profiles of 25 genes correlated with GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone, a LASSO–Cox regression analysis [19] generated the predictive genes. This process was repeated 1000 times with the glmnet R package to ensure the stability of the gene [28]. In the LASSO regression, the partial likelihood of deviance reached the minimum value to obtain the optimal λ (Figure S4A). Six, four, and three genes from GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone, respectively, with positive LASSO coefficients were subjected to the log-rank test and the Kaplan–Meier curve (Figure 3A), which identified a final set of four genes. There were four genes with the maximum coefficient of GC in the three zones, including APOB (p-value = 0.028, FDR = 0.0042) correlated with GC in the cardiac zone, VCAN (p-value = 9.2 × 10−25, FDR = 0.0004) correlated with GC in the fundus/corpus zone, and ABCA6 (p-value = 0.0028, FDR = 0.0042) as well as CTSF (p-value = 0.023, FDR = 0.023) correlated with GC in the pyloric zone. For each of these four genes, the increased expression level was vastly associated with a worse OS for gastric cancer patients (Figure 3A). Besides, these four genes (APOB, VCAN, ABCA6, and CTSF) are included in the DEG lists in the GC data of respective zones in dataset GSE66229. We next depicted the PRGS expression level at a single-cell resolution. We analyzed the scRNA-seq data derived from 29 gastric cancer and 11 normal gastric tissues [29]. After the quantity control and removal batch effect, we obtained a total of 200,954 cells majorly comprising lymphoid cells (CD8A and KLRD1 positive), plasma (TNFRSF17 positive), epithelial cells (CDH1 positive), macrophages (CD163 positive), fibroblasts (FN1 and LUM positive), B cells (MS4A1 positive), mast cells (KIT positive), and pericytes (NOTCH3 positive) (Figures S4B–E and S5A). As shown in Figure S5B, VCAN was expressed in macrophages and fibroblasts; ABCA6 and CTSF were mainly expressed in fibroblasts; and APOB was expressed in epithelial cells (Figure S5B). We also observed that GC samples in different Lauran classifications (intestinal, diffuse, and mixed types) exhibited higher PRGS scores than normal samples (Figure S6A–D). We computed global PRGS scores for all cell types and found that fibroblasts had the highest PRGS scores compared to other cell types (Figure S6D). We also identified the chromatin accessibility of these four genes; thus, we analyzed the relative enriched proportions of coding regions, intergenic regions, introns, exons, and upstream and downstream regions (Figure S7A–C) with ATAC-seq data from TCGA. The peak annotation demonstrated that the peaks of these genes were more likely to be located in promotor regions (Figure S7D). We also performed motif enrichment and calculated potential regulatory TFs within the 200 bp range of gene loci based on genes from the PRGS model with HOMER (Figure S8A). A KEGG analysis of the peaks was also performed (Figure S8B–E). The risk score for each patient was then calculated using the expression matrix of these four genes (APOB, VCAN, ABCA6, and CTSF) weighted by their regression coefficients in the Cox model (Table S7). All patients were divided into high- and low-PRGS groups by the survminer package [30]. As can be seen from Figure 3B, the overall survival (OS) was significantly lower in the high-PRGS group relative to the low-PRGS group in the TCGA target GTEx training dataset and the two validation datasets (all with p < 0.05) (Figure 3B and Table S8). In Figure S9A, we see the distribution of the PRGS scores for the patient group, as well as the relationship between the PRGS and survival time. The cut-off for the high- and low-PRGS groups was 50%. We also evaluated the PRGS in Lauren and WHO histotypes. The results shown in Figure S9B–D also indicate that the overall survival (OS) of all classifications was significantly lower in the high-PRGS group relative to the low-PRGS group in the TCGA target GTEx training dataset and the two validation datasets (all with p < 0.05) (The GSE66229 and GSE15459 only provide the information of Lauran classification). Then, we assessed the OS status in different TNM stage groups. As shown in Figure S10A,B,D,E, the high-PRGS group was tightly correlated with a worse OS status in late stages in TCGA target GTEx (T3, T4, N2, and N3) and in GSE66229 (T2, T3, N1, N2, and N3). The high-PRGS group was vastly associated with a worse OS status in the M0 stage and most of the patients in these datasets were in the M0 stage (Figure S10C,F and Table S4). We noticed that patients with a high PRGS were distributed in all different stages and OS statuses (Figure S11), indicating that our PRGS model is prognosis-specific and capable of assessing the risk of GC patients regardless of stage and status, though clinically, it would be potentially used at later stages for precision. To investigate whether the prognostic value of the PRGS based on the four genes was an independent risk factor, a multivariate Cox regression analysis was performed; the results indicated that the PRGS was notably associated with OS compared with other clinical characteristics (age, sex, clinical stages, and T, N, and M stages), thus validating that the PRGS is robust in independently predicting the GC prognosis (Figure 3C). Similarly, the PRGS was still an independent risk factor for OS in the validation datasets (GSE66229 and GSE15459) (Figure 3D). We further explored the performance of the PRGS with other characters and found that the performance of the PRGS was better than that of other factors, including gender (whether male or female), age, pathological stages (T1~4, N0~3, M0~M1), and clinical stages (stage I~IV) in TCGA target GTEx, GSE66229, and GSE15459 (Figure S12A–C). The discrimination of the PRGS was measured by an ROC analysis, with 1-, 3-, and 5-year AUCs of 0.601, 0.663, and 0.717 in TCGA target GTEx; 0.607, 0.709, and 0.707 in GSE66229; and 0.657, 0.674, and 0.711 in GSE15459 (Figure 4A and Table S9). The C-index (95% confidence interval) of the PRGS was the highest compared with other factors (gender; age; T, N, and M; clinical stage) in the TCGA target GTEx cohorts (Figure S13). To further validate the performance of our PRGS model, we conducted multiple analyses to evaluate the robustness of these four prognostic genes in the aforementioned independent datasets (GSE66229 and GSE15459). First, through consensus clustering based on the four prognostic genes, we applied a consensus cluster analysis to all GC samples, resulting in the division of the samples into k clusters (k = 2–9). The cumulative distribution function (CDF) curves of the consensus score matrix and proportion of ambiguous clustering (PAC) statistic indicated that the optimal number was obtained when k = 2 [35]. We classified GC patients in GSE66229 and GSE15459 into Cluster 1 and Cluster 2 (Figure 4B,C and Tables S10 and S11), respectively. The Kaplan–Meier curve indicated significant OS differences between the two clusters via the log-rank test, and the OS of the patients in Cluster 1 was significantly worse than that of Cluster 2 (Figure 4D). As shown in Figure S14A,E, the overall expression levels of the PRGS in Cluster 1 were higher than in Cluster 2 for both datasets. When k = 3 and 4, the Kaplan–Meier curve indicated that patients in Cluster 1 with the lower PRGS expression level had a vastly better OS than that of the other clusters (Figure S14B–D,F–H). Thus, for these four genes, the increased expression level was associated with a worse survival rate for gastric cancer patients. The observed consistency suggests that the expression levels of these four genes are vastly correlated with the OS of patients. There are many prognostic gene expression signatures that have been developed based on bioinformatics methods. To compare the performance of the PRGS with other signatures, we selected five cancer stem cell-related feature genes in GCscore risk models (FANCA, DUSP3, HIST1H3B, CLNS1A, and FANCC) [6], three common clinical GC biomarkers (CEACAM1, CEACAM5, and CEACAM6) [21], seven immune-related signatures (TGFB1, NOX4, F2R, TLR7, CIITA, RBP5, and KIR3DL3) [31], two cadherin gene signatures (CDH2 and CDH6) [32], six metastasis-related gene signatures (TMEM132, PCOLCE, UPK1B, PM20D1, FLJ35024, and SLITRK2) [33], eight methylation-based gene signatures (TREM2, RAI14, NRP1, YAP1, MATN3, PCSK5, INHBA, and MICAL2) [34], and three hypoxia-immune-based gene signatures (CXCR6, PPP1R14A, and TAGLN) [36] as features to classify gastric cancer patients with different machine-learning (ML) classifiers. We utilized the logistic regression (LR) classifier and random forest (RF) classifier ML models to predict whether patients had GC in GSE66229 (GSE15459 did not have normal samples). We performed binary classification with them at the same time. The results indicated that the PRGS had a low sensitivity to different classifiers while it had the highest and most robust AUC (Figure 4E and Figure S14A,B). Additionally, when performing classification tasks on the patients in different clinical stages (stages I, II, III, and IV), the PRGS had the highest and most robust accuracy (Figure 4F, Figures S15A,B and S16A). Hence, the PRGS signature had optimized effects in classifying GC samples from control samples, which could serve as a potential feature for examining patients’ prognoses. Together, we believe that the expression matrix based on these four genes (APOB, VCAN, ABCA6, and CTSF) as features for screening GC samples could properly assist classifiers in distinguishing cancerous samples from normal samples, acquiring satisfactory precision from test datasets and predicting whether patients have gastric cancer in each stage accurately. To examine the protein expression levels of the four genes (APOB, VCAN, ABCA6, and CTSF) in gastric cancer, we performed immunohistochemistry (IHC) on the pathological section of the samples from patients of different stages, including normal, paracancerous tissue, early gastric cancer (EGC), and advanced gastric cancer (AGC) (Figure S16B–D). Compared with the samples from the normal tissues, the expression levels of the four proteins (APOB, VCAN, ABCA6, and CTSF) were much higher in EGC (Figure 5A–D). These four genes showed strong expression in almost all of the AGC samples and showed weak expressions in a portion of the paracancerous areas (Figure 5E–H). These results indicated a higher expression level for the four genes (APOB, VCAN, ABCA6, and CTSF) in GC compared to normal and paracancerous lesions. Furthermore, these proteins have already reached high expression levels in EGC samples compared to the matching normal samples. To investigate how these four highly-expressed genes influence tumorigenesis, we further performed anti-phosphorylated histone 3 (pH3) immunostaining and flow cytometry-based cell cycle assays (Figure 6A–D). Consistent with the results in human cancer tissues, all four genes were highly expressed in GES-1 and BGC803 gastric cancer cells (Figure 6E,F). Knocking down any of these four genes independently led to reduced phosphorylated histone 3, a well-established biomarker [37] of cell proliferation (Figure 6A,B). Consistently, the cell cycles of GC cells shifted from the S/G2/M to the G1 state upon the knockdown of APOB, VCAN, ABCA6, and CTSF, as demonstrated in flow cytometric analyses of the cell cycle with propidium iodide staining (Figure 6C,D). Note that the knockdown efficacy of the siRNAs used in our experiments were validated by quantitative real-time PCR (Figure 6E,F). These data unraveled that APOB, VCAN, ABCA6, and CTSF play important roles in the incidence of GC. We detected the gene expression of the PRGS using an RT-PCR assay in a clinical cohort that included 19 normal gastric samples, 18 EGC patients, and 8 AGC patients by conducting qRT-PCR experiments (Figure 6G,H). The result indicated that the expression level in GC patients was significantly higher than in normal samples. These assays supported that our PRGS model was robust. Immune-infiltrating cells in the tumor microenvironment (TME) can modulate the tumor phenotype. We assessed tumor purity as well as stromal and immune scores using the ESTIMATE algorithm in TCGA target GTEx samples [23]. ESTIMATE generates three types of scores, namely stromal scores indicating the presence of stroma, immune scores representing the infiltration of immune cells, and an ESTIMATE score, which infers tumor purity. Samples with a low tumor purity show high ESTIMATE scores [23]. The results demonstrated that stromal and immune cells significantly increased along with malignancy progression (from stages I to IV). The ESTIMATE score also increased from stage I to stage IV, whereas tumor purity decreased in higher grades (Figure 7A and Table S12) in accordance with previous studies [38], which illustrated that lower tumor purity correlates with severer malignancy. As expected, a high abundance of stromal cells and immune cells and a low tumor purity were shown in the high-PRGS group (Figure 7A). Hence, the high-PRGS group was positively correlated with malignancy. To further chart the underlying immune cells, we implemented the CIBERSORT algorithm to infer the differential abundance between the high- and low-PRGS patients [24]. ]. The high-PRGS group had a very high level of naïve B cells (p = 0.00014), monocytes (p = 0.0312), M2 macrophages (p = 1.6 × 10−5), and resting mast cells (p = 5.6 × 10−5) (Figure 7B and Table S13), whereas T follicular helper cells (p = 3.9 × 10−7), Treg (p = 2.1 × 10−6), resting NK cells (p = 0.00716), and activated mast cells (p = 0.03602) exhibited a consistent negative correlation with the low-PRGS group. Then, we determined whether the infiltrating immune cells mentioned above could be associated with patient survival. In agreement with previous studies indicating the promoting roles of M2 macrophage cells in tumor progression [39] and naïve B cells when they differentiate into Breg cells in the tumor microenvironment, participating in tumor metastasis [40], we discovered that the naïve B cells and M2 macrophage cells appeared to be associated with poor survival (Figure 7C,D), consistent with the PRGS groups. In addition, the regulatory T cells (Treg), T follicular helper (Tfh) cells, and M0 macrophage cells showed a negative correlation with patient survival (Figure 7E,F), which accords with the low-PRGS group, where there were more Treg and Tfh cells than in the high-PRGS group. Together, our data indicate that the high- PRGS group was characterized by a TME with a high immune cell infiltration and a low tumor purity. To investigate PRGS-related mechanisms in GC, we also analyzed somatic mutations. When comparing the mutant frequency between samples of the low- and high-PRGS groups, we detected more mutations in the low-PRGS group than the high-PRGS group, indicating that more mutations led to a lower PRGS and GC risk (Figure 7G). Past studies have revealed sophisticated correlations between mutations and tumor prognoses, e.g., TP53 mutations have significant associations with poor outcomes in kidney renal clear cell carcinoma, head and neck squamous cell carcinoma, and acute myeloid leukemia, as well as improved outcomes in ovarian serous cystadenocarcinoma [41]. Besides, IDH1 and MUC16 mutations are associated with an improved prognosis in gastric cancer [41,42,43,44,45]. All of the top 20 frequent mutations were significantly enriched in cases with patients in both groups (Figure 7G). As a reference, we utilized the GCscore [6] as feature genes (FANCA, DUSP3, HIST1H3B, CLNS1A, and FANCC) to classify GC patients from the TCGA dataset into high- and low-risk groups (Figure S17A). The top 20 genes with the highest mutation frequencies also had relatively higher mutation frequencies in the low-risk group than in the high-risk group (Figure S17B). This result exhibited a similar tendency as our PRGS. We also found that MUC16 (p = 0.051), CSMD1 (p = 0.054), and FAT4 (p = 0.033) mutants had better outcomes than wild type (WT), while TP53 and TTN, genes with the highest mutation frequencies, did not show differences in the outcomes between their mutants and the WT (Figure S17C–G). Frequent mutations in TNN, TP53, and MUC16 were significantly enriched in the high- and low-PRGS groups, which were within expectations according to previous reports [46]. Additionally, APOB portrayed a mutation percentage of 8% and 18% in patients of the high- and low-PRGS groups, respectively. The mutation percentage for VCAN was 6% and 10% in the high- and low-PRGS groups; for ABCA6, it was 2% and 3%; and for CTSF, it was 1% and 3% (Figure S18A). Previous work [47] has reported APOB and VCAN as mutation driver genes. Moreover, we observed significant co-occurrences among mutations of these genes. Among the top 20 genes with the highest mutation probabilities in gastric cancer, the majority of these gene mutations were co-occurring. The gene mutations in the low-PGRS group displayed more significant co-occurrences than those in the high-PRGS group (Figure S18B). There were also gene pairs that were mutually exclusive. For example, previous studies have reported that ARID1A mutant tumors display p53 pathway activation, and that ARID1A directly regulates TP53 target genes [48,49]; our analysis also displayed that in the high-PRGS group, TP53 and ARID1A mutations were significantly mutually exclusive. On the other hand, within the low-PRGS group, KMT2D and FAT4 were also significantly mutually exclusive along with the previous two. These results also indicate a possible pair of mutually exclusive mutations in patients within the low-PRGS group. Somatic mutations were further explored on the basis of oncogenic signaling pathways. We summarized the gene expression levels and mutations of common oncogenic signaling pathways from patients of the high-PRGS group and low-PRGS group. For instance, disturbing the hippo pathway promoted GC proliferation and metastasis [50], and dysregulation of the MAPK pathway promoted cell metabolism, proliferation, apoptosis, and migration [51]. Our results also indicated that both the hippo and MAPK pathways had high mutation percentages for the samples from the high-PRGS and low-PRGS groups (Figure S18C). We discovered that for the most investigated cancer pathways, the patients from the high-PRGS group had higher pathway enrichment scores than the patients in the low-PRGS group (Figure S18D). Yet, the percentage of patients with mutations was lower in the high-PRGS group than in the low-PRGS group, which might have arisen from other molecular mechanisms. To date, there have rarely been effective prognostic genes identified for detecting and predicting gastric cancer prognoses. There is a noticeable difference among GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone. However, researchers might neglect some significant genes when considering mixed data. Hence, we constructed co-expression networks of GC in these three zones separately to select prognostic genes and build the PRGS. Different treatment options mean that patients need better-individualized evaluations when implementing clinical decisions. These can be used as reliable biomarkers for the diagnosis of “high-risk” GC patients. In this study, WGCNA and Cytoscape were applied to identify the gene co-expression networks of GC in the cardiac zone, the fundus/corpus zone, and the pyloric zone. With the expression profiles of these genes in TCGA target GTEx and two independent datasets, the LASSO–Cox regression model was applied to develop a prognostic gene signature. The prognostic analysis demonstrated that this PRGS was a deleterious indicator of the OS. Besides, the PRGS demonstrated a high accuracy and consistent performance for TCGA target GTEx. We also performed cross-validation using two independent GEO datasets (GSE66229 and GSE15459), which indicated great potential for the clinical application of the PRGS. The common tools for evaluating clinical outcomes and making therapeutic schedules include T, N, M, and the clinical stage [4]. Remarkably, our signature worked independently of these factors and had a vastly superior performance in predicting prognoses. Besides, we took the PRGS as features to classify GC and normal samples with machine-learning classifiers. All of the classification accuracy based on the PRGS signature as features were highest with independent datasets (GSE66229) and for several clinical stages (stages I, II, III, and IV). There are a number of prognostic gene signatures based on characteristic genes, such as stem cell-related gene signatures [6] and DNA methylation-related gene signatures [34]. These gene signatures overlook the differences among tumor locations. Although several of these signatures have been developed, few have been implemented in clinical experiments and even fewer have undergone rigorous validation. We compared the PRGS with these signatures to classify GC patients, and the PRGS had a better performance than the other signatures. To further confirm the clinical significance of the PRGS, we conducted a validation assay using qRT-PCR on 45 frozen gastric cancer tissues. The results further supported the validity of the PRGS as a clinical marker. Consequently, our signature has the potential to be a useful clinical tool for the prognosis determination of GC. To further test the clinical explanation of the PRGS, the experimental validation was based on the IHC results from different clinical stages of independent GC patients, validating our prior findings and assessing their feasibility. This indicated that the protein expression level of the PRGS signature was significantly higher in GC patients. This conclusion was further experimentally validated in GES-1 and BGC803 gastric cancer cells, as the knockdown of these four genes led to cell growth inhibition by regulating the cell cycle. Hence, the PRGS signature could serve as a promising surrogate for assessing the prognosis of GC in clinical settings. Pathologists generally determine tumor purity by visual evaluation, which is affected by the sensitivity of histopathology, interobserver bias, and variability in accuracy [52]. Our results show that the high-PRGS-group patients had a lower tumor purity and higher levels of immune and stromal cell infiltration compared with patients in the low-PRGS group. Previous studies have reported that tumor cells can dominate the microenvironment [52], which has given rise to the hypothesis that malignant GC recruits abundant surrounding cells and subjugates them to compose a protective shield. Therefore, a lower tumor purity and correlated cellular heterogeneity may contribute to a worse prognosis for GC. In our study, we found that the PRGS was positively correlated with several infiltration cells, such as M2 macrophages and naïve B cells, which are positively correlated with the prognosis of GC patients; furthermore, the PRGS was negatively correlated with follicular helper T cells, M0 macrophages, and regulatory T cells, which are negatively correlated with the prognosis of GC patients. We found that GC patients in the low-PRGS group had a higher rate of oncogenic mutations. It should be noted that the association of gene mutations with cancer outcomes is sophisticated. Take MUC16 and IDH1 as examples: past GC studies have illustrated that MUC16 mutations could activate the p53 pathway and DNA repair pathway, which are all tumor suppressor pathways [42,43]; thus, improved outcomes of GC might be expected from mutated MUC16 [34,36]. Mutations in IDH1, as a tumor suppressor in human glioma cells through the negative regulation of Wnt/β-catenin signaling, improves survival conditions [44,45]. In addition, mutations in the top 20 genes had a high frequency of co-mutations. However, further investigations are needed for a deep understanding of the mechanisms of mutations in GC. In a word, based on a series of bioinformatics, machine-learning-based algorithms, and experimental validation, we developed a powerful and robust signature for assessing the prognosis of GC patients. This PRGS model may be a promising tool for screening and monitoring individual GC patients.

PMC10000517

Sanjive Qazi,Fatih M. Uckun

CD22 Exon 12 Deletion as an Independent Predictor of Poor Treatment Outcomes in B-ALL

04-03-2023

B-ALL,CD22,mRNA,relapse,aberrant splicing,SIGLEC-2

Simple Summary We previously reported that cancer cells from the most common type of childhood cancer, namely, a form of acute leukemia known as B-ALL, are characterized by an abnormality known as CD22 exon 12 deletion. The purpose of the present study was to evaluate the clinical significance of the CD22 exon 12 deletion. Our findings provide the first evidence that CD22 exon 12 deletion is associated with a poor treatment outcome in B-ALL. The reported results also support the notion that the further evaluation of the clinical potential of new strategies targeting this abnormality in B-ALL is warranted. Abstract We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 (“CD22E12low”), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.

CD22 Exon 12 Deletion as an Independent Predictor of Poor Treatment Outcomes in B-ALL We previously reported that cancer cells from the most common type of childhood cancer, namely, a form of acute leukemia known as B-ALL, are characterized by an abnormality known as CD22 exon 12 deletion. The purpose of the present study was to evaluate the clinical significance of the CD22 exon 12 deletion. Our findings provide the first evidence that CD22 exon 12 deletion is associated with a poor treatment outcome in B-ALL. The reported results also support the notion that the further evaluation of the clinical potential of new strategies targeting this abnormality in B-ALL is warranted. We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 (“CD22E12low”), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL. The inhibitory B-cell co-receptor CD22 (Siglec-2) regulates several signaling pathways related to the proliferation and survival of B-lineage lymphoid cells [1,2,3,4]. The inhibitory function of CD22 is mediated by the interactions of its cytoplasmic domain with the protein tyrosine phosphatase SHP-1 [5,6]. Any CD22 mutation impairing or preventing this protein–protein interaction would hamper the inhibitory function of CD22 and thereby result in abnormally augmented proliferation as well as prolonged survival of B-lineage lymphoid cells. We previously reported a splicing defect associated with the deletion of CD22 Exon 12 (CD22E12) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL) [7]. We demonstrated that this CD22E12 deletion (“CD22ΔE12”) causes a truncating frameshift mutation and yields a C-terminal truncated dysfunctional CD22 protein that lacks most of its cytoplasmic domain, including the signal transduction elements that are required for the interaction of CD22 with SHP-1 [7]. CD22ΔE12 is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models [7]. Furthermore, forced overexpression of the mutant human CD22ΔE12 in transgenic mice caused fatal B-ALL, demonstrating that CD22ΔE12 alone may be sufficient as a driver lesion for the leukemic transformation and aggressive in vivo growth of BCPs [7,8]. We previously reported that CD22E12 mRNA expression levels, as measured via multiprobe transcriptome profiling using the microarray platform, are selectively and significantly reduced in B-ALL cells with the CD22ΔE12 splicing defect [8,9,10,11,12,13,14]. Using Western blot analysis and RT-PCR, we demonstrated that CD22 exon 12 deletion is not observed in normal human pro-B and pre-pre-B cells [7,10]. Further, our comparison of matched-pair diagnostic vs. post-induction remission bone marrow specimens from B-ALL patients showed a marked reduction of CD22ΔE12 mRNA levels after chemotherapy. These findings demonstrate that normal hematopoietic cells in the remission bone marrow of CD22ΔE12+ B-ALL patients do not express the aberrant CD22ΔE12 mRNA associated with the CD22ΔE12 genetic defect [12]. Although CD22ΔE12 was detected in a high percentage of newly diagnosed and relapsed B-ALL patients [7,8,9,10,11,12,13,14], its clinical significance has yet to be deciphered. The purpose of the present study was to evaluate the clinical prognostic significance of CD22ΔE12 in B-ALL. We postulated that residual wildtype CD22 with undeleted exon 12 could compensate for the missing inhibitory function of the truncated CD22 molecules and thereby mitigate the net effect of the CD22ΔE12 splicing defect on B-ALL cells [12,13]. Indeed, we previously demonstrated that lentiviral-based overexpression of full-length wildtype CD22 in CD22ΔE12-positive B-ALL cells virtually abrogates their clonogenic growth in vitro [13]. We hypothesized that the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by very low levels of wildtype exon 12-containing CD22. Therefore, we set out to test the hypothesis that B-ALL patients with very low levels of CD22E12 (“CD22E12low”), as measured by RNAseq-based CD22E12 mRNA levels, would have a worse prognosis than other B-ALL patients. Our findings provide unprecedented evidence that CD22E12low B-ALL patients have worse leukemia-free survival (LFS) and overall survival (OS) outcomes than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in univariate and multivariate Cox proportional hazards models. We used the publicly available archived gene expression profiling datasets GSE13159, GSE11877, and GSE13351, which were generated in the GeneChip Human Genome U133 Plus 2.0 Array platform (Thermo Fischer Scientific, Waltham, MA, USA), to examine the relative expression levels of CD22 exons 11−14 in primary leukemia cells from 421 newly diagnosed pediatric B-ALL patients. The B-ALL patient population included Ph-like B-ALL patients (n = 154; GSE11877 and GSE13351); TCF3-PBX1+/E2A-PBX1 B-ALL patients (n = 25; GSE11877 and GSE13351); KMT2A/MLL-R+ B-ALL patients (n = 25; GSE11877 and GSE13351); BCR-ABL/Ph+ ALL patients (n = 123; GSE13159 and GSE13351); other B-ALL patients (n = 94; GSE11877 and GSE13351). BLAT analysis of CD22 probe sequences for the Affymetrix probe set 217422_s_at (9 probes covering exons 11–14; Human Genome U133 Plus 2.0 Array platform) that were mapped onto specific CD22 exons was visualized using the UCSC genome browser. Expression of each probe was log2-transformed and median-centered across all probes in each sample. Similarly, the expression levels of CD22E11, CD22E13, and CD22E14 were estimated by determining the mean expression values for 6 probes mapped to these CD22 exons in B-ALL samples mean-centered to the corresponding expression values in non-leukemic CON samples. The perfect match (PM) signal value for each probe was background-corrected, robust multiarray analysis (RMA)-normalized, log2-transformed, and median-centered across all probes in each sample. The expression level of CD22E12 in the B-ALL samples (n = 421) was estimated from the mean signal value for the 3 CD22E12 probes, namely HG-U133_PLUS_2:217422_S_AT_7 aligned to chr19:35836590-35836614, HG-U133_PLUS_2:217422_S_AT_6 aligned to chr19:35836566-35836590, HG-U133_PLUS_2:217422_S_AT_5 aligned to chr19:35836535-35836559, and mean-centered to the mean signal value for the same probes in non-leukemic control (CON) samples (n = 74, GSE13159). A CD22E12 index was calculated by subtracting the mean expression values for the 6 probes for CD22E11, CD22E13, CD22E14 in the 217422_s_at probe set, namely, HG-U133_PLUS_2:217422_S_AT_11 aligned to chr19: 35837525-35837549/CD22E14), HG-U133_PLUS_2:217422_S_AT_10 aligned to chr19: 35837478-35837502/CD22E14, HG-U133_PLUS_2:217422_S_AT_8 aligned to chr19: 35837100-35837124/CD22E13), HG U133_PLUS_2:217422_S_AT_9 aligned to chr19: 35837117-35837139/CD22E13), HG-U133_PLUS_2:217422_S_AT_4 aligned to chr19:35835979-35836003/CD22E11, and HG-U133_PLUS_2:217422_S_AT_3 aligned to chr19:35835811-35835960/CD22E11, from the expression values for the 3 CD22E12 probes, as previously reported [8,9,10,11,12,13,14]. Expression levels for CD22E12 versus the mean of CD22E11, CD22E13, and CD22E14 were visualized using density graphs (fitted using a Gaussian smoothing kernel density estimation) superimposed on histograms of the CD22E12 index values and were plotted for CON and B-ALL samples (ggplot2_3.3.5 R package). Cellular RNA was extracted from Ficoll–Hypaque-separated leukemia cells of 12 de-identified pediatric patients with newly diagnosed high-risk B-ALL and 12 de-identified pediatric patients with newly diagnosed standard-risk ALL using the Qiagen RNeasy Mini Kit (Cat# 74104, Qiagen, Valencia, CA). The secondary use of de-identified leukemia cells for subsequent laboratory studies did not meet the definition of human subject research per 45 CFR 46.102 (d and f) because it did not include identifiable private information, and it was approved by the IRB (CCI) at the Children’s Hospital Los Angeles (CHLA) (Protocol #’ CCI-09-00304 and CCI-10−00141; Human Subject Assurance Number: FWA0001914). One-step real-time quantitative (q) RT-PCR was performed using the One-Step PrimeScript RT-PCR kit (Cat. # RR064B, Takara/Clontech, Mountain View, CA) and the Applied Biosystems 7900HT Fast Real-Time PCR System housed in the CHLA/USC Stem Cell Core Facility to compare the expression levels of the CD22ΔE12 mRNA in pediatric B-ALL samples, as previously described in detail [12]. The PCR primer pair (viz.: forward primer E11-F2: 5′-CAGCGGCCAGAGCTTCTT-3′ and reverse primer E13-R2: 5′-GCGCTTGTGCAATGCTGAA-3′) was selected to amplify a 113-bp fragment spanning from Exon 11 to Exon 13 of the human CD22ΔE12 cDNA. The amplified fragment was then specifically annealed to a pre-mixed oligo DNA probe (5′-TGTGAGGAATAAAAAGAGATGCAGAGTCC-3′) conjugated with 5′ FAM reporter and 3′BHQ quencher on the CD22ΔE12-specific unique junction region between exon 11 and exon 13. The FAM reporter fluorescence intensity was recorded by applying the sequence detection system of the real-time qPCR system, expressed as threshold cycle threshold (Ct) value for quantification, as reported [12]. Each sample was also subjected to a qRT-PCR reaction for the housekeeping gene beta (β)-actin with a primer set amplifying a 234-bp region at the junction between exon 4 and exon 5 of the human β-actin gene for normalization of the Ct values, as previously reported [12]. The results were visualized in box plots superimposed with a kernel density plot showing the peaks, median, and inter-quartile range in the numerical distribution of the data (ggplot2_3.3.5 R package). We downloaded the RNAseq data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program (https://target-data.nci.nih.gov/Public/ALL/mRNA-seq/Phase2/L3/expression/BCCA/ accessed on 28 January 2022). Summary files were named using a coding system specific to Office of Cancer Genomics (OCG) characterization programs (https://ocg.cancer.gov/sites/default/files/OCG-Project-Codes_Tissues-and-Samples_03-02-2021.pdf accessed on 28 January 2022), allowing for the identification of data files that contained exon-level mRNA expression levels for each sample. These data files reported the exon locations for the CD22 gene (Human (GRCh37/hg19) build), raw read counts, length of exons, and reads per kilobase million (RPKM) values. Read count, alignment, and within-sample-level normalization of mRNA expression levels were detailed in previous study reports that contributed to the data deposited in the TARGET repository (4; summarized in https://ocg.cancer.gov/programs/target/target-methods#3202 (accessed on 28 September 2022)). Briefly, exon-level quantification was performed by aligning Illumina paired-end RNA sequencing reads (fasta files) to GRCh37-lite genome-plus-junctions and exon–exon junction sequences, whereby the corresponding coordinates were based on annotations of transcripts in the Ensembl (v59) reference using BWA version 0.5.7. Mapped reads between these junction regions were positioned according to the reference genome (GRCh37/hg19) build). The raw count reports the number of reads that overlapped each of the CD22 exon junctions in each sample. The corresponding data files with exon-level quantification for the CD22 exons 11, 12, 13, and 14 were manually downloaded from the TARGET repository. The exon locations, which were all on the positive strand, were as follows: CD22E11:35835954-35836029; CD22E12:35836505-35836623; CD22E13:35837054-35837138; CD22E14:35837469-35838258. We utilized the normalized RPKM metric for the exon-level quantification of CD22 mRNA. In this summarization, the total read count in a sample was divided by 106 to obtain the scaling factor. The read counts were divided by the scaling factor to normalize the read counts for sequencing depth (reads per million (RPM)). The RPM value was then divided by the length of the exon in kilobases to calculate RPKM. We calculated the CD22E12 expression to measure the relative level of CD22E12 expression compared to the mean level of expression of 3 surrounding exons with similar RPKM values, namely, CD22E11, CD22E13, and CD22E14. Mean RPKM values were used for cases with duplicate samples. The sample information was obtained from annotation files deposited on the TARGET website: TARGET_ALL_SampleMatrix_Phase2_Validation_20190606.xlsx and TARGET_ALL_SampleMatrix_Phase2_Discovery_20190606.xlsx. The archived database contained the data from the B-ALL patients (n = 141) that were used in our analyses, including 90 pediatric or young adult patients with NCI high-risk ALL treated in the Children’s Oncology Group (COG) studies P9906 [14] or AALL0232 [15,16,17] and 51 pediatric patients with standard risk ALL treated in COG study AALL0331 [18,19]. Within the 141-patient RNA-seq subset, 89 high-risk patients were treated in the COG study AALL0232 [15,16,17], and 51 standard-risk patients were treated in the COG study AALL0331 [18,19]. CD22-targeting therapies were not part of these protocols. No patient received inotuzumab-ozogamicin. Treatment details for the AALL0232 and AALL0331 protocols are described in detail and can be accessed via https://clinicaltrials.gov/ct2/show/NCT00075725 and https://clinicaltrials.gov/ct2/show/NCT00103285, respectively. In brief, AALL0232 was a randomized, multicenter study (see https://clinicaltrials.gov/ct2/show/results/NCT00075725 for treatment details, accessed on 1 January 2023). Patients were stratified according to early response (slow early response (SER) vs. rapid early response (RER)). For Induction therapy, patients were randomized to 1 of 4 treatment arms: (i) ARM I: patients received cytarabine intrathecally (IT) on day 1, vincristine intravenously (IV) and daunorubicin IV on days 1, 8, 15, and 22, dexamethasone orally (PO) or IV twice daily (BID) on days 1–14, methotrexate (MTX) IT on days 8 and 29*, and pegaspargase intramuscularly (IM) once on day 4, 5, or 6. Patients with CNS3 disease (WBC > 5/mL in cerebrospinal fluid and positive for blasts on cytospin) also received MTX IT on days 15 and 22. (ii) ARM II: patients received induction therapy as in ARM I. (iii) ARM III: patients received cytarabine, vincristine, daunorubicin, and pegaspargase as in ARM I. Patients also received prednisone PO or IV BID on days 1–28 and MTX IT on days 8 and 29. (iv) ARM IV: patients received induction therapy as in ARM III. Patients in all arms were evaluated at day 29 of induction therapy. Patients with M3 disease were removed from the study. Patients with M1 disease and less than 1% minimal residual disease (MRD) proceeded to consolidation therapy beginning on day 36. Patients with M2 disease or with MI disease and at least 1% MRD received extended induction therapy for 2 additional weeks. Patients with SER disease and MLL rearrangements were removed from the study. For extended induction therapy, patients continued to receive therapy according to the arm to which they were originally randomized: ARMS I and II: patients received dexamethasone PO or IV BID on days 1–14, vincristine IV on days 1 and 8, daunorubicin IV on day 1, and pegaspargase IM on day 4, 5, or 6 and were then reevaluated; ARMS III and IV: patients received prednisone PO or IV BID on days 1–14 and vincristine, daunorubicin, and pegaspargase as in Arms I and II, and they were then reevaluated. Patients in all arms who had M1 disease and less than 1% MRD after extended induction proceeded to consolidation therapy and continued as SER patients. All other patients were removed from the study. For consolidation therapy, all patients received cyclophosphamide IV over 30 min on days 1 and 29, cytarabine IV or subcutaneously (SC) on days 1–4, 8–11, 29–32, and 36–39, mercaptopurine (MP) PO on days 1–14 and 29–42, vincristine IV on days 15, 22, 43, and 50, pegaspargase IM on days 15 and 43, and MTX IT on days 1, 8, 15, and 22. Patients with testicular disease also received radiotherapy to the testes. Patients with CNS3 disease received MTX on days 1 and 8 only. For interim maintenance therapy 1, patients continued to receive treatment according to the arm to which they were originally randomized: (i) ARM I: (escalating-dose MTX) patients received vincristine IV and escalating-dose MTX IV on days 1, 11, 21, 31, and 41, pegaspargase IM on days 2 and 22, and MTX IT on days 1 and 21. (ii) ARM II: (high-dose MTX) patients received vincristine IV and high-dose methotrexate IV over 24 h on days 1, 15, 29, and 43, MP PO on days 1–56, and IT MTX on days 1 and 29. Patients also received leucovorin calcium IV every 6 h for at least 3 doses, beginning 42 h after start of each MTX infusion. (iii) ARM III: (escalating-dose MTX) patients received interim maintenance 1 therapy as in ARM I. (iv) ARM IV: (high-dose MTX) patients received interim maintenance therapy as in ARM II. For delayed intensification therapy 1, all patients received vincristine IV on days 1, 8, 15, 43, and 50, dexamethasone PO or IV BID on days 1 to 21 for patients ages 1 to 12 OR on days 1–7 and 15–21 for patients ages 13 and over, doxorubicin IV on days 1, 8, and 15, pegaspargase IM on day 4, 5, or 6 as well as day 43, cyclophosphamide IV over 30 min on day 29, cytarabine IV or SC on days 30–33 and 37–40, thioguanine PO on days 29–42, and MTX IT on days 1, 29, and 36. After delayed intensification I, SER patients proceeded to interim maintenance 2 and delayed intensification 2. RER patients proceeded directly to maintenance. For interim maintenance therapy 2, all patients received vincristine IV and MTX IV on days 1, 11, 21, 31, and 41, pegaspargase IM on days 2 and 22, and MTX IT on days 1 and 21. Patients then proceeded to delayed intensification 2. For delayed intensification therapy 2, all patients received therapy as in delayed intensification 2, ARM I. CNS3 patients also received radiotherapy for 3–10 days, beginning on day 29. All other SER patients, patients with MLL rearrangements, and some patients pretreated with steroids (>48 h within the week prior to diagnosis) received prophylactic cranial radiotherapy (CRT) for 8 days, beginning on day 29. Patients then proceeded to maintenance therapy. For maintenance therapy, all patients received vincristine IV on days 1, 29, and 57, dexamethasone PO BID on days 1–5, 29–33, and 57–61, MP PO on days 1–84, MTX IT on day 1*, and MTX PO on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 78. RER patients (who did not undergo CRT) also received MTX IT on day 29 for maintenance courses 1–4. In all arms, maintenance therapy was repeated every 12 weeks until the total duration of therapy was 2 years from the start of interim maintenance 1 for female patients and 3 years from the start of interim maintenance 1 for male patients. Patients with testicular disease could receive testicular radiotherapy for 8 days during one of the first 3 courses of maintenance therapy. Patients were followed monthly for 1 year, every 2 months for 1 year, every 3 months for 1 year, every 6 months for 1 year, and then annually thereafter. In the AALL0331 study, which tested whether intensified postinduction therapy that improves survival in children with high-risk B-cell acute lymphoblastic leukemia (ALL) would also improve outcomes for those with standard-risk (SR) ALL, patients received a 3-drug induction with IT cytarabine on day 1; weekly IV vincristine for 4 doses; oral dexamethasone for 28 days; 1 dose of intramuscular PEG on day 4, 5, or 6; IT MTX for 2 to 4 doses (see https://www.slideshare.net/AlfredYeung2/aall0331-protocol, accessed on 28 September 2022). Bone marrow (BM) aspiration was performed on days 8 and 15 (if the day-8 marrow was M2/M3) to determine the hematologic response by examining morphology. MRD testing was performed at day 29 using flow cytometry. Rapid early response (RER) was defined as <5% BM blasts (M1) by day 15 based on local morphologic interpretation and an M1 BM with MRD < 0.1% at day 29. Slow early responders (SERs) had an M2 (5–25%) or M3 (>25%) BM on day 15 and/or positive MRD (≥0.1% to <1%) at day 29. For patients with M3 marrow at day 29, induction was considered to have failed, and they were taken off protocol therapy. Patients with an M2 marrow or an M1 marrow with MRD ≥ 1% at day 29 received an extended induction with 2 additional weeks of therapy and continued on study as SERs if they achieved day-43 M1 marrow and MRD < 1%. Those not achieving these criteria were removed from protocol therapy. All patients were initially required to have central testing for triple trisomies of chromosomes 4, 10, and 17 (TT) and BCR-ABL1, ETV6-RUNX1, or KMT2A rearrangement (KMT2A-R) using fluorescence in situ hybridization. After induction, patients were classified into 1 of 3 risk groups: SR low (RER, CNS1, and favorable cytogenetics of TT or ETV6-RUNX1 fusion), SR average (no unfavorable genetic features (BCR-ABL1, KMT2A-R, or hypodiploidy with < 44 chromosomes), RER, and CNS1 or 2 (patients with favorable genetics who were RERs and CNS2)), or SR high (KMT2A-R and RER, anyone with CNS3 at diagnosis, and SERs by morphology or MRD). Patients with overt testicular leukemia were not eligible. Patients with BCR-ABL1 fusion or hypodiploidy did not continue to receive therapy after induction. Patients with SR-low disease were randomly assigned to regimens with or without 4 additional doses of PEG at approximately 3-week intervals, with the backbone of standard consolidation (SC) and initially standard IM with weekly oral MTX. All patients with SR-low disease received standard DI and maintenance. Patients with SR-average disease were randomly assigned initially in a 2-by-2 factorial design to 1 of 4 treatment regimens: SS (SC and standard IM and DI), SA (SC with intensified IM (AIM) and DI (ADI)), IS (intensified consolidation (IC) and standard IM/DI), and IA (IC, AIM, and ADI). IC was identical to the augmented Berlin–Frankfurt–Münster (BFM) consolidation used in COG AALL0232, AIM was identical to the Capizzi-style escalating IV MTX and PEG, and DI incorporated additional doses of VCR and PEG, as used in AALL0232. Patients with SR-high disease were nonrandomly assigned to receive full augmented BFM therapy, as administered in CCG 1961, including IC, AIM1, ADI1, AIM2, ADI2, and maintenance. CNS3 patients underwent 18-Gy cranial irradiation. In all arms of AALL0331, the length of therapy from the start of IM1 was 2 years for girls and 3 years for boys. In 2008, the results of CCG 1991 became available, showing that escalating IV MTX without leucovorin rescue improved EFS compared to standard IM with oral MTX. AALL0331 amendment 2C replaced the oral MTX IM phase with escalating IV MTX for all patients with SR-average disease. All patients with SR-average disease received IM with IV escalating MTX and standard DI. This amendment also changed dexamethasone administration in DI to discontinuous dosing (days 1–7 and 15–21) rather than a continuous schedule (days 1–21) because of increased rates of osteonecrosis in AALL0232 with continuous dexamethasone during DI. When the results of AALL0232 demonstrated that high-dose MTX was superior to Capizzi MTX, amendment 7 (May 2011) changed therapy for patients with SR-high disease who had not yet begun maintenance cycle 2. They then received an additional IM phase with high-dose MTX. 26 patients out of the 1126-patient complete set (2.3%) of the TARGET database for B-ALL received a transplant compared to 6 patients out of 141 RNAseq subset (4.3%) who received a transplant (p = 0.2). Within the 141-patient RNAseq subset, none of the 21 CD22E12low patients received transplants compared to 6 out of 120 (5%) remaining patients from the RNAseq subset (Fisher’s Exact Test, p = 0.6). No information is available regarding the type or timing of the transplants. No patient received inotuzumab-ozogamicin. We excluded the data from 43 additional patients with an unknown cell of origin/immunophenotype information for leukemia cells. In addition, data files reporting the RPKM metric for CD22 gene exon-level quantification of mRNA for CD22 exons 1-4 were downloaded from the TARGET phase 2 project (https://target-data.nci.nih.gov/Public/ALL/mRNA-seq/Phase2/L3/, accessed on 28 September 2022). The expression level of each exon was mean-centered to the average RPKM values across exons 1 to 4 (CD22E1–4) for each of the 141 B-ALL patients (RPKM-normalized). A one-way hierarchical clustering technique was used to organize the RNAseq-based mRNA expression patterns for the CD22 exons 11, 13, and 14 side-by-side with the expression of CD22E12 across 141 B-ALL patients. The expression level of each CD22 exon was mean-centered to the average RPKM values across CD22E11–14 such that patient level and exon-level expression patterns displayed similar expression profiles that were grouped together using the average distance metric (default Euclidean distance and Wards linkage implemented using the heatmap.2 function in the R package gplots_3.1.1). The cluster analysis revealed a subset of patients whose cells exhibited a markedly and selectively reduced expression level of CD22E12 compared to CD22E11, CD22E13, and CD22E14 (“CD22E12low subset”). To confirm the selective reduction of CD22E12 relative to CD22E11 and CD22E13 in CD22E12low patients, the corresponding normalized RPKM expression values were compared utilizing a two-factor ANOVA model: patient grouping and exon ID were used as fixed variables, and patient grouping × exon ID was an interaction term (p-values were adjusted for multiple comparisons by controlling the false discovery rate to less than 0.01 (multcomp_1.4-17 and emmeans_1.7.0 packages ran in R version 4.1.2 (1 November 2021) with Rstudio front end (RStudio 2021.09.0 + 351 “Ghost Orchid” Release)). Bar chart graphics were constructed using the ggplot2_3.3.5 R package. We downloaded gene-level RNAseq data from the TARGET program (https://target-data.nci.nih.gov/Public/ALL/mRNA-seq/Phase2/L3/expression/BCCA/ accessed on 9 July 2022) using the web-scraping utility implemented in R version 4.1.2 (1 November 2021) (rvest_1.0.2 and stringr_1.4.0). These data files reported the Ensembl gene IDs, raw read counts, median length of each gene, and RPKM values. Gene identifications were converted from ensemble IDs to gene symbols using the Bioconductor database org.Hs.eg.db_3.14.0, which was interrogated using the functions provided in AnnotationDbi_1.56.1. We compared the gene level RNAseq data for B-ALL samples that exhibited substantially reduced levels of CD22E12 mRNA (n = 21; CD22E12low) versus all other B-ALL samples (n = 120) using the DESeq2 package (DESeq2_1.34.0) obtained from http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html (accessed on 28 September 2022) and implemented using R version 4.1.2 (1 November 2021) [20]. DESeq2 employs a generalized linear model for each gene that fits raw read counts to negative binomial distribution to calculate mean and variance estimates, whereby the mean is taken as a quantity proportional to the concentration of cDNA fragments from the gene in the sample and scaled by a normalization factor across all samples. The normalization method in the DESeq2 algorithm determines the counts divided by sample-specific size factors calculated from the median ratio of gene counts relative to the geometric mean per gene across all samples that accounts for the sequencing depth and RNA composition of each gene. This method allows for fold change comparisons across treatment groups in the GLM model [21,22,23]. The statistical significance of differences in gene expression levels was assessed by testing the null hypothesis that there is no differential expression across the two sample groups (Log2 fold change = 0) using the Wald test [20], reporting the test statistic and p-value for each gene. To visualize the gene expression profiles in heatmaps, we calculated the normalized log2 values from the RNAseq count data using the statistical package implemented in R, vsn_3.62.0 [24]. This method uses a robust variant of the maximum likelihood estimator for the stochastic model of count data that employs data calibration, accounting for the dependence of variance of mean intensity and variance stabilizing data transformation. Low-count values tend to generate large fold changes; therefore, to calculate a more accurate log2 fold change estimate, we applied a shrinkage of the log2 fold change estimates toward zero when the read counts were low and variable (“normal” function in the DESeq2 package) [25]. Data files reporting raw read counts determined using gene-level quantification of mRNA were downloaded from the TARGET phase 2 project (https://target-data.nci.nih.gov/Public/ALL/mRNA-seq/Phase2/L3/expression/BCCA/ (accessed on 9 July 2022)). We determined the differential expression of genes comparing patients that exhibited low CD22 exon 12 expression (n = 21; CD22E12low) with all other patients (n = 120) using DESeq2 package (DESeq2_1.34.0 implemented in R). We used GSEA (fgsea_1.20.0 [26] implemented in R) and the rank-ordered Wald statistic for the comparison of the 21 CD22E12low patients versus the remaining 120 patients regarding representation patterns of reactome pathways (reactome.db_1.77.0 obtained from Bioconductor: https://www.bioconductor.org/packages/release/data/annotation/html/reactome.db.html, accessed on 9 July 2022). We focused our initial analysis on pathways grouped under transcription, translation (including mRNA processing, mRNA transport, and post-translational modification), and cell cycle because our previously published cluster analysis of highly enriched gene sets revealed that transcriptional, translational and cell cycle processes were significantly upregulated in all three comparisons of CD22ΔE12 Tg mice compared to BCR-ABL Tg, Eμ-MYC Tg, and WT mice (GSE58874 and GSE58872) [9]. In addition, pathways were analyzed and grouped by signal transduction based on our previously published phosphoproteome data for CD22ΔE12-Tg mice (GSE58873 and GSE 58874). The GSEA evaluated the enrichment score (ES) values, representing observed rankings compared to the expected null distribution calculated from the permutation of gene assignments to the ranking scores. Nominal p-values were computed by comparing the tails of the ES scores for observed and permutation-generated null distributions following 100,000 permutations. The significance of the association was assessed using weighted Kolmogorov–Smirnov statistics. In order to compare the differences in gene expression levels across gene sets, normalized enrichment scores (NES) were calculated based on the number of genes in the gene set. Low-expression genes (base mean values calculated in the Dseq2 procedure > 10 normalized counts across all samples) were filtered before GSEA analysis for representation in reactome pathways. This resulted in 22,068 genes that were processed for GSEA, comparing CD22E12low B-ALL patients with all other patients for gene set enrichment in 1256 in reactome pathways (gene sets ranged from 10–100 genes; 100,000 permutations were performed to calculate enrichment scores and associated p-values). The expression of significantly affected genes was visualized using heatmaps and dendrograms represented in a cluster figure (R package gplots_3.1.1) depicting normalized expression levels in CD22E12low B-ALL samples mean-centered to all other samples. The outcome data were retrieved from the TARGET clinical annotation files TARGET_ALL_ClinicalData_Phase_II_Discovery_20211118.xlsx and TARGET_ALL_ClinicalData_Phase_II_Validation_20211118.xlsx. The Kaplan–Meier (KM) method, log-rank chi-square test, and the software packages survival_3.2-13, survminer_0.4.9, and survMisc_0.5.5, which were operated in the R environment, were used to compare the treatment outcomes of patients, including time to relapse, relapse-free survival (LFS), and overall survival (OS). Graphical representations of the treatment outcomes were generated using three graph-drawing packages implemented in the R programming environment: dplyr_1.0.7, ggplot2_3.3.5, and ggthemes_4.2.4. We compared the outcomes of CD22E12low patients (n = 21) vs. all other patients (n = 120) in an effort to evaluate the prognostic significance of the CD22E12low status. The statistical significance of differences in the outcomes of the compared patient subsets was examined using the log-rank chi-square test, and p-values less than 0.05 were deemed significant. Multivariate analysis of the poor prognostic impact of CD22ΔE12 was performed using a Cox proportional hazards model in which we examined if the CD22E12low status observed in 21 of 141 B-ALL patients remained a significant predictor of adverse outcomes after controlling for other patient characteristics of established prognostic significance. We compared the hazard ratios (HR) from the multivariate Cox model with univariate Cox models for each of the prognostic factors used as covariates in the multivariate model. Estimates of the life table HR were calculated using the exponentiated regression coefficient for Cox regression analyses implemented in R (survival_3.2-13 was run with R version 4.1.2 (1 November 2021)). Forest plots were utilized to visualize the HR values obtained in the Cox proportional hazards model (survminer_0.4.9 was run with R version 4.1.2 (1 November 2021)). A total of 141 patients were evaluable in the Cox proportional hazards regression model. Analyses were performed both for all 141 patients as well as for the high-risk subset of 90 patients. The patient characteristics analyzed in the Cox proportional hazards model included the following: (i) CD22E12low status, (ii) age, (iii) gender, (iv) cytogenetic features including molecular markers, (v) WBC at diagnosis (both linear and categorized as ≥20 × 109/L or < 20 × 109/L), and (vi) measurable residual disease (MRD) burden at the end of induction therapy on day 29 (0% vs. >0%; 0% defined as < 0.001% or <1 × 10−5), as measured by using 6-color flow cytometry. CD22ΔE12 is associated with a selective reduction in expression levels of CD22E12 mRNA [8,9,10,11,12,13,14]. We first examined the distribution of CD22E12 mRNA expression levels in primary leukemia cells from 421 B-ALL patients, as measured by the CD22E12 index values based on the transcriptome profiling data obtained using the genome expression microarray platform (Figure 1A). The CD22E12 index values for B-ALL samples showed interpatient variability (mean ± SE = −0.29 ± 0.03; median = −0.29; range = −1.92–1.92; n = 421) (Figure 1A). The density graph of the CD22E12 index values for B-ALL cells showed broad multipeak distributions consistent with marked patient-to-patient heterogeneity in CD22E12 expression levels. Next, we used qRT-PCR to examine the CD22ΔE12 expression levels in primary leukemia cells from 24 pediatric B-ALL patients (Figure 1B–D). The displayed ΔCt values demonstrated marked interpatient heterogeneity and broad multipeak distributions in CD22ΔE12 expression levels. The data on the microarray-based CD22E12 index values combined with the qRT-PCR-based data on CD22ΔE12 Ct and ΔCt values indicate that the biological impact of CD22ΔE12 may vary from patient to patient due to varying levels of normal CD22 and truncated CD22 levels. We next examined the relative expression levels of CD22E12 in primary leukemia cells from 141 patients with B-ALL by interrogating the archived exon-level quantitative RNAseq data from the TARGET program. In accordance with the results obtained with the microarray platform and qRT-PCR data, the RNAseq data showed marked heterogeneity in the magnitude of the selective reduction of CD22E12 mRNA expression levels, as evidenced by the heat map of the cluster figure (Figure 2A) and the broad distribution of the normalized RPKM values for CD22E12 (Figure 2B). By using the method of hierarchical clustering, a subset of 21 patients (“CD22E12low”) with selective reduction CD22E12 levels relative to CD22E11, CD22E13, and CD22E14 levels was identified, in which the normalized RPKM value for CD22E12 mRNA was <0.8 (Figure 2B). The mean CD22E12 expression level (in normalized RPKM) for the CD22E12low patients was 0.714 ± 0.014 (median = 0.728; range = 0.572–0.785), which was significantly lower than the mean CD22E12 expression level of 0.934 ± 0.006 (median = 0.935; range = 0.805–1.133) for the remaining 120 patients (two-way ANOVA, linear contrast, p-value < 10−15) (Figure 2B). The CD22E12 expression level for this subset was significantly lower than the expression levels for exons CD22E11 (mean = 1.101 ± 0.018; median = 1.109; range = 0.839–1.224; p-value < 10−15), CD22E13 (mean = 1.073 ± 0.011; median = 1.076; range = 0.961–1.151; p-value < 10−15), and CD22E14 (mean = 1.112 ± 0.015; median = 1.103; range = 1.026–1.279; p-value < 10−15). (Figure 2C). In addition to CD22ΔE12, CD22 exon 2 (CD22E2) skipping that results in very low CD22E2 mRNA levels also occurs in B-ALL and is associated with resistance to CD22-directed immunotherapies due to reduced expression of the target CD22 protein. As both events are caused by aberrant splicing, we next evaluated CD22E2 mRNA levels in CD22E12low patients in an effort to determine if CD22E12low and CD22E2low subsets overlap in the studied B-ALL patient population (n = 141). A subset of 34 patients (24.1%) with CD22E2 reductions (CD22E2low) were identified via examination of the RPKM values for CD22E2 and the adjacent CD22 exons 1 (CD22E1), 3 (CD22E3), and 4 (CD22E4) (Figure S1). The normalized RPKM value for each patient in this CD22E2low subset was less than 0.397. The mean CD22E2 expression level for the CD22E2low patients was 0.295 ± 0.016, which was significantly lower than the mean CD22E2 expression level of 0.565 ± 0.014 for the remaining 107 patients (Welch two-sample t-test, T = 12.673, df = 83.3, p-value < 10−15) (Figure S1). Among the 21 CD22E12low patients and 120 others, 8 (38%) and 26 (22%) were CD22E2low (Fisher’s exact test, p = 0.16). The density plots for CD22E2 mRNA expression for CD22E12low vs. other patients displayed a near-complete overlap (Figure S2). The mean CD22E2 expression level for the CD22E12low patients was 0.46 ± 0.039, which was not significantly different from the mean CD22E2 expression level of 0.507 ± 0.016 for the remaining 120 patients (Welch two-sample t-test, T = 1.13, df = 27.2, p-value = 0.27). The CD22E2 expression levels among the 34 CD22E2low patients were very similar for the 8 CD22E12low subset vs. the remaining 26 patients (0.298 ± 0.032 vs. 0.295 ± 0.019). Likewise, the CD22E2 expression levels for the 107 non-CD22E2low patients did not show significant differences between 13 CD22E12low patients and the remaining 94 patients (0.56 ± 0.039 vs. 0.566 ± 0.015) (two-way ANOVA, FDR-adjusted p-value = 0.96 for both CD22E2low and all other patients comparisons) (Figure S2). These results demonstrate that CD22E12low status applies to a smaller fraction of B-ALL patients (14.9% vs. 24.1%, p = 0.16, taking into account eight patients (6%) who were both CD22E12low and CD22E2low) and does not show an apparent relationship to the more frequent CD22E2low status. We next sought to determine if the CD22E12low patient subset is characterized by an enrichment of high-risk prognostic markers within the confines of a relatively small sample size (n = 141). As shown in Table 1, the patient characteristics exhibited similar ages and gender/race/ethnicity distribution, and they did not show marked differences between CD22E12low patients and the remainder of the patient population (Table S1 and Table 1). The mean age was 8.2 ± 1.2 years (median = 7.6 years) for CD22E12low patients vs. 7.9 ± 0.5 years (median = 6.4 years) for other patients (p = 0.8). A total of 11 of 21 (52.4%) CD22E12low patients vs. 53 of 120 (44.2%) other patients were in the high-risk age category (age < 2 years or ≥10 years) (p = 0.6, Table 1). There was no enrichment for adult patients (viz.: >18 years of age; 1/21 (4.8%) CD22E12low patients versus 4/120 (3.3%) others; p = 0.6). The mean WBC was 45.5 ± 13.1 × 109/L (median = 15.9 × 109/L) for CD22E12low patients and 76.8 ± 12.0 109/L (median = 33 × 109/L) for other patients (p = 0.2). A total of 11 of 21 (52.4%) CD22E12low patients vs. 79 of the remaining 120 patients (65.8%) had NCI high-risk ALL (Fisher’s Exact, 2-tailed test, p = 0.3, Table 1). There was no enrichment for patients with higher WBC (viz.: WBC ≥ 20 × 109/L (10/21 (47.6%) vs. 73/120 (60.8%), p = 0.3), CNS2 or CNS3 category (5 of 21 CD22E12low patients (23.8%) versus 23 of 120 (19.2%) other patients, p = 0.6), poor-risk cytogenetics assessed by karyotyping (pseudodiploidy, hyperdiploidy, or hypodiploidy with structural chromosomal abnormalities (SCA) (i.e., 7 of 12 (58.3%) evaluable CD22E12low patients versus 71 of 94 (75.5%) evaluable other patients, p = 0.3). None of the evaluable CD22E12low patients were t(9;22)/BCR-ABL1+ or MLL-R/t(4;11)+. Three out of twenty (15%) evaluable CD22E12low patients were TCF3-PBX1+ compared to 11/112 (9.8%) evaluable other patients (p = 0.4). We noticed that none of the 20 evaluable CD22E12low patients had the favorable prognosis markers ETV6-RUNX1 or hyperdiploidy with trisomy 4 and 8. By comparison, among the remaining 112 evaluable other patients, 11 (9.8%) were ETV6-RUNX1+ (p = 0.2), and 10 (8.9%) had trisomy 4 and 10 (0/21 (0%) vs. 21/112 (18.8%), p = 0.042). Notably, there was no apparent evidence for intrinsic resistance to induction chemotherapy. There were only two induction failures in this group of patients; none of the 21 CD22E12low patients and 2 of the 119 other patients in the RNA-seq subset experienced an induction failure. At the end of the induction therapy, 57.1% of CD22E12low patients and 46.7% of the remaining patients had no measurable residual disease (MRD), as measured with 6-color flow cytometry (p = 0.5). The mean day 29 MRD values were 0.07 ± 0.03% for CD22E12low patients and 0.87 ± 0.35% for the remaining patients (p = 0.4). End-of-consolidation MRD data was available for only three CD22E12low patients, and they all were zero (Table 1). Within the NCI high-risk subset of 90 patients, only 3 of 52 patients (6%) with MRD > 0 and 8 of 38 patients (21%) with MRD = 0 were CD22E12low (Fisher’s exact p-value = 0.048). We used GSEA to examine the transcriptomes of primary leukemia cells from CD22E12low B-ALL patients for selective disruptions of reactome pathways that are selectively affected in murine B-ALL cells from CD22ΔE12-Tg mice [9]. Notably, the expression of several genes represented in reactomes involved in transcription (Figure S3, Table S2), mRNA processing/mRNA transport (Figure S4, Tables S3 and S4), translation (Figure S5A, Table S5), post-translational protein modification (Figure S5B, Table S6), signal transduction (Figure S6, Table S7), and cell cycle (Figure S7, Table S8) were dysregulated in primary leukemia cells from CD22E12low patients. Table 2 compares the affected reactome pathways in CD22E12low human B-ALL cells vs. murine B-ALL cells originating from CD22ΔE12-Tg mice [9]. The most significantly upregulated genes in primary leukemia cells from CD22E12low B-ALL patients, according to the affected reactome pathways they are represented in, exhibited fold-increase values over the expression values in primary leukemia cells from other patients ranging from 1.5 to 2.3 and included (i) CCNK, NELFA, GATAD2A, and NUDT21 (transcription); (ii) DDX20, HNRNPD, SMN1, and KHSRP (mRNA processing); (iii) SRRM1, SRSF3, RAE1, and POLDIP3 (mRNA transport); (iv) ETF1, EIF5, EIF4E, and EIF4H (translation); (v) EP300, CREBBP, UBE2G1, and RTF1 (post-translational protein modification); (vi), PPP2CA, SKP1, PSMC2, and NFKB1 (signal transduction); (vii) MAPRE1, DYNC1LJ1, CHMP4B, and FZR1 (cell cycle) (Figure 3). We next evaluated the potential impact of reduced CD22 exon 12 expression on the treatment outcomes in B-ALL by comparing the outcomes of CD22E12low patients to the outcomes of the remaining patients. The median follow-up time was 963 days (range = 77–4175 days; interquartile range = 549–1948 days) for the 141 B-ALL patients with RNA-seq data. Among these patients, the median follow-up times were 867 days (range = 77–3830 days; interquartile range = 528–1220 days) for the 21 CD22E12low patients and 1017 days (range = 98–4175 days; interquartile range = 558–2426 days) for the remaining 120 patients. A total of 101 of 141 patients experienced a relapse after attaining remission. CD22E12low patients had a significantly higher incidence of relapse (19/21; 90.4%) than other patients (82/120 (68%) (Fisher’s exact test, p = 0.039). The probability of relapse within five years was 90.5 ± 6.4% for CD22E12low subset and 69.2 ± 4.4% for other patients (p = 0.023) (Figure 4A). Further, the 685-day median time to relapse (95% CI: 531–1129) in CD22E12low patients was significantly shorter than the median time to relapse in the remaining patients (median: 1012, 95% CI: 819–1191 days; log-rank chi-square = 5.19, p-value = 0.023) (Figure 4A). CD22E12low patients had significantly worse LFS outcomes than other patients (Figure 4B). The probability of surviving leukemia-free at 5-years was 9.5 ± 6.4% for the CD22E12low subset and 29.8 ± 4.3% for other patients (p = 0.039). The median LFS time for CD22E12low patients was 685 (95% CI: 531–1129) days, which was significantly shorter than the median LFS time of 958 (95% CI: 801–1178) days for other patients (log rank chi-square = 4.48, p-value = 0.034) (Figure 4B). The OS outcome of CD22E12low patients was significantly worse than the OS outcome of other patients (Figure 4C). The probability of being alive after five years was 19.0 ± 8.6% for the CD22E12low subset and 53.9 ± 4.6% for other patients (p = 0.004). The median OS time for CD22E12low patients was 1008 days (need 95% CI = 883–1715), which is significantly shorter than the median OS time of 2029 days (95% CI = 1456-NA) for the remaining patients (log-rank chi-square = 7.82, p-value = 0.005) (Figure 4C). We next asked if CD22E12low status remained a poor prognostic indicator for LFS and OS in the 90-patient subset of NCI high-risk B-ALL patients. CD22E12low patients (n = 11) within the high-risk subset of patients had worse treatment outcomes than the remaining patients (n = 79) (Figure S8). CD22E12low high-risk B-ALL patients had a shorter time to relapse (median values: 625 days (95% CI = 520–NA) vs. 1083 days (95% CI = 881–NA); log-rank chi-square = 4.19, p-value = 0.04) (Figure S8A), shorter LFS (median values: 625 days (95% CI = 520–NA) vs. 1040 days (95% CI = 863–NA); log rank chi square = 3.7, p-value = 0.05) (Figure S8B), and shorter OS (median values: 1008 days (95% CI = 736–NA) vs. 2029 days (95% CI = 1413–NA); log-rank chi-square = 4.58, p-value = 0.03) than other high-risk B-ALL patients (Figure S8C). The probability of high-risk B-ALL patients to be alive and leukemia-free after five years was 18.2 ± 11.6% for the CD22E12low subset and 46.4 ± 5.6% for other patients (p = 0.1). The probability of high-risk patients to be alive after five years was 18.2 ± 11.6% for the CD22E12low subset and 54.3 ± 5.7% for other patients (p = 0.025). We also examined the effects of CD22E12low status as an indicator of poor prognosis associated with a poor LFS as well as poor OS in univariate and multivariate Cox proportional hazards models. These models include as variables the following candidate poor prognostic characteristics for hazard ratio (HR) determinations: (i) CD22E12low status; (ii) age < 2 y or ≥ 10 y; (iii) male; (iv) poor risk classification based on molecular markers (BCR-ABL1+, MLL-R+, or TCF3-PBX1+); (v) WBC at diagnosis (as a linear covariate); and (vi) end-of-induction day 29 MRD burden > 0 (defined as MRD ≥ 0.001%). CD22E12low status was associated with an increased hazard ratio for shorter LFS in both univariate (HR = 1.7 ± 0.3, 95% CI: 1.0–2.8, p-value = 0.04) and multivariate models (HR = 1.8 ± 0.3, 95% CI: 1.1–3.0, p-value = 0.03) (Figure 5A,B). Similarly, CD22E12low status was associated with an increased hazard ratio for shorter OS in both univariate (HR = 2.1 ± 0.3, 95% CI: 1.2–3.6, p-value = 0.006) and multivariate models (HR = 2.3 ± 0.3, 95% CI: 1.3–4.0, p-value = 0.004) (Figure 5C,D). In the multivariate Cox model, the HR values for CD22E12low status (viz.: 1.8 for LFS and 2.3 for OS) were not as high as those associated with a poor-risk molecular marker profile (viz.: 4.0 for LFS and 3.3 for OS) but higher than those associated with any other poor risk variable analyzed. Notably, the comparison of the LFS and OS outcomes for the subset of 90 evaluable high-risk B-ALL patients also showed a significant increase in HR for patients with CD22E12low status (n = 11) compared to other patients (n = 79) (Figure S9). In the multivariate Cox regression model, CD22E12low status was found to be a significant and independent predictor of poor LFS outcomes (HR = 3.1 ± 0.4, p-value = 0.01) as well as poor OS outcomes (4.0 ± 0.5, p-value = 0.003). The HR values for CD22E12low status (viz.: 3.1 for LFS and 4.0 for OS) were higher than those associated with any other poor risk variable included in the multivariate Cox model. We had originally hypothesized that CD22ΔΕ12 might act as an oncogenic protein by competitively binding to the cis ligands of CD22 and preventing residual wildtype CD22 from in cis ligand binding, thereby contributing to the increased proliferation and defective apoptosis of leukemic B-cell precursors from B-ALL patients caused by the CD22ΔE12-associated impaired regulatory function of CD22 [7,8]. Expression of a truncated CD22ΔE12 protein was indeed associated with aggressive in vivo growth of primary B-ALL cells in immunodeficient NOD/SCID mice [7]. Lentiviral-based overexpression of truncated CD22ΔE12 protein but not full-length CD22 in B-ALL cells resulted in a marked increase in the sizes of blast colonies in vitro, consistent with increased self-renewal and clonogenicity [13]. Further, CD22ΔE12 depletion via CD22ΔE12-specific small interfering RNA (siRNA) and their liposomal formulations inhibited the in vitro and in vivo clonogenicity of B-ALL cells [8,9,13]. These experimental findings collectively support our hypothesis regarding the oncogenic function of CD22ΔE12 protein and demonstrate that CD22ΔE12 expression is associated with the selective survival and growth advantage of B-ALL cells [7,8,9,10,11,12,13,14]. Our initial studies demonstrated that CD22ΔE12, with the selective reduction of CD22E12 levels, is a characteristic splicing defect in primary leukemia cells from newly diagnosed high-risk and relapsed B-ALL patients [10]. We now report for the first time that in newly diagnosed B-ALL, CD22E12low patients with very low CD22E12 levels, who represent 14.9% of the patient population, had significantly worse LFS and OS outcomes than other patients. Our study thereby fills a significant gap in our understanding of the clinical significance of CD22ΔE12 in B-ALL. The cumulative proportion of patients in the analyzed population of 141 newly diagnosed B-ALL patients who survived after five years was 19.0 ± 8.6% for the CD22E12low subset (n = 21) and 53.4 ± 4.6% for the remaining 120 patients (p = 0.004). The median OS time for CD22E12low patients was 1008 days, which is significantly shorter than the median OS time of 2029 days for the remaining patients (log-rank chi-square = 7.8, p-value = 0.005). Likewise, in the 90-patient subset of NCI high-risk B-ALL patients, CD22E12low patients (n = 11) had significantly worse treatment outcomes than the remaining 79 patients (n = 79). Importantly, CD22E12low status was associated with a significantly increased hazard ratio for shorter PFS and OS in both univariate and multivariate models. In the multivariate Cox regression model for the NCI high-risk subset that also included patient age, sex, WBC at presentation, end-of-induction MRD burden, and poor-risk cytogenetics/FISH markers (BCR-ABL1+, MLL-R+ or TCF3-PBX1+) as covariables, we found CD22E12low status to be a significant and independent predictor of poor LFS outcomes (HR = 3.1 ± 0.4, p-value = 0.01) as well as poor OS outcomes (HR = 4.0 ± 0.5, p-value = 0.003). These new results significantly extend a previous study that implicated CD22ΔE12 as a driver lesion, contributing to the aggressive biology of relapsed pediatric B-ALL [10]. CD2E12low status at presentation may have clinical utility as a poor prognostic biomarker and may help guide the early allocation of risk-adjusted, patient-tailored treatment regimens as well as refine risk classification in high-risk B-ALL. Forced overexpression of the mutant CD22ΔE12 in transgenic mice caused fatal B-ALL, demonstrating that CD22ΔE12 alone may be sufficient as a driver lesion for the leukemic transformation and aggressive in vivo growth of B-cell precursors [7,8]. Notably, genes related to transcriptional, translational, signal transduction-related, and cell cycle-related reactome pathways were significantly upregulated in B-ALL cells from CD22ΔE12 Tg mice [9]. We previously reported that the CD22ΔE12 signature transcriptome encodes a phosphoproteome that is differentially overexpressed in CD22ΔE12-Tg B-ALL cells, confirming that CD22ΔE12 corrupts the regulation of multiple signaling networks. Several anti-apoptotic proteins such as mTOR, AKT, NFκB, transcription factors implicated in oncogenesis, and serine kinase signaling pathway proteins such as MAPK, PKC, and PKD were among the most significantly overexpressed members of the CD22ΔE12 signature phosphoproteome [9,13]. In the current study, we used GSEA in gene sets obtained from the Bioconductor Reactome Database to examine the transcriptomes of primary leukemia cells from CD22E12low B-ALL patients for selective disruptions of reactome pathways we had previously discovered to be selectively dysregulated in murine B-ALL cells from CD22ΔE12-Tg mice [9,13]. Notably, the expression of several genes represented in reactomes involved in transcription, mRNA processing/mRNA transport, translation, post-translational protein modification, signal transduction, and cell cycle were selectively amplified in primary leukemia cells from CD22E12low patients. These findings demonstrate that the regulation of gene expression is corrupted in CD22E12low B-ALL cells across multiple reactomes, which is reminiscent of the CD22ΔE12-associated transcriptome changes in transgenic mice [9,13]. We propose that the observed differences in gene expression profiles of CD22E12low vs. other B-ALL patients may contribute to the seemingly more aggressive biology of the CD22E12low subset. RNAi therapeutics targeting CD22ΔE12 may disrupt the signaling networks that promote the proliferation and survival of CD22ΔE12+ B-ALL cells [8,9,13]. We previously reported preclinical data regarding the in vitro and in vivo anti-leukemic activity of nanoformulations of CD22ΔE12-specific siRNA as an innovative new treatment platform for CD22ΔE12+ B-ALL [8,9,13]. Further development and optimization of this experimental platform may have clinical potential. The clinical development of personalized nanomedicines against the CD22ΔE12 poor-risk B-ALL might address an unmet challenge in treating B-ALL by improving the landscape of effective treatment modalities. The observed poor prognosis of newly diagnosed B-ALL patients with CD22ΔE12-associated CD22E12low status, as reported here, supports the notion that further exploration of the clinical potential of CD22ΔE12-targeting RNAi therapeutics is warranted. Black et al. reported mutations in splicing factor genes, including the genes for hnRNPs, as a possible mechanism for aberrant splicing in B-ALL [27]. Pathogenic intronic mutations were implicated in aberrant splicing and human disease [28,29]. In patients with CD22ΔE12, we previously reported multiple homozygous mutations within a 132-bp mutational hotspot segment of the intronic sequence between exons 12 and 13, some of which involved locations of known single nucleotide polymorphisms (SNP) [7]. Within this intronic segment, we identified multiple accessible potential binding sites for the heterogenous nuclear ribonucleoprotein (hnRNP) family of splicing factors that act as global regulators of alternative splicing [7,30,31]. The documented mutations within the hot spot region were associated with secondary structure conformation and folding patterns that affected the target motifs for hnRNP-E2/PCBP, hnRNP-I/PTB, and hnRNP-L as well as the surrounding structure of the predicted pre-mRNA [7]. Therefore, we proposed that these mutations might contribute to aberrant pre-mRNA splicing by affecting the recognition of the 5′ splice site of CD22E12 via the splicing factors and preventing an orderly assembly of the splicesome assembly [7]. In addition to CD22ΔE12, CD22E2 skipping by aberrant splicing [32,33] also occurs in B-ALL, which results in very low CD22E2 mRNA levels and is associated with inherent resistance to CD22-directed immunotherapies such as inotuzumab, ozogamicin, and CAR-T cells [34,35,36] due to reduced expression of the target CD22 protein. As both events are caused by aberrant splicing, we evaluated CD22E2 mRNA levels in CD22E12low patients in an effort to determine if CD22E12low and CD22E2low subsets overlap in the studied B-ALL patient population. Our findings indicate that the CD22E12low status applies to a smaller fraction of B-ALL patients and does not show an apparent relationship to the more common CD22E2low status. Therefore, CD22-targeting immunotherapeutics remain a viable treatment modality for a significant portion of CD22E12low patients without evidence of CD22E2 skipping and/or a marked reduction of CD22 expression levels. The present study has a significant limitation owing to a patient selection bias caused by the availability of RNA-seq data for only 12.5% of patients in the TARGET database who had worse leukemia-free survival and overall survival outcomes than the total patient population in the database. A number of statistically significant differences in patient characteristics suggests that the 141-patient RNA-seq subset was likely enriched for cases with a biologically more aggressive disease, including a higher mean WBC at diagnosis in the RNA-seq subset (72.2 ± 10.4 63.7 ± 3.2, p = 0.046), a higher portion of patients with CNS 2 or CNS 3 at diagnosis in the RNA-seq subset (19.9% vs. 11.6%, p = 0.003), a higher incidence of CNS relapse in the RNA-seq subset (10.6% vs. 5.1%, p = 0.003, odds ratio: 2.6), a higher portion of patients with MRD > 0 on day 29 in the RNA-seq subset (51.8 vs. 41.8%, p = 0.014), and a higher portion of patients with structural chromosomal abnormalities in the RNA-seq subset (73.6% vs. 64.4%, p = 0.04, odds ratio: 1.6) (Table S9). Although these differences might have contributed to poor survival outcomes, it was not possible to decipher the exact reasons of the worse-than-expected survival outcomes in the analysis population, as patient-specific treatment information was not archived in the TARGET database. Another limitation of the study relates to the fact that some of the prognostically relevant data, such as the MRD values at the end of consolidation, were not collected for all patients. Although the characteristics of the CD22E12low patients within the RNA-seq subset were very similar to those of the remainder of patients in the RNA-seq subset, which allowed an accurate comparison of the outcomes of the CD22E12low and CD22E12high patients (Table 1, Table S1), a hypothesis-testing prospective validation study will be necessary to confirm the poor prognostic effect of CD22E12low status, preferably with RT-qPCR and RNA-seq in a larger B-ALL patient population treated according to a contemporary standard of care regimen. Our study demonstrates that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 (“CD22E12low”), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse LFS as well as OS than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.

PMC10000519

Emily L. Chávez-Delgado,Daniel A. Jacobo-Velázquez

Essential Oils: Recent Advances on Their Dual Role as Food Preservatives and Nutraceuticals against the Metabolic Syndrome

03-03-2023

essential oils,metabolic syndrome,diabetes,obesity,neuroprotection,antioxidant,antimicrobial,nanoencapsulation

Essential oils (EO) are compounds synthesized by plants as secondary products and are a complex mixture of volatile molecules. Studies have demonstrated their pharmacological activity in the prevention and treatment of metabolic syndrome (MetS). Moreover, they have been used as antimicrobial and antioxidant food additives. The first part of this review discusses the role of EO as nutraceuticals to prevent metabolic syndrome-related disorders (i.e., obesity, diabetes, and neurodegenerative diseases), showing results from in vitro and in vivo studies. Likewise, the second part describes the bioavailability and mechanisms of action of EO in preventing chronic diseases. The third part presents the application of EO as food additives, pointing out their antimicrobial and antioxidant activity in food formulations. Finally, the last part explains the stability and methods for encapsulating EO. In conclusion, EO dual role as nutraceuticals and food additives makes them excellent candidates to formulate dietary supplements and functional foods. However, further investigation is needed to understand EO interaction mechanisms with human metabolic pathways and to develop novel technological approaches to enhance EO stability in food systems to scale up these processes and, in this way, to overcome current health problems.

Essential Oils: Recent Advances on Their Dual Role as Food Preservatives and Nutraceuticals against the Metabolic Syndrome Essential oils (EO) are compounds synthesized by plants as secondary products and are a complex mixture of volatile molecules. Studies have demonstrated their pharmacological activity in the prevention and treatment of metabolic syndrome (MetS). Moreover, they have been used as antimicrobial and antioxidant food additives. The first part of this review discusses the role of EO as nutraceuticals to prevent metabolic syndrome-related disorders (i.e., obesity, diabetes, and neurodegenerative diseases), showing results from in vitro and in vivo studies. Likewise, the second part describes the bioavailability and mechanisms of action of EO in preventing chronic diseases. The third part presents the application of EO as food additives, pointing out their antimicrobial and antioxidant activity in food formulations. Finally, the last part explains the stability and methods for encapsulating EO. In conclusion, EO dual role as nutraceuticals and food additives makes them excellent candidates to formulate dietary supplements and functional foods. However, further investigation is needed to understand EO interaction mechanisms with human metabolic pathways and to develop novel technological approaches to enhance EO stability in food systems to scale up these processes and, in this way, to overcome current health problems. Obesity is the leading risk factor for metabolic syndrome (MetS) due to energetic imbalance; this can cause impaired glucose tolerance, insulin resistance, type 2 diabetes, dyslipidemia, hypertension, and a chronic proinflammatory state [1,2]. Non-pharmaceutical alternatives, such as nutraceuticals, phytotherapy, and functional foods, have been explored to prevent and treat common diseases, and their use is increasing in the public domain by up to 50–70% [2]. Nevertheless, their long-term safety, efficacy, and dose schemes are being actively researched [3,4]. Essential oils (EO) are composed of a mixture of natural, volatile, aromatic compounds characterized by a strong odor and are produced as secondary metabolites by aromatic plants in different plant organs, including buds, flowers, seeds, leaves, roots, fruits, wood, twigs or bark [5,6,7]. Extraction methods are divided into conventional and non-conventional techniques. Common conventional techniques include hydrodistillation, Soxhlet extraction, water distillation, steam distillation, and organic-solvent extraction. Non-conventional extraction processes include ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), high-pressure (HP), pressurized liquid extraction (PLE), negative pressure cavitations-assisted extraction (NPCE), subcritical water extraction (SWE), supercritical fluid extraction (SFE), enzyme-assisted extraction (EAE), pulsed electric field-assisted extraction (PEF), and accelerated solvent extraction (ASE). These extraction techniques and non-thermal and are categorized as “green”, since they use lower amounts of solvents, energy, and time, giving higher yields compared to conventional methods [6,8]. Organic compounds present in EO vary between 20–200 different types, with the vast majority present in traces, although two or three of these compounds are the most representative ones (20–70%) and are thought to be responsible for the biological activities of the EO [6,7]. Despite that, terpene hydrocarbons are the primary chemical group found in EO [6]. Terpene hydrocarbons are classified as monoterpenes (C10), which are the major constituents of EO; sesquiterpenes (C15); diterpenes (C20); terpenoids (oxygenated terpenes); and aromatic compounds, such as phenylpropanoids, derived from phenylpropane [7]. Traditional medicinal herbs and their derived EO are phytochemical-rich sources of health-promoting bioactive compounds [9]. As nutraceuticals, some reported health benefits of EO include antioxidative, antimicrobial, antitumor, anticarcinogenic, anti-inflammatory, antiatherosclerosis, antimutagenic, antiplatelet aggregation, and angiogenesis inhibitory activities [1,3,4,6,7,9]. Nowadays, EO have been used in the food industry as food additives due to their antioxidant and antimicrobial properties. A large variety of EO from different plants have been incorporated into food systems, such as basil, chamomile flowers, cardamom seeds, and rosemary [10]. There is active research in the study of EO as natural potential candidates to prevent and treat MetS. Their incorporation in proper food vehicles to achieve this goal is a new and interesting approach that deserves particular interest. For this reason, several studies focused on these fields are recapitulated herein. However, to the best of our knowledge, few or no studies are focused on using EO as nutraceuticals and food additives to treat metabolic and non-communicable diseases. Therefore, this review is intended to give a new approach for the production of functional foods using EO as promising molecules. This review is divided into four main sections; the first part discusses the effects of EO in MetS and its comorbidities, specifically in obesity, diabetes, and neurodegenerative diseases, including in vitro and in vivo studies and clinical trials. The second section describes the bioavailability and EO mechanisms of action of their common administration routes (i.e., oral, dermal, and pulmonary administration). The third part presents the incorporation of EO as food additives and discusses current applications in food systems as antimicrobial and antioxidant agents. Finally, the last part focuses on the stabilization and common methods for encapsulating EO in other to preserve their bioactivity and how they could be incorporated into food matrixes. In this review article, EO bioactivities and additive properties reported were searched in several databases such as Elsevier, Google Scholar, PubMed, and SpringerLink via Tecnológico de Monterrey library system. In these databases, keywords were: “essential oils, anti-obesity, anti-diabetes, metabolic syndrome, adipogenesis inhibition, postprandial hyperglycemia control, neuroprotection, microencapsulation, and antimicrobial and antioxidative properties”. Research and review articles were selected based on the use of EO in controlling these pathologies and their incorporation into food matrixes. In the case of anti-obesity, anti-diabetes, and neuroprotection activities, those studies focused only on individual or majority components of EO were excluded since it was intended to show the bioactivity of EO per se. Because this field has been relatively little explored, we aimed to explore extensively what has been reported over the past 18 years (2005–2023). MetS is a combination of metabolic disorders that comprises central obesity, insulin resistance, hypertension, and atherogenic dyslipidemia. These factors propitiate chronic inflammation, leading to cardiovascular disease (CVD) development. Because obesity rates have been increasing worldwide, MetS has become highly relevant; thus, early prevention and treatment are crucial factors in decreasing mortality rates [1,11]. It has been reported the use of EO for treating obesity and diabetes. The following sections present in vitro and in vivo studies and clinical trials regarding the use of EO for preventing and treating metabolic syndrome-related disorders. Adipocytes are cells in charge of maintaining energetic homeostasis. These cells are present in white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is important in energy storage, while BAT uses energy to produce heat. Morphologically, WAT is characterized by one single lipid droplet, whereas BAT comprises many multilocular liquid droplets and mitochondria [12]. Excessive fat accumulation results in inflammation and oxidative stress in adipose tissue as a result of a constant elevation of plasma-free fatty acids (FFAs) caused by a growing release from enlarged adipose tissue that activates and upregulates the expression of several proinflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin (IL)-1β and IL-6, which aggravates metabolic alterations [13,14]. For instance, the constant liberation of FFAs promotes the synthesis of very low-density lipoprotein, cholesterol, and gluconeogenesis in the liver, thus provoking impaired insulin signaling and glucose metabolism and, by this process, causing insulin resistance [15]. Fat homeostasis is driven by two principal metabolic pathways: lipogenesis and lipolysis. The former is accountable for packaging esterified triglycerides in the liquid droplet when there is an excess of nutrients, thus expanding adipose tissue, which may play a central role in obesity comorbidities. At the same time, the latter hydrolyze lipid triglycerides into glycerol and three fatty acids [16,17,18]. Lipogenesis is driven by two principal transcriptional regulators, the Sterol Response Element Binding Protein 1c (SREBP1c) and the Carbohydrate Response Element Binding Protein (ChREBP). The activation of both pathways is due to increased insulin signaling in response to high glucose levels [14]. Lipogenesis inhibition is a promising strategy to prevent fat storage in adipocytes; therefore, WAT and BAT are crucial targets for obesity treatment and related diseases [12]. Adipocyte differentiation, also known as adipogenesis, is a process in which preadipocytes are transformed into mature adipocytes [14]. It plays a vital role in regulating obesity; for instance, combined with adipocyte hypertrophy, it is is the primary mechanism leading to this disorder [19,20]. In vitro and in vivo experiments on the anti-obesity potential of different EO are presented in Table 1. Regarding adipogenesis, this process compromises several steps guided by different transcription factors regulating adipogenic gene expression. There are two important families, the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs). Adipogenesis initiates with the expression of C/EBPβ and C/EBPδ, which in turn activates C/EBPα and PPARγ mRNA. These molecules trigger the transcription of adipogenic genes, resulting in phenotypically and functionally different fat cells. Likewise, extracellular signal-regulated kinases (ERKs), members of the mitogen-activated protein kinases (MAP-Ks), also participate in the signaling cascades of C/EBPα and PPARγ expression [19]. As presented in Table 1, many studies demonstrated that using EO led to suppressed lipid droplet accumulation and adipogenesis in a dose-dependent manner. Yen et al. [1], Ngamdokmai et al. [18], Hwang et al. [19], Lee et al. [20], Ko et al. [21], Cheng et al. [22], and Sprenger et al. [23] investigated the effect of various EOs, such as lemon balm, peppermint, lavender, bergamot, cypress, niaouli nerolidol, geranium-rose, revensara, lemon grass, ginger, black pepper, Artemisa annua L., calamus, Pinus koraiensis, and cinnamon. These EOs were all tested in 3T3-L1 preadipocytes treated with different concentrations during the differentiation process, followed by Oil-Red O (ORO) assay to assess lipid accumulation. The outcomes of these studies exhibited that these EOs significantly inhibited lipid accumulation through the downregulation of 3T3-L1 adipocyte differentiation. Adipogenesis inhibition is due to the suppression of adipogenic transcription factors expression, mentioned above. It is concluded that these EO have anti-obesogenic and hypolipidemic potential via inhibition of PPARγ-related signaling. For instance, the studies performed by Ngamdokmai et al. [18], Lee et al. [19], Cheng et al. [22], Sprenger et al. [23], Lai et al. [24], and Lai et al. [25], besides testing EOs per se, also isolated the major component of each and tested their action in the model of study (in vitro or in vivo). It was concluded that those components exerted anti-obesity effects. In the case of olfactory stimulation, the study performed by Hong et al. [15] demonstrated that citronellol is a volatile and prominent patchouli EO (PEO) compound that is accountable for diminishing food intake, thus preventing obesity. Moreover, α-patchoulene and β-patchoulene release the PEO odor, stimulating the hypothalamus and regulating serum leptin levels, lowering food intake. However, as Russo et al. [7] mentioned, further investigation is required for individual components’ action in adipocyte metabolism since EOs are phytochemically complex molecules in which each component is thought to take part in the overall outcome and could regulate the effect of the others, either synergistically or antagonistically. Therefore, it could be uncertain which individual components are the only ones responsible for conferring anti-obesity effects, thus it is preferable to assert results taking into consideration the EO as a whole. Studies in animals have demonstrated that certain EOs exert anti-obesogenic activity. Ko et al. [21], Cheng et al. [22], Lai et al. [24], Lai et al. [25], Asnaashari et al. [26], and Ciftci et al. [27] investigated the effect of garlic, Pinus koraiensis, lime, cinnamon, ginger and a mix of thyme, orange peel, bay leaf, and eucalyptus, respectively, on body weight, food intake, serum biochemical metabolites (glucose, insulin, free fatty acids, cholesterol, and triglycerides), and adipose tissue of standard or high-fat diet (HFD) fed animals (mice, rats or quails). EOs tested suppressed—in a dose-dependent manner—increases in fat pads, body weight, and serum biochemical parameters induced by HFD. Scientific reports evaluating the anti-obesity effects of EO compared to orlistat—a specific gastrointestinal lipase inhibitor commonly used in obesity treatment that inhibits the absorption of fat, resulting in weight loss—concluded that sweet orange and cumin EOs are potential candidates to replace pharmacological obesity treatments through downregulation of PPARγ expression, consequently preventing preadipocytes [28,29,30,31]. Diabetes mellitus (DM) is a chronic, lifelong progressive metabolic disorder caused by impaired insulin secretion or insulin resistance, resulting in chronic hyperglycemia. Metabolic abnormalities in carbohydrates, lipids, and proteins arise as a result of low levels of insulin to achieve adequate response or insulin resistance in target tissues [32,33,34]. Two primary factors are involved in the development of type 2 diabetes (T2D): impaired insulin secretion by pancreatic β-cells or a lowered number of β-cell mass, which may also contribute to insufficient secretion of insulin and the inability of insulin-sensitive tissues to respond appropriately to this hormone [35]. Insulin resistance in T2D increases the demand for insulin in insulin-target tissues. However, this increased demand for insulin could not be met by the pancreatic β cells due to defects in the function of these cells, which in turn decreases insulin secretion due to the gradual destruction of β cells, resulting in a vicious cycle of metabolic state worsening that could transform some type 2 diabetes patients from being independent to becoming dependent on insulin [33]. Postprandial hyperglycemia has been described in the etiology of T2D and cardiovascular disease (CVD); moreover, it is a significant risk factor for the development of atherosclerosis in nondiabetic people [36,37]. Postprandial hyperglycemia is an excessive plasma glucose concentration after eating, characterized by hyperglycemic spikes that induce oxidative stress. Even in healthy subjects, short-term postprandial hyperglycemia is accompanied by endothelial dysfunction, elevated adhesion molecules, and proinflammatory cytokines in the blood. Postprandial hyperglycemia is driven by many factors such as timing, quantity, meal composition, carbohydrate content, insulin and glucagon secretion, among others [37,38,39]. Regarding adipocytes, they are the principal targets for postprandial glucose uptake [1]. Carbohydrates are the main dietary component that affects glycemia [36]. Once a meal rich in carbohydrates is ingested, starch is hydrolyzed quickly by digestive enzymes such as α-amylase and α-glucosidase, which results in a high rise in blood glucose and insulin level [40]. Starch contributes to 40–60% of the total energy intake in the human diet [41]. This complex carbohydrate comprises two glucose polymers: amylose, a linear polymer composed of glucose units linked by alpha-(1→4) bonds, and amylopectin, which is a large branched molecule that also has glucose chains linked by alpha-(1→4) bonds and also has glucose chain branches with alpha-(1→6) bonds [42]. In humans, α-amylases are found in the salivary glands that secrete the enzyme in the mouth and the pancreas, which secretes the enzyme in the small intestine. It hydrolyzes the α-(1→4) glycosidic bonds in the starch molecule leading to the production of maltose, maltotriose, maltotetraose, maltodextrins, and glucose [42]. For its part, α-glucosidase is found on the luminal surface of enterocytes and is secreted in the small intestine. It is a key enzyme that catalyzes the hydrolysis of disaccharides (maltose and sucrose) into monosaccharides (glucose and fructose) and acts predominantly on α-amylase digestion products, rapidly converting them to glucose. Likewise, α-glucosidase can hydrolyze α-(1→6) bonds, which cannot be attacked by α-amylase, removing dextrins and allowing starch digestion to complete [41]. Diabetes management includes glycemic control, reducing body weight, changes in lifestyle, prevention of micro and macrovascular damage, and others. Glycemia in type 2 diabetes patients can be controlled by pharmacological therapy. Four main groups of antidiabetic drugs act through different mechanisms: (i) biguanides: reduce gluconeogenesis in the liver (e.g., metformin); (ii) insulin secretagogues: stimulate insulin secretion of the pancreas (e.g., sulfonylureas); (iii) insulin sensitizers: improve the sensitivity of peripheral tissues to insulin (e.g., thiazolidinediones); and (iv) insulin or its analogs which provide insulin exogenously in the form of recombinant insulin [43]. In the case of thiazolidinediones, they act via the activation of peroxisome proliferator-activated receptors (PPARs), decreasing insulin resistance and regulating adipocyte differentiation. For instance, biguanides, such as metformin, act by activating adenosine monophosphate-activated protein kinase (AMPK), which plays a significant role in energetic balance, insulin signaling, and metabolism of fats and glucose [1]. Additionally, metformin affects the translocation of GLUT4 in insulin-targeted cells. GLUT4 is an ATP-independent glucose transport protein prevalent in adipose and muscle tissues and enhances glucose uptake. Metformin also activates AMPK phosphorylation in adipose and muscle tissues; this mechanism compromises phosphorylation of insulin receptor substrate 1 (IRS-1) Ser789, which, via cascade signaling, activates phosphoinositide 3 kinase/protein kinase B (PI3K/PKB) signaling, thus increasing blood glucose balance and decreasing insulin resistance. In adipocytes, AMPK activation inhibits lipogenesis while enhancing energy consumption, leading to an anti-obesity effect [44]. Both AMPK activators and PPARs ligands regulate glucose homeostasis and decrease insulin resistance in adipose tissue [1]. Control of postprandial hyperglycemia is an essential factor in diabetes treatment. Currently, there are three main oral antidiabetic drugs: acarbose, miglitol, and voglibose, which regulate glucose availability for intestinal absorption by modifying carbohydrate digestion. All of these drugs are 𝛼-glucosidase inhibitors that reversibly and competitively reduce the hydrolytic activity of these enzymes, thereby regulating the availability of glucose for intestinal absorption and the speed and extent of postprandial hyperglycemia [45]. Acarbose has been used as a pharmacological prescription to manage postprandial glucose. It has been reported that it can decrease diabetes progression by 25% [36]. Drug combination therapeutic management has shown better results than drug monotherapy; therefore, acarbose and metformin treatment has been reported to improve effects on patients with T2D [43]. However, acarbose has common gastrointestinal adverse effects, including abdominal pain, diarrhea, and bloating [46]. These side effects result from maltose fermentation, accumulating due to α-glucosidase inhibition [47]. The difference in the mechanism of action of acarbose to miglitol and voglibose is that the former reduces polysaccharides digestion in the upper small intestine. In contrast, the latter reduces disaccharide digestion, thus in the lower small intestine there is a higher polysaccharide content when consuming acarbose; with miglitol and voglibose, there is a higher disaccharide content in the lower small intestine [45]. Postprandial hyperglycemia in nondiabetic people is a predictor of insulin resistance and cardiovascular disease. In the case of patients with T2D, it has a relationship with micro and macrovascular disease. Moreover, sharp long-term changes in blood insulin levels in normal individuals may cause insulin resistance in organs and tissues, a central mark of hyperglycemia and type 2 diabetes. Regulating postprandial hyperglycemia early is a feasible strategy for preventing and managing T2D [40]. Hence, common pharmaceutical approaches in the management and treatment of T2D compromise AMPK activators, PPARs ligands, and α-amylase and α-glucosidase inhibitors, which moderate the metabolism of dietary carbohydrates [48]. Nevertheless, undesirable effects are displayed by these treatments, which could be attenuated by EOs exerting antidiabetic effects (Table 2). In the case of carbohydrate-related enzymes, which regulate carbohydrate digestion and glucose absorption in the small intestine, it has been reported that partial inhibition of α-amylase and α-glucosidase by EO is a natural alternative in the control of T2D. In the study performed by Radünz et al. [49], among all the EOs evaluated, thyme offered the most significant α-glucosidase inhibition (98.9%), while sweet orange EO showed the most potently inhibitory effect in α-amylase (95.4%). Their major components, thymol and D-limonene, respectively, are thought to be responsible for inhibitory capacity. All EO evaluated in this study exhibited a better capacity for enzyme inhibition than acarbose, the conventional drug prescribed. Moreover, incomplete enzyme inhibition, and medium and high range inhibition for α-amylase and α-glucosidase, respectively, is proposed for clinical treatment since these ranges allow the control of T2D without compromising nutrients or glucose absorption in the small intestine. According to Rahali et al. [50], some important factors need to be considered respecting the biological activity of plant EO. Even though its chemical composition is responsible for conferring bioactivities, it is influenced by plant genotype, organ type, extraction type, phenological stage, and environmental conditions. Their study analyzed the chemical composition of different plant organs, such as leaves, flower buds, flowers, and fruits, in terms of the EO of Hertia cheirifolia, and how these differences influenced α-amylase inhibitory activities. It was reported that leaves and fruits EO possessed the highest activity of α-amylase inhibition with 8.32 and 8.84 mg Eq acarbose/g EO, respectively. In this regard, the study performed by Pavlić et al. [51] evaluated different extraction techniques and experimental conditions in peppermint leaves. Extraction methods included conventional hydrodistillation (HD), microwave-assisted hydrodistillation (MWHD), soxhlet extraction (SOX), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE). HD and MWHD were applied to obtain the volatile fraction, that is, pure EO. It is expected that polyphenols and flavonoids were not present in these samples. The rest of the techniques aimed to recover lipophilic compounds, which are mixtures of volatile and non-volatile lipids. Its chemical composition varied depending on the extraction method since some monoterpene hydrocarbons (α-pinene, camphene, myrcene, and terpinolene) were absent in SOX, MAE, and UAE. For instance, SFE allows the extraction of terpenoids (oxygenated compounds) and other lipophilic bioactive compounds. Results of this study showed that peppermint EO, obtained by HD and MWHD, was the most potent α-amylase inhibitor, with an activity range of 1.24–1.76 ACEs/g. However, EOs did not exhibit α-glucosidase inhibition, while most lipophilic extracts were potent inhibitors with a 57.96–58.89 mmol ACEs/g activity range. Studies in animals have demonstrated that lemon balm EO has antihyperglycemic effects. The study carried out by Chung et al. [52] demonstrated that supplementation to mice fed with lemon balm EO showed a decrease in glucose concentration and an increment in glucose tolerance. These results indicated that lemon balm EO stimulates glucokinase (GCK) activity and inhibits glucose-6-phosphatase (G6Pase) activity in the liver of mice. The former is stimulated by insulin and enhances glucose consumption and uptake in the liver, while the latter controls hepatic gluconeogenesis and glucose output in the liver; it is inhibited by insulin. When its activity is reduced, it decreases hepatic glucose production. As presented in Table 2, some EO exhibited a lower carbohydrate-enzymatic-related inhibition activity when using acarbose as a positive control [53,54,55,56,57]. However, the side effects of synthetic drugs used to treat obesity and diabetes are not expected to happen with natural compounds like EO. Diabetes is a risk factor for developing Alzheimer’s disease (AD) and other types of dementia [67]. In this context, untreated diabetes can cause memory disorders [68]. Because of chemical properties or monoterpenes, they can travel quickly across the single epithelial nasal mucosa, be incorporated into blood circulation, and cross the blood-brain barrier. For those reasons, aromatherapy with EO has been an alternative to AD treatment [67]. Furthermore, cholinesterase inhibitors are the target for preventing and treating AD. These inhibitors impede the cholinergic deficit associated with cognitive dysfunction [63]. Two principal cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are associated with AD [58]. An increase in these cholinesterases leads to reduced levels of acetylcholine neurotransmitter, which is involved in memory and learning [67]. For instance, AChE is related to β-amyloid plaques and neurofibrillary tangles (NFT) [69]. This inhibition promotes an increase in the level of acetylcholine in neuronal synapsis, which leads to improved stimulation of the cholinergic receptors [62,63]. Wild mint EO has been evaluated as a potential cholinesterase inhibitor. The study performed by Asghari et al. [58] showed that wild mint (Mentha longifolia var. calliantha) EO has an intense AChE activity. Enzymes’ inhibitory activity was expressed as equivalents of galantamine (GALAEs). In the case of AChE, IC50 was 1.82 mg GALAEs, and 2.57 mg GALAEs for BChE. This study mentioned that oxygenated monoterpenes present in the EO were accountable for neuroprotection since 1,8-cineol, the most abundant component, and carvacrol have been reported to be acetylcholinesterase inhibitors. The study by Sarikurkcu et al. [62] suggested that Origanum species are recommended for AD treatment. It was reported that O. vulgare subsp. vulgare and O. vulgare subsp. hirtum exhibited a similar inhibitory action on both AChE and BChE. It is thought that inhibitory properties are due to the high concentration of thymol and carvacrol in O. vulgare subsp. vulgare, while for O. vulgare subsp. hirtum it is because of the high concentration of linalool. Another study conducted by Aumeeruddy-Elalfi et al. [63] proposed that citrus species such as Citrus grandis (CGp), Citrus hystrix (CH), and Citrus reticulata (CR) have comparable activity as galantamine, a common drug used to treat mild to moderate AD. These EO are categorized as an uncompetitive type of inhibitor since there is a decrement in Km and Vmax parameters in their presence. Discovering the inhibition type would be helpful for further investigations to achieve, so as to elucidate the interaction of the EO with cholinesterases. EOs are a complex mixture of volatile and non-volatile compounds, such as hydrocarbons, fatty acids, sterols, carotenoids, waxes, and flavonoids. In the case of volatile constituents, there are hydrocarbons (e.g., pinene, limonene, bisabolene), alcohols (e.g., linanol), acids (e.g., benzoic acid), aldehydes (e.g., citral), cyclic aldehydes (e.g., cuminal), ketones (e.g., camphor), lactones (e.g., bergaptene), phenols (e.g., eugenol), phenolic ethers (e.g., anethole), oxides (e.g., 1,8 -cineole), and esters (e.g., geranyl acetate) [70,71]. The biological properties of EO are commonly attributed to the main molecules at the highest concentrations. However, it is thought there is a synergistic relationship between the molecules in the EO. Hence, the other minor molecules could regulate the activity of major components [5]. It has been suggested that crude plant extract administration of EO is better for their bioavailability than purified compounds [44]. Investigation about the absorption, distribution, and metabolism of EO is necessary to extrapolate in vitro to in vivo studies results since therapeutic activities depend on the availability of EO compounds reaching specific target organs [72]. Some studies about the bioavailability and pharmacokinetics of monoterpenes present in EO have been published recently. For instance, oral administration of thymol and carvacrol, which are monoterpene aglycones, have a slow absorption in the bloodstream. Monoterpene aglycones have a nonpolar ending that signifies they can easily travel across cell membranes; nevertheless, their hydrophobicity is challenging. Therefore, future studies are needed to understand these compounds’ receptor interaction, activity, and specificity to elucidate their therapeutic potential [44,68]. It has been reported that EO can be easily absorbed via pulmonary, dermal, or oral administration (Figure 1). Regarding the pulmonary mechanism of action, smells stimulate the olfactory bulb, a part of the limbic system involved in behavioral and emotional responses, which comprises the hippocampus, amygdala, and hypothalamus. When an aromatic compound binds to cilia in olfactory receptor cells, they activate adenylate cyclase, promoting the increment in cAMP concentration. This second messenger binds to Ca2+ channels, causing the entrance of Ca2+ into the cell and depolarizing the cell membrane. Moreover, intracellular Ca2+ activates Cl− channels and causes further membrane depolarization. These signals produce action potentials that are transferred to the glomeruli in the olfactory bulb, which eventually will be transmitted to the limbic system and cerebral cortex [70,73]. The hypothalamus oversees the autonomic nervous, endocrine, and immune systems. For instance, the autonomic nervous system (ANS) has three different divisions in terms of anatomy: sympathetic (SNS); parasympathetic (PNS); and enteric (ENS) nervous systems. In this way, studies in rats have demonstrated that some fragrances enhance sympathetic nervous activity and suppress parasympathetic activity, while others have the opposite effect on the ANS [70,74]. Olfactory stimulation studies done by Hong et al. [15], Batubara et al. [30], and Shen et al. [31] proved that citronella, grapefruit, and patchouli EO reduce body weight, as well as food intake, appetite, and plasma biochemical parameters (glucose, cholesterol, triglycerides), via sympathetic nerve stimulation in brown adipose tissue, since it promotes thermogenesis (heat production) that converts fatty acids into fuel (energy consumption). This heat generation has been reported to reduce body fat since there is an increment in mitochondria respiration and fatty acid oxidation related to AMPK activation or adipogenesis inhibition. Also, heat production can be enhanced through uncoupling protein 1 (UCP1) activation; consequently, energy consumption and body temperature are raised by uncoupling oxidation from ATP production in mitochondria. In the case of grapefruit EO, in addition to what was previously mentioned, it was also reported that it suppresses parasympathetic gastric nerve activity; as a result, it inhibits nutrient digestion and absorption. Pulmonary absorption depends on subjects’ breathing mechanisms, mucosal compound deposition, metabolism, and the type of compound evaluated. Elimination of EO occurs mainly exhaled as CO2 [71,75]. Regarding dermal absorption, EO can easily be absorbed due to their lipophilic character and can easily penetrate through the skin into the bloodstream. For instance, the absorption rate in cells increases with their hydrophobicity and decreases as their molecular weight increases. Some studies have reported that after dermal administration of linalyl acetate, terpinen-4-ol, citronellol, and α-pinene, they reached their highest level 15–20 min after application and decreased gradually for 2 h [70,71,75,76,77]. The stratum corneum (SC) is the top layer of the epidermis and is a barrier to the penetration of substances. SC mainly consisted of lipids and protein keratin. EOs have been used as penetration enhancers (PE) in transdermal drug delivery systems. The role of PE is to temporarily provoke a reversible reduction in the barrier function of SC in order to allow safe and effective drug delivery via skin. EO as PE can achieve this through different mechanisms such as (i) intracellular lipid structure rearrangement between corneocytes in SC, (ii) intracellular proteins conformational modifications due to interactions, (iii) enhance drug partitioning into SC, and (iv) enhancement of desmosome connections between corneocytes or metabolic activity alteration within the skin. In this way, EOs and their active constituents can penetrate the epidermis by two different pathways: (1) transcellular (intracellular) permeation across the corneocytes of SC by appendage penetration through hair follicle, sebaceous and sweat glands; and (2) intercellular permeation through intercellular spaces of the SC. Briefly, a drug has to travel across continuous layers of intracellular lipids and proteins to reach blood circulation via the skin [77,78,79]. When orally administered, EOs interact with digested food. The kinetic rate depends on digestive enzymes to hydrolyze EO compounds from the fatty acid linkages. In the case of terpenoids and steroids present in EO, they can be digested in the small intestine along with other lipids due to their lipophilic properties. On the other hand, hydrophilic EO compounds, such as polyphenols, flavones, flavanols, lignans, and aromatic acids, are bound to saccharides metabolized in the small intestine. Aglycones that are not absorbed cross to the liver, where they are absorbed and enzymatically degraded. Free hydrophilic molecules are transported into enterocytes via passive diffusion or active transport in the duodenum [71]. Intravenous administration suggests that the elimination half-life of EO in humans is about one hour. However, it has been reported that the highest concentration of active compounds from EO is two hours after administration, and after five hours, the substances have already been effectively eliminated from the bloodstream. Evidence shows that the half-life of carvacrol, thymol, eugenol, and trans-cinnamaldehyde was between 1.84 and 2.05 h. Elimination of EO occurs mainly by renal secretion in the form of glucuronides or exhaled as CO2. EO are non-toxic molecules since they are fast and quickly metabolized; thus, they are not accumulated in the organism and are excreted from the body with urine and feces [71,75,77,78]. EO have been cataloged as Generally Recognized as Safe (GRAS) for food additives and flavorings. Some EO that are GRAS include basil, cinnamon, clove, coriander, ginger, lavandin, menthol, nutmeg, oregano, rose, sage, and thyme [75]. There is active research on in vitro study of the antimicrobial and antioxidant activities of EO (Figure 2). In the following sections, some studies are presented as novel developments in incorporating EO in different food systems [80]. Active packaging aims to extend food shelf-life and maintain or improve the packaged food’s properties. Unlike conventional food packaging, active packaging interacts with the product by incorporating compounds that could be released into the food or absorb substances responsible for the deterioration of the product. Incorporating EOs and their components into food packaging has been reported to increase food shelf-life since they have exhibited antimicrobial and antioxidant activities that can eradicate the presence of pathogen microorganisms and reduce lipid oxidation. Hence, EOs are an alternative to reduce or replace synthetic additives [10]. Several studies demonstrated the antimicrobial activity of EO in food matrices. EO compounds can disrupt the bacterial membrane, damage their metabolic pathways, and prevent the synthesis of bacterial toxins [71]. Nevertheless, Gram-positive bacteria are more vulnerable to attaining antimicrobial effects, since hydrophobic molecules can easily pass through the cell membrane. In contrast, the outer membrane of Gram-negative bacteria can act as a barrier due to lipopolysaccharides’ presence, so they are more resistant toward hydrophobic compounds like those presented in EO; however, some phenolic compounds present in EO (i.e., thymol, eugenol, and carvacrol) can interfere with the cell wall outer membrane [81,82]. Due to their low molecular weight and lipophilic properties, EO can easily pass through the cell membrane and can inhibit bacteria growth by disrupting cell membranes, enzyme systems, and cell division, preventing biofilm formation, inducing bacterial membrane to produce clumps and auto-aggregation, hyperpolarization of cell membrane, altering lipid profile by formation of fatty acid hydroperoxidases caused by the oxygenation of unsaturated fatty acids within the cytosol, and by formation of cell membrane channels which cause leakage of ions, cellular material, and nucleic acids. Cell damage can lead to disruption of proton motive force and can cause ATP loss or affect ATP synthesis, changing the conformation of ATPase and inhibiting the expression of ATPase-related subunits interfere [82,83]. For instance, it has been reported that Mentha species contain hydrogen peroxide which can damage biomolecules of microorganisms, such as proteins, lipids, nucleic acids, and carbohydrates [84]. In terms of antimicrobial properties related to fungi, it has been reported that interaction of cinnamaldehyde, a major compound of cinnamon oil, with Aspergillus flavus caused elevated Ca2+ and ROS, decrease in mitochondrial membrane potential, release of cytochrome c, activation of metacaspase, and DNA damage. This compound increased the expression levels of apoptosis-related genes [82]. Moreover, EO exert antifungal effects through cell wall disruption causing leakage of cellular contents; it is thought that this disruption is caused by interactions of EO with ergosterol, which is the principal sterol present in fungi cell membranes and which controls permeability and fluidity [85]. Nonetheless, instead of synthetic antibiotics, EOs are commonly used in food systems as natural antiseptics to ensure food safety. The in vitro antimicrobial activity of particular EOs, such as oregano and thyme, against many Gram-positive and Gram-negative bacteria, yeasts, and molds has been thoroughly analyzed and documented [86]. For example, Siroli et al. [87] evaluated the efficacy of oregano and thyme essential oils for lamb’s lettuce decontamination and compared it to the efficacy of chlorine. The results showed that by applying EO, a product shelf-life similar to that obtained with chlorine was achieved [86]. Some studies reported by Ribeiro-Santos et al. [9] demonstrated that low-density polyethylene films with linalool and methyl chavicol exhibited antimicrobial activities against Escherichia coli and Listeria innocua in Cheddar cheese packaging previously inoculated with those organisms. Moreover, another polyethylene film with cinnamon EO and cinnamaldehyde, inhibited the growth of fungi (Penicillium islandicum, Penicillium roqueforti, Penicillium nalgiovense, Eurotium repens, Aspergillus flavus, Candida albicans, Debaryomyces hansenii, and Zigosaccharomyces rouxii) and bacteria (Bacillus cereus) at 4% (w/w) of active compounds. In comparison, Listeria monocytogenes and Staphylococcus aureus were inhibited at 8% (w/w), and E. coli, Yersinia enterocolitica, Salmonella choleraesuise, and Pseudomonas aeruginosa at >10% (w/w). Furthermore, Masyita et al. [75] mentioned that L. monocytogenes is one of the major pathogens responsible for diseases in humans and animals. They reported a study in which clove and cinnamon EOs were evaluated in ground beef. Results demonstrated that 10% clove EO could decrease the growth of L. monocytogenes. Additionally, it has been reported that eucalyptus EO reduces Saccharomyces cerevisiae in Orangina juice. Antioxidant capacity exerted by EO is due to the double bonds present in alcohols, ethers, ketones, aldehydes, and phenolic compounds. Both terpenoid and phenylpropanoid families compromise phenolic compounds; these have high reactivity with peroxyl radicals, which are disposed of by hydrogen-atom transfer [83,88]. In the case of food products, lipid oxidation is the primary source of oxidation which produces rancidity; the use of EO polyphenolic compounds (i.e., terpenoids and phenolic acids) can act as oxygen and free radical scavengers to reduce lipid oxidation [83]. Some EO have been reported to exert in vitro antioxidant capacity. Routinary assays are used to measure radical scavenging properties against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid radical cation (ABTS). These methods are commonly used in the food industry to evaluate the antioxidant activity of specific compounds within the food matrix. Radünz et al. [49] evaluated the effect of clove, thyme, oregano, and sweet orange EOs using DPPH assay. Results exhibited that clove EO had the highest inhibition percentage (94.3%). The primary compound found in clove EO was eugenol, to which authors attributed the remarkable ability to interact with free radicals compared to the major components of the other EO evaluated. For instance, wild mint (Mentha longifolia var. calliantha) was evaluated by Asghari et al. [58] using DPPH and ABTS radicals to determine the free radical scavenging ability of the EO. It was demonstrated that wild mint EO had moderate antiradical potential in DPPH assay (5.8 mmol TEs/g oil), in contrast to a very high potential in ABTS assay (186 mmol TEs/g oil), expressed as equivalents of standard antioxidant compound Trolox (TEs). It is thought that this strong antioxidant capacity is due to the principal monoterpenes present in the EO, such as 1,8-cineol, linalool, and carvacrol, which are capable of donating hydrogens. Moreover, two different species of oregano were evaluated by Sarikurkcu et al. [62], Origanum vulgare subsp. vulgare and subsp. hirtum. Besides performing DPPH and ABTS methods to measure radical scavenging, the β-carotene bleaching method was used to measure lipid peroxidation inhibition. In this assay, β-carotene is oxidized by radicals formed by linoleic acid oxidation in an emulsion, in which, eventually, the system loses its chromophore and is monitored spectrophotometrically. The results of this study exhibited that O. vulgare subsp. vulgare obtained 57.23 mg TEs/g oil for DPPH and 176.41 mg TEs/g oil for ABTS. This species exerted higher activity than that of O. vulgare subsp. hirtum in both assays. Due to differences in the chemical profile, the free radical capacity is influenced by the major components of the EO; in this regard, O. vulgare subsp. vulgare has a higher concentration of thymol and carvacrol (58.31 and 16.11%, respectively), which have been reported to be efficient scavengers of free radicals, as reported by Asghari et al. [58]. In terms of the β-carotene bleaching method, it obtained similar results since O. vulgare subsp. vulgare is the better inhibitor of linoleic acid oxidation (99.89%) compared to O. vulgare subsp. hirtum (23.54%). Hence, O. vulgare subsp. vulgare can be helpful for the management of lipid oxidation in the food industry. Regarding the application of EOs as antioxidants in food products, there is active research in the meat industry to prevent oxidation reactions in meat and meat products. Studies reported by Pateiro et al. [89] suggested that oregano EO added at a concentration of 3% w/w significantly reduced oxidation reactions in raw and cooked porcine and bovine ground meat., Sage EO was also evaluated in fresh pork sausages, and there was a protective effect against lipid oxidation; furthermore, it had a higher antioxidant value than synthetic BHT. However, these antioxidant properties can be converted into prooxidants at high concentrations. An example of this was a study in which 150 ppm of rosemary was added to meat, and inhibition of lipid and protein oxidation was achieved. However, at 300 and 600 ppm concentrations, it promoted oxidation reactions because of interactions with fatty acids or concentration of tocopherols present in the product. Hence, it is essential to consider doses and meat matrix components interactions since their activity depends on the concentration utilized. In active packaging, EOs are recommended to be introduced as micro or nanoemulsions. Incorporating EOs as micro or nanoemulsions prevents intense aroma [75]. In this regard, several strategies exist to incorporate EO into packaging materials. These techniques include a direct addition into polymeric materials, incorporation into coatings, immobilization with substrates, trapping into physical carries, insertion into headspace, or micro/nanoencapsulation in carriers, followed by incorporation into food matrices [90]. EOs are quite volatile and sensitive in certain conditions of illumination, temperature, and humidity, which are common during food processing. Therefore, researchers have used various encapsulation systems with different shell materials to protect EO from volatilization, oxidation, instability, and insolubility. The most common and effective encapsulation systems include liposomes, chitosan nanoparticles, cyclodextrin, silicon dioxide, nanoemulsions, solid lipid nanoparticles, nanofibers, and edible films. Furthermore, certain packaging methods, including food wraps and nanofibers, have also been proven to protect EO [91]. Recently, Reis et al. [92] published a review that addressed the conventional and most innovative encapsulation methods and the most relevant shell materials used in food systems. For instance, some essential factors have to be considered beforehand when selecting an appropriate encapsulation technique, such as desired particle size, shell materials’ physical properties, the core material’s solubility, controlled release, layer permeability, and costs. In this way, wall materials have to accomplish three different stages in order to be considered successful: (i) formation of a wall around the core; (ii) core components have to be kept inside the capsule without any release or degradation; and (iii) incorporation of the capsule in food systems and correct release of oil components. Encapsulation techniques are divided into two different categories: physical and chemical methods. Physical methods do not involve polymerization reactions, and microcapsule formation occurs mechanically. This classification includes extrusion, fluidization, lyophilization, solvent removal, spray drying, and supercritical fluid techniques. For its part, chemical methods involve polymerization reactions, and techniques include coacervation, ionic gelation, liposomes, and miniemulsion polymerization. Moreover, common shell materials used in food matrixes include polysaccharides (i.e., starch, dextrin, maltodextrin, modified starch, cyclodextrin, chitosan), gums (i.e., arabic gum, sodium alginate), proteins (i.e., whey, soy, casein, gelatin), cellulose (i.e., modified cellulose), and lipids (i.e., waxes, paraffin, fats) (Figure 3). There is a discussion about emulsification as an encapsulation technique or as a step before encapsulation since the latter guarantees an improvement in storage stability because droplets are immobilized in a solid matrix. At the same time, the former comprises a liquid wall material that could disfavor the core compounds’ physical and chemical resistance and retention. Nevertheless, emulsification methods are divided into conventional methods (i.e., colloid mill, high-speed mixer, high-pressure homogenizer, ultrasonic homogenizer) and membrane emulsification methods. Some advantages regarding the membrane emulsification method as a novel technique are lower energy demand, low shear rates, lower temperature elevation, more control of droplet size, and ease of scaling up. However, some disadvantages of this method are fouling phenomena on the membrane surface and pores, the membrane’s short lifetime, and more resistance in mass transfer regarding the membrane. Research has shown that nanoencapsulation boosts the preservative potential of plant essential oils in vitro and in food systems. For instance, Jamil et al. [93] investigated the antimicrobial efficacy of cardamom oil encapsulated in chitosan-based nanoparticles. The results demonstrated that the nano-encapsulated EO exhibits excellent antimicrobial potential against Escherichia coli and Staphylococcus aureus [94]. This encapsulation system has been used to protect EO effectively and improve their functional performance in food systems [95]. Amiri et al. [96] used conventional nanoemulsion and fortified nanoemulsion as delivery systems for Zataria multiflora in corn starch and analyzed its effect on the sensory properties of ground beef patties. The results showed that the fortified nanoemulsion had the highest scores for all the sensory parameters, while the control showed the lowest scores [95]. Another study by Viacava et al. [97] also analyzed the effect nanoencapsulation has on sensory attributes. The researchers studied the impact of free and β-cyclodextrin encapsulated thyme EO on the quality of minimally processed Romaine lettuce. The results demonstrated that the lettuce treated with the nano-encapsulated EO exhibited better organoleptic quality scores than the control and free EO-treated lettuce [95]. Therefore, the authors concluded that nanoencapsulation could help improve the organoleptic attributes of food items. Other delivery systems, including surfactant-based systems, films, fibers, and oleogels, are being investigated for their effectiveness. For instance, Chen & Yang [98] have used Quillaja saponin, a natural triterpene, to stabilize orange oil via oleogel [99]. The authors concluded that the oleogels with high gel strength had good thixotropic recovery and reversibility to reconstituted emulsions. Therefore, oleogels show great potential for food, cosmetics, and pharmaceutical applications [99]. Quillaja saponin has also been used by Sedaghat Doost et al. [100] to stabilize thymol nanoemulsions. With this system, the authors could create thymol emulsions with long-term stability that contained a relatively low content of green solvents. Furthermore, the authors reported that, compared to free thymol, emulsification improved its antioxidant activity. Another innovative strategy has been investigated by Silva et al. [101], as they encapsulated coriander essential oil in cyclodextrin nanosponges to achieve a controlled oil release. The authors reported that cyclodextrin polymers can effectively incorporate and release coriander essential oil and that including this oil inside the nanosponge improves the crystallinity of the polymer, which leads to a more effective controlled release. For instance, in a study performed by Siahbalaei et al. [54], some EO (Oliveria decumbens, Thymus kotschyanus, Trachyspermum ammi, and Zataria multiflora) were individually encapsulated into a gelatin-pectin nanocomposite consisting of gelatin (7 g), pectin (3 g), 100 mL acetic acid (60%), glycerol (100 mg/g of total polymer), and glutaraldehyde (10 mg/g of total polymer), resulting in a 500 to 700 nm size range. Each composite displayed several bioactivities such as glucose autoxidation inhibition, lipid peroxidation inhibition, protein oxidation, glycation inhibition, and α-amylase and α-glucosidase inhibition activity. EOs have been demonstrated to have anti-obesity, antidiabetic activities, and neuroprotective effects. They can be used as a natural alternative to treat these disorders even though further investigation is needed to elucidate the interaction between EO and metabolic pathways involved in developing these diseases at both the cellular level and in living organisms. EO are an attractive alternative for substituting synthetic additives since they show antimicrobial and antioxidant activities that extend food products’ shelf-life and guarantee food safety to consumers. EO micro/nanoencapsulation is a novel technological approach for their stabilization and incorporation into different food systems. Nevertheless, the interaction of these compounds with food matrices needs to be studied in detail before their incorporation to obtain the expected results. The scientific information herein presented demonstrates the dual role of EO in preventing and treating metabolic syndrome-related disorders and their well-demonstrated role as antioxidant and antimicrobial food additives. This dual role makes EO excellent candidates to formulate dietary supplements and functional foods. Over the course of this review, there are several research gaps and perspectives in the field that have been identified and need to be further explored. The first of these is that there are several factors, with respect to the raw material used, that must be required when carrying out in vitro or in vivo studies. Among them are the quality, the extraction method, the part of the plant used, and its stage of maturation. Since, depending on this, primary compounds may vary, so too will bioactive properties be impacted as well. Another point to consider is the nanoencapsulation of EO. Besides preserving and/or enhancing bioactivity, it needs to ensure a benefit in the organoleptic properties of food systems, such as flavor, color, aroma, and texture, so that in this way, a high consumption of functional foods fortified with EO is guaranteed. Furthermore, studies about synergistic combinations of EO to exert therapeutical and technological properties in food systems need to be performed, especially regarding the quality and quantity used of each one in food matrixes, in order to offer the promised bioactivity. In this regard, novel technological approaches to enhance EO stability in food systems deserve more research to scale up processes in an industrial manner and, in this way, to overcome current health problems.

PMC10000521

Roberto Fernández-Acosta,Behrouz Hassannia,Jurgen Caroen,Bartosz Wiernicki,Daniel Alvarez-Alminaque,Bruno Verstraeten,Johan Van der Eycken,Peter Vandenabeele,Tom Vanden Berghe,Gilberto L. Pardo-Andreu

Molecular Mechanisms of Nemorosone-Induced Ferroptosis in Cancer Cells

24-02-2023

nemorosone,ferroptosis,mitochondrial uncoupling,fibrosarcoma,neuroblastoma

Ferroptosis is an iron-dependent cell death-driven by excessive peroxidation of polyunsaturated fatty acids (PUFAs) of membranes. A growing body of evidence suggests the induction of ferroptosis as a cutting-edge strategy in cancer treatment research. Despite the essential role of mitochondria in cellular metabolism, bioenergetics, and cell death, their function in ferroptosis is still poorly understood. Recently, mitochondria were elucidated as an important component in cysteine-deprivation-induced (CDI) ferroptosis, which provides novel targets in the search for new ferroptosis-inducing compounds (FINs). Here, we identified the natural mitochondrial uncoupler nemorosone as a ferroptosis inducer in cancer cells. Interestingly, nemorosone triggers ferroptosis by a double-edged mechanism. In addition to decreasing the glutathione (GSH) levels by blocking the System xc cystine/glutamate antiporter (SLC7A11), nemorosone increases the intracellular labile Fe2+ pool via heme oxygenase-1 (HMOX1) induction. Interestingly, a structural variant of nemorosone (O-methylated nemorosone), having lost the capacity to uncouple mitochondrial respiration, does not trigger cell death anymore, suggesting that the mitochondrial bioenergetic disruption via mitochondrial uncoupling is necessary for nemorosone-induced ferroptosis. Our results open novel opportunities for cancer cell killing by mitochondrial uncoupling-induced ferroptosis.

Molecular Mechanisms of Nemorosone-Induced Ferroptosis in Cancer Cells Ferroptosis is an iron-dependent cell death-driven by excessive peroxidation of polyunsaturated fatty acids (PUFAs) of membranes. A growing body of evidence suggests the induction of ferroptosis as a cutting-edge strategy in cancer treatment research. Despite the essential role of mitochondria in cellular metabolism, bioenergetics, and cell death, their function in ferroptosis is still poorly understood. Recently, mitochondria were elucidated as an important component in cysteine-deprivation-induced (CDI) ferroptosis, which provides novel targets in the search for new ferroptosis-inducing compounds (FINs). Here, we identified the natural mitochondrial uncoupler nemorosone as a ferroptosis inducer in cancer cells. Interestingly, nemorosone triggers ferroptosis by a double-edged mechanism. In addition to decreasing the glutathione (GSH) levels by blocking the System xc cystine/glutamate antiporter (SLC7A11), nemorosone increases the intracellular labile Fe2+ pool via heme oxygenase-1 (HMOX1) induction. Interestingly, a structural variant of nemorosone (O-methylated nemorosone), having lost the capacity to uncouple mitochondrial respiration, does not trigger cell death anymore, suggesting that the mitochondrial bioenergetic disruption via mitochondrial uncoupling is necessary for nemorosone-induced ferroptosis. Our results open novel opportunities for cancer cell killing by mitochondrial uncoupling-induced ferroptosis. The natural product nemorosone was isolated in 1996 through the extraction of floral resins from Clusia rosea, an evergreen tropical plant with several medicinal applications [1]. Its chemical structure was unraveled in 2001 as a type A polyisoprenylated benzophenone [2]. The high interest in nemorosone mainly resides in its cytotoxic anti-cancer activity, as shown in a broad spectrum of different human cancer models such as leukemia, colorectal, pancreatic, hepatic, and breast cancer [3,4,5,6,7,8,9,10,11]. In most cases, apoptosis was reported as its mode of cytotoxicity along with the arrest of cell cycle progression. Recently, its antimetastatic potential through the modulation of molecules related to the epithelial-mesenchymal transition (EMT) was also described [11]. Furthermore, several research works have reported antimutagenic activity without a genotoxic effect, selectivity toward cancer cells, and the capacity to circumvent multidrug-resistance mechanisms [4,5,12,13,14]. Altogether, these previous studies show that nemorosone can be considered as a lead compound for the development of novel antiproliferative drugs for cancer therapy. On the other hand, we found that nemorosone disrupts the mitochondrial bioenergetic status by acting as a potent protonophoric mitochondrial uncoupler [6,9]. Therefore, despite the well-established capacity of nemorosone to induce apoptosis [4,5], other modes of regulated cell death could be induced depending on the cell type. Since the conceptualization of ferroptosis in 2012, several compounds previously described as inducers of apoptosis, such as cisplatin, sorafenib, and withaferin A, have been found to elicit ferroptosis [15,16,17]. Moreover, the induction of ferroptosis has also been identified in a myriad of natural products [18]. Ferroptosis is a form of regulated necrosis mediated by iron-catalyzed excessive lipid peroxidation [19,20], often referred to as biological rust of lipid membranes [21]. Ferroptosis can be induced by inactivating glutathione peroxidase 4 (GPX4), which detoxifies lipid hydroperoxides. GPX4 can be inactivated through direct targeting and inhibition, by class II and III inducers, or alternatively by indirect mechanisms through the depletion of intracellular GSH, an essential co-factor of GPX4, by class I inducers [22]. Furthermore, ferroptosis can be activated by the increase in the cellular labile iron pool (LIP), which is the intracellular non-protein bound redox-active iron, or iron oxidation mediated by class IV inducers [21]. Recently, Gao et al. showed that mitochondria play a central role in cysteine-deprivation-induced and erastin-induced ferroptosis (Class I FINs), but not in the case of the ferroptosis induced by GPX4 inhibition (Class II FINs). Mechanistically, cysteine deprivation leads to a transient hyperpolarization of the mitochondrial membrane potential and lipid peroxide production [23]. On the other hand, cell death induced by mitochondrial uncoupling is accompanied by depolarization of the mitochondrial membrane potential, reduced ATP levels, increased ROS, and diminished antioxidant defense by decreasing GSH levels [24]. The latter may resemble a similar cellular effect observed in erastin-treated cells. Therefore, we hypothesized and examined whether mitochondrial uncoupling by nemorosone could initiate a ferroptotic response. Using different cell lines, we found that nemorosone triggers ferroptosis, as detected by lipid peroxidation. We elucidated that nemorosone-induced ferroptosis involves a double-edged mechanism. Nemorosone decreases the glutathione levels by blocking the cystine/glutamate antiporter and induces lipid peroxidation as an early event. At later time points, nemorosone administration results in activation of the KEAP1–NRF2–HMOX1 axis, causing an increase in the intracellular labile Fe2+ pool and consequent reactive oxygen species (ROS) production. Methylnemorosone, a structural variant of nemorosone that has lost the capacity to uncouple mitochondrial respiration, does not trigger cell death anymore. The classical mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) has a similar biological effect as nemorosone, in inducing ferroptosis. In summary, our results demonstrate that nemorosone as well as other mitochondrial uncouplers drive intrinsic ferroptosis. The present findings could open new perspectives for a better insight into ferroptosis initiated by mitochondrial dysfunction and for the development of novel ferroptosis inducers for cancer treatment. The following antibodies were used in this study: β-tubulin (Abcam, Cambridge, UK, ab21058), GPX4 (Abcam, ab41787), HMOX1 (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-896-F), NRF2 (Abcam, ab62352), and KEAP1 (Proteintech, Rosemont, IL, USA, 10503-2-AP). The following chemicals were used: SytoxGreen (Thermo Fisher Scientific, Waltham, MA, USA, S7020): 1.7 μM, BODIPY 581/591 C11 probe (Invitrogen, Waltham, MA, USA, D-3861): 2 μM, SytoxBlue (Thermo Fisher Scientific, S11348): 1.25 μM, DRAQ7 (BioStatus, Loughborough, UK, DR71000): 0.3 μM, TMRE: tetramethylrhodamine ethyl ester (Thermo Fisher Scientific, T669): 200 nM, FeRhoNox-1 (Goryo Chemical, Sapporo, Hokkaido, Japan, GC901): 10 μM, MitoSOX Red (Thermo Fisher Scientific, M36008): 5 μM, erastin (Selleckchem, Houston, TX, USA, S7242): 10 and 20 μM, CCCP: carbonyl cyanide 3-chlorophenylhydrazone (Sigma, St. Louis, MO, USA, Cat#C2759), Nec-1s (Calbiochem, San Diego, CA, USA, 480065): 10 μM, Fer1 (Xcess Biosciences, Chicago, IL, USA, 053224): 1 μM, DFO (Sigma-Aldrich, St. Louis, MI, USA, D-9533): 50 μM, and CPX (Sigma-Aldrich, C0415): 5 μM. Z-VAD-FMK (Bachem, Bubendorf, Switzerland, N-1510), a caspase peptide inhibitor, was used at a concentration of 10 μM, while DEVD-AMC (Pepta Nova, Sandhausen, Germany, 3171-V), a fluorogenic substrate for caspase-3, was used at 20 μM. The mitochondrial complexes inhibitors rotenone (Sigma, Cat#R8875), antimycin A (Sigma, Cat#A8674), and oligomycin (Sigma, Cat#75351) were used at 0.5 and 10 μM, 0.5 and 50 μM, and 1.5 μM, respectively. Hemin (Sigma-Aldrich, H9039) was used at 5 and 10 μM, zinc protoporphyrin: ZnPP (Enzo Life Sciences, ALX-430-049-M025) was used at 1 μM, and ferrous ammonium sulfate: Fe(NH4)2(SO4)2·6H2O (Sigma-Aldrich, FX0245) was used at 1 mM. Trimethylsilyldiazomethane (Sigma-Aldrich, 362832): a 2.0 M solution in hexanes was used. Toluene, methanol, n-hexane, and diethyl ether were purchased from Chem-Lab NV (Zedelgem, Belgium, HPLC grade). TLC was performed using precoated silica gel plates (Macherey-Nagel SIL G-25 UV254). Chemical shifts for the 1H NMR and 13C NMR spectra, recorded on a Bruker Avance 400 spectrometer, were reported in parts per million with reference to the residual solvent signal (CDCl3: 7.26 ppm; CD3OD: 3.30 ppm and 49.00 ppm). Coupling constants (J) are expressed in hertz. Electrospray mass spectra were recorded by means of an Agilent 1100 series single quadrupole MS detector type VL, with APCI and API-ES sources, and provided with a Phenomenex Luna C18 (2) column (5 µm 250 mm × 4.60 mm). An Agilent 1100 series connected to a 6220A TOF-MS detector, equipped with an APCI-ESI multi-mode source, was used to conduct high resolution mass spectrometry (HRMS). A Perkin-Elmer 1000 FT-IR infrared spectrometer (HATR) was utilized to record the infrared spectra. A Perkin Elmer 241 polarimeter was used to measure optical rotation. Nemorosone was isolated, as previously described [2], from the floral resin of Clusia rosea. Concisely, an EtOH-H2O solution was used to crystallize nemorosone from the resin of the flowers of this plant species. Figure S1A shows the structure of nemorosone: C33H42O4, a mixture of tautomers (1S,5R,7R)-5-benzoyl-4-hydroxy-6,6-dimethyl-1,3,7-tris(3-methylbut-2-en-1-yl)bicyclo [3.3.1]non-3-ene-2,9-dione and (1S,5S,7R)-1-benzoyl-4-hydroxy-8,8-dimethyl-3,5,7-tris(3-methylbut-2-en-1-yl)bicyclo[3.3.1]non-3-ene-2,9-dione. Purity of isolated nemorosone was >99%, as determined via reversed-phase HPLC, with detection at 214/254 nm (retention time: 6.9 min; see Figure S4). Eluting conditions: eluent A/eluent B (50/50) for 30 s, followed by gradient elution (A/B from 50/50 to 0/100) over 6 min (eluent A: 0.1% HCOOH in water; eluent B: acetonitrile) on a Phenomenex Luna C18 (2) column (5 µm 250 mm × 4.60 mm). Analytical data are in agreement with the literature and identical to recorded data of commercially available nemorosone (purchased from Cayman Chemical, Ann Arbor, MI, USA, 24256). Rf 0.22 in hexane/EtOAc 8/2 [Lit: ≈0.21 [25]]. 1H NMR (CD3OD, 400 MHz): 7.53 (br d, J = 7.7 Hz, 2H), 7.43 (app tt, J = 7.4 Hz/1.2 Hz, 1H), 7.22–7.28 (m, 2H), 4.96–5.11 (m, 3H), 3.13 (dd, J = 15.0 Hz/7.8 Hz, 1H, A-part of ABX-system), 3.07 (dd, J = 14.8 Hz/7.4 Hz, 1H, B-part of ABX-system), 2.54 (dd, J = 14.7 Hz/7.8 Hz, 1H, A-part of ABX-system), 2.47 (dd, J = 14.5 Hz/7.3 Hz, 1H, B-part of ABX-system), 2.09–2.19 (m, 1H), 2.01 (br dd, J = 13.1 Hz/2.7 Hz, 1H), 1.65–1.85 (m, 2H), 1.68 (s, 3H), 1.64 (app s, 12H), 1.58 (s, 3H), 1.39–1.50 (m, 1H), 1.33 (s, 3H), 1.10 (s, 3H) ppm. 13C NMR (CD3OD, 100 MHz): 209.26 (C), 194.88 (C), 138.23 (C), 135.25 (C), 134.24 (C), 133.66 (C), 133.12 (CH), 129.61 (CH), 128.78 (CH), 123.99 (CH), 122.12 (CH), 121.18 (C), 120.79 (CH), 44.64 (CH), 30.40 (CH2), 28.19 (CH2), 26.30 (CH3), 26.02 (CH3), 24.35 (CH3), 22.32 (CH2), 18.30 (CH3), 18.11 (CH3), 17.97 (CH3), 16.20 (CH3) ppm. [a]D20 +99° (c 0.095, MeOH) [Lit: −98.3° (ent-nemorosone, c 1.19, MeOH) [26]]. IR (HATR): 3534 (m), 3418 (m), 2966 (m), 2916 (m), 2882 (m), 1711 (s), 1699 (s), 1582 (vs), 1446 (m), 1391 (m), 1368 (vs), 1317 (m), 1262 (m), 1242 (m), 1215 (s), 1197 (m), 1185 (m), 1172 (m), 1157 (m), 1128 (m), 1101 (m), 1063 (m), 1032 (w), 1019 (m), 1003 (w), 959 (w), 936 (w), 922 (w), 897 (w), 839 (s), 799 (m), 772 (w), 751 (m), 730 (w), 690 (m), 666 (m) cm−1. HRMS (ESI, positive mode): calculated for C33H43O4+ [M+H+]: 503.3156, found: 503.3149. Data on isolated natural nemorosone: [2]; data on synthetic ent-nemorosone: [26]; data on synthetic racemic nemorosone: [25]. Copies of 1H and 13C (APT) spectra can be found in Supplementary Materials (Figures S5 and S6, respectively). For the synthesis of O-methylated nemorosone, to a solution of nemorosone (200 mg, 0.398 mmol, 1 eq) in toluene/methanol (10 mL, 4/1), trimethylsilyldiazomethane (1.2 mL, 2.0 M in hexanes, 2.4 mmol, 6 eq) was added dropwise. After stirring the reaction mixture (a pale yellow solution) at room temperature for 30 min, silica was added to quench the excess of reagent. The resulting suspension was filtered and, under reduced pressure, the filtrate was concentrated. The 1H NMR spectrum of the crude mixture of both formed isomers was consistent with findings in the literature data on pure individual compounds [25]; integration of diagnostic methyl ester signals showed an isomer ratio of 78/22 (see Figure S7). The residue was partially purified using flash chromatography (gradient elution: hexane/ether 99/1–93/7), affording pure major isomer (48 mg, 0.093 mmol, 23% yield) and a mixture of major and minor isomers (153 mg, 0.296 mmol, 74% yield, ratio major/minor: 72.4/27.6, as determined via RP-HPLC/MS, integration of peaks at 214 nm, retention times 6.3 min and 6.5 min; see Figure S8). Eluting conditions: eluent A/eluent B (100/0) during 30 s, followed by gradient elution (A/B from 100/0 to 0/100) over 6 min (eluent A: 5 mM NH4OAc in water; eluent B: acetonitrile) on a Phenomenex Luna C18 (2) column (5 µm 250 mm × 4.60 mm). Major isomer: Rf 0.25 in hexane/ether 9/1. HRMS (ESI, positive mode): calculated for C34H45O4+ [M + H+]: 517.3312, found: 517.3335. Minor isomer: Rf 0.17 in hexane/ether 9/1. HRMS (ESI, positive mode): calculated for C34H45O4+ [M + H+]: 517.3312, found: 517.3330. DMEM medium supplemented with 10% (v/v) fetal calf serum (FCS), sodium pyruvate (1 mM), l-glutamine (1 mM), and non-essential amino acids (1 mM) was used to cultivate U87MG and U373MG human glioblastoma cells and HT22 cells (non-tumorigenic mouse hippocampal neuronal cell line); while IMR-32 (human neuroblastoma cell line) and HT1080 human fibrosarcoma cells were cultured in RPMI 1640 and EMEM medium, respectively, both supplemented in the same way as DMEM medium. Each cell line was obtained from ATCC. Every 3–4 days, cells cultures were split using a trypsin/EDTA solution and maintained at 37 °C in a humid 5% CO2 environment. It is important to highlight that these cancer cell lines were chosen both for their clinical relevance (they are representative cell lines of tumors refractory to conventional therapy) and for their reported sensitivity to the induction of non-apoptotic regulated cell death [17,19,27,28]. In addition, nemorosone had not been tested in any of these cell lines. Using the FLUOstar Omega fluorescence plate reader (BMG Labtech GmbH), cell death and caspase-3 activity were measured as previously reported [29,30]. Briefly, cells were seeded in a 96-well plate, and all experiments were carried out in triplicate. The following day, after being preincubated with the selected inhibitors, cells were treated with stimuli at desired concentrations in the presence of SytoxGreen and DEVD-AMC. At 1 h intervals, the fluorescence intensity of both fluorescent probes was measured. Percent cell death was calculated using Triton X-100 (0.05%) as a reference for 100% cell death. Following this same procedure, live cell images of seeded cells were obtained using a Zeiss LSM780 confocal microscope. The ImageJ program was used to merge the images. To analyze the induction of cell death, SytoxBlue staining was also used in conjunction with flow cytometry (BD LSR-Fortessa, BD Biosciences, Franklin Lakes, NJ, USA). Lipid ROS generation was determined by a previously described methodology [30]. In short, in a 6-well plate, HT1080 (300,000 cells/well) and IMR-32 (500,000 cells/well) cells were seeded. Cells were stimulated the following day and harvested. Fluorescent probes, C11-BODIPY and DRAQ7, were added to the wells 10 min prior to each time point, and lipid ROS accumulation was measured by flow cytometry (BD LSRFortessa, BD Biosciences). B530 (C11-BODIPY) and R780 (DRAQ7) channels were used to measure fluorescence. Only non-permeable live cell fluorescence was evaluated. Per condition, a minimum of 10,000 cells were examined. Mitochondrial ROS generation was determined using MitoSOX Red. In brief, HT1080 cells (300,000 cells/well) were seeded in a 6-well plate and incubated overnight. Afterward, cells were exposed to the test compounds according to the instructions of the experiment. After being washed with pre-warmed HBSS (Thermo Fisher Scientific, 14025076), cells were then incubated with fresh medium containing MitoSox Red for 15 min at 37 °C. Subsequently, cells were washed with HBSS and collected in PBS (Thermo Fisher Scientific, 10010023) containing SytoxBlue for measurement using BD FACSVerse (BD Biosciences). B586 (MitoSOX Red) and V448 (SytoxBlue) channels were used to measure fluorescence. Only non-permeable live cell fluorescence was evaluated. Per condition, a minimum of 10,000 cells were examined. FeRhoNox-1 dye was used to measure iron levels as previously described [17]. HT1080 cells (300,000 cells/well) were seeded in a 6-well plate and incubated overnight. Cells were harvested the next day and centrifuged at 300× g for 5 min. Then, cells were centrifuged at 300× g for 5 min after being washed with PBS buffer. The collected cells were stained with FeRhoNox-1 in PBS and kept in a CO2 incubator for 30 min. Following HBSS washing, cell culture was dissolved in 300 μL of HBSS containing SytoxBlue, and examined using BD LSRFortessa (BD Biosciences). Y585 (FeRhoNox-1) and V450 (SytoxBlue) channels were used to measure fluorescence. Only non-permeable live cell fluorescence was evaluated. Per condition, a minimum of 10,000 cells were examined. In a 6-well plate, HT1080 (300,000 cells/well) and IMR-32 (500,000 cells/well) cells were seeded. The following day, after cells had received the indicated treatment, TMRE (200 nM) was added, and the mixture was incubated for 30 min. The cells were washed with PBS to remove extra TMRE before being collected for analysis with the BD LSRFortessa (BD Biosciences). B575 (TMRE) and V450 (SytoxBlue) channels were used to measure fluorescence. Only non-permeable live cell fluorescence was evaluated. Per condition, a minimum of 10,000 cells were examined. As previously reported [17], glutathione levels were determined using QuantiChrom Glutathione Assay Kit (BioAssay Systems, Hayward, CA, USA, DIGT-250). Concisely, 1,000,000 cells per condition (HT1080 or IMR-32 cells) were seeded in a 6-well plate. The next day, cells were treated as indicated in each experiment. After that, cells were gathered, transferred to a new tube, and centrifuged at 425× g for 5 min at 4 °C. After being resuspended in 300 μL of PBS, each cell pellet was lysed using ultrasound. Each lysate was centrifuged at 14,000 rpm for 10 min at 4 °C. The cleared lysate was then used to calculate the amount of GSH present in each sample following the kit descriptions. Intracellular glutamate levels were measured using Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (Thermo Fisher Scientific, A12221). HT1080 cells (400,000 cells/well) were seeded in a 6-well plate and incubated overnight. The following day, cells were treated according to the conditions described in each experiment and collected by centrifugation at 300× g for 5 min. After removing the supernatant, the pellet was resuspended in PBS buffer. Afterward, each sample was centrifuged again at 300× g for 5 min, and the pellet was resuspended in 100 μL of Tris HCl buffer (0.1 M, pH = 7.5). Then, cells were lysed by sonication, and each sample was diluted (2×) with Tris HCl buffer. Next, 50 μL of the diluted samples were transferred into separate wells of a microplate (OptiPlate 96-well plate), and the amount of intracellular glutamate was calculated following the kit descriptions. ATP levels were determined using CellTiter-Glo 2.0 Assay Kit (Promega, Madison, WI, USA, Cat# G9242/3) based on the firefly luciferin–luciferase assay system. Briefly, HT1080 cells (400,000 cells/well) were seeded in a 96-well plate in the absence (control) or presence of nemorosone, CCCP, or oligomycin, in line with the conditions described in the experiment legend. The measurement was performed in accordance with the instructions of the kit. Intact HT1080 or IMR-32 cells were added to a 2 mL chamber at a concentration of 1,000,000 cells/mL. Oxygen consumption was measured at 37 °C using a high-resolution respirometer (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria). Oxygen flow per cell (pmol·s−1·mL−1) was recorded continuously using DatLab software 6 (Oroboros Instruments). After approximately 10 min of monitoring oxygen consumption, corresponding sequential injections of selected compounds and inhibitors were performed as indicated by the phosphorylation control protocol [31]. The Seahorse XFe96 Analyzer (Agilent) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). HT1080 cells (200,000 cells/well) were seeded into 96-well plates and incubated for 24 h. Before the assay, the culture medium was changed to a similar medium without phenol red and with 25 mM glucose, 1 mM sodium pyruvate, and 1 mM glutamine, and the cells were equilibrated for 30 min at 37 °C. During the assay, the compounds of interest were added, and the OCR and ECAR values were measured at intervals of approximately 6 min. An RNA 6000 nano chip (Agilent Technologies, Santa Clara, CA, USA) as well as an RNA labchip (Caliper GX-Perkin Elmer) were used to assess the total RNA quality of gall and control samples. Concentrations were determined using a Quant-it Ribogreen RNA assay (Life Technologies). Then, 265 ng of RNA were employed for the library prep through the QuantSeq 3′ mRNA libr prep FWD kit (Lexogen). Library prep was carried out in accordance with the recommendations of the manufacturer. In brief, first-strand cDNA synthesis was performed, followed by an RNA removal step. Then, second-strand synthesis was performed with the use of UMIs, after which the cDNA was purified using beads (Lexogen). In addition to being purified with beads, the cDNA was used for 13 cycles of enrichment PCR. Using a high sensitivity DNA chip from Agilent Technologies, the quality was examined. To enable equimolar library pooling, a qPCR assay was used to quantify the libraries in accordance with the Illumina protocol. Finally, sequencing was carried out on a Nextseq500 using 20% Phix spike-in (single-end reads, 76 cycles). Through the use of FastQC (version 0.11.9), the quality of the reads was confirmed [32]. The following parameters were used to trim reads with Trimmomatic (version 0.39): ILLUMINACLIP:<TruSeq3-SE adapter file>:3:30:10, SLIDINGWINDOW:5:20, MINLEN:20 [33]. STAR (version 2.7.8a) was used for mapping with the subsequent parameters: readFilesCommand zcat, outFilterMultimapNmax 1 and outSAMtype BAM SortedByCoordinate using the GRCh38.106 genome build [34]. These R packages were utilized to create a count table: GenomicFeatures (version 1.44.2), to convert the GRCh38.106 GTF file into a Granges object, and GenomicAlignments (version 1.28.0), for the summarizeOverlaps function to create the count table [35]. The counting options were as follows: mode = ‘Union’, singleEnd = TRUE, and ignore.strand = FALSE. To find differentially expressed genes, DESeq2 (version 1.32.0) was used with a Benjamini–Hochberg FDR cutoff of 0.05 [36]. Lists of differentially expressed genes were used for downstream analysis using Ingenuity pathway analysis (Qiagen). An R (v 4.1.3) environment was used for the analysis. Total RNA from treated HT1080 cells was extracted according to the NucleoSpin® RNA Plus protocol (fifth revision, corresponding to January 2021) prepared by MACHEREY-NAGEL GmbH & Co. KG (Düren, Germany). For DNA synthesis, a C1000 Touch® thermocycler (Bio-Rad) was used. The qPCR analysis was performed under the following conditions: 95 °C for denaturation, 60 °C for hybridization, and 70 °C for elongation. qbase+ software (Biogazelle) was used to calculate the expression levels of mRNA (HMOX1: Bio-Rad, Hercules, CA, USA, qHsaCIP0033307) from the structural genes (housekeeping genes), HMBS (Bio-Rad, qHsaCID0038839) and RPL3 (Bio-Rad, qHsaCED0038656), which were used as internal references. At designated times, test compound-treated HT1080 cells were harvested and subjected to two washes with cold PBS solution. A cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) was used to extract the total cytosolic proteins, and their concentrations were determined by the Bradford method. In the wells of the 10% SDS-PAGE gel, 25 µg of protein were loaded along with the molecular weight marker. After performing the run (1 h, 100 V), the transfer of the proteins from the gel to nitrocellulose membranes was carried out. Subsequently, the membranes were blocked with 5% skim milk powder prepared in TBST saline (0.05% Tween 20). The membranes were incubated for 24 h at 4 °C with each primary antibody of interest (except in the case of β-tubulin, with which they were incubated for 1 h). Peroxidase-labeled secondary antibodies (PerkinElmer Life Sciences) were used to detect immunoreactive proteins. Unpaired Student’s t-test was carried out, using GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA, USA), to calculate p values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; see figure legends for more information), with the exception of Figure 3C, where a two-way ANOVA test was employed. Unless otherwise stated, data are displayed as the mean ± SD of three separate experiments. First, we analyzed the cytotoxic effect of nemorosone (Supplemental Figure S1A) in a panel of cancer cell lines. Nemorosone was highly potent in killing HT1080 fibrosarcoma cell lines and high-risk MYCN-amplified IMR-32 neuroblastoma cells (Figure 1A). The most potent effect was exerted on HT1080 cells, reaching 100% of cell death in 12 h (Figure 1A,B and Figure S1B), while in IMR-32 cells around 70% of cell death was reached in 24 h (Figure 1A). However, nemorosone did not show any cytotoxic effect in glioblastoma (U87MG and U373MG) and the non-tumorigenic mouse neuronal cell lines (HT22) after 24h (Figure 1A). The EC50 (half-maximal effective concentration) of nemorosone on HT1080 cells was determined to be 26.9 μM at 12 h (Figure 1B) and 16.7 μM at 24 h. To determine the type of cell death induced by nemorosone and prior to nemorosone exposure, HT1080 and IMR-32 cells were treated with a variety of apoptotic and non-apoptotic cell death inhibitors. The cell death induced by nemorosone was prevented by the tested ferroptosis inhibitors, the iron chelators deferoxamine (DFO) and ciclopirox olamine (CPX) and the lipophilic radical trap ferrostatin-1 (Fer1) (Figure 1C and Figure S1C), but it was not affected by the pan-caspase inhibitor Z-VAD-FMK and the RIPK1-kinase inhibitor necrostatin-1 (Nec-1s) (Figure 1C and Figure S1C). Moreover, analysis of caspase-3 activity with fluorescent caspase-activity probe (DEVD-AMC) did not show any caspase activity in HT1080 or IMR-32 cells. Considering that ferroptosis is characterized by high levels of lipid peroxidation compared to other cell death modalities [20], we examined this parameter after challenging HT1080 and IMR-32 cells with nemorosone using the fluorochrome C11-BODIPY. We observed that nemorosone triggers a time-dependent increase in lipid peroxidation in both fibrosarcoma and neuroblastoma cells (Figure 1D,E, Figure 3D and Figure S1D). Moreover, we noticed that DFO and Fer1 completely prevented the production of lipid hydroperoxide (Figure 1D,E and Figure S1D). Inactivation of GPX4 is one of the canonical ways of induction of ferroptosis and is exerted by class II ferroptosis inducers. Depletion of intracellular glutathione, which acts as a cofactor for GPX4 to reduce lipid hydroperoxides, is a second canonical way of ferroptosis induction, for example, by blocking the System xc cystine/glutamate antiporter by class I ferroptosis inducers [19,22,37,38,39,40]. Similar to erastin, a class I FIN, we observed a substantial decrease in GSH levels in the fibrosarcoma and neuroblastoma cells after nemorosone treatment (Figure 2A and Figure S1E). We did not detect any GPX4 depletion at protein level after incubation with nemorosone or erastin, apart from the expected reduction in the signal due to the protein degradation exerted by the occurrence of cell death at the highest time points (Figure 2B). Consistent with the GSH levels’ decrease, we found that nemorosone, like erastin, increases the intracellular glutamate levels, suggesting the inhibition on the cystine/glutamate exchange mediated by the System xc cystine/glutamate antiporter (Figure 2C). If the blockade of cystine import through the System xc antiporter can trigger ferroptosis, then providing this metabolite to cells through an alternative means should rescue the cells from death [19]. Therefore, we pretreated cells with β-mercaptoethanol (β-ME), which reduces extracellular cystine to cysteine and bypasses the inhibition of the System xc antiporter, since cysteine can be imported via other pathways [41]. As shown in Figure 2D, β-ME inhibited nemorosone- and erastin-induced cell death in HT1080 cells, as previously reported [42]. However, β-ME inhibited 100% of the cell death induced by erastin, whereas it inhibited the cell death induced by nemorosone by approximately 60% (Figure 2D). The above results indicate not only that nemorosone partially acts as an erastin-like class I ferroptosis inducer but also that nemorosone additionally induces ferroptosis by another mechanism that circumvents the protection exerted by β-ME. Considering the central role of mitochondria in erastin-induced ferroptosis [23], we evaluated the involvement of the Electron Transport Chain (ETC) in nemorosone-induced ferroptosis using the mitochondrial complex I inhibitor (rotenone) and the mitochondrial complex III inhibitor (antimycin A) (Figure 2E). We found that both inhibitors suppressed cell death and the lipid ROS accumulation triggered by nemorosone and erastin (Figure 2F,G). These results show that functional ETC are required for nemorosone to induce ferroptosis. Altogether, these results suggest that nemorosone acts as a class I FIN and as an ETC-dependent ferroptosis inducer. To examine whether the uncoupling effect of nemorosone is required to induce ferroptosis, we first verified its uncoupling potency compared to the classical protonophoric mitochondrial uncoupler CCCP by measuring the oxygen consumption rate (OCR) increase, the drop of mitochondrial membrane potential (MMP), ATP levels reduction, extracellular acidification rate (ECAR) increase (which can indicate higher rates of glycolysis), and mitochondrial ROS production (mitoROS). We found that nemorosone is at least an equally potent uncoupler compared to CCCP in both the neuroblastoma (Figure S1F–H) and fibrosarcoma contexts (Figure S2A–G). Next, we prepared an analogue of nemorosone by a reaction of the vinylogous carboxylic acid moiety with trimethylsilydiazomethane, giving O-methylated nemorosone (hereafter named “methylnemorosone”, isolated as a ~3/1 mixture of isomers resulting from both tautomeric forms of nemorosone), which lacks the uncoupling effect (Figure S2H and Figure 3A,B). We identified that, in the absence of its mitochondrial uncoupling effect, nemorosone shows no more cytotoxic effect nor lipid peroxidation increase in HT1080 cells (Figure 3C,D). These results indicate, for the first time, the reactive moiety that is crucial for nemorosone-induced protonophoric mitochondrial uncoupling and ferroptosis induction. Furthermore, this association between nemorosone-induced ferroptosis and mitochondrial uncoupling is consistent with the previously stated requirement of nemorosone dependency on a functional ETC to trigger ferroptosis. Finally, we checked whether CCCP could also induce ferroptotic cell death. We found that both CCCP-induced cell death and lipid peroxidation were inhibited by canonical inhibitors of ferroptosis (Fer1 and DFO) and by ETC inhibitors (rotenone at complex I and antimycin A at complex III) (Figure 3E,F). Moreover, we observed that CCCP decreases GSH levels, which is associated, similar to nemorosone, with an increased level of intracellular glutamate (Figure 3G,H). These results show, for the first time, the capacity of not only nemorosone but also CCCP to induce ferroptosis, indicating that other protonophoric uncouplers could induce ferroptotic cell death. To further characterize the mechanism of nemorosone-induced ferroptosis, we performed a genome-wide transcriptome analysis using RNA-Seq in HT1080 cells. We observed a significant transcriptional change after 2 and 8 h of nemorosone treatment (Figure 4A). Remarkably, we found that heme oxygenase-1 (HMOX1) was the most upregulated gene by nemorosone, and one of the most upregulated by CCCP and erastin, which is in line with both the NRF2 upregulation and downregulation of the components of the KEAP1–CUL3–RBX1 E3 ubiquitin ligase protein complex (Figure 4B,D, Table S1). HMOX1 expression is controlled by the transcription factor NRF2, which is kept in check by KEAP1-dependent degradative ubiquitination [43]. Correspondingly, one of the main signaling pathways induced by nemorosone, CCCP, and erastin, according to data analysis by Ingenuity Pathway Analysis (IPA), is the NRF2-mediated oxidative stress response (Figure 4C and Table S2). Nemorosone-induced upregulation of HMOX1 was confirmed at both the mRNA (Figure 5A) and protein (Figure 5B) levels. Consistently, we found that when HMOX1 is upregulated, KEAP1 levels are reduced, while NRF2 levels are elevated (Figure 5B). The breakdown of heme molecules by HMOX1 is a major source of free Fe2+ [44]. In line with this, we observed a time-dependent increase in the intracellular levels of the labile iron pool (LIP) upon nemorosone treatment, measured using an Fe2+-selective probe (FeRhoNox-1) (Figure 5C). To check whether the increase in LIP is sufficient to induce ferroptosis in HT1080 cells, we treated cells with ferrous ammonium sulfate [Fe(NH4)2(SO4)2]. We revealed that the increase in LIP by the ferrous ammonium sulfate triggers ferroptotic cell death, which can be inhibited by Fer1 (Figure 5D). Of note, pharmacological inhibition of HMOX1 with zinc protoporphyrin (ZnPP), a metalloporphyrin that competitively inhibits the HMOX1 activity [45], prevents both lipid peroxidation and ferroptotic cell death after exposure to nemorosone (Figure 5E,F). Moreover, the combination of nemorosone with the HMOX1 substrate hemin increased labile Fe2+ levels, lipid peroxidation, and cell death (Figure 5G–I). These findings propose that nemorosone targets the KEAP1–NRF2–HMOX1 axis to promote ferroptosis by increasing the LIP through excessive activation of heme oxygenase-1. Therefore, it can also act as a class IV FIN [17]. To further confirm the role of HMOX1 upregulation in nemorosone-induced ferroptosis, we treated the cells with combinations of non-toxic concentrations of hemin and nemorosone. Interestingly, we observed that such a combination is sufficient to induce synergistically lipid peroxidation and cell death (Figure 5J–M). In the current work, nemorosone, a phytochemical isolated from the floral resin of the C. rosea plant, was identified to induce ferroptosis in fibrosarcoma and neuroblastoma cells. In 20 years of nemorosone anticancer-effect research, this is the first report that expands the potential of nemorosone by showing its capacity to induce another mechanism of cell death than apoptosis [4,11]. Notably, while nemorosone was cytotoxic against the neuroblastoma and fibrosarcoma cell lines, it did not show any effect on glioblastoma U87MG and U373MG cells. In general, the sensitivity to ferroptosis depends on the different endogenous mechanisms that protect cells against the lipid peroxidation that drives ferroptosis [46]. Recently, ferroptosis suppressor protein 1 (FSP1) was shown to play an essential role in the resistance of U373MG cells to erastin-induced System xc inhibition, while some unidentified mechanisms that support GPX4 function, independent of System xc activity, were also observed [47]. In line with this, a higher methionine uptake has been reported in gliomas than in normal astrocytes, which positively correlated with tumor viability and aggressiveness and indicated a greater reliance on transsulfuration, a metabolic pathway that connects methionine with glutathione biosynthesis independent of the System xc antiporter [48,49]. These reports allow us to explain a priori the resistance of glioblastoma cell lines to nemorosone-induced ferroptosis, although new experimental results are required to corroborate the aforementioned hypotheses. Conversely, based on the literature data, HT1080 and IMR-32 cells appear to be sensitive to the ferroptosis induced by decreased GSH levels through System xc inhibition and the increased labile iron pool (LIP) [17,19]. In this investigation, it was found that nemorosone induces ferroptosis by modulating these two parameters (Section 3.2 and Section 3.4). On one hand, nemorosone-induced ferroptosis in HT1080 and IMR-32 cells involves the drop in the glutathione levels that can be associated with a blockade of the System xc cystine/glutamate antiporter or SLC7A11, which resembles the canonical ferroptosis-inducing pathway triggered by a class I FIN, such as erastin, also known as cysteine-deprivation-induced ferroptosis [19,23]. Inhibition of cystine import, which is required for GSH synthesis, results in depletion of intracellular GSH levels [16,33,37], an important cofactor for selenium-dependent GPX4. Therefore, GSH depletion by nemorosone could indirectly inactivate GPX4, leading to production of lipid ROS, which in turn results in lipid peroxidation and ferroptotic cell death [19,42]. It should be mentioned that more direct experimental approaches, such as the [14C]-cystine uptake assay [19], are required to confirm nemorosone-induced blockade of the System xc antiporter. Furthermore, inhibition of cystine entry is not necessarily the only pathway by which nemorosone could be lowering GSH levels. In fact, the decrease in the levels of NADPH, an electron-donor agent, relevant in the reduction of oxidized substrates, was reported as a common phenotypic effect for different structurally divergent uncoupling compounds [24]. It has been suggested that the dissipation of the mitochondrial membrane potential renders nicotinamide nucleotide transhydrogenase (NNT) incapable of maintaining the reduced NADPH state, which in turn can affect GSH regeneration via glutathione reductase (GR) [50,51]. In effect, the abundance of NADPH functions as a biomarker that is inversely correlated with the sensitivity of cells to the inducers of ferroptosis [52]. This important experimental issue should also be studied in future research. On the other hand, nemorosone induces a non-canonical mechanism of ferroptosis by increasing the LIP, in response to the excessive activation of heme oxygenase-1 by targeting KEAP1 and NRF2, which is sufficient to trigger toxic lipid peroxidation. This result is comparable with the effect of withaferin A (WA), a natural FIN isolated from Withania somnifera roots, which at a medium dose induces ferroptosis through a massive upregulation of HMOX1 [17]. Likewise, Tagitinin C, another natural compound, induces ferroptosis in colorectal cancer cells through the PERK–NRF2–HMOX1 signaling pathway, and again the significant overexpression of HMOX1 led to the increase in the LIP, which promoted lipid peroxidation and ferroptosis [53]. As can be seen, these results with nemorosone add to a still small, but apparently growing, list of natural compounds that modulate the NRF2–HMOX1 axis to induce ferroptotic cell death, which could be related to some natural protection mechanism of plants (not yet reported) against pathogenic microorganisms. Similarly, HMOX1 was also shown as an essential enzyme that is involved in iron supplementation and lipid peroxidation in erastin-induced ferroptosis [54]. By giving cancerous cells antioxidant and cytoprotective effects and by removing toxic intracellular heme, the inducible intracellular enzyme HMOX1 was also shown to play a role in cancer progression [55]. This is in line with the fact that HMOX1 is elevated in various human malignancies such as, for example, fibrosarcoma tumors and HT1080 cells [56,57]. In consequence, HMOX1 inhibition was explored to reduce tumor growth [43,44,46]. Nevertheless, based on the current data, a massive activation of HMOX1 is important to kill HT1080 cells, strongly suggesting the efficacy of an opposite strategy: making tumor cells sensitive to the induction of ferroptosis via the therapeutic overactivation of HMOX1. At the same time, the active role of HMOX1 in tumor cells constitutes a significant difference compared to the healthy tissue and is, consequently, a way by which compounds such as nemorosone could induce a selective ferroptosis mechanism in cancer cells such as fibrosarcoma. However, nemorosone-induced activation of the NRF2-mediated oxidative stress response pathway (Figure 4C), with the consequent modulation of NRF2 target genes (Table S1), shows that nemorosone activated the NRF2 pathway as an antioxidant and antiferroptotic response. It is the disproportionate upregulation of HMOX1 compared to other genes that results in a pro-ferroptotic effect. It is notable that HMOX1 is the gene most upregulated by nemorosone: more than 90 times compared to the control, while FTH1 is upregulated less than 3 times (Figure 4B and Table S1). That is, nemorosone generates an expression of HMOX1 30 times higher than the induced expression of FTH1, which, similar to what was reported for WA, suggests the induction of ferroptosis by raising the LIP in a context of insufficient ferritin buffering capacity [17]. In other words, the effect of nemorosone reveals a hormetic response associated with the NRF2–HMOX1 axis: a protective effect after moderate activation (classical and most common reports) vs. a cytotoxic effect after excessive activation (reported for some naturally occurring ferroptosis-inducing compounds). It remains to be answered why nemorosone and other natural compounds generate such an overactivation of heme oxygenase-1. First of all, it must be taken into account that activation of HMOX1 by pathways other than NRF2 cannot be excluded. Several classes of stress-responsive transcription factors that activate HMOX1 gene have also been identified, such as members of the heat-shock factor (HSF), nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) families [58]. On the other hand, nemorosone was identified as a natural activator of the p300 histone acetyltransferase that enhanced histone acetylation in cells [59]. At the same time, p300-mediated NRF2 acetylation was shown to be essential for the maximal binding of NRF2 to specific ARE (antioxidant response element)-containing promoters [60]. Moreover, p300 was recently reported to compete with KEAP1 for the regulation of NRF2, enhancing the protein level of NRF2 and allowing NRF2 to translocate to the nucleus to upregulate the transcription of target genes [61]. This possible nemorosone-induced epigenetic regulation of the KEAP1–NRF2–HMOX1 axis could also explain the capacity of nemorosone to induce non-canonical ferroptosis through excessive activation of HMOX1. Consistently, we observed that nemorosone also induces the downregulation of KEAP1, CUL3, and RBX1, while it upregulates SQSTM1 and EIF2AK3 (PERK) (Figure 4B,D), all of which suggests the activation of the SQSTM1–KEAP1–NRF2–HMOX1 and PERK–NRF2–HMOX1 pathways as part of HMOX1 overactivation-mediated cytotoxicity. It is important to highlight that the regulation of the expression at gene level of the KEAP1–CUL3–RBX1–NRF2 complex does not exclude the possibility of regulation at the protein level by a direct binding between nemorosone and KEAP1, as was reported in the aforementioned case of withaferin A [17]. All these factors need to be addressed in future experimental activities. The time relation existing between the two ferroptosis mechanisms triggered by nemorosone is also noteworthy: the drop of GSH, resulting in a lipid peroxidation increase, appears from 2 h (an early event), while HMOX1 overexpression, with its consequent increase in the intracellular labile Fe2+ levels, only begins at 6 to 8 h (a later event). Moreover, before the execution of the later event, there is already cell death induction in some cells. However, the cell death level is accelerated and enhanced at the time points in which HMOX1 expression can be associated with labile Fe2+ and an additional lipid peroxidation increase. It is unclear whether this difference in cell death by early lipid peroxidation due to blockage of cystine import and by the later event represent two distinct responding populations, in which cell resistance to cell death during the early lipid peroxidation wave receives a second ferroptotic hit due to HMOX1 upregulation, hemin degradation, and the increase in the labile Fe2+ pool. Importantly, high sensitization values were achieved by combining nemorosone and the substrate of HMOX1 hemin, which confirms the cytotoxic role of nemorosone-induced HMOX1 activation and points out a possible therapeutic approach to be experimentally tested in in vivo experiments. The aforementioned results allow for the conclusion that nemorosone exerts an erastin-like ferroptosis (intrinsic ferroptosis) in fibrosarcoma cells that is characterized by the concurrence of both canonical (decreasing GSH levels) and non-canonical (increasing LIP through HMOX1 upregulation) mechanisms. This may confer more therapeutic efficacy to nemorosone by circumventing the resistance mechanisms of the tumor cells that bypass the System xc blockade or the depletion of GSH levels, an effect suggested by the persistence of the induction of cell death, unlike erastin, in the presence of β-ME (Figure 2D). On the other hand, erastin-induced cell death and, in general, cysteine-deprivation-induced (CDI) ferroptosis are exerted by transient mitochondrial membrane potential (MMP) hyperpolarization, in such a way that low concentrations (10 μM) of the mitochondrial uncoupler CCCP can prevent (by the drop of MMP) CDI lipid ROS accumulation and protect against ferroptosis [23]. However, we confirmed that nemorosone acts as a mitochondrial uncoupler, dissipating (similar to CCCP) the transmembrane proton gradient prior to cell death execution. The possible involvement of mitochondrial uncoupling in ferroptosis induced by nemorosone was approached by using a high concentration of CCCP (50 μM) and methylnemorosone. While CCCP acted similarly to nemorosone regarding ferroptosis induction, methylnemorosone, which cannot exert mitochondrial uncoupling activity anymore, completely lost cytotoxicity. Altogether, this suggests that mitochondrial uncoupling is indeed required for nemorosone to trigger ferroptosis in fibrosarcoma cells. Furthermore, Figure S3 shows a possible link between HMOX1 over-activation and mitochondrial uncoupling: CCCP, a classic mitochondrial uncoupler, also increases Fe2+ levels upon HMOX1 upregulation. In addition, the obtained results at a high concentration of CCCP and the reported capacity to protect against erastin-induced ferroptosis at a low concentration [23] show a dual role as uncoupler compounds to induce ferroptosis or protect against it by varying the concentration. The protective mechanism could be an important approach to treat several ferroptosis-associated diseases such as ischemic organ injury, brain damage, and kidney failure [62,63], expanding the potential application of mitochondrial uncouplers. To sum up, here, we connect, for the first time, mitochondrial uncoupling with ferroptotic cell death induction by the use of two closely related agents: proficient (nemorosone) and deficient (methylnemorosone). The cascade of cellular effects leading to ferroptosis induced by the mitochondrial uncoupler compounds in cancer cells is still an unexplored and emerging area of research and therapeutic opportunities. Here, we show, for the first time, that nemorosone can induce intrinsic ferroptosis in fibrosarcoma and neuroblastoma cells by a double-edged targeting mechanism consisting of the drop of GSH as an early event and the increase in labile Fe2+ levels through the overexpression of HMOX1 as a later event. The work also expands the current knowledge about the role of mitochondria in ferroptosis by showing that compounds with an uncoupling action can trigger an erastin-like ferroptosis mechanism linked to MMP dissipation.

PMC10000525

Rina Bandopadhyay,Ariana Gatt,Tammaryn Lashley

Advances in the Understanding of Frontotemporal Dementia

01-03-2023

Advances in the Understanding of Frontotemporal Dementia Frontotemporal dementia (FTD) encompasses a group of clinically, genetically and pathologically heterogeneous neurodegenerative disorders that mainly affect people under the age of 64 years. However, around 25% of those affected have a later age of onset. FTD represents 10–20% of all dementia cases [1]. It is predominantly characterized by the progressive atrophy of the frontal and temporal lobes [2]. Disease duration ranges between 2 and 20 years, with 8 years being the mean following the onset of symptoms. FTD treatment is restricted to symptom control, and no disease-modifying treatments are available. The clinical hallmarks of FTD include gradual yet progressive deficits in behaviour and/or language with the relative preservation of memory. Subtypes of FTD are identified clinically according to the symptoms that appear prominently at presentation. Clinical diagnoses include behavioral variant FTD (bVFTD), which accounts for nearly 60% of cases; primary progressive aphasia (PPA), which affects language; and the movement disorders progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS) [2]. With disease progression, the debilitating symptoms cause marked impairments of social and/or occupational functioning. Early and accurate diagnosis is crucial for the streamlining and development of any disease-modifying treatment therapies. A third of FTD cases are genetically linked with mutations occurring in C9orf72, progranulin (GRN) and MAPT, with C9orf72 repeat expansions being the most common. Neuropathologically, TAR DNA binding protein-43 (TDP-43), fused in sarcoma (FUS) and tau are three major proteins that cause pathological deposits in FTD post-mortem brains [2]. Those with C9orf72 expansions also have an additional pathology where di-peptide repeat proteins are also found deposited in patients. It is thought that disease pathogenesis is caused either by a gain of toxic function or a loss of nuclear function associated with protein dislocation from the nucleus, which in turn may lead to neuronal degeneration. FUS regulates the transcription of multiple genes, including the MAPT gene [3,4]. Recent studies have highlighted the molecular pathways associated with lysosomal dysfunction, lipid dysregulation, RNA splicing aberrations, synaptic loss and neuroinflammation as putative causes of sporadic forms of FTD. The disease mechanisms are far from understood in FTD, especially the cellular changes occurring in the early disease stages. Continued research with improved animal models, iPS technologies, clinicopathological correlations with donated human brain tissue and the discovery of early biomarkers of disease progression should enable us to rationalize the mechanisms involved in these neurodegenerative diseases and identify much-needed therapeutic targets. This Special Issue will collect articles relating to all advances in FTD research, both clinical and non-clinical.

PMC10000527

Jordi Duran

Role of Astrocytes in the Pathophysiology of Lafora Disease and Other Glycogen Storage Disorders

24-02-2023

glycogen,aggregation,Lafora disease,neuroinflammation,neurodegeneration,epilepsy

Lafora disease is a rare disorder caused by loss of function mutations in either the EPM2A or NHLRC1 gene. The initial symptoms of this condition are most commonly epileptic seizures, but the disease progresses rapidly with dementia, neuropsychiatric symptoms, and cognitive deterioration and has a fatal outcome within 5–10 years after onset. The hallmark of the disease is the accumulation of poorly branched glycogen in the form of aggregates known as Lafora bodies in the brain and other tissues. Several reports have demonstrated that the accumulation of this abnormal glycogen underlies all the pathologic traits of the disease. For decades, Lafora bodies were thought to accumulate exclusively in neurons. However, it was recently identified that most of these glycogen aggregates are present in astrocytes. Importantly, astrocytic Lafora bodies have been shown to contribute to pathology in Lafora disease. These results identify a primary role of astrocytes in the pathophysiology of Lafora disease and have important implications for other conditions in which glycogen abnormally accumulates in astrocytes, such as Adult Polyglucosan Body disease and the buildup of Corpora amylacea in aged brains.

Role of Astrocytes in the Pathophysiology of Lafora Disease and Other Glycogen Storage Disorders Lafora disease is a rare disorder caused by loss of function mutations in either the EPM2A or NHLRC1 gene. The initial symptoms of this condition are most commonly epileptic seizures, but the disease progresses rapidly with dementia, neuropsychiatric symptoms, and cognitive deterioration and has a fatal outcome within 5–10 years after onset. The hallmark of the disease is the accumulation of poorly branched glycogen in the form of aggregates known as Lafora bodies in the brain and other tissues. Several reports have demonstrated that the accumulation of this abnormal glycogen underlies all the pathologic traits of the disease. For decades, Lafora bodies were thought to accumulate exclusively in neurons. However, it was recently identified that most of these glycogen aggregates are present in astrocytes. Importantly, astrocytic Lafora bodies have been shown to contribute to pathology in Lafora disease. These results identify a primary role of astrocytes in the pathophysiology of Lafora disease and have important implications for other conditions in which glycogen abnormally accumulates in astrocytes, such as Adult Polyglucosan Body disease and the buildup of Corpora amylacea in aged brains. Cells store glucose in the form of glycogen—a branched polymer of glucose—to minimize the increase in osmotic pressure that would be associated with the accumulation of free glucose. Glycogen molecules can store up to 55,000 glucose units in a water-soluble form that can be rapidly degraded when energy is required. This polysaccharide is synthesized through the coordinated action of glycogen synthase (GS), which joins glucose units by α-1,4-glycosidic linkages, and glycogen branching enzyme (GBE), which introduces branching points via α-1,6-glycosidic linkages [1]. Similarly, glycogen is broken down by the coordinated action of glycogen phosphorylase and glycogen debranching enzyme, which digest α-1,4- and α-1,6-glycosidic linkages, respectively. In normal glycogen, branches are introduced at even intervals and this branched structure is important for its function and solubility, generating a spherical molecule that can be rapidly degraded when the cell needs glucose. In contrast, in poorly branched glycogen, long α-1,4 linked glucose chains can form single or double helices [2,3,4] that exclude water, thereby impeding the degradation of glycogen and decreasing its solubility. Some tissues, such as skeletal muscle and particularly the liver, accumulate high amounts of glycogen (up to 2% and 8% of wet weight, respectively) [5]. Muscle glycogen provides energy for contraction during intense exercise, while liver glycogen is mobilized during fasting periods to produce glucose, which is then released into the blood to be used by the rest of the body. All other tissues and cell types contain glycogen, although at lower concentrations than those found in the liver and skeletal muscle. Within the brain, glycogen concentration is relatively low—about 0.1% of wet weight—and it is present mostly in astrocytes [6,7,8,9,10]. However, neurons also have an active glycogen metabolism that plays an essential role in the function of these cells [11,12]. Given the low concentration of glycogen in the brain, its role in this organ was traditionally overlooked and widely presumed to serve as an emergency reservoir for pathophysiological conditions like hypoglycemia or ischemia [13]. However, it is now clear that glycogen plays key roles in the normal functioning of the brain under physiological conditions [14,15]. Brain glycogen is mobilized as supplemental fuel when energy needs increase due to neuronal activity [16,17], and substantial deficits in learning and memory arise when its use is blocked [12,18,19,20,21]. To support neuronal function, and according to the astrocyte-neuron lactate shuttle hypothesis, astrocytes degrade glycogen to generate lactate, which they then release and which is taken up and oxidized by neurons [22]. Another simpler hypothesis is that, in situations of high-energy demand, astrocytes use glucose obtained from their own glycogen store, thereby sparing interstitial glucose for neurons [23]. Despite the key physiological role of glycogen, this polysaccharide can also participate in brain pathology. In some conditions, glycogen abnormally accumulates in the nervous tissue. Lafora disease (LD, OMIM 254780) is probably the most striking example of the consequences of the abnormal buildup of glycogen in the brain. In LD, poorly branched glycogen accumulates in the brain and other tissues in the form of aggregates known as Lafora bodies (LBs). Glycogen in LBs is not accessible to glycogen phosphorylase and glycogen debranching enzyme. Thus, it cannot be degraded and progressively accumulates. Glycogen in LBs also contains high levels of covalently bound phosphate, which had been hypothesized to participate in glycogen insolubility in LD [24]. However, it seems now clear that abnormal glycogen branching, not hyperphosphorylation, underlies glycogen insolubility in LD [25,26]. In addition to poorly branched, hyperphosphorylated glycogen, LBs contain several proteins, including GS, ubiquitin, and the autophagy adaptor p62 [27,28,29,30]. The onset of LD occurs in adolescence, in previously healthy children, normally in the form of epileptic seizures that are difficult to distinguish from idiopathic generalized epilepsies. Seizures escalate over time, together with a rapid decline in cognitive function. The patient develops severe dementia and eventually enters a vegetative state with continuous seizures. Death invariably comes 5 to 10 years after the onset as a consequence of status epilepticus or complications derived from neurodegeneration [31,32,33]. LD is a rare disease with an estimated prevalence of ~4 cases per million individuals in the world [31]. However, the number of undiagnosed cases might be higher, particularly in developing countries. Current treatment remains palliative, with limited success in the modulation of symptoms. LD is an autosomal recessive disease caused by mutations in two genes: EPM2A, encoding laforin, a dual phosphatase that contains a carbohydrate-binding domain [34,35], and EPM2B/NHLRC1, encoding malin, an E3-ubiquitin ligase [36]. Mutations in either of these genes cause an indistinguishable disease. The exact roles of malin and laforin in glycogen metabolism are not yet fully understood, but it is widely accepted that malin uses laforin as a scaffold to bind to glycogen and ubiquitinate proteins involved in glycogen metabolism [37,38,39]. The accumulation of poorly branched glycogen in LD suggests that malin and laforin form this functional complex to regulate glycogen synthesis and prevent glycogen insolubility [40]. To minimize the toxic consequences of the accumulation of poorly branched glycogen, proteins like the autophagy adaptor p62 promote its compaction in the form of LBs [29] (Figure 1). This protective mechanism is reminiscent of the condensation of ubiquitinated, misfolded proteins into larger structures to be degraded by autophagy [41,42,43]. To study LD, several mouse models of the disease lacking laforin or malin have been generated [44,45,46,47]. These animals present similar pathophysiological phenotypes that recapitulate the human disease; i.e., they accumulate LBs in several tissues, including the brain, show a progressive neuronal loss, behavioral impairments, neuroinflammation with reactive astrocytes and microglia, altered autophagy, and increased susceptibility to epileptogenic drugs such as kainate and pentylenetetrazole [27,29,44,45,46,47,48]. In these LD models, the accumulation of LBs increases with age and the pathological phenotypes also worsen progressively as the animals age [27,28,44,49,50]. The role of LBs in the pathophysiology of LD has been unclear for many years. For instance, it was hypothesized that the primary cause of LD was an impairment in autophagy and that the accumulation of LBs was a consequence of this defect [48,51,52,53]. However, several groups, including ours, took advantage of mouse models of LD to demonstrate that excess glycogen underlies the pathology of this disease. Indeed, impeding or reducing glycogen synthesis in malin- or laforin-deficient mice prevents LB formation and prevents all the pathologic traits of the disease [27,45,54,55]. These models also showed that autophagy impairment is secondary to LB accumulation since autophagy markers are also normalized when glycogen accumulation is prevented [27,50]. Furthermore, we also used Drosophila and mouse models to demonstrate that forced accumulation of glycogen in neurons induces their death by apoptosis [56]. All these findings identified excessive glycogen accumulation as an inducer of neurodegeneration, and glycogen synthesis therefore became a putative target for the treatment of LD. Therapies based on the inhibition of glycogen synthesis are the focus of current research efforts [28,57,58,59,60]. In the first description of LD in 1911, Dr. Gonzalo Rodriguez-Lafora reported the presence of LBs in neurons [61]. Until recently, it was widely believed that LBs accumulated exclusively in this cell type, and thus, all the pathologic traits of the disease were attributed to the toxic effects of neuronal LBs [31,62,63,64]. However, the premise that LBs are present exclusively in this cell population was inconsistent with the fact that, as mentioned before, brain glycogen is present mainly in astrocytes in normal conditions. It is now clear that LBs also accumulate in astrocytes. In 2011, we first reported the presence of LBs in these cells in a malin-deficient mouse model [46], but the significance of this discovery was underestimated. Several years later, we [65] and others [66] demonstrated that most LBs are present in astrocytes, particularly in regions like the hippocampus. We classified these bodies into neuronal (nLBs), and Corpora amylacea-like (CAL), the latter present in astrocytes, which were named this way because of their resemblance to Corpora amylacea, which are glycogen aggregates that accumulate in aged brains [67] (see below). Interestingly, CAL and nLBs differ not only in the cell type in which they accumulate but also in their shape and subcellular localization. CAL are polymorphic and present predominantly in astrocytic processes, and they show a patchy distribution, each patch corresponding to an individual astrocyte. In contrast, nLBs are normally present in the form of a single spherical aggregate close to the neuronal nucleus, and they resemble inclusion bodies formed by protein aggregates such as Lewy bodies [68]. The progressive accumulation of CAL and nLBs takes place in parallel, and both types of LB are already present at early stages in mouse models of LD [28,66]. Astrocytes have been shown to have phagocytic activity and can engulf apoptotic cells [69]. Thus, LBs present in astrocytes might not have originated in astrocytes themselves, but instead, they may have a neuronal origin; i.e., proceeding from the phagocytosis of an apoptotic body derived from a dead neuron. To decipher the origin of astrocytic LBs, as well as to understand their contribution to the pathology of LD, we generated a malin-deficient mouse in which GS was specifically deleted from astrocytes (malinKO + GSGfap-KO mice), thus preventing the synthesis of glycogen specifically in this cell type. The brains of these animals contained nLBs but were devoid of CAL, thereby unequivocally demonstrating that the latter originate in astrocytes [50] (Figure 2). The demonstration that LBs are also present in astrocytes opened up the possibility that these astrocytic LBs contribute to the pathology of LD. In fact, the quantification of CAL and nLBs showed that in brain regions like the hippocampus, CAL are clearly predominant over nLBs [65,66]. The analysis of malinKO+GSGfap-KO brains confirmed this quantification since the hippocampi of these mice are largely free of LBs [50] (Figure 2). Furthermore, RNA-Seq studies indicated that most of the upregulated genes in the brains of malin- and laforin-deficient mice encode pro-inflammatory mediators and that reactive glia, including astrocytes, are responsible for the expression of these inflammatory genes [70]. To understand the contribution of astrocytic LBs to the pathology of LD, the characteristic pathologic traits of the disease were analyzed in malinKO + GSGfap-KO mice. Neuroinflammation is considered one of the initial determinants of LD [71]. The brains of LD mouse models present clear astrogliosis, microgliosis, and increased expression of inflammatory genes [50,70]. In contrast, the analysis of malinKO + GSGfap-KO brains revealed normal levels of all these markers of neuroinflammation, thereby indicating that astrocytic LBs underlie neuroinflammation in LD [50] (Figure 2). The link between the excessive accumulation of astrocytic glycogen and neuroinflammation was further confirmed with another mouse model in which a constitutively active form of GS was expressed specifically in astrocytes. These mice, which accumulate high amounts of glycogen in astrocytes, present profound astrogliosis and microgliosis, as well as a marked increase in the expression of inflammatory genes [50]. These results confirmed that the excessive accumulation of glycogen in astrocytes induces neuroinflammation. However, the mechanism that links excessive astrocytic glycogen accumulation with neuroinflammation is currently unknown and is the focus of current research efforts. As mentioned before, the initial symptom of LD is most commonly the presence of epileptic seizures, which worsen progressively with age. Animal models of LD reproduce this pathologic trait of the disease in the form of increased susceptibility to epileptogenic drugs like kainic acid. Astrocytes play essential roles in brain function, including the regulation of extracellular potassium and glutamate homeostasis, thus making them crucial actors in epilepsy [72]. In this regard, astrocytic glycogen has been shown to fuel potassium uptake into astrocytes, since the astrocytic sodium/potassium pump uses ATP obtained from glucose 6-phosphate originating from glycogen breakdown [73,74]. The non-clearance of extracellular potassium would result in neuronal hypersynchronization and burst firing, which would result in seizure generation and propagation. Thus, it has been suggested that alterations in glycogen metabolism contribute to the imbalance of glutamatergic and GABAergic neurotransmission associated with epileptic seizures [75]. Accordingly, it was reasonable to hypothesize that the impairment of astrocytic glycogen metabolism in LD compromises potassium uptake, which would increase excitability and thus be responsible for the epileptic phenotype of the disease. In line with this hypothesis, the presence of glycogen aggregates in astrocytes has also been described in patients with temporal lobe epilepsy [76]. Surprisingly, malinKO + GSGfap-KO mice do not show a significant amelioration of susceptibility to epilepsy [50], thereby indicating that astrocytic glycogen accumulation is not the main factor responsible for the epileptic phenotype of LD. Importantly, the deletion of GS specifically in astrocytes does not increase susceptibility to epilepsy per se [19]. Thus, the epileptic phenotype of LD might be attributable to neuronal LBs, most likely to those present in GABAergic interneurons, which would impair their function and generate an imbalance of glutamatergic and GABAergic transmission. In line with this notion, we described that parvalbumin interneurons of the hippocampus accumulate LBs [46] and this buildup is accompanied by damage to GABAergic neurons in mouse models of LD [77]. In summary, astrocytic glycogen accumulation drives the neuroinflammatory phenotype of LD but not the increased susceptibility to epilepsy, which might be attributable to neuronal LBs. The accumulation of glycogen in the nervous tissue is not exclusive to LD. As mentioned before, the presence of glycogen aggregates known as Corpora amylacea (“starch bodies” in Latin, due to their resemblance to starch) has also been observed in aged human brains [78]. Interestingly, these aggregates accumulate to a greater extent in neurodegenerative conditions like Alzheimer’s, Parkinson’s, Huntington’s, and Pick’s diseases, as well as in patients with temporal lobe epilepsy [67,79]. Although the cellular localization of Corpora amylacea has been a source of debate, several articles have described their presence in astrocytes [80,81,82,83,84]. Similar aggregates progressively accumulate with age in the astrocytes of control mice [30,65,67] (Figure 2). The composition of Corpora amylacea greatly resembles that of LBs, consisting of insoluble, poorly branched glycogen and a minor content of protein, including GS, ubiquitin, and p62 [67]. Interestingly, these glycogen aggregates are not found in the brains of aged GS knockout mice [30]. This observation thus indicates that, as for LBs, glycogen synthesis is a prerequisite for the formation of Corpora amylacea. In contrast, the formation of these aggregates is enhanced in models of accelerated aging, such as the Senescence Accelerated Mouse-Prone 8 (SAMP8) mouse [65]. The overexpression of protein targeting to glycogen (PTG), an activator of GS, also resulted in an increase in the formation of these glycogen aggregates [65]. This observation suggests that an imbalance between GS activity and GBE activity favors the formation of poorly branched glycogen, which would accumulate in the form of Corpora amylacea-like structures (see adult polyglucosan body disease below). Strikingly, the brains of control, SAMP8, and PTG-overexpressing animals show the presence of CAL but not nLBs [65]. Therefore, the latter seem to be exclusive to LD models. Collectively, all of points explained above suggest that the progressive accumulation of Corpora amylacea in the nervous system contributes to the neurological decline associated with aging [79,85]. Mutations in malin and laforin would drastically increase the rate of this process; i.e., LD could be considered an accelerated aging process with respect to the consequences of glycogen accumulation in the brain. Furthermore, the increased presence of Corpora amylacea in neurodegenerative conditions like Alzheimer’s and Parkinson’s disease opens up the possibility that the toxicity induced by glycogen accumulation also participates in the pathology of these disorders. Alternatively, the presence of waste elements in Corpora amylacea has led some authors to hypothesize that these structures are waste containers in which deleterious or residual products are isolated for later removal by the natural immune system [78,86,87]. Another rare genetic condition in which glycogen accumulates abnormally in the nervous tissue is adult polyglucosan body disease (APBD, OMIM 263570). This is an autosomal recessive neurodegenerative disorder with onset normally in the 5th or 6th decade of life and slow progression, affecting the central and peripheral nervous system with severe leukodystrophy, atrophy of the spine and medulla, and cognitive impairment [88,89,90]. The disease is caused by mutations in GBE that result in the formation of glycogen with low solubility due to the lack of branching. Consequently, there is a progressive intracellular accumulation of glycogen aggregates (the so-called polyglucosan bodies), which are similar to LBs. In fact, the term “polyglucosan body” is also used to refer to LBs and Corpora amylacea [78,91] APBD is caused by mutations that generate a partial loss of GBE activity (with 5–20% residual activity), the most common of which is p.Y329S, found in patients of Ashkenazi Jewish descent [92]. Other mutations in GBE cause a clinically heterogeneous disorder collectively known as glycogen storage disease IV (OMIM 232500), with hepatic and neuromuscular presentations [93]. A mouse model with the GBE mutation p.Y329S presents a phenotype that is reminiscent of APBD in humans, with the accumulation of polyglucosan bodies and neurological dysfunction [94]. This model has allowed researchers to demonstrate that, like LD, APBD can be rescued by inhibiting GS [95], again evidencing the role of glycogen accumulation in the etiopathogeny of neurodegeneration. In this regard, strategies targeting glycogen synthesis have proven effective in mouse models of both LD and APBD [59,60]. Similarly to LD, polyglucosan bodies have been reported to accumulate in astrocytes and neurons in APBD [96,97]. The exact contribution of astrocytic polyglucosan bodies to the pathophysiology of this disease is not clear. To unequivocally dissect this contribution, GS should be ablated specifically in astrocytes in the APBD mouse model, in a similar fashion as malinKO + GSGfap-KO mice for LD [50]. However, after all of the considerations above, it is reasonable to hypothesize that astrocytic polyglucosan bodies participate in the pathophysiology of APBD, probably by inducing neuroinflammation in a similar fashion as LBs in LD. In fact, the presence of polyglucosan bodies in astrocytes is sufficient to cause APBD [98]. RANBP2-Type and C3HC4-Type Zinc Finger Containing 1 protein (RBCK1, also known as HOIL1) is another E3 ubiquitin ligase that is related to glycogen metabolism. Mutations in the RBCK1 gene result in polyglucosan body myopathy with or without immunodeficiency (OMIM 615895). This disease affects children, and courses with progressive proximal muscle weakness and dilated cardiomyopathy, accompanied in some cases by severe immunodeficiency and a hyperinflammatory state [99,100]. The muscle and the heart of these patients show extensive polyglucosan body accumulation [100]. Although the disease is primarily a skeletal and cardiac myopathy, a mouse model of this condition also shows the presence of profuse aggregates of poorly branched glycogen in the nervous tissue, especially in the hippocampus, cerebellum, and spinal cord [101]. Interestingly, these polyglucosan bodies are localized mainly in astrocytes, and this accumulation is accompanied by astrogliosis and microgliosis, again linking neuroinflammation to the excessive accumulation of glycogen in astrocytes [101]. These observations indicate that the accumulation of polyglucosan bodies in the nervous tissue might also play a role in the pathophysiology of human RBCK1 deficiency. As with LD and APBD, the inhibition of glycogen synthesis rescues the pathological traits of the mouse model of RBCK1 deficiency, once again demonstrating that glycogen synthesis is a prerequisite for the formation of the polyglucosan bodies that underlie the disease [101]. Of note, a recent report has shown that the substrate of RBCK1 ubiquitination is not a protein but glycogen itself. More specifically, RBCK1 targets unbranched glucosaccharides, participating in a mechanism aimed at preventing polyglucosan body accumulation [102]. Thus, different E3 ubiquitin ligases (malin and RBCK1) are involved in preventing abnormal glycogen accumulation. Interestingly, ubiquitin is present in glycogen aggregates both in the absence of malin and of RBCK1 [48,103]. The genuine substrate of malin is still not clear, but the restoration of malin expression in a malin-deficient mouse model results in the degradation of the accumulated GS and laforin [104], thereby indicating that these two proteins are targets of malin ubiquitination. These experiments also showed that, once LBs have accumulated in the CNS, malin restoration is not able to promote their removal. This observation thus indicates that the role of malin is related to preventing the accumulation of abnormal glycogen (Figure 2) rather than eliminating it after its accumulation. Comparison of the pathology of LD, APBD, and RBCK1 deficiency opens up a number of questions. Why do LD and RBCK1 deficiency affect children while APBD affects adults? Why are the neurological presentations so different between the three diseases? Do LBs, Corpora amylacea and the polyglucosan bodies that accumulate in APBD have any distinguishing features amongst them that would offer insights into the differences among glycogen storage diseases? The observation that APBD patients do not present epilepsy reinforces the idea that the epileptic phenotype of LD is due to the accumulation of LBs in GABAergic interneurons. But then, if the malin-laforin complex, GBE, and RBCK1 are all important to prevent the accumulation of abnormal glycogen, why does the lack of one or the other result in the accumulation of polyglucosan bodies with different cell type-specificity? Over recent years, the role of astrocytic dysfunction in neurodegenerative diseases previously thought to have an exclusively neuronal origin is becoming apparent. LD is not only an example of such diseases but also one in which the pathology is due in part to a defect originated primarily in astrocytes. In conclusion, the study of LD has allowed the identification of the toxic consequences of the excessive accumulation of glycogen in astrocytes, a process that plays a key role in the pathophysiology of LD. This pathologic mechanism might have important implications for other conditions in which glycogen abnormally accumulates in astrocytes, such as in other rare conditions like APBD and RBCK1 deficiency, and more common neurodegenerative conditions like Alzheimer’s, Parkinson’s, Huntington’s, and Pick’s diseases, or even during normal aging. Further research is needed to understand the molecular mechanisms that link excess glycogen in astrocytes with neuroinflammation.

PMC10000528

Abdullah Alfaifi,Mohammed Y. Refai,Mohammed Alsaadi,Salem Bahashwan,Hafiz Malhan,Waiel Al-Kahiry,Enas Dammag,Ageel Ageel,Amjed Mahzary,Raed Albiheyri,Hussein Almehdar,Ishtiaq Qadri

Metabolomics: A New Era in the Diagnosis or Prognosis of B-Cell Non-Hodgkin’s Lymphoma

23-02-2023

metabolomics,B-cell non-Hodgkin’s lymphoma,biomarkers,metabolites,early diagnosis,therapeutic

A wide range of histological as well as clinical properties are exhibited by B-cell non-Hodgkin’s lymphomas. These properties could make the diagnostics process complicated. The diagnosis of lymphomas at an initial stage is essential because early remedial actions taken against destructive subtypes are commonly deliberated as successful and restorative. Therefore, better protective action is needed to improve the condition of those patients who are extensively affected by cancer when diagnosed for the first time. The development of new and efficient methods for early detection of cancer has become crucial nowadays. Biomarkers are urgently needed for diagnosing B-cell non-Hodgkin’s lymphoma and assessing the severity of the disease and its prognosis. New possibilities are now open for diagnosing cancer with the help of metabolomics. The study of all the metabolites synthesised in the human body is called “metabolomics.” A patient’s phenotype is directly linked with metabolomics, which can help in providing some clinically beneficial biomarkers and is applied in the diagnostics of B-cell non-Hodgkin’s lymphoma. In cancer research, it can analyse the cancerous metabolome to identify the metabolic biomarkers. This review provides an understanding of B-cell non-Hodgkin’s lymphoma metabolism and its applications in medical diagnostics. A description of the workflow based on metabolomics is also provided, along with the benefits and drawbacks of various techniques. The use of predictive metabolic biomarkers for the diagnosis and prognosis of B-cell non-Hodgkin’s lymphoma is also explored. Thus, we can say that abnormalities related to metabolic processes can occur in a vast range of B-cell non-Hodgkin’s lymphomas. The metabolic biomarkers could only be discovered and identified as innovative therapeutic objects if we explored and researched them. In the near future, the innovations involving metabolomics could prove fruitful for predicting outcomes and bringing out novel remedial approaches.

Metabolomics: A New Era in the Diagnosis or Prognosis of B-Cell Non-Hodgkin’s Lymphoma A wide range of histological as well as clinical properties are exhibited by B-cell non-Hodgkin’s lymphomas. These properties could make the diagnostics process complicated. The diagnosis of lymphomas at an initial stage is essential because early remedial actions taken against destructive subtypes are commonly deliberated as successful and restorative. Therefore, better protective action is needed to improve the condition of those patients who are extensively affected by cancer when diagnosed for the first time. The development of new and efficient methods for early detection of cancer has become crucial nowadays. Biomarkers are urgently needed for diagnosing B-cell non-Hodgkin’s lymphoma and assessing the severity of the disease and its prognosis. New possibilities are now open for diagnosing cancer with the help of metabolomics. The study of all the metabolites synthesised in the human body is called “metabolomics.” A patient’s phenotype is directly linked with metabolomics, which can help in providing some clinically beneficial biomarkers and is applied in the diagnostics of B-cell non-Hodgkin’s lymphoma. In cancer research, it can analyse the cancerous metabolome to identify the metabolic biomarkers. This review provides an understanding of B-cell non-Hodgkin’s lymphoma metabolism and its applications in medical diagnostics. A description of the workflow based on metabolomics is also provided, along with the benefits and drawbacks of various techniques. The use of predictive metabolic biomarkers for the diagnosis and prognosis of B-cell non-Hodgkin’s lymphoma is also explored. Thus, we can say that abnormalities related to metabolic processes can occur in a vast range of B-cell non-Hodgkin’s lymphomas. The metabolic biomarkers could only be discovered and identified as innovative therapeutic objects if we explored and researched them. In the near future, the innovations involving metabolomics could prove fruitful for predicting outcomes and bringing out novel remedial approaches. B-cell non-Hodgkin’s lymphomas (B-NHLs) are a genetically, metabolically, and clinically heterogeneous group of neoplasms, with most emerging from B lymphocytes in the germinal centre (GC). B-NHLs account for approximately 90% of all non-Hodgkin’s lymphomas [1]. Diffuse large B-cell lymphomas (DLBCLs), follicular lymphoma (FL), Burkitt lymphoma (BL), and B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) are typical B-NHL subtypes [2]. Myc amplification [3] and metabolic heterogeneity in B-NHL are important biologically because they influence therapy responses and can predict clinical outcomes [4,5]. As cells are driven to grow, proliferate, or die, their metabolic needs fluctuate, and it is essential that cellular metabolism correspond to these needs [6]. B-cell lymphoma and cancer cells have dysregulated metabolisms that promote uncontrolled proliferation [7,8]. This altered metabolism leads to metabolic phenotypes that can be utilised for earlier cancer detection and/or therapy response biomarkers [9]. Fluorodeoxyglucose-PET imaging is an essential tool for the management of many malignancies, including B-cell lymphomas [10]. Other metabolites in biological samples have been in the limelight for diagnosis, monitoring, and therapy [11]. Metabolomics is a comprehensive evaluation of both qualitative and quantitative parameters of all the metabolites present in cells, tissues, and bodily fluids, which can reveal crucial information about the cancer state that would not be obvious otherwise. Metabolomics-based diagnosis investigates the metabolites present in the human body and how they react under stress conditions, like various diseases and disorders [12,13]. Metabolomics is a powerful tool that can identify cancer biomarkers and drivers of tumorigenesis. An example includes the de novo synthesis of phospholipid compounds in malignant tissues, which increases at the time of the progression of the tumour [14,15]. Worthy, LDH-A was the first metabolic target demonstrated to be directly regulated by an oncogene (MYC), and genetic or pharmacologic inhibition of LDH-A diminishes MYC-dependent tumours [16]. Even now, it is a challenging task to detect and treat the lymphoma at an initial stage. This review provides an overview of existing and future metabolomics prospects to improve B-cell non-Hodgkin’s lymphoma diagnosis, monitoring, and treatment. First, we review B-cell non-Hodgkin’s lymphoma metabolism. We then introduce general metabolomics techniques, including their analytical advantages and disadvantages. In the final section, we present instances where metabolomics has been employed in the clinical and research areas as a way to lead prospective future applications for the prognosis and diagnosis of B-cell non-Hodgkin’s lymphoma. In practice, metabolomics has been widely covered. For more information on best practises in metabolomics analysis, the reader is referred to other excellent reviews cited throughout this article. Cell metabolism is a well-defined set of metabolic activities that generate and store energy equivalents, maintain redox homeostasis, synthesise biologically active macromolecules, and eliminate organic waste [17]. Catabolism breaks down carbon sources into simpler intermediates, which are then employed as building blocks in the production of lipids, amino acids, carbohydrates, and nucleotides (anabolism) [18]. Tumour cells are able to survive, grow, and divide because of their metabolic versatility and plasticity, which allow them to produce ATP as an energy source while maintaining the reduction–oxidation (redox) balance and devoting resources to biosynthesis [19]. Recent sequencing approaches have not discovered significant metabolic genes as direct lymphoma driver mutations (Figure 1) [20,21]. Metabolic alterations in B-NHL are characterised by the production of enough energy and maintenance of anabolism for survival, growth, and division in the face of low levels of nutrients and oxygen (such as HIF1 and MYC), deregulation of metabolic regulators (like mTORC1), and rewiring of metabolic pathways (e.g., BCR signalling) [22,23]. The Warburg effect promotes aerobic glycolysis over aerobic oxidation [24], and this is supported by HIF1-alpha and MYC. This leads to the production of lactate and poor producing ATP, but helps create biomass. As a result, the body’s reaction to hypoxia-induced metabolic abnormalities may promote anabolism in GC-derived B-cell lymphoma [22]. MYC oncogene aberrations, including translocations or overexpression, are characteristics of B-cell lymphoma aetiology [25]. B-cell lymphomas require higher MYC levels to maintain their rapid proliferation rate. MYC upregulates nucleoside metabolism, which is essential for cell development. Glutamine metabolism is similarly regulated by MYC expression [25,26]. Glucose uptake, glycolysis, and lipid biosynthesis are all controlled by MYC as well [27]. On the other hand, alpha-ketoglutarate (αKG) synthesis can be inhibited by hypoxia and mitochondrial dysfunction, which in turn reduces the activity of αKG-dependent enzymes, leading to increased DNA and histone hypermethylation and stabilisation of HIF1α. HIF1α is the primary transcriptional regulator of the adaptive response to hypoxia and is constitutively stabilised in a significant proportion of DLBCLs and FLs [22]. HIF1α and MYC promote anaerobic glycolysis by activating genes for glucose transporter (GLUT), hexokinase (HK), monocarboxylate transporter (MTC), pyruvate dehydrogenase (PDK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK), pyruvate kinase (PK), and lactate dehydrogenase (LDHA) [27]. mTORC1 is essential for generating metabolic precursors via the tricarboxylic acid cycle (TCA) and stimulating cellular proliferation. Activation of mTORC1 thereby enhances the survival of B-cell lymphoma. T-cell-selected GC B cells in the light zone necessitate mTORC1 activation in order to proliferate and mutate in the dark zone. mTORC1 may be aberrantly activated in GCB-DLBCL through activating mutations of PI3K/Akt/mTOR pathway genes [22]. A further marker of B-cell lymphoma is altered B-cell receptor (BCR) signalling, which is essential for the maintenance and creation of both healthy and malignant B cells [28]. PI3K/AKT/mTORC1 is one of the BCR signalling pathway’s downstream branches. PI3K regulates glycolysis and energy generation, and consequent AKT signalling influences the cellular metabolome. AKT promotes glucose uptake and glycolysis by increasing the expression and translocation of GLUT1 and glycolytic enzymes, including hexokinase (HK) expression and activation [28]. In a subset of DLBCL and MCL, PTEN mutations lead to AKT/mTORC1 pathway gene expression [29]. RagC mutations in FL enhance mTORC1 signalling by eliminating amino acid dependence [30]. Numerous anabolic and energy-generating processes, including protein synthesis, pyrimidine synthesis, HIF1α expression, glycolysis, the oxidative portion of the pentose phosphate pathway (PPP), lipid and mitochondrial metabolism, and glutaminolysis, are stimulated by mTORC1 expression [23]. There is an urgent need for biomarkers based on non-invasive sampling procedures (e.g., blood, urine, etc.) that can help in the diagnosis of lymphoma, such as metabolite profiling. The perfect test should be easy, reliable, and accurate. “What simple, non-invasive, painless, and convenient tests can be used to detect cancer early?” ranked as the most important research priority for the early detection of cancer in the UK-focused research gap survey performed by the James Lind Alliance, which includes patients and doctors [31]. Accordingly, serum biomarkers of lymphoma activity have been studied extensively over the last decade [32], and we conclude that they are clinically relevant for the diagnosis, prognosis, and therapeutic monitoring of lymphomas. In this review, we shed light on the major metabolic dysregulation described in B-cell non-Hodgkin’s lymphoma research (Table 1 and the 3rd Table in Section 3.5). The most prevalent B-cell non-Hodgkin’s lymphoma is DLBCL. Over 40% of DLBCL patients are refractory and have a worse prognosis for survival [40]. The International Prognostic Index (IPI) is currently used as the primary risk-stratification tool for prognosis in the clinic, and higher IPI scores indicate a worse outcome [41]. However, IPI cannot identify high-risk individuals [42]. Multiple investigations have failed to replicate the predictive power of the molecular heterogeneity of DLBCLs [43], which is widely regarded as a crucial factor influencing the response to therapy [42]. Therefore, additional research is required to identify new prognostic biomarkers to enhance the current DLBCL stratification system and direct the optimisation of therapeutic strategies. Increased uptake of the glucose analogue 18F-fluoro-2-deoxy-D-glucose (18F-FDG) and up-regulated expression of GLUT1 and HK are indicative of the robust metabolism of DLBCL cells [41,44]. Metabolic heterogeneity, as shown by malignancies’ varied substrate dependency, is common among tumour types and subtypes [4]. DLBCL is metabolically heterogeneous and categorised into oxidative phosphorylation (OxPhos) and BCR groups [20,45]. Few studies to date have identified specific metabolic indicators involved in the diagnosis and prognosis of DLBCL (the 3rd Table in Section 3.5); the reader is directed to previous studies on these topics [46,47,48,49,50,51,52,53,54,55,56,57,58]. An indolent lymphoma originating from germinal centre B cells is called follicular lymphoma (FL) [59]. It is the second most prevalent lymphoid malignancy, and accounts for 20% of non-Hodgkin’s lymphomas and is a disease of adults [60]. Transformation into DLBCL is related to increased glycolytic enzyme expression, which is in line with higher glucose uptake by 18F-FDG PET/CT [61]. Banoei et al. found higher levels of ADP, AMP, GTP, NADHP, glucose, and uridine diphosphate glucose (UDP-glucose) in FL compared with controls; and this was linked to aggressive cases of FL [62]. Regrettably, little is known about the metabolism of FL. MCL represents about 5–10% of B-NHLs. MCL is classified as indolent, but the disease progresses quite aggressively [63]. Many studies have pointed to a disruption of the upstream PI3K/AKT pathway as a driver of mTOR in MCL. Supporting this idea is the finding that PTEN, an intrinsic PI3K/AKT inhibitor, is often absent or at low levels in MCL [64]. Evidence from clinical trials shows that mTOR inhibition effectively targets MCL metabolism, and so it is authorised for the relapsed/refractory (r/r) setting [61,65]. Glycolysis, PPP, and lipid biosynthesis are all stimulated by mTOR signalling [66]. Higher quantities of lactic acid, TCA metabolites, and amino acids were found in MCL compared with controls, which may suggest a cancer-specific energy metabolic mechanism to ensure ongoing proliferation within the constrained resources of their microenvironment, as reported by Sekihara et al. [64]. By analysing the metabolic processes of MCL cells and their response to the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib (IBR), Lee et al. proposed imaging biomarkers (lactate and alanine) to detect response and resistance to IBR in MCL and suggested pathways to overcome IBR resistance, most notably glutaminolysis, the major oxidative ATP-producing pathway in these cells [67]. Dennis Burkitt discovered the rare and aggressive Burkitt lymphoma (BL) [2]. BL is characterised by chromosomal rearrangements of the c-Myc proto-oncogene, which stimulates the expression of multiple enzymes in serine biosynthesis [46]. Serine is necessary for one-carbon metabolism and nucleotide synthesis [68]. Yang et al. studied BL mice serum metabolomics. Glucose, glutamate, and unsaturated lipids were significantly different in BL and controls. Abnormal metabolism and metabolites of BL were found. These discoveries may help create noninvasive approaches for BL diagnosis and prognosis based on these biomarkers [69]. Chronic lymphocytic leukaemia (CLL) is characterised by the heterogeneous malignant proliferation of mature monoclonal B cells in the blood, bone marrow, and lymphoid organs [70]. Alterations in carbohydrate metabolism, lipid metabolism, and OXPHOS are all part of the dynamic metabolic reprogramming of CLL that occurs at different stages of the tumour [71]. Furthermore, TP53, ATM, and MYC, among others, are tumour-suppressor genes that regulate the metabolic reprogramming that occurs during CLL [21]. CLL cells are highly glycolytic, but not as much as DLBCL cells [72]. CLL cells exploit altered lipid metabolism to promote mitochondrial function via activating STAT3 [73]. High FDG uptake in a PET/CT scan is an indication of a glycolytic phenotype in CLL cells, which may predict Richter’s transformation into an aggressive lymphoma, most often DLBCL [74]. Metabolomics uses nuclear magnetic resonance (NMR) or mass spectrometry (MS) to look at global, dynamic, and endogenous metabolites [77,78]. Metabolomics has been used to explore disease pathogenesis and discover novel biomarkers. Thus, metabolomics can be utilised not just to identify new biomarkers but also to develop noninvasive diagnostic and prognostic tools for medical conditions [69,79]. In the study of cancer, introducing certain novel technologies such as metabolomics is found to impart fruitful and reliable information regarding cancer metabolism, particularly for the main mechanism in tumour proliferation [77]. The impact of B-cell lymphoma on the patient’s metabolomics is still not fully known. Little research has been conducted on the treatment response and prognosis of B-cell lymphoma [80]. When looking for alternative methods to improve the rate of detection and compliance in the assessment of B-cell non-Hodgkin’s lymphoma, the study of metabolomics with its comprehensive and unbiased exploration for changes in the metabolic profile has been found to be an effective approach [12]. Thus, developing metabolomics technology and functional metabolic assessment in B-cell non-Hodgkin’s lymphoma remains an interesting subject for enhanced diagnosis and therapy [23]. As discussed below, there are a variety of steps to metabolomics analysis (Figure 2), each with their own set of benefits and drawbacks [81,82]. Metabolomics studies can be divided into two classes: targeted and non-targeted. Targeted analysis is used for the identification and quantification of pre-defined metabolites and can be used for quantitative as well as qualitative analysis [83]. Non-targeted analysis consists of analysing all accessible metabolites in a given sample and is the first choice for cancer biomarker discovery studies [84]. Therefore, non-targeted metabolomics research necessitates advanced analytical methodologies, computerised spectral data processing, biological data elucidation, and hypothesis generation [85,86,87]. In the DLBCL studies, non-targets were the most frequently applied, with an average of 61.5% compared with targeted methods (the 3rd Table in Section 3.5). The collection of the sample, its preparation, and storage are the second step in the metabolomics study plan. The most common samples for conducting clinical metabolomics research are blood and urine [88]. It is important to design the research based on metabolomics to reduce the influence of certain constituents such as age, gender, state of fasting, diet, physical activity, exercise, and the day and time of sample collection. Before starting the actual research, it is important to conduct a pilot study of healthy individuals and report it as part of the research to validate the results’ reproducibility. The samples (particularly plasma, serum, and urine) must be kept in various aliquots soon after collection to avoid the production of compounds from the many freeze–thaw cycles used for different metabolomics studies [89]. The factors used for processing the sample, such as pH buffering and extraction, should also be uniform and follow standard operating procedures (SOPs) [81,82,90,91]. The samples that are non-invasive in nature, such as blood or urine, are the best for regular clinical analysis [85]. Comparing the serum metabolomics of high-risk individuals with those who have been cured by standard chemotherapy can provide useful information about the prognosis of DLBCL as well as the mechanisms involved in failed treatment procedures [92,93]. For best practices in metabolomics, the reader is directed to previous reviews on these topics [94,95,96,97]. The study of metabolomics is regarded as one of the most trustworthy and comprehensive tools for investigating the physiological parameters of an individual, analysing the metabolic pathways, and discovering new biomarkers [98] by employing mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy technologies [85,92]. The MS technique has the ability to isolate the intricate mixture of compounds for their detection and quantification with elevated sensitivity and specificity, and can also demonstrate information regarding molecular structures [99]. MS separation techniques are essential for reducing sample complexity and minimising ionisation suppression effects [100]. A preceding separation stage, such as high-performance liquid chromatography (HPLC), or ultra-performance liquid chromatography (UPLC), and capillary electrophoresis (CE), is frequently required. There are three main components in a mass spectrometer: an ion source, a mass analyser, and a detector. The ion source is used for converting the sample molecules into ions, which are then resolved into an electromagnetic field or time-of-flight tube by the mass analyser, while the detector is employed for measuring the end results. For maximising the coverage of the metabolome, it is advisable to conduct the analysis of biological samples in the m/z 50–1000 scan range and in both positive and negative ionisation forms [101,102]. Electrospray ionisation (ESI) is used in metabolomics trials due to its “soft ionization” competency and ability to produce unbroken molecular ions [102]. Medriano et al. examined the metabolomics of two types of blood cancers, myeloma and non-Hodgkin’s lymphoma, using plasma samples from both cancer patients and healthy individuals to detect all the potential metabolites and pathways that were affected by employing metabolomics based on LC–MS. Their results revealed a significant metabolomic difference between the healthy control individuals and the myeloma and NHL patients, with disturbed metabolic pathways such as choline metabolism and oxidative phosphorylation being associated with the progression and growth of tumours [77]. GC–MS is a technique that combines great separation efficacy with sensitive, selective, and versatile mass evaluation and is suitable for comprehensive analysis. It is a combination of MS and GS that is used for the detection and quantification of a wide range of chemical compounds, such as natural products, blood, and urine. GS–MS is used in many fields of study, such as detecting drugs, amino acid evaluation, doping control, and the detection of natural materials like food products [103]. EI, or electron ionization, is used for combining MS with GC in almost all the metabolomics applications that are based on GC. The EI–MS method works well for chemical compounds that do not change when heated and that are volatile and are separated by chromatography at high temperatures [104]. Bueno Duarte et al. collected urine samples from NHL patients and conducted their metabolic analysis by employing untargeted GC–MS, which was found to be a valuable tool for distinguishing the population under study. Their GC–MS results indicated the presence of as many as 18 metabolites in the urine sample that contributed to differentiating healthy subjects from DLBCL patients with an accuracy of about 99.8% (p < 0.001). GC–MS is considered a valuable option for studying metabolomics due to its operational simplicity, low cost, reliable identification of metabolites, robustness, and easy availability [105]. NMR spectroscopy is a universal metabolite detection method that allows for direct analysis of samples with little sample preparation and simultaneous measurement of numerous types of tiny metabolites [106,107]. However, it has limitations, such as high equipment costs, high maintenance costs, and decreased sensitivity [108,109]. Mass spectrometry is better than NMR in several ways, although NMR has its own advantages (Table 2). The B-NHL study design determines the optimal analysis. When the metabolomics data are produced, it is important to ensure that they are reproducible [89,110]. Quality standardisation and quality control are considered for the optimisation of the reproducibility of results. Data analysis and bioinformatics are used to process the data, which are then subjected to statistical analysis. There are two classical approaches to the statistical analysis of multivariate data: unsupervised learning and supervised learning. A popular unsupervised learning method is principal component analysis (PCA). The second main approach is supervised learning, such as with artificial neural networks (ANN), partial least squares discriminate analysis (PLS-DA), etc., which can be used for excavating the data further to obtain the biomarkers [111,112]. The discovery process of biomarkers can be driven through supervised models that can be linked with clinical results, histopathological scores, and various other omics data. It is important to test the supervised models with precise internal cross-validation processes or external tests to obtain trusted biomarkers and models and to decrease the chances of data overfitting [113]. Profiling the metabolites in each biological entity is incomplete without accurate data measurement and precise interpretation. To identify the features of potent spectral biomarkers, attempts are made to recognise the unidentified spectral biomarkers. The peaks can be identified with the help of public metabolomics databases and in-house spectral databases such as the Golm database, LIPID MAPS, human metabolome database (HMDB), METLIN database, etc. Following the identification of metabolomics biomarkers, additional experiments are required to validate or test the biomarkers [82,112,114]. DLBCL is the most common non-Hodgkin’s lymphoma, and therefore 13 publications of relevance to our research interest are included in this review (search query in PubMed: “Metabolomics” and “DLBCL”), and summarised in Table 3. In the clinical setting, metabolomics is finding an expanding number of applications, including disease diagnosis and understanding, the discovery of novel drug targets, the customisation of medication treatment, and the monitoring of therapeutic results [115]. In this last section, we discuss the clinical applications of metabolomics and offer examples to clarify how metabolism will open a new era in lymphoma research and how this will positively influence diagnosis and treatment. Metabolism in B-NHL plays a crucial role in established therapeutic approaches (Table 4). Antimetabolites were the name given to the chemical compounds that were first used to treat cancer. The reason for choosing this name was that these compounds were found to resemble endogenous metabolites in their chemical structure and disrupt the process of normal metabolism. In comparison to other omics, metabolomics is best for evaluating the potential of these cancer treatment regimens. This study was carried out to discover whether the therapies could cause alterations in the metabolic pathways and detect the pharmacokinetics of drugs simultaneously or not. In the coming time, it will become crucial to combine the study of pharmacometabolomics with other biological systems knowledge, such as mRNA, genetics, miRNA, and imaging. This will help in determining the correlation of the metabolomics response with the cancer stage, undesirable incidents, and the growth or recession of the tumour. The study of pharmacometabolomics is capable of monitoring a patient’s metabolic response to a drug; thus, it is very interesting to use metabolomics in detecting cancerous growth, prognosis, and therapy management [82]. Moreover, the therapies based on metabolomics can not only enhance the responses of immune cells to extremely immunogenic tumours but can also elevate the immunogenicity of cancer cells, thereby increasing the ability of immunotherapy to cure a vast variety of carcinomas. For further information, the reader is directed to previous reviews on these topics [23,116,117,118,119,120]. A recent metabolomics study suggested a methodology for discovering novel biomarkers that can be used for the diagnosis and characterisation of various lymphoma subtypes. The GC–MS method was used for the investigation and evaluation of plasma samples taken from individuals with different subtypes of lymphomas. The results showed a significant prevalence of elaidic acid and hypoxanthine (HX) in patients suffering from Hodgkin’s lymphoma, MM, CLL, and DLBCL compared with healthy control individuals in all the study groups [50]. Yoo et al. analysed the urine samples taken from lymphoma patients and translated the data into ions of low mass, i.e., less than 1000 m/z. They chose three peaks of high intensity and low mass ions for the analysis, of which the peak in the range of 137.08 m/z ion was detected as HX. The levels of HX and xanthine inside the cells are found to be inversely proportional to the energy modifications of adenylate and thus to the ATP of the cells. Additional research is required as abnormal metabolic processes are detected as initial lymphoma biomarkers [127]. For further information, the reader is directed to previous reviews on these topics [53,80,118,128]. Genetic mutations accumulate sequentially during tumour development, eventually resulting in malignant tumours. However, it has also been shown that metabolic processes and inflammatory factors indirectly contribute to the development of the tumour [11]. In their study, Pettersena et al. proved that the cell line of B-cell lymphoma surrounds numerous amplified genomic uracil concentrations in comparison with non-lymphoma cell lines or normal lymphocytes. They utilised a method based on liquid chromatography combined with mass spectrometry (LC/MS) for quantifying the genomic sequence of 2-deoxyuridine and proving their study. In harmony with uracil generated by activation-induced cytidine deaminase (AID), they discovered a distinctive mutational signature of an AID hotspot in the lymphoma area where there was clustered mutation. They also presented an important revelation about the expression of SMUG1 and uracil-DNA glycosylases UNG along with the excision capacity of uracil by stating its negative correlation with the concentration of genomic uracil, which somewhat decreased the AID effect [129]. Another study was also conducted on the metabolomic pattern of Burkitt lymphoma that was induced by MYC glucose deprivation, as well as hypoxic and aerobic conditions. They used a [U-13C, 15N]-glutamine tracer to detect glutamine import and metabolism via the TCA cycle under hypoxia conditions and discovered that glutamine is significantly precipitated to citrate carbons. The deficiency of glucose leads to the significant augmentation of citrate, fumarate, and glutamine-derived malate. Their arrangements showed a different pathway for the generation of energy called glutaminolysis, which is associated with the glucose-independent TCA cycle. Under the conditions of hypoxia and scarcity of glucose, the critical role of glutamine in the proliferation of cells makes them susceptible to BPTES (glutaminase inhibitors), which in turn can be used for treating tumours [130]. As the use of metabolomics is continuously increasing in clinical trials, it may soon become one of the most successful tools for detecting and healing cancerous growths. The changes related to metabolic pathways may occur in a broad range of B-cell non-Hodgkin’s lymphomas. Researching and knowing about them can help in identifying new remedial targets and discovering novel metabolic biomarkers. In the near future, the study of metabolomics will become crucial for outcome prediction and the revelation of new treatment regimens. There is a need for conducting metabolomics research on B-cell lymphomas in large cohort trials to discover new biomarkers, which will thus prove to be an influential step in the path of clinical integration of biomarkers that are discovered by metabolomics.

PMC10000530

Juanjuan Wang,Ningning Zhu,Xiaomin Su,Yunhuan Gao,Rongcun Yang

Gut-Microbiota-Derived Metabolites Maintain Gut and Systemic Immune Homeostasis

02-03-2023

gut microbiota,SCFAs,tryptophan metabolites,bile acid metabolites,tolerogenic macrophages,regulatory T cells

The gut microbiota, including bacteria, archaea, fungi, viruses and phages, inhabits the gastrointestinal tract. This commensal microbiota can contribute to the regulation of host immune response and homeostasis. Alterations of the gut microbiota have been found in many immune-related diseases. The metabolites generated by specific microorganisms in the gut microbiota, such as short-chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites, not only affect genetic and epigenetic regulation but also impact metabolism in the immune cells, including immunosuppressive and inflammatory cells. The immunosuppressive cells (such as tolerogenic macrophages (tMacs), tolerogenic dendritic cells (tDCs), myeloid-derived suppressive cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Breg) and innate lymphocytes (ILCs)) and inflammatory cells (such as inflammatory Macs (iMacs), DCs, CD4 T helper (Th)1, CD4Th2, Th17, natural killer (NK) T cells, NK cells and neutrophils) can express different receptors for SCFAs, Trp and BA metabolites from different microorganisms. Activation of these receptors not only promotes the differentiation and function of immunosuppressive cells but also inhibits inflammatory cells, causing the reprogramming of the local and systemic immune system to maintain the homeostasis of the individuals. We here will summarize the recent advances in understanding the metabolism of SCFAs, Trp and BA in the gut microbiota and the effects of SCFAs, Trp and BA metabolites on gut and systemic immune homeostasis, especially on the differentiation and functions of the immune cells.

Gut-Microbiota-Derived Metabolites Maintain Gut and Systemic Immune Homeostasis The gut microbiota, including bacteria, archaea, fungi, viruses and phages, inhabits the gastrointestinal tract. This commensal microbiota can contribute to the regulation of host immune response and homeostasis. Alterations of the gut microbiota have been found in many immune-related diseases. The metabolites generated by specific microorganisms in the gut microbiota, such as short-chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites, not only affect genetic and epigenetic regulation but also impact metabolism in the immune cells, including immunosuppressive and inflammatory cells. The immunosuppressive cells (such as tolerogenic macrophages (tMacs), tolerogenic dendritic cells (tDCs), myeloid-derived suppressive cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Breg) and innate lymphocytes (ILCs)) and inflammatory cells (such as inflammatory Macs (iMacs), DCs, CD4 T helper (Th)1, CD4Th2, Th17, natural killer (NK) T cells, NK cells and neutrophils) can express different receptors for SCFAs, Trp and BA metabolites from different microorganisms. Activation of these receptors not only promotes the differentiation and function of immunosuppressive cells but also inhibits inflammatory cells, causing the reprogramming of the local and systemic immune system to maintain the homeostasis of the individuals. We here will summarize the recent advances in understanding the metabolism of SCFAs, Trp and BA in the gut microbiota and the effects of SCFAs, Trp and BA metabolites on gut and systemic immune homeostasis, especially on the differentiation and functions of the immune cells. The gut microbiota is established at birth and evolves with age, and also maintains a commensal relationship with the host, being an integral part of the human body. The mammalian gastrointestinal tract harbors large amounts of different gut microbiota [1], including bacteria, archaea, fungi, viruses and phages. These gut microorganisms not only participate in food and energy metabolism but also contribute to the host immune response and homeostasis [2,3]. The alteration of the gut microbiota can lead to the occurrence and development of many diseases [4]. In recent years, with the rapid development of molecular biology, genomics, bioinformatics analyses and high-throughput sequencing techniques, great progress has been made in understanding the gut microbiota with diseases [5] such as neurodegenerative diseases (Parkinson’s disease and Alzheimer’s disease), cardiovascular diseases (hypertension and atherosclerosis), metabolic diseases (obesity, diabetes, and non-alcoholic fatty liver disease (NAFLD)), and gastrointestinal diseases (inflammatory bowel diseases (IBD) and colorectal cancer (CRC)). These effects on the health of the host can occur through many ways such as energy absorption [5] and the microbiota–gut–brain axis [6]. However, the roles of altered gut microbiota in diseases are related to gut microbiota metabolites such as short-chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites from different microorganisms. The effects of gut microbiota metabolites on the local and systemic immunity have already attracted much attention. A growing body of clinical evidence has suggested an intricate relationship between the gut microbiota and the immune system. Most altered-gut-microbiota-mediated diseases are related to impaired immune responses [7]. Gut-microbiota-derived metabolites not only affect genetic and epigenetic regulation but also impact the metabolism of the immune cells via their receptors in the immune cells [8,9,10]. These metabolites from different microorganisms can not only promote the differentiation and function of immunosuppressive cells but also inhibit the inflammatory cells, together maintaining the gut and systemic immune homeostasis of the individuals (Figure 1). Since there are three main specific classes of metabolites, namely SCFAs, Trp and BA metabolites, that have been found in the gut microbiota so far, we here will summarize the recent advances in understanding the metabolism of SCFAs, Trp and BA in different microorganisms and the effects of SCFAs, Trp and BA metabolites on the gut and systemic immune homeostasis, especially on the differentiation and functions of immune cells. Short-chain fatty acids (SCFAs) are carboxylic acids produced from dietary fiber fermentation in the cecum and colon by gut bacteria [11,12] (Table 1), mainly including acetate (C2), propionate (C3) and butyrate (C4). Tryptophan (Trp) metabolism in the gut microbiota has been reviewed by us [18] and others [19,20,21]. Trp can be converted into various metabolites by the gut microbiota (Table 2) such as indole, indole-3-aldehyde (IAld), indole-3-acid-acetic (IAA), tryptamine, indoleacrylic acid (IA), indole ethanol (IE), indole-3-propionic acid (IPA), indole-3-acetaldehyde (IAAld) and 3-methylindole (skatole). Trp also produces kynurenine (Kyn) and downstream metabolites such as 3-hydroxykynurenine (3H-Kyn) and 3-hydroxyanthranilic acid (3-HAA) [19,20,21]. Two primary bile acids (BAs), i.e., cholic acid (CA) chenodeoxycholic acid (CDCA) are generated in the liver. These primary BAs can be conjugated, deconjugated and transformed into other metabolites in the gut microbiota (Table 3). Primary BAs are conjugated with glycine, taurine or other amino acids in hepatocytes and also in the gut microbiota. Conjugated BAs derived from the liver can be deconjugated in the gut microbiota through bile salt hydrolases (BSHs) in the small intestine [30]. While BAs are deconjugated, BAs can be converted into secondary BAs, i.e., DCA and lithocholic acid (LCA). There are four distinct ways to transform BAs, including deconjugation, dehydroxylation, oxidation and epimerization in human [31]. A range of oxo-, epi- and iso-derivatives by microbes [32] is found, such as the oxo-bile acid metabolites 3-oxoLCA, 7-oxoCA, 7-oxoCDCA, 12-oxoCA and 12-oxoDCA [33], and others such as iso-LCA, 3-oxo-LCA, allo-LCA, 3-oxoallo-LCA, isoalloLCA, 3-ketoLCA, LCA acetate and LCA propionate [31,34,35]. Chenodeoxycholic acid (CDCA) can be converted to UDCA [36] and DCA to iso-DCA by 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-HSDH dehydrogenate [37]. The metabolites of DCA, a 3β-hydroxydeoxycholic acid (isoDCA) has been also identified [38]. However, more gut bacterium species that produce BA metabolites still need to be identified. Several different ways such as passive diffusion, transporters and receptors help SCFAs enter cells. SCFA absorption can be promoted by the proton-coupled monocarboxylate-transporter 1 (MCT1) and the sodium-coupled monocarboxylate-transporter 1 (SMCT1) promote. Free-fatty acid receptor (FFAR) 2, G-protein coupled receptor (GPR) 43, FFAR3 (GPR41), hydroxycarboxylic acid receptor 2 (HCAR2) (also called GPR109a), Olfr-78 (OR51E2) in humans and Olfr-87 in mice can be activated by SCFAs [53,54]. The SCFAs acetic, propionic and butyric acids mainly activate GPR43 and/or GPR41, whereas butyric and β-hydroxybutyric acids are stimulators of GPR109a. In addition, SCFAs, mainly propionic and butyric acids, also participate in the activation of the peroxisome proliferator-activated receptor γ (PPARγ) [55]. The aryl hydrocarbon receptor (AhR) can be activated by various endogenous and exogenous polycyclic aromatic hydrocarbon ligands such as Trp metabolites [56]. This AhR can sense a wide range of intestinal signals, maintaining homeostasis between the gut microbiota and host [57,58]. After activation, a complex of inactive AhRs located in the cytoplasm with the AhR nuclear translocator protein (ARNT) is formed and translocated to the nucleus to control transcriptional activity. Notably, AhR interactions with other proteins are only triggered by specific AhR ligands. This indicates that the specific protein complexes may be induced by different AhR ligands. For AhR activation, indole, skatole, IA, tryptamine, IPyA and indole-3-acetamide (IAM) are the most effective, but IAA, IAID, IPA and ILA are less active [59,60]. Additionally, pregnant X receptor (PXR) can also be recognized by Trp metabolites [61]. The Trp metabolite indole and its derivatives through AhR and PXR contribute to anti-inflammatory activities [62]. The receptors of BAs and their metabolites include nuclear and membrane receptors, which have been reviewed by Biagioli et al. [63]. These receptors include nuclear receptors such as farnesoid X receptor (FXR), liver-X-receptor (LXR), vitamin D receptor (VDR), PXR, retinoid related orphan receptor (RORγt), constitutive androstane receptor (CAR) and cell membrane receptors such as G-protein BA receptor 1 (GPBAR1) (or Takeda G protein-coupled receptor 5 (TGR5)), cholinergic receptor muscarinic 2 and 3 (CHRM2, CHRM3), sphingosine-1-phosphate receptor 2 (S1PR2) and MAS-related GPR family member X4 (MRGPRX4) [64]. Gut-microbiota-derived SCFAs, Trp and BA metabolites exert a critical role in maintaining gut and systemic homeostasis through inhibiting inflammatory immune cells and promoting the differentiation and function of immunosuppressive cells (Figure 2) Macrophages (Macs) are heterogeneous. Their phenotypes and functions can be regulated by the surrounding microenvironments. These cells are generally divided into two kinds, inflammatory (i) and tolerogenic (t, immunosuppressive) macrophages (tMacs). IMacs are involved in inflammatory immune responses, whereas tMacs suppress inflammation and retain homeostasis by producing a large amount of IL-10 and TGF-β. In the resting intestine, mature resident (tolerogenic) ly6clow/-CX3CR1hiMHCIIhi Macs from inflammatory Ly6chigh monocytes/Macs reside either within the lamina propria (LP) or the muscle layer to maintain intestinal homeostasis [65]. LP Macs can be further subdivided into mucosal and submucosal Macs [66]. The intestinal epithelium and vasculature in the intestines are lined by Mucosal Macs [67,68]. Gut-microbiota-derived metabolites such as SCFAs, Trp and BA metabolites can promote the differentiation from iMacs to tMacs (Figure 3). SCFAs. SCFAs such as acetate (C2), propionate (C3) and butyrate (C4) exert an important role in maintaining immune homeostasis. Lipopolysaccharide (LPS)-mediated proinflammatory cytokines such as IL-6 could be inhibited by SCFAs. SCFAs could significantly reduce the histone deacetylase (HDAC) mRNA expression in monocytes and Macs [69]. SCFAs, especially butyrate, also negatively regulate the inflammatory signaling pathway mediated by NLRP3 (NOD-like receptor thermal protein domain associated protein 3) to inhibit the activation of Macs [70]. In addition, butyrate but not acetate or propionate can reprogram Mac metabolism toward oxidative phosphorylation to lead to an anti-inflammatory tolerogenic phenotype [71]. Trp metabolites. Trp metabolites (Trps) have an important role in the differentiation and function of Macs through the receptor AhR [72]. AhR-deficient mice were more sensitive to LPS-induced lethal shock [73] and produced higher amounts of tumor necrosis factor (TNF)α, interleukin (IL)-6 and IL-12. AhR was also required for Streptococcus- and Salmonella typhimurium-caused immunopathology in LPS tolerant mice [74]. In vitro studies showed that Trps-mediated suppression of inflammatory responses occurred through suppressing histamine production in the macrophages [75]. Through inhibiting LPS-induced SP1 (specificity protein 1) phosphorylation in macrophages, the AhR-SP1 complex represses histidine decarboxylase expression [75]. SP1 can bind to GC box elements (5′-GGGCGG-3′) in the promoter region [76] and is particularly important to TATA-less genes involved in the immune response [77]. It has also been found that AhR down-regulation in human disease is related to an abnormal interaction between SP1 and the AhR promoter [78]. The activation of AhR also results in a mitigated inflammatory response by LPS through a Ras-related protein Rac1 (ras-related C3 botulinum toxin substrate 1) ubiquitination-dependent mechanism, which can attenuate AKT (protein kinase B) signaling [79]. In addition, the Kyn downstream metabolite 3-HAA inhibits the LPS-mediated PI3K (phosphatidylinositol 3 kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) and NF-κB (nuclear factor κ gene binding) signaling pathways and decreases the production of pro-inflammatory cytokines in the macrophages [80]. The Trp metabolite receptor AhR can also inhibit the proliferation of myeloid precursor cells [81], drive DC differentiation over Macs [72] and suppress human CD34+ hematopoietic precursor cells to differentiate into monocytes and Langerhans cells [82]. BA metabolites. BA metabolites (BAs) are essential to maintain a tolerogenic phenotype of Macs via the BA receptor TGR5 (GPBAR1) [83,84,85]. TGR5 can inhibit the release of cytokines from Macs after exposure to LPS. LPS-induced inflammation in the liver could be accelerated in TGR5-deficient mice, whereas the suppressive effects of TGR5 agonist on inflammatory cytokines could be abolished [86]. TGR5 can also block NLRP3 inflammasome-dependent inflammation [87,88]. Indeed, the TGR5 ligands and secondary BAs DCA and LCA can function as endogenous inhibitors of NLRP3 activation by activating TGR5-cAMP (adenosine monophosphate)-PKA (protein kinase A)-dependent ubiquitination of NLRP3 [87,88]. The elevated intracellular cAMP levels can induce the phosphorylation and the ubiquitination of NLRP3 to block NLRP3-dependent inflammation and NLRP3-related metabolic disorders. TGR5 activation also promotes macrophage polarization to tolerogenic-phenotype Macs [89]. The hierarchy is LCA > DCA > CDCA > UDCA > CA for TGR5 activation [90]. In addition, FXR is also essential to maintain a tolegeronic phenotype of Macs as demonstrated in FXR knockout mice [83], and it is an important negative regulator of NLRP3 by directly interacting with NLRP3 and caspase-1 [91]. FXR is recruited to iNOS (nitric oxide synthase) and IL-1β promoters and stabilizes nuclear receptor corepressor 1 (NCOR1) complexes on the promoters of these genes [92]. Several pro-inflammatory genes such as iNOS, TNFα and IL-1β are marked by NCoR1 in promoter regions, which are linked to an NF-κB responsive element. FXR also activates SOCS3 (suppressor of cytokine signaling 3), CYP450 (Cytochrome P450) and FGF19 (fibroblast growth factor 19) to inhibit inflammation and SHP (Src homology-2 containing protein tyrosine phosphatase) to inhibit NF-κB, AP-1 (activator protein-1) and NLRP3 [93,94,95,96]. PXR, a nuclear receptor, also binds to LCA [90]. PXR activation can decrease the expression of IL6, TNFα and IL8 [97]. Dendritic cells (DCs) link the innate and adaptive immune responses. DCs are divided into monocyte DCs (moDCs), plasmacytoid DCs (pDCs) and conventional DCs (cDCs). The cDCs can be further divided into two subsets, cDC1 and cDC2. DCs are the most efficient antigen-presenting cells and are necessary for the effective activation of naïve T cells. However, DCs can also acquire tolerogenic functions such as conventional CD11c+ DCs expressing perforin (perf-DCs) and CD103+ DCs, which participate in the central and peripheral tolerance and the resolution of immune responses. Although DCs play distinct roles in shaping T cell development, differentiation and function, tolerogenic DCs (tDCs) mainly contribute to Treg differentiation and homeostasis (Figure 3). SCFAs. The SCFAs butyrate and propionate inhibit the activation of bone-marrow-derived DCs (BMDC) via suppressing the LPS-mediated expression of co-stimulatory molecules such as CD40 and the production of cytokines such as IL-6 and IL-12p40 [98]. Through modulating DCs, the SCFA butyrate also suppresses colonic inflammation and carcinogenesis [99]. Trp metabolites. Trp-metabolite-mediated AhR activation induces tDCs. These tDCs can limit T cell effective responses and promote the generation of Tregs. This may be because of NF-κB activation controlled by AhR, such as NF-κB expression and NF-κB/AhR protein interactions [100]. However, the molecular mechanisms involved are mostly unknown. Notably, AhR activation can indeed boost DCs to foster FoxP3+ Treg differentiation. BA metabolites. The secondary BA DCA suppresses the LPS-induced expression of pro-inflammatory genes such as IL-6 in DCs [101]. TGR5-deficient mice could recover LPS-induced expression of pro-inflammatory genes. TGR5 activation was found to induce the differentiation of human monocytes into IL-12 and TNF-α hypo-producing DCs [102]. Studies showed that BA receptor TGR5-mediated inhibition occurred through the repression of NF-κB by TGR5–cAMP–PKA (protein kinase A) signaling [101]. In addition, the secondary BA derivative isoDCA can also limit FXR activity in DCs and confer upon them an anti-inflammatory phenotype [52]. INT-747/obeticholic acid, which could activate FXR [83], greatly attenuated the differentiation of CD14+ monocytes into mature DCs [103]. A reduced number of activated DCs in the colon of mice administered with INT-747/obeticholic acid was also observed. The activation of the BA receptor VDR also inhibited the production of inflammatory cytokines and the maturation of DCs [104]. Regulatory T cells (Tregs) play key roles in maintaining immune homeostasis. The differentiation and function of Tregs can be regulated by gut-microbiota-derived metabolites such as SCFAs, Trps and BAs (Figure 4). Tregs express transcription factor forkhead box protein 3 (Foxp3) and differentiate in the thymus or the periphery. These cells are the main obstacles in successful immunotherapy and active vaccination. However, other T regulatory cells such as Foxp3 negative interleukin (IL)-10 producing type 1 regulatory T cells (Tr1 cells) also play an important role in homeostasis. SCFAs. SCFAs can regulate T cell function through G-protein coupled receptor (GPR) [105,106], are crucial in maintaining intestinal epithelium physiology and have a direct role in inducing Tregs in the gut. They can promote the naïve T cells toward Tregs [107]. Since SCFAs can be transported into the circulation, SCFAs also have wider systemic effects. Indeed, increased Foxp3+Tregs can be observed in mice provided with SCFAs [108]. The main mechanisms for SCFA-mediated Tregs include the G-protein coupled receptors (GPCRs) GPR41, GPR43 and GPR109A on the target cell surface mediating signaling and the inhibition of histone deacetylases (HDACs) to regulate gene expression [109]. The inhibition of HDAC activity can enhance gene transcription by increasing histone acetylation. Butyrate also upregulates histone H3 acetylation of Foxp3 to promote the differentiation of Tregs [108]. In addition, SCFAs such as butyrate can also condition mouse and human DCs to promote the differentiation of Tregs. After exposure to butyrate, DCs facilitate Foxp3+Treg differentiation and inhibit interferon (IFN)-γ-producing cells through indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2) [110]. Notably, SCFAs also promote the production of IL-10 in Th1, Th17 and Treg cells [111]. Trp metabolites. Indole and its derivatives from Trp can regulate the differentiation and function of Tregs [112,113]. The transcription factor Foxp3’s expression in Tregs can be promoted, whereas RORγ (retineic-acid-receptor-related orphan nuclear receptor gamma) in Th17 cells is inhibited by Trp metabolites. The AhR ligands 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and 4-n-nonylphenol are linked not only to differentiation but also to the functions of Tregs in mice and humans [114,115,116,117]. The AhR activated with ITE could suppress IBD [118] and improve encephalomyelitis (EAE) symptoms [119]. Notably, AhR in the Tregs of spleen and lymph nodes is lower than that in the intestinal Tregs [120]. In addition, Kyn in the gut microbiota could promote differentiation of Tregs [74,121,122,123]. Mechanically, Kyn metabolites work through direct transactivation and epigenetic modifications to support Treg differentiation [123,124,125]. Indeed, 3-HAA promotes the generation of Foxp3+Treg cells via nuclear coactivator 7 (NCOA7) [126]. The Trp metabolite receptor AhR also promotes the development of Tr1 cells [127]. During Tr1 cell differentiation, AhR is physically associated with c-Maf to activate IL-10 and IL-21 promoters to promote the differentiation of Tr1 cells [128]. AhR activation also promotes hypoxia inducible factor-1 (HIF1)-α degradation and takes control of Tr1 cell metabolism [127]. In addition, AhR can initiate the differentiation of mucosal-homing Tim3+Lag3+Tr1 cells [129]. BA metabolites. BA metabolites (BAs) modulate the differentiation and function of Tregs [130]. The bile acid derivatives isoalloLCA and 3-oxoLCA can promote the differentiation of Tregs. Mechanically, these derivatives promote the generation of mitochondrial reactive oxygen species (mitoROS) [131]. Indeed, for their energy production, Tregs mainly rely on oxidative phosphorylation (oxPhos) after exposure to BA derivatives. The mitochondrial activities also promote Treg generation [132]. Nuclear receptor subfamily 4, group A, member 1 (NR4A1) is also required for the isoalloLCA-induced Treg cells [133]. IsoalloLCA can increase the binding of NR4A1 at the Foxp3 locus to enhance the expression of the Foxp3 gene [134]. The composition of the gut BA pool also modulates the colonic Tregs expressing RORγt [135]. Decreased RORγt+Tregs could be observed in the colon while BA metabolic pathways were genetically abolished in individual gut symbionts, whereas rescuing the intestinal BA pool increased colonic RORγ+Treg cells and meanwhile also ameliorated the host susceptibility to colitis. Notably, the stability of the lineage-determining transcription factors RORγ and Foxp3 in Th17 and Treg cells can be regulated by post-transcriptional modifications. In addition, the BA receptor VDR’s activation promotes the induction of Tregs [136] and reduces Th17 cell production [137]. IsoDCA also induces the generation of Foxp3+Tregs after reducing DC stimulatory properties by ablating FXR in DCs [52]. Differentiation of T helper (Th) 17 cells from naïve T cells is related to professional antigen-presenting cells (APCs) and cytokines including IL-6, IL-21 and TGFβ. However, the differentiation of these cells is also affected by gut microbiota metabolites. These Th17 cells produce interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 21 (IL-21) and interleukin 22 (IL-22) [138]. SCFAs. SCFAs are crucial factors of the mucosal immune responses [139]. The gut microbiota can influence the differentiation of Tregs and Th17 cells [140]. The disequilibrium of SCFAs from the gut microbiota can damage the balance of Treg/Th17 [132]. The SCFA butyrate also decreased the proliferation and reduced the cytokine production of Th1, Th17 and Th22 cells [141]. The peroxisome proliferator-activated receptor gamma (PPARγ) and reprogrammed energy metabolism are involved in SCFA-mediated function in these cells [142]. Trp metabolites. Trp metabolites suppress Th1 and Th17 [143]. AhR of Trp metabolites plays a key role in Th17 cell differentiation. Indeed, IAA can decrease Th17 cells through activating AhR, downregulating RORγt and STAT3 (signal transducer and activator of transcription 3) [144]. However, studies also showed that 6-formylindolo(3,2-b) carbazole (FICZ), a Trp product, could promote T cells into Th17 cells [145]. BAs metabolites. Th17 and Treg cell differentiation can be controlled by BA metabolites (BAs) [131]. 3-oxoLCA and isoalloLCA can reduce Th17 cell differentiation and increased Tregs in mice[131]. Th17 cell differentiation can be inhibited by 3-oxoLCA through blocking the function of RORγt [131,146] and directly binding to RORγt [131]. Similar to 3-oxoLCA, isoLCA also suppressed Th17 cell differentiation by inhibiting RORγt [47]. RORγt is selectively expressed by Th17 and innate lymphoid cell group 3 (ILC3). It is a critical for these cells’ differentiation in chronic inflammation and autoimmune diseases [147]. Indeed, RORγt inhibition not only reduces the frequencies of Th17 cells but also provides therapeutic benefits in intestinal inflammation [148]. CD4+Th1 cells are mainly responsible for cell-mediated immunity and produce interferon (IFN)-γ, IL-2 and TNF-α, whereas Th2 cells are involved in antibody production and produce IL-4, IL-5, IL-10 and IL-13 cytokines. Although T-bet and GATA binding protein 3 (GATA3) are master transcription factors for the differentiation of Th1 and Th2 cells, respectively, their differentiation and heterogeneity are usually determined by combinatorial transcription factors. SCFAs. DCs from mice treated with the SCFA propionate have an impaired ability to initiate Th2 cells [149]. These DCs have a reduced expression of CD40, programmed cell death ligand 2 (PD-L2) and CD86. Notably, SCFAs can promote the microbiota’s antigen-specific IL-10 production in Th1 cells through GPR43. Mechanistically, SCFAs upregulate transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1). However, SCFAs also have the potential to induce inflammatory responses [150]. SCFAs can induce Th1 and Th17 cells upon exposure to immunological challenges. A high concentration of butyrate also induces Th1 transcription factor T-bet expression. Trp metabolites. Many patients with cancer often show decreased plasma Trp levels in parallel with an elevated Th1 type immune activation marker. Oral Trp supplementation suppresses antigen-specific Th1 responses at subtoxic concentrations [143]. Through IDO1-mediated Trp catabolism, synovial fibroblasts can also selectively suppress Th1 cell responses [151]. BA metabolites. Upon exposure to BAs, CD4+ T cells can maintain gut homeostasis [152]. Pols et al. revealed that unconjugated LCA inhibited the activation of primary human and mouse CD4+ Th1 cells to reduce TNFα and INFγ production through a BA receptor VDR-dependent mechanism [153]. A shift from Th1 to Th2 cells could be promoted by BA receptor VDR activation through c-Maf and GATA-3 [154]. A decreased number of liver-infiltrating CD4+ Th1 cells is associated with a good response of patients with primary biliary cholangitis to UDCA treatment. In addition, PXR activation also inhibits T cell proliferation in both mouse and human T cells in vitro. However, CXCR5+CD4+ T follicular helper cells could be induced by BA metabolism to cause neuromyelitis optica spectrum disorder [155]. Regulatory B (Breg) cells are immunosuppressive cells that support immunological tolerance. Breg cells have multiple subsets, including immature and mature B cells, which can express IL-10, IL-35 and/or TGF-β and surface molecules such as CD9, CD1d, CD21, CD23, CD24, CD5, CD138, TIM (T cell immunoglobulin and mucin domain-1) and/or PD-L1/L2. In addition, other Breg cell subsets have also been reported such as CD1dhighCD21highCD23+IgMhighIgD− T2 MZ (marginal zone) precursor B cells, CD1dhighCD5+ CD1dhigh CD21highCD23IgMhighIgD−MZ B cells and CD25+CD69+ CD72highCD185−CD196+IgM+IgD+B cells. These cells suppress immunopathology through the production of IL-10, IL-35 and TGF-β cytokines. SCFAs. Rosser and colleagues recently showed that butyrate could divert Trp metabolism toward the serotonin pathway and the production of 5-hydroxyindole-3-acetic acid (5-HIAA) [156]. 5-HIAA activates AhR in Bregs, mediating the suppressive effect in a rheumatoid arthritis model in vivo [156]. The administration of SCFAs also improved rheumatoid arthritis (RA) symptoms and increased the Breg frequency [157]. Trp metabolites. B cell differentiation, maturation and activation can be regulated by the Trp metabolite receptor AhR [158,159]. AhR activation regulates the differentiation and function of IL-10-producing CD19+CD21highCD24highBregs [160]. AhR-deficient mice develop exacerbated arthritis with significant reductions in IL-10-producing Bregs. Our study showed that in the presence of LPS, IAA by gut microbiota could activate the transcription factors PXR, CAR and NF-κB to induce the generation of IL-35+ Breg cells [161]. Others also found that LPS increased the expression of p35 and Ebi3 in B cells isolated from mice [162]. The transcription factor NF-κB promoted influenza A virus (IAV)-mediated IL-35 [163]. B cells play a key role in the responses to microbial infections and pathogen clearance. These B cells not only produce antibodies but also release a broad variety of cytokines. BA-metabolite-mediated VDR activation reduces the ongoing proliferation of B lymphocytes [164], induces activated B cell apoptosis [165] and inhibits Ig production [166]. However, SCFAs can also stimulate glycolysis in B cells via mTOR activation. SCFA-derived acetyl-CoA is crucial for plasma cell differentiation and antibody production [167]. SCFAs also can promote the secretion of IgA by B cells [168]. The activation of Trp metabolism is related to flavivirus-mediating B cell differentiation into antibody-secreting cells in humans [169]. Myeloid-derived suppressor cells (MDSCs) are most commonly immunosuppressive cells during chronic inflammation, especially late-stage cancers. These cells consist of two large groups of cells termed granulocytic or polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. In humans, the total MDSCs are characterized by HLA-DRlow/negLinlow/negCD33posCD11bpos. PMN-MDSCs are identified with negative CD14 or positive CD15, whereas M-MDSCs are identified with positive CD14 or negative CD15 [170]. They use different mechanisms for immunosuppression. PMN-MDSCs mainly suppress T cell responses by producing ROS (reactive oxygen species), whereas M-MDSCs produce high amounts of NO (nitrogen oxide), Arg-1 and immunosuppressive cytokines such as IL-10, which suppress both antigen-specific and non-specific T cell responses [171]. M-MDSCs have higher suppressive activity than G-MDSCs. Taurodeoxycholate (TDCA) can increase the number of PMN-MDSCs in the spleen of septic mice [172]. There are three different groups of innate lymphoid cells (ILCs), namely, ILC1s, ILC2s and ILC3s, but only ILC3s are IL-22 producers [173]. IL-22 is crucial for the maintenance of intestinal epithelial cells (IECs) and the defense against pathogens [174]. It belongs to an IL-10 family cytokine [175]. The gut microbiota has profound effects on the differentiation and functions of ILCs. Trp metabolites. Trp metabolites play a critical role in the development of ILC3s. AhR activation is essential for IL-22 production in ILC3s through AhR ligands from the microbiota [176,177]. Trp metabolites are involved in mucosal immunity through AhR modulation. An impaired AhR activity in AhR knockout mice was related to reducing ILC3 and aggravating inflammatory diseases [23]. The disruption of gut-microbiota-related Trp metabolism results in reduced IL-22 in the intestinal tract, whereas the activation of AhR in ILC3 promotes IL-22 production, thereby modulating the intestinal immune response and protecting the function of the intestinal barrier. AhR also plays an important role in the differentiation of ILC3s [178,179]. Especially in the early stage after birth, AhR ligands are required for the differentiation of IL-22-producing ILC3s [180]. Mechanically, AhR not only participates in Runx3- and RORγt-mediated ILC3 development [181] but also mediates Notch and c-Kit expression [178,182]. Notably, reduced AhR signaling can cause alterations between ILC3 and ILC1 cellular populations. In addition, AhR can also cause IL-22 expression in the Th17 cells [183]. Naïve CD8+ T cells can produce a large number of effector cells to fight infections or tumors after antigen stimulation. SCFAs. The SCFAs butyrate and propionate regulate CD8+ T cell activation via inhibiting IL-12 production in DCs. However, microbiota-derived SCFAs can boost CD8+ T cell functions by modifying the cellular metabolism [184]. The anti-tumor functions of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells can be significantly enhanced by pentanoate and butyrate [185]. Through regulating mTOR activity and cellular metabolism, acetate also promotes IFN-γ production in CD8+ T cells. Trp metabolites. Kyn can upregulate the expression of PD-1 in CD8+T cells through interacting with the ligand-activated AhR [186], which mediates immunosuppressive responses. 3-HAA from the Kyn pathway causes immune suppression by inducing apoptosis in T cells through glutathione depletion [187]. However, Trp metabolites can promote CD8+T cells to induce apoptosis of co-cultured cancer cells, increase cancer-infiltrating CD8+T cells and suppress tumor growth of lung cancer in mice [188]. BA metabolites. BA metabolites can disrupt intracellular calcium homeostasis, which is essential for NFAT (nuclear factor of activated T cells) signaling and T cell activation [189]. 24-Norursodeoxycholic acid (NorUDCA) changes immunometabolism in CD8+ T cells and alleviates hepatic inflammation [190]. It has strong immunomodulatory efficacy in CD8+T cells, which affect lymphoblastogenesis, expansion, glycolysis and target of rapamycin complex 1 (mTORC1) signaling. BA receptor VDR activation also reduces the ongoing proliferation of T lymphocytes [164]. Natural killer (NK) cells, as a first line of defense against cancer, are powerful effectors of innate immunity. These cells can express an array of receptors to eliminate tumor cells. Kyn metabolites, particularly Kyn itself, can suppress the activity of NK cells [191] and cause cell death via a ROS pathway in NK cells [192]. These Kyn metabolites can prevent the cytokine-mediated upregulation of the specific triggering receptors responsible for NK-cell-mediated killing [193]. NKT cells, an unusual population of T cells, can recognize lipids presented by CD1d. Gut-microbiome-mediated BA metabolism regulates liver cancer via NKT cells [194]. CXCL16 expression of liver sinusoidal endothelial cells regulated by BA can control the accumulation of NKT cells [194]. The activation of the BA receptor FXR can result in a profound inhibition to produce a potent pro-inflammatory mediator osteopontin in NKT cells [195]. Neutrophils play a critical role in the host defense against infection. SCFA-mediated activation of GPR43 can induce the neutrophils to inflammatory sites and enhance their phagocytosis [196]. However, pro-inflammatory cytokine production such as TNFα in neutrophils can be inhibited by SCFAs [197]. SCFAs also affect neutrophil-mediated anti-HIV responses [198]. Serum BAs in liver cirrhosis promote neutrophil dysfunction [199]. Sphingosine-1-phosphate receptor (S1PR) can reduce neutrophil aggregation [200]. In addition, the Trp metabolite indole suppresses neutrophil myeloperoxidase to diminish bystander tissue damage [201]. The intestinal epithelium contains a unique population of CD4+CD8αα+ T cells [26]. These CD4+CD8αα+ T cells can promote gut tolerance to dietary antigens [127]. They can be found in the intestine of mice colonized with L. reuteri. Through Trp-metabolite-mediated AhR activation, L. reuteri can reprogram CD4+ T cells into CD4+CD8αα+ cells in the gut [26]. CD4+CD8αα+ IELs can resist apoptosis and upregulate IL-15 and IL-10 in a colitis model [202]. Gut-microbiota-derived SCFAs, Trp and BA metabolites have been widely related with intestinal and extra-intestinal disorders such as inflammatory bowel diseases (IBDs), chronic liver diseases, metabolic syndrome, diabetes and cancer [203,204,205,206]. Gut-microbiota-derived metabolites play a key role in inflammatory bowel disease (IBD) [204]. Metabolite disturbances including BAs and short-chain fatty acids (SCFAs) have been reported in patients with IBDs [204]. Ursodeoxycholic acid reduces the severity of intestinal inflammation in a DSS-induced mouse model of colitis [207]. Longitudinal analyses also demonstrated that certain metabolites such as tryptophan metabolites were decreased in coeliac disease [208]. The indole metabolites are dysregulated in patients with active IBD and in mouse models of colitis, and the restoration of depleted indoles reduces disease severity [209]. The metabolites from the gut microbiota can modulate the development and progression of non-alcoholic fatty liver disease (NAFLD) [210]. Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity [211]. Interventional studies with certain bacterial strains such as Akkermansia muciniphila have shown effects on obesity-related parameters [206]. The tryptophan-derived metabolite IAA induces the generation of IL-35+B cells through PXR and TLR4 to inhibit obesity in mice [161]. Thus, the manipulation of the gut microbiota may impact the immune system and improve immune-mediated disorders. An increasing number of studies has reported the use of fecal microbiota transplantation (FMT) for the treatment of diseases such as metabolic syndrome, diabetes, multiple sclerosis, psoriasis, Crohn’s disease, cancer and Parkinson’s disease [212,213]. Typically, the modulation of the gut microbiota with the FMT method has successfully cured patients with refractory immune-checkpoint-inhibitor-associated colitis [214]. Notably, the composition of the gut microbiota in immunosuppressed patients such as allogeneic hematopoietic-cell transplantation is changed, which is characterized by a loss of diversity and domination by single taxa [215,216]. However, a large body of evidence has also shown that the importance of the intestinal microbiota in immunosuppressed patients. Fecal microbiota transplantation (FMT) in immunocompromised cohorts can provide protection against bacterial translocation via the introduction of a diverse microbiome and restoration of epithelial defenses [217]. Promoting microbial diversity via FMT is also likely to enhance natural barrier defenses, including anti-microbial peptides, tight junction assembly/integrity, mucus production and epithelial proliferation [217]. In addition, exclusive enteral nutrition may also cultivate the presence of beneficial microbiota and improve BA metabolism, possibly influencing disease and immune activity [218]. Several nutritional therapies have been designed not only to treat the nutritional deficiencies seen in children with active Crohn’s disease (CD) but also to correct dysbiosis and reduce intestinal inflammation [219]. Multi-donor FMT with an anti-inflammatory diet effectively induced deep remission in mild–moderate ulcerative colitis [220]. The gut microbiota harbors trillions of microorganisms in the human digestive system. These microorganisms affect the gut and systemic immunity via their metabolites such as SCFAs, Trp and BA metabolites to maintain gut and systemic homeostasis. The alteration of the gut microbiota/metabolites can lead to the onset of many diseases ranging from gastrointestinal and metabolic conditions to neuropsychiatric diseases and cancers. The effects of gut microbiota metabolites on different immune cells have important consequences not only in the onset and development of diseases but also in the diagnosis and therapy of these diseases and predictions of clinical outcomes, prognosis and immunotherapy responses such as cancer immune checkpoint blockade. With the rapid development of recent techniques, more bacterial strains to produce the metabolites (including SCFAs, Trp and BA metabolites) remain to be identified. This will be beneficial for understanding different diseases and designing targeted strategies to control the production of the metabolites for the therapy of these diseases. However, several critical techniques need to be overcome to find more gut-microbiota-derived metabolites that are potentially related to diseases. (1) Discovery of new culture method(s) for gut microbiota. A key question for gut microbiota metabolites is whether gut microorganisms can be successfully cultured in vitro. The discovery of any new culture technique will be beneficial to the identification of gut microbiota metabolites. (2) Improvement of the metabolite analyses. For currently targeted metabolomics, the restricted standard samples have limited application, whereas for untargeted metabolomics, it is easy to produce “false positive” data. (3) Synthesis of gut microbiota metabolites. Some metabolites from the gut microbiota need to be synthesized for their functions and application. (4) Determination of immune cell subset function. With the development of single-cell sequencing techniques, more immune cell subpopulations related to the gut microbiota or metabolites will be identified. However, the functional potential of these immune cell subsets remains to be determined. (5) Establishment of new animal models. Some gut microbiota metabolites may exert their function through new mechanism(s), including receptor, signal pathway, genetic and epigenetic modification and metabolism. All of these need new animal models to explain how the metabolites exert their effects on the immune cells and/or diseases.

PMC10000531

Marcin Czop,Karolina Gasińska,Ewa Kosior-Jarecka,Dominika Wróbel-Dudzińska,Janusz Kocki,Tomasz Żarnowski

Twenty Novel MicroRNAs in the Aqueous Humor of Pseudoexfoliation Glaucoma Patients

24-02-2023

miRNA,aqueous humor,pseudoexfoliation glaucoma,PEXG,glaucoma,pseudoexfoliation syndrome,PEX

The microRNAs (miRNAs) are short non-coding RNAs (19–25 nt) that regulate the level of gene expression at the post-transcriptional stage. Altered miRNAs expression can lead to the development of various diseases, e.g., pseudoexfoliation glaucoma (PEXG). In this study, we assessed the levels of miRNA expression in the aqueous humor of PEXG patients using the expression microarray method. Twenty new miRNA molecules have been selected as having the potential to be associated with the development or progression of PEXG. Ten miRNAs were downregulated in PEXG (hsa-miR-95-5p, hsa-miR-515-3p, hsa-mir-802, hsa-miR-1205, hsa-miR-3660, hsa-mir-3683, hsa -mir-3936, hsa-miR-4774-5p, hsa-miR-6509-3p, hsa-miR-7843-3p) and ten miRNAs were upregulated in PEXG (hsa-miR-202 -3p, hsa-miR-3622a-3p, hsa-mir-4329, hsa-miR-4524a-3p, hsa-miR-4655-5p, hsa-mir-6071, hsa-mir-6723-5p, hsa-miR-6847-5p, hsa-miR-8074, and hsa-miR-8083). Functional analysis and enrichment analysis showed that the mechanisms that can be regulated by these miRNAs are: extracellular matrix (ECM) imbalance, cell apoptosis (possibly retinal ganglion cells (RGCs)), autophagy, and elevated calcium cation levels. Nevertheless, the exact molecular basis of PEXG is unknown and further research is required on this topic.

Twenty Novel MicroRNAs in the Aqueous Humor of Pseudoexfoliation Glaucoma Patients The microRNAs (miRNAs) are short non-coding RNAs (19–25 nt) that regulate the level of gene expression at the post-transcriptional stage. Altered miRNAs expression can lead to the development of various diseases, e.g., pseudoexfoliation glaucoma (PEXG). In this study, we assessed the levels of miRNA expression in the aqueous humor of PEXG patients using the expression microarray method. Twenty new miRNA molecules have been selected as having the potential to be associated with the development or progression of PEXG. Ten miRNAs were downregulated in PEXG (hsa-miR-95-5p, hsa-miR-515-3p, hsa-mir-802, hsa-miR-1205, hsa-miR-3660, hsa-mir-3683, hsa -mir-3936, hsa-miR-4774-5p, hsa-miR-6509-3p, hsa-miR-7843-3p) and ten miRNAs were upregulated in PEXG (hsa-miR-202 -3p, hsa-miR-3622a-3p, hsa-mir-4329, hsa-miR-4524a-3p, hsa-miR-4655-5p, hsa-mir-6071, hsa-mir-6723-5p, hsa-miR-6847-5p, hsa-miR-8074, and hsa-miR-8083). Functional analysis and enrichment analysis showed that the mechanisms that can be regulated by these miRNAs are: extracellular matrix (ECM) imbalance, cell apoptosis (possibly retinal ganglion cells (RGCs)), autophagy, and elevated calcium cation levels. Nevertheless, the exact molecular basis of PEXG is unknown and further research is required on this topic. Glaucoma is a group of multifactorial eye diseases that result in progressive irreversible damage to the optic nerve and cause vision loss, and is considered the second leading cause of blindness in the world [1]. The basic process in the pathophysiology of glaucoma is the loss of retinal ganglion cells (RGCs) and their axons, which leads to changes in the morphology of the optic disc and consequently to visual field defects. The main risk factor for glaucoma is increased intraocular pressure (IOP), caused by an imbalance between the production and outflow of aqueous humor (AH) from the anterior chamber [2]. The most important outflow pathway is trabecular meshwork (TM), which consists of trabecular cells surrounded by the extracellular matrix (ECM). Dysfunction in the normal homeostatic process leads to increased outflow resistance and elevated IOP [3]. Lowering IOP is the only treatment that prevents the progression of vision loss [4]. Pseudoexfoliation glaucoma (PEXG) is a type of secondary open-angle glaucoma, developing usually over 50 years of age in the course of pseudoexfoliation syndrome (PEX). The prognosis is worse than for primary open-angle glaucoma (POAG) due to higher IOP values and greater fluctuations. PEX was first described in 1917 by Finnish ophthalmologist, John Linberg [5,6]. It’s a chronic, age-related systemic disorder, with variable prevalence among men and women in different studies [7,8,9,10,11]. Pathognomonic eye symptoms are gray-white deposits in the anterior segment of the eyeball (corneal endothelium, trabecular meshwork, pupillary margin, iris pigment epithelium, dilator muscle of the iris, iris blood vessels, ciliary epithelium, lens epithelium, lens capsule, zonules) and periorbital tissues (conjunctiva, Tenon’s capsule, orbital septa, extraocular muscles) [12,13,14,15]. PEX material can also be found in many internal organs (heart, lungs, liver, kidneys, gallbladder, blood vessels, cerebral meninges) and skin [5]. The deposits are the result of increased production or reduced turnover of ECM [16]. They are composed of elastic fibers (elastin, tropoelastin, amyloid P, fibrillin-1, fibulin-2, vitronectin, fibronectin, lysyl oxidase, clusterin, LTBP-1, LTBP-2, and other proteins) and noncollagenous basement membrane materials (laminin) which form fibrils. They are coated with glycosaminoglycans (heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronic acid) [17,18]. Mechanisms leading to the development of PEX may include oxidative stress, hypoperfusion, and hypoxia [19]. Previous studies revealed many genes that can be related to PEX (APOE, CACNA1A, CLU, CNTNAP2, FBLN5, GST, LOXL1, MMP1, MMP3, TNF, TMEM136) by participating in the formation of PEX material, regulation of trabecular cells and ECM proteins [18,20,21,22,23]. Gene expression is a complex process in which microRNAs (miRNAs) are involved. Many miRNAs show a tissue-specific expression pattern and can be identified in eye tissues and fluids, but the role of miRNAs in glaucoma and PEXG is not well understood [24,25,26]. MiRNAs are short non-coding RNAs (19–25 nt) that regulate the level of protein expression at the post-transcriptional level. These molecules are responsible for the regulation of many physiological cellular processes, including division, cell differentiation, and regeneration, but also for many pathological processes such as neoplastic transformation. MiRNAs are found both in the cell and in extracellular fluids, i.e., plasma, urine, and aqueous humor [27,28,29,30]. MiRNAs are transcribed in a similar way to protein genes. The first step in the expression of a gene encoding miRNA is the synthesis of primary miRNA (several hundred nucleotides long), which are then modified in the nucleus at both ends of the RNA. The next stage of miRNA maturation takes place in the cytoplasm and, as a consequence, a pre-miRNA of about 70 nucleotides is formed, followed by the production of mature micro RNA. The functioning of micro RNA is closely related to the RNA-induced silencing complex (RISC). Both molecules linked together to interact with the target mRNA through partial complementarity of the sequence of one of the miRNA strands, leading to degradation of the target mRNA through the activity of the RISC complex. By analyzing the level of miRNA expression, it is possible to draw conclusions about the pathomechanism of the studied diseases and to use them as biomarkers of these diseases [27,31,32]. The most important features of biomarkers are: specificity for the disease entity, the possibility of quick, easy, and safe determination, and the possibility of obtaining accurate results [33,34,35,36]. The aim of the study was to assess the levels of miRNA expression in a group of patients with PEXG and to look for mechanisms caused by altered expression of miRNA that may be related to this disease. Eighteen western descent patients underwent routine cataract surgery at the Department of Diagnostics and Microsurgery of Glaucoma, Medical University of Lublin (Poland) (nine PEXG and nine age-matched control patients). Prior to the surgery, all of the patients underwent the ophthalmic examination including best-corrected visual acuity (BCVA) assessed with Snellen charts, an IOP test measured by Goldmann applanation tonometry (GAT), gonioscopy, a slit-lamp examination, indirect ophthalmoscopy after pupil dilation with a stereoscopic optic nerve head assessment, optical coherence tomography (Carl Zeiss Cirrus HD-OCT 5000), and standard automated perimetry (SAP) (24-2 strategy using the Humphrey perimeter). Inclusion criteria in the study group: (1) Incipient senile cataract: cloudy area in the lens that causes decreased vision. (2) PEX syndrome: typical deposits of white powdery material observed along the pupillary margin and on the peripheral lens capsule. (3) Glaucomatous optic neuropathy: elevated IOP of greater than 21 mmHg, documented in medical history; open-angle grade III/IV according to Schaffer’s classification; glaucomatous optic nerve head damage (excavation, neuroretinal rim thinning or notching, and localized or diffuse retinal nerve fiber layer [RNFL] defect); and glaucomatous defect in SAP in at least two consecutive tests, with three reliability indices better than 15% (results were considered abnormal if the Glaucoma Hemifield Test result was outside normal limits and at least three contiguous points were present within the same hemifield on the pattern deviation [PD] plot at p < 1%, with at least 1 point at p < 0.5%). (4) Advanced stages of PEXG: severe visual field loss with MD in visual field test > −18.0 dB. Inclusion criteria in the control group: (1) Incipient senile cataract: cloudy area in the lens that causes decreased vision. (2) No clinical signs of glaucoma, nor PEX. (3) Normal IOP: from 10 mmHg to 21 mmHg. In both groups, other causes of RNFL thinning (i.e., myopia, optic disc anomalies, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, traumatic optic neuropathy, multiple sclerosis, and degenerative diseases such as Alzheimer’s and Parkinson’s disease) were excluded. Moreover, all of the participants had no previous intraocular surgeries, no other systemic or ocular diseases known to affect the visual field (e.g., pituitary lesions, demyelinating diseases, diabetes mellitus, etc.), and no previous eye or head trauma in their medical history. The patients’ demographic and clinical data are presented in Table 1. Approximately 100 µL of AH were obtained from the eye’s anterior chamber at the beginning of the cataract surgery with special care to avoid contamination with blood or tears. The total RNA was isolated from the AH samples using an miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The isolated RNA was stored at −80 °C for further analysis. The RNA concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In addition, RNA analysis was performed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and a Pico RNA Kit according to the manufacturer’s protocol. A microarray system (GeneChip miRNA 4.0 Array chip, Affymetrix, Santa Carla, CA, USA) was used to determine the miRNA expression profiles. The RNA preparation and hybridization were performed according to the manufacturer’s protocol, with one modification to extend the hybridization time to 42 h. The gene chips were scanned with an Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Carla, CA, USA). The raw data, in a CEL format, were analyzed as log2-transformed intensities using the Affymetrix Transcriptome Analysis Console (TAC), following the software’s guidelines to determine differentially expressed genes (DEGs) between the PEXG and control patients. The miRNAs that showed at least a one-fold difference between the control and PEXG groups, with a p-value of less than 0.01, were considered statistically significant and differentially expressed. The functional analysis of selected miRNA was performed using the Diana mirPATH v3.0 (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath (accessed on 2 September 2022)) [37]. The visualization of the regulatory network with obtained interactions was carried out by using Cytoscape v3.9.1 software (https://cytoscape.org (accessed on 2 September 2022)) [38]. The analysis of the interactions of the selected miRNAs with genes was carried out in an R environment (version 4.1.2, https://www.r-project.org (accessed on 1 December 2021)) using the multiMiR 1.16.0 package (https://bioconductor.org/packages/release/bioc/html/multiMiR.html, (accessed on 2 September 2022)) [39]. The enrichment analysis was performed using the mirNET database (https://www.mirnet.ca/miRNet/home.xhtml (accessed on 2 September 2022)), where a list of selected miRNAs and a list of genes with which they interact were introduced [40]. Terms of the Kyoto Encyclopedia of Genes and Genomes (KEGG), REACTOME, and Gene Ontology (GO) categories were searched for in PEX-related pathways. The plot visualizing the enrichment analysis was generated using the ggplot2 3.3.0 package in the R environment. Cytoscape was also used to perform protein–protein interactions (PPI). StringApp v2.0 (https://apps.cytoscape.org/apps/stringapp (accessed on 5 January 2023)) [41] was used for this purpose, which uses the Search Tool for the Retrieval of Interacting Genes (STRING). Molecular Complex Detection v2.0.2 (MCODE) (https://apps.cytoscape.org/apps/mcode (accessed on 7 January 2023)) [42] and cytoHubba v0.1 (https://apps.cytoscape.org/apps/cytohubba (accessed on 7 January 2023)) [43] plugins were then used to create clusters and extracting hub genes in a PPI network. Results at p < 0.05 were considered statistically significant. The following software was used: GraphPad Prism 9 (Graph Pad Software, San Diego, CA, USA) and Statistica 13.3 (StatSoft, Krakow, Poland). The analyzes included the Shapiro–Wilk test (to assess the compliance of the examined variables with the normal distribution), the student’s t- test, and ROC curves (area under the curve (AUC) were calculated) for variables on a quantitative scale (data presented as an average). Data on a qualitative scale are presented as numbers and percentages and were analyzed using the Chi^2 test. Analysis comparing the expression level of miRNAs using expression microarrays in the PEXG group compared to the cataract group showed 20 miRNAs as DEG’s. Ten miRNAs were downregulated in PEXG (hsa-miR-95-5p, hsa-miR-515-3p, hsa-mir-802, hsa-miR-1205, hsa-miR-3660, hsa-mir-3683, hsa -mir-3936, hsa-miR-4774-5p, hsa-miR-6509-3p, and hsa-miR-7843-3p) (Table 2, Figure 1) and ten miRNAs were upregulated in PEXG (hsa-miR-202-3p, hsa-miR-3622a-3p, hsa-mir-4329, hsa-miR-4524a-3p, hsa-miR-4655-5p, hsa-mir-6071, hsa-mir-6723-5p, hsa-miR-6847-5p, hsa-miR-8074, and hsa-miR-8083) (Table 3, Figure 2). In the group of DEG’s miRNAs whose expression was lower in PEXG compared to the group (p < 0.01; AUC > 0.852; p < 0.01), a mean reduction in the level of PEXG expression was found at the level of 29.31% for hsa-miR-95-5p, 36.86% for hsa-miR-515-3p, 29.13% for hsa-mir-802, 28.35% for hsa-miR-1205, 13.15% for hsa-miR-3660, 43.64% for hsa-mir-3683, 42.14% for hsa-mir-3936, 14.35% for hsa-miR-4774-5p, 22.13% for hsa-miR-6509-3p, and 27, 37% for hsa-miR-7843-3p. In the group of DEG’s miRNAs whose expression was higher in PEXG compared to the group (p < 0.01; AUC > 0.840; p < 0.01), a mean increase in the level of PEXG expression was found at the level of 28.73% for hsa-miR-202-3p, 29.65% for hsa-miR-3622a-3p, 20.05% for hsa-mir-4329, 21.64 for hsa-miR-4524a-3p, 34.06% for hsa-miR-4655 -5p, 23.50% for hsa-mir-6071, 24.61% for hsa-mir-6723-5p, 24.30% for hsa-miR-6847-5p, 21.57% for hsa-miR-8074, and 23.06% for hsa-miR-8083. Using the DIANA miRPath v3.0 database, a functional analysis of each of the selected miRNAs was performed (the analysis was performed separately for miRNAs with reduced and increased expression in PEXG). Next, networks were constructed visualizing possible functional relationships between the selected miRNAs (Figure 3 and Figure 4). The functional network for downregulated miRNA functionally linked together nine miRNAs (leaving one type of miRNA without functional linkage—hsa-miR-3683). The terms with the most associations between the selected miRNAs were: “Ion binding”, “organelle”, “biosynthetic process”, “cellular nitrogen compound metabolic process”, “molecular function” and “molecular function”. Functional network for upregulated miRNA functionally linked eight miRNAs (leaving two types of miRNAs (has-miR-6723-5p, and hsa-miR-4655-5p) without functional links between other miRNAs and with each other). The terms that obtained the most connections between the selected miRNAs were: “Ion binding”, “organelle”, “biosynthetic process”, “cellular nitrogen compound metabolic process”, “steroid hormone biosynthesis” and “gene expression”. The next step was to perform an enrichment analysis to evaluate the biological processes regulated by the selected miRNAs and interacted genes using the mirNET database. Five categories were selected: KEGG (Kyoto Encyclopedia of Genes and Genomes), GO: Biological Processing (GO:BP), GO:Cellular Compartment (GO:CC), GO: Molecular Function (GO:MF), and Reactome. Genes indicated as targets of the expressed miRNAs were associated with intracellular and extracellular structures in addition to various processes. The top 10 terms for each category that may be potentially related to glaucoma and/or PEX are presented in Figure 5 and Figure 6, separately for downregulated and upregulated miRNAs in PEXG. Within the group of downregulated miRNAs, the most frequent terms are related to the regulation of transcription and translation, internal cellular transport (“trans-Golgi network transport vesicle”, “endocytosis”), and signal transduction (Figure 5). In the upregulated miRNA group, the most frequently repeated terms are related to the regulation of transcription and translation, the regulation of the cell cycle, and the activity of cations (mainly calcium and zinc) (Figure 6). A PPI network of the genes regulated by selected miRNAs were constructed and clustering analysis was performed based on these PPI networks. In total, six clusters (sub-networks) were created for each PPI network of the genes regulated by downregulated miRNAs (Figure 7, Table 4). Clusters I and IV are functionally related to chromatin organization and epigenetic regulation of gene expression, moreover, the key genes in them are the EZH2 gene (in cluster I) and BAZ1B (in cluster IV). Cluster II is functionally related to nuclear chromosome segregation and cellular response to DNA damage (the key gene: TIMELESS). Cluster III is functionally related to the voltage-gated calcium channel complex and MAPK signaling pathway (the key gene: CACNA2D1). Cluster V is functionally related to protein transport (the key gene: RAB7A). Cluster VI is functionally related to ubiquitinization (the key gene: UBE2D1). In total, six clusters (sub-networks) were created for each PPI network of the genes regulated by upregulated miRNAs (Figure 8, Table 5). Cluster VII is functionally related to cytokine-cytokine receptor interaction and cell–cell adhesion (the key gene: IL10). Cluster VIII is functionally related to the activity of RNA polymerases II and III (the key gene: PLR2D). Cluster IX is functionally related to ubiquitinization (the key gene: PHC1). Cluster X is functionally related to L-type voltage-gated calcium channel complex (the key gene: CACNA1G). Cluster XI is functionally related to the regulation to the jnk cascade (the key gene: AXIN1). Cluster XII is functionally related to Rho GTPases activate ROCKs and phospholipase D signaling pathway (the key gene: RHOA). The hub genes identified using the cytoHubba app included the EZH2, H2AFX in hub I, CACNA1D in hub IIa, and MYC in hub IIb (Figure 9). Enrichment analysis for these networks indicated that hub I is related to generic transcription pathway and cellular responses to stress, hub IIa is related to voltage-gated channel activity and MAPK signaling pathway, hub IIb is related to cellular senescence and Wnt, Hippo, JAK-STAT signaling pathways (Table 6). MiRNA molecules are found both inside the cell and in extracellular fluids. The miRNA in the AH may be derived from blood plasma and may be secreted by intraocular cells. These molecules have been shown to be of great importance in processes such as retinal homeostasis and its development [44,45]. Current knowledge about the pathological mechanisms of PEXG is still unknown. Many studies suggest the role of non-coding RNAs, which, through altered expression levels, may be associated with the development of PEXG. The role of miRNAs [26,46,47,48] and snoRNA [49] is mainly suggested. Current literature suggests a role for certain miRNAs in the pathogenesis of PEXG. These are hsa-miR-671, hsa-miR-374a-5p, hsa-miR-1307-5p, hsa-miR-708-5p [48], and hsa-miR-30d-5p, hsa-miR-320a, hsa- miR-3156-5p, hsa-miR-4458, hsa-miR-6717-5p, hsa-miR-6728-5p, hsa-miR-6834-5p, hsa-miR-6864-5p, hsa-miR-6879- 5p, hsa-miR-877-3p, hsa-miR-548e-3p, and hsa-miR-6777-5p [26]. The main mechanisms associated with the altered expression of these miRNAs is cell apoptosis by suppressing the Wnt3a/β-catenin pathway and the PI3K/AKT pathway [48] and the TGF-beta signaling pathway [26]. In our work, we examined the level of miRNA expression in a group of patients with PEXG and cataracts. After selecting the 20 DEGs of miRNAs (10 upregulated and 10 downregulated), we performed an enrichment and functional analysis. One of the pathological mechanisms related to PEX contains too high of a concentration of calcium cations, which is directly related to the activity of the channels for these cations. Currently, three types of calcium channels are cited, i.e., CACNA1A [21], CACNB3 [46,49,50], and CACNB4 [50]. The calcium channel is composed of subunits, of which the α subunit forms a transmembrane channel, while the remaining subunits (β and α2/δ) perform regulatory functions. Additionally, in some channels, there may be a γ subunit. The calcium channels are divided into three subfamilies, i.e., CaV1, CaV2, and CaV3. The activity of the CaV1 subfamily channels are responsible for the initiation of contraction and regulates gene expression and transmission in synapses. The CaV2 subfamily channels are associated with synaptic transmission, while the CaV3 subfamily channels are associated with the production of action potentials in myocytes and neurons. The CaV1.4 transporter has been shown to be present in the retinal cell membranes of the eye and its activity is related to visual signaling [51,52]. The results of functional analysis in our study showed that hsa-miR-8074 influences the transport of calcium cations across biological membranes through the regulation of the ATP2B2 gene. Moreover, we found that the voltage-gated calcium channel activity is being regulated by the genes targeted by selected miRNAs. Additionally, the Wnt signaling pathway is regulated by the genes targeted by selected miRNAs, where one of the signaling pathways is the Wnt/Ca2 + pathway, the activity of which is related to G proteins and phospholipases. An important regulatory structure of the IOP is HTM (human trabecular meshwork), which is made up of trabecular cells surrounded by the ECM. HTM is responsible for the outflow of AH—in a situation of difficult outflow, the IOP increases [53,54]. The literature provides two pathways (TGFβ1 and TGFβ2), in which the changes lead to an imbalance in the eye’s ECM and, consequently, to an increase in IOP [53,54]. Among the results of the functional analysis in our work, we obtained the term “ECM receptor interactions” for four miRNAs that are regulated by genes: hsa-mir-802 (COL5A1, LAMC1, FN1, CD47), hsa-mir-3660 (LAMA3), hsa-mir -3662a-3p (ITGB8, SV2B, COL24A1, COL4A6, TNR), and hsa-miR-4774-5p (COL11A1, COL24A1). In addition, proteins are a very important element of the ECM. Disruption of protein metabolism (synthesis, post-translational modification, or transport) may lead to the development of PEX [55]. Functional analysis of selected miRNAs showed that a relatively large number of these miRNAs influence genes related to protein metabolism. The process of protein modification in the cell is regulated by genes whose expression is influenced by six out of twenty selected miRNAs (hsa-miR-95-5p, hsa-miR-1205, hsa-miR-3660, hsa-miR-515-3p, and hsa- miR-6071,22-3p). In addition, subsequent miRNAs affect the expression level of genes related to protein metabolism, i.e., “protein complex” (hsa-miR-4329, hsa-miR-6071, hsa-miR-1205, and hsa-miR-3660), “cytoskeletar protein binding” (hsa-miR-6847-5p, 1205), “post-translational protein modification” (hsa-miR-7843-3p), “endoplasmic reticulum-Golgi intermediate compartment” (has-miR-802), and “cytoplasmic sequestering of protein “(Hsa-miR-4655-5p). Moreover, hsa-miR6723-5p regulates the expression level of the UBE2H gene related to ubiquitin-mediated proteolis. Among the enriched functional terms of genes targeted by selected miRNAs in AH in PEXG patients, it is worth mentioning the “trans-Golgi network transport vesicle” as a process that influences protein transport. In addition, many times the results of this analysis were terms related to the process of protein biosynthesis (“positive regulation of translation”, “regulation of translation”) and transcription (“negative regulation of transcription from RNA polymerase II promoter”, “negative regulation of transcription, DNA- dependent “). In a previous work, we suggested that some snoRNA molecules are related to PEXG [49]. One of the terms obtained in enrichment analysis is “spliceosomal complex” which may suggest that the selected miRNAs may also be responsible for this process. The mechanisms discussed above largely explained where the elevated IOP comes from. Elevated IOP results in RGC apoptosis and, furthermore, optic nerve degeneration which is often observed in late PEXG [56,57,58]. Another mechanism that can lead to IOP increase and cell death in glaucoma is oxidative stress [59,60,61,62]. The hsa-miR-802 molecule by regulating the expression of MCL1, PPP2CB, and SOD2 genes influences the processes taking place in the mitochondria during apoptosis. Among the enriched functional terms of genes targeted by selected miRNAs in AH in PEXG patients, it is worth mentioning “G0 and early G1” and “G2/M transition of the mitotic cell cycle”. In addition, “AKT phosphorylates targets in the cytosol” and “PI3K/AKT activation” are terms that suggest that selected miRNAs may regulate the survival of ocular neurons and thus may lead to optic nerve degeneration. The role of the PI3K/Akt pathway was previously mentioned [63,64,65,66,67]. Hsa-miR-802 shows an increased expression in primary human umbilical vein endothelial cells (HUVECs) in response to induced hypoxia [67]. As above, hsa-miR-202-3p is one of the miRNAs whose expression levels have been altered in lung adenocarcinoma A549 cells by hypoxia [68]. Additionally, a correlation between the risk of developing normal-tension glaucoma (NTG) and the level of retinol in the blood serum was demonstrated [69]. Moreover, another group of scientists reports that retinoic acid (a retinol derivative) lowers the expression level of the gene which codes the small heat shock protein B8 (HSPB8) [70], which is a chaperone protein functionally related to chaperone-assisted selective autophagy (CASA) [71]. One of the miRNA molecules selected as DEGs in our work, hsa-miR-6509-3p regulates the expression level of the HSPB8 gene. The above data may suggest a potential influence of the autophagy process in the development of PEXG. The results of the PPI analysis suggest and confirm that a very important mechanism of PEXG may be disturbances in the transport of calcium cations in the eye. One of the hub genes that have been selected is the CACNA1D gene. Our research had some limitations. First, the study groups were small, consisting of nine patients each. A study involving a larger group of patients is needed to confirm the results. Furthermore, PEXG patients had advanced neuropathy, which suggests that miRNA molecules may be the result of the pathological process and not a causative factor. However, we precluded that the same stage of the disease within the group would enable more unique results. Since the advanced stages of PEXG, comparison of miRNAs in the early stages of PEXG is needed. In addition, all enrolled patients were diagnosed with senile cataracts. Therefore, the impact of cataracts on the expression of miRNA in the PEXG group should be taken into consideration, but in the studied group all patients also had similar stages of cataracts. However, it is ethically impossible to collect aqueous humor in healthy eyes due to the invasive nature of the procedure. Until now, the scientific literature has not provided such information, which means that this is the first time we are doing it. These mechanisms are possible, but their role in the pathogenesis of PEXG/PEX must be carefully studied. Our results suggest 20 new miRNAs whose altered expression in the aqueous humor may have an influence on the pathomechanisms of PEXG. Functional and enrichment analysis confirms the mechanisms, such as high levels of calcium cations, ECM imbalance, apoptosis of RGC cells, and the optic nerve damage or autophagy as possible causes of PEXG. Moreover, the PPI analysis suggests that CACNA1D, EZH2, and MYC may be hub genes for PEXG. However, confirmation of these conclusions requires further study.

PMC10000532

Yao Song,Lei Wang,Kaidong Wang,Yuhua Lu,Pengcheng Zhou

COL12A1 Acts as a Novel Prognosis Biomarker and Activates Cancer-Associated Fibroblasts in Pancreatic Cancer through Bioinformatics and Experimental Validation

26-02-2023

pancreatic cancer,cancer-associated fibroblasts,COL12A1,TCGA,prognosis

Simple Summary Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers. The cancer-promoting effect was reversed with COL12A1 knockdown. These findings indicate that COL12A1 acts as a novel prognosis biomarker and provides new opportunities for TME-targeted therapies in pancreatic cancer. Abstract Pancreatic cancer remains one of the most challenging malignancies to date and is associated with poor survival. Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Herein, we report our discoveries in this research area. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. Survival and COX regression analyses revealed the significant clinical prognostic value of COL12A1 expression in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. This was verified with our PCR analysis in cancer cells and CAFs. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers actin alpha 2 (ACTA2), fibroblast activation protein (FAP), and fibroblast-specific protein 1 (FSP1). Meanwhile, the interleukin 6 (IL6), CXC chemokine Ligand-5 (CXCL5), and CXC chemokine Ligand-10 (CXCL10) expressions were inhibited, and the cancer-promoting effect was reversed by COL12A1 knockdown. Therefore, we demonstrated the potential prognostic and target therapy value of COL12A1 expression in pancreatic cancer and elucidated the molecular mechanism underlying its role in CAFs. The findings of this study might provide new opportunities for TME-targeted therapies in pancreatic cancer.

COL12A1 Acts as a Novel Prognosis Biomarker and Activates Cancer-Associated Fibroblasts in Pancreatic Cancer through Bioinformatics and Experimental Validation Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers. The cancer-promoting effect was reversed with COL12A1 knockdown. These findings indicate that COL12A1 acts as a novel prognosis biomarker and provides new opportunities for TME-targeted therapies in pancreatic cancer. Pancreatic cancer remains one of the most challenging malignancies to date and is associated with poor survival. Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Herein, we report our discoveries in this research area. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. Survival and COX regression analyses revealed the significant clinical prognostic value of COL12A1 expression in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. This was verified with our PCR analysis in cancer cells and CAFs. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers actin alpha 2 (ACTA2), fibroblast activation protein (FAP), and fibroblast-specific protein 1 (FSP1). Meanwhile, the interleukin 6 (IL6), CXC chemokine Ligand-5 (CXCL5), and CXC chemokine Ligand-10 (CXCL10) expressions were inhibited, and the cancer-promoting effect was reversed by COL12A1 knockdown. Therefore, we demonstrated the potential prognostic and target therapy value of COL12A1 expression in pancreatic cancer and elucidated the molecular mechanism underlying its role in CAFs. The findings of this study might provide new opportunities for TME-targeted therapies in pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is difficult to treat and has a 5-year survival rate of less than 5% [1]. As the early diagnosis of pancreatic cancer is difficult, 80% of patients are diagnosed in the advanced stage of the disease or have locally invasive tumors. Only 20% of patients have the opportunity for curative resection. These patients have an overall 5-year survival rate of 10–25% [2,3]. Moreover, local recurrence or distant metastasis is common, even in patients who have undergone surgery. In such cases, adjuvant therapy consisting of chemotherapy and/or radiation therapy is necessary. However, the outcomes of combined chemotherapy and radiotherapy remain poor [4]. Therefore, the identification of molecular prognostic biomarkers and targeted molecular therapies are essential to improve the outcomes of PDAC patients. The extracellular matrix (ECM) performs a crucial role in tumor progression invasion and chemo-resistance in pancreatic cancer [5]. The most abundant protein component in the ECM is collagen, which can directly bind to the cancer cell receptors discoidin domain receptor 1 (DDR1) and discoidin domain receptor 2 (DDR2), regulate immune cell infiltration, and TGFβ expression indirectly associated with the cancer cell to induce tumor growth and metastasis [6,7]. Thirty-two types of collagens have been identified in ECM [8]. The different types of collagens expressed in PDAC have critical roles in cancer genesis and progression. Even some types of collagens have a prognosis value for PDAC patients as confirmed with serum, tissues, or bioinformatic analyses [9,10,11,12]. COL12A1, a member of the major collagen family of Fibril-Associated Collagens (FACIT) collagens, assumes a key role in tumor growth [6]. High COL12A1 expression has been correlated with poor survival and cancer metastasis in gastric cancer and colon cancer [13,14]. Bioinformatic analysis indicated COL12A1 has a prognosis effect in pancreatic cancer [15,16,17,18], but the mechanism underlying the effect of COL12A1 in PDAC progression is still unelucidated. In this study, using the Gene Expression Omnibus (GEO) dataset, we found the pathway of differentially expressed genes (DEGs) that mainly focuses on the ECM receptor interaction and collagen catabolic process. The collagen family was focused on finding the key genes associated with pancreatic cancer progression. Analysis of the TCGA dataset implied that COL12A1 has an important role in pancreatic cancer prognosis. CAFs that expressed COL12A1 make a crucial contribution to PDAC genesis and progression. This was explored with bioinformatic analysis and validated using in vitro and in vivo experiments. The findings of this study might provide new opportunities for TME-targeted therapies in pancreatic cancer. RNA sequencing and the related clinical data of TCGA and Genotype-Tissue Expression (GTEx) were downloaded from the UCSC XENA website (https://xenabrowser.net/datapages/, accessed on 5 December 2021). GSE16515, GSE15471, GSE60979, GSE62452, GSE71989, and GSE91035 gene expression profiles were retrieved from the GEO database (https://www.ncbi.nlm.nih.gov/geo/, accessed on 5 December 2021) in microarray platform (GLP570). These data were analyzed using the Affymetrix Human Genome U133 Plus 2.0 Array (transcript (gene) version; Santa Clara, CA, USA). After downloading the datasets for GSE16515, GSE15471, GSE60979, GSE62452, GSE71989, and GSE91035, the GEO2R online tool was used to identify DEGs. Tumor and normal tissues were selected to evaluate gene expression. The threshold value for the screening of DEGs was p < 0.05 and |log fold-change| > 1. The Online Venn diagram tool was used to visualize the DEGs in the six data sets. The DAVID online tool (http://david.ncifcrf.gov/, version 6.8, accessed on 19 December 2019) [19] was used to check gene function (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO describes genes in terms of their biological process (BP), molecular function (MF), and cellular component (CC) [20]. The KEGG pathway was used to check the indicated genes, including their reference pathways [21]. p < 0.05 was considered to indicate statistical significance. GEPIA (http://gepia.cancer-pku.cn/detail.php, accessed on 19 December 2019) is an interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and GTEx projects. The GEPIA expression module was used to visualize mRNA expression in TCGA combined with GTEx. The Oncomine database, the largest oncogene chip database, was used to demonstrate the COL12A1 mRNA expression level difference between tumors and normal tissues. Additionally, we used the R package “ggplot2” to explore the relationship between COL12A1 mRNA expression and clinical parameters in TCGA. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset was used for COL12A1 protein expression analysis. The Human Protein Atlas (HPA) database (http://v13.proteinatlas.org/, accessed on 19 December 2019) is designed to map all the human proteins in cells, tissues, and cancers. It was used to demonstrate the differential expression of COL12A1 protein using immunohistochemistry staining in normal and cancer tissues. Tumor Immune Single-cell Hub (TISCH) is a scRNA-seq database (http://tisch.comp-genomics.org/home/, accessed on 21 May 2022) focusing on TME. In the “dataset” and “gene” modules, we visualized the expression levels of COL12A1 at the single-cell level in the pancreatic cancer RA001160 and GSE111672 datasets. We evaluated the association of COL12A1 expression with overall survival (OS) using the GEPIA survival module. In addition, the R packages “survminer” and “survival” were used to visualize the COL12A1 expression, Disease Specific Survival (DSS), and Progress Free Interval (PFI). The log-rank test was used to compare differences in survival between the low and high levels of COL12A1 groups using the R package “ggrisk”. The R package “timeROC” was used to compare the predictive accuracy of COL12A1 mRNA. We established a nomogram combining COL12A1 expression and key clinical factors to predict the 1-, 3-, and 5-year survival of pancreatic cancer patients using the R packages “rms” and “survival”. Additionally, we conducted a calibration analysis to check the nomogram. Mutation analysis was performed on the cBioportal online web (http://www.cbioportal.org, accessed on 21 May 2022). Gene methylation analysis was performed on GSCA: Gene Set Cancer Analysis online web (http://bioinfo.life.hust.edu.cn/GSCA/, accessed on 21 May 2022) and UALCAN. To explore the abnormal changes in downstream pathways caused by the enhanced expression of COL12A1, we identified DEGs between pancreatic cancer samples with COL12A1 high and low mRNA expression based on the TCGA data using the R packages “DESeq2” and “ggplot2”. To further clarify the potential mechanisms of COL12A1 in pancreatic cancer progression, GO and KEGG enrichment was performed to predict the functions and pathways of the COL12A1-related DEGs using the R package “clusterProfiler”. In addition, we analyzed some important pathways involved in cancer using the R package “GSVA”, choosing parameter as method = ‘ssGSEA’. The correlation between genes and pathway scores was analyzed using Spearman correlation. Tumor Immune Estimation Resource (TIMER) is a comprehensive resource for the systematical analysis of immune infiltrates across diverse cancer types (http://timer.comp-genomics.org/, accessed on 1 April 2022). CAFs play a key role in the development and maintenance of the stromal cancer compartment, mediating an increase in the synthesis of the extracellular matrix [22]. To explore the correlation between COL12A1 expression and cancer-associated fibroblast (CAF) infiltrates, we applied the immune gene module and selected the CAFs for analysis. The correlation between COL12A1 expression and the cell markers of CAFs was also analyzed. PANC-1, BxPC-3, CFPAC-1, PATU-8988, ASPC-1, and MIA PaCa-2 were obtained from Procell. PANC-1, PATU-8988, and HPDE6-C7 were cultured with DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). ASPC-1 was cultured with RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). CFPAC-1 was cultured with IMDM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). MIA PaCa-2 cells were cultured with DMEM (Gibco) supplemented with 10% FBS (Gibco), 5% HS (Gibco), and 1% penicillin-streptomycin (Gibco). To measure COL12A1 expression, 31 pairs of fresh pancreatic cancer tissues and matched para-cancer tissues were obtained between November 2019 and November 2021 at the Affiliated Hospital of Nantong University. The study protocol was approved by the Human Ethics Review Committees of Affiliated Hospital of Nantong University (approval no. 2019-L034). CAFs were isolated from fresh pancreatic cancer tissues following the method described by Bachem et al. [17]. Pancreatic cancer tissues were obtained surgically and cut into small pieces and cultured in DMEM (Gibco) plus 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) in T25 flasks. The medium was changed every three days. After 7–10 days, CAFs migrated out of the tissues. The cells were maintained in a humidified incubator at 37 °C in an atmosphere of 5% CO2. All resected tissues were postoperatively diagnosed with pancreatic cancer. All patients provided written informed consent, and the Ethics Committee of the Affiliated Hospital of Nantong University approved this study. CAFs were identified by the detection of the CAF-specific markers ACTA2, FAP, and FSP1 with immunofluorescence. CAFs at the logarithmic growth phase were digested and seeded into a 6-well plate. When cell confluence reached 60–70%, CAFs were transfected with siRNA NC and siRNA COL12A1 (GCAAUAAACACCUUCCCUUTT) using Lipofectamine 2000 reagents. Total RNA was extracted from cells or tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted RNA was reverse transcribed into complementary DNA (cDNA) following the instructions of the Reverse Transcription Kit (Takara, Kusatsu, Japan). Thereafter, the cDNA was subjected to real-time PCR using the SYBR Green PCR kit (Takara, Kusatsu, Japan) and the Step One instrument (Applied Biosystems, Carlsbad, CA, USA). The primers used in the study were provided in Table 1. For the EdU assay, CAFs were seeded into a 24-well plate. Each well was incubated with EdU medium for 3 h and then fixed with 4% paraformaldehyde. The cells were further incubated with EdU regent in the dark for 30 min. Finally, cells were incubated with Hoechst 3334 for 10 min. Cells stained with EdU and with Hoechst 33342 were counted. EdU positive rate (%) = the number of EdU cells/the number of total cells × 100%. For the wound healing assay, CAFs were seeded in 12-well plates. When the confluent monolayer was formed, the cells were starched with a sterile 200 µLpipette tip to create a wound gap. The medium was replaced with an FBS-free medium and cultured for another 24 h. For the transwell assay, an 8 μm transwell chamber (Corning, kennebunk, ME, USA) was used. A CAF suspension (100 µL, 4 × 104 cells) was added to the upper chamber and a medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C for 18 h, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. Eight fields of view were randomly selected, and the cell number was counted under the microscope. For the clone formation assay, the conditional medium of CAFs transfected with siNC and siCOL12A1 was collected, respectively. Pancreatic cancer cells PANC-1 were seeded into 12-well plates at a density of 500 cells/well with different conditional medium. Finally, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The number of colonies in each well was counted. For western blotting, total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (SolarBio Science & Technology Co., Ltd., Beijing, China). Protein concentration was quantified using a bicinchoninic acid (BCA) kit (Beyotime Biotechnology, Nantong, China). The protein was separated using polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes using the wet transfer method. The membrane was blocked with NcmBlot blocking buffer (New cell & Molecular Biotech Co., Ltd., Suzhou, China) for 10 min and incubated with primary antibodies against ACTA2 (1:1000, Servicebio, Wuhan, China), FAP (1:500, Beyotime Biotechnology, Nantong, China), and FSP (1:1000, Peoteintech, Wuhan, China) at 4 °C overnight. Next, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:10,000, Biosharp, Hefei, China) at room temperature for 1 h and then developed. For immunofluorescence, cells were seeded onto slides and fixed with 4% paraformaldehyde. Then, they were incubated with primary antibody against ACTA2 at 4 °C overnight. Next, cells were incubated with a secondary antibody goat anti-rabbit Alexa Fluor 488 IgG (1:200, Servicebio, Wuhan, China) at room temperature for 1 h in the dark. Finally, the cells were stained with Hoechst 33342. The slides were visualized under a confocal microscope. We performed univariate Cox proportional hazard analysis to identify hub genes significantly related to patient survival (p < 0.05). Genes that significantly correlated with patient survival in univariate analysis were included in multivariate Cox regression analysis. All data were statistically analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA), and all experiments were independently repeated at least thrice. Measurement data were expressed as mean ± standard deviation (SD). Two groups of data were compared using an independent sample test-test. A value of p < 0.05 was regarded as statistically significant. Six datasets, namely GSE16515, GSE15471, GSE60979, GSE62452, GSE71989, and GSE91035, were obtained from the National Center for Biotechnology Information GEO database, which contains data for pancreatic cancer and normal tissue samples. A total of 1769 DEGs in GSE15471, 1277 DEGs in GSE16515, 2029 DEGs in GSE60979, 294 DEGs in GSE62452, 1966 DEGs in GSE71989, and 3020 DEGs in GSE91035 were identified using the criteria p < 0.05 and |log fold-change| > 1 (Figure 1A,B). The Venn diagram shows that 123 genes were regulated (Figure 1B). The DAVID online tool was used to analyze the biological function of overlapping DEGs. GO in terms of BP, CC, and MF of overlapping DEGs among the regulated genes were analyzed. The most extensive BP enrichment was observed during extracellular matrix organization and collagen fibril organization; CC enrichment was the highest in the extracellular space and extracellular region; MF enrichment was the highest in extracellular matrix structural constituents and calcium ion binding. The investigation of the signaling pathway of the overlapping DEGs revealed that the protein digestion, absorption, and ECM-receptor interaction were the most important KEGG pathways (Figure 1C–F). We collected the genes common for DEGs and collagen family genes. The Venn diagram shows that six collagen genes, COL1A1, COL3A1, COL5A2, COL8A1, COL10A1, and COL12A1, were identified between 123 DEGs and 32 collagen genes (Figure 2A). The TCGA and GETx datasets indicated that COL1A1, COL3A1, COL5A2, COL8A1, COL10A1, and COL12A1 genes were expressed much higher in tumor tissues than in the normal ones (Figure 2B). The survival rate was much worse for patients with high COL12A1 expression than that in patients with low expression. However, this is not the case for COL1A1, COL3A1, COL5A2, COL8A1, and COL10A1 (Figure 2C and Figure S1). The Oncomine online tool indicated a higher COL12A1 expression in tumor tissues than that in normal tissues (Figure 3A). COL12A1 expression was much higher in cancer tissue than in para-cancer tissue as indicated by the qPCR results (Figure 3B). CPTAC and HPA indicated that the protein level of COL12A1 was significantly higher in tumor tissues than in normal tissues (Figure 3C,D). Much higher COL12A1 expression was observed in stages III/IV than in stages I/II (Figure 4A). The expression of COL12A1 was significantly associated with the T stage, higher in T3/4 than that in T1/2 (Figure 4B), but not in the N and M stages (Figure 4C,D). In histologic grades, II/III/IV, much higher COL12A1 expression was observed than that in grade I (Figure 4E) (histologic grade in TCGA means the numeric value to express the degree of abnormality of cancer cells; it is a measure of differentiation and aggressiveness). High COL12A1 expression was positively correlated with a worse prognosis (Figure 5A). The COL12A1 expression was much higher in dead patients than in alive people (Figure 5B). The ROC curve indicated that COL12A1 might be a predictor for the survival rate of pancreatic cancer patients. The area under the curve was 0.579 for 1-year survival, 0.603 for 2-year survival, and 0.669 for 3-year survival (Figure 5C). Moreover, based on the TCGA dataset, the progression-free interval (PFS) was much worse for patients with high COL12A1 expression than that for those with low expression (HR = 1.54, 1.04–2.29, p = 0.031) (Figure 5D). The disease-specific survival (DSS) was much longer in low-COL12A1 expression patients than that in high-expression ones (HR = 1.98, 1.23–3.19, p = 0.005) (Figure 5E). Univariable and multivariable cox regression analyses showed that the COL12A1 expression level was an independent determinant to predict the outcome of pancreatic cancer patients. (Figure 5F,G). COL12A1 expression was combined with age, gender, and the T and N stages to build a nomogram for OS prediction (Figure 6A). The nomogram is predictive of the OS for pancreatic cancer patients and demonstrates comparatively high accuracy, as shown by the calibration curves (Figure 6B). We investigated the complex molecular properties of COL12A1 in PDAC tissues. The TCGA dataset was used to analyze genetic alterations, which were found to be 1.6% for COL12A1 in PDAC tissues. The COL12A1 methylation was negatively correlated to the COL12A1 mRNA expression in PDAC patients. However, the expression of methylated COL12A1 expression was much higher in tumor tissue than in normal tissue in the pancreas. In the pancreatic cancer tissues with P53 and KRAS mutation, COL12A1 expression is much higher than that in the wild-type pancreatic cancer tissues, as shown in Figure S2A–F. TISCH checking indicated that COL12A1 is primarily expressed in cancer-associated fibroblasts but not in tumor cancer cells or other immune cells in TME for pancreatic cancer (Figure 7A). The expression of COL12A1 was significantly higher in CAF than in tumor cells as evidenced using qPCR (Figure 7B). The different methods indicated COL12A1 was crucially related to cancer-associated fibroblast infiltration. Furthermore, COL12A1 expression correlated with the expression of the genes associated with fibroblast activation (Figure 7C–E). COL12A1 co-expression networks were studied using the TCGA database to verify the potential function of COL12A1 in tumor tissues. A total of 616 genes were significantly upregulated to COL12A1, and 1586 genes were significantly downregulated to COL12A1 (Figure 8A). LRRC53, TCP11X2, FGL1, C6orf58, and CALY represented the genes downregulated with COL12A1 expression. Conversely, EPYC, MAB21L2, CASP14, COL11A1, and APELA represented the genes upregulated with COL12A1 expression (Figure S3A,B). The most enriched BP, CC, and MF were the extracellular matrix structural constituents, collagen-containing extracellular matrix, and extracellular matrix organization. The investigation of the signaling pathway of the overlapping DEGs revealed that the PI3K/AKT pathway was the most important KEGG pathway (Figure 8B,C). Further, the pathway correlation results indicated that COL12A1 expression correlated with collagen formation, ECM-related genes, the TGF-β pathway, and the inflammation signature (Figure 8D). Finally, CO112A1 expression was positively correlated with 27 out of 41 chemokines (Figure 8E). We used a small interfering RNA (siRNA) to knock down COL12A1 in CAFs and explored the potential bio-function. COL12A1 mRNA expression was significantly downregulated in CAFs transfected with siCOL12A1 compared with siNC (Figure 9A). The findings from the EDU assay indicated that COL12A1 knockdown inhibited the proliferation ability of CAFs (Figure 9B). Transwell migration and wound healing activities were compromised after COL12A1 knockdown (Figure 9C,D). In addition, the immunofluorescence intensity of ACTA2 and the number of ACTA2 fibers were reduced after COL12A1 knockdown in CAFs (Figure 9E). The western blot demonstrated that COL12A1 knockdown can decrease the protein level of ACTA2, FAP, and FSP (Figure 9F). Moreover, we observed a decrease in IL6, CXCL5, and CXCL10 expression in siCOL12A1 CAFs compared to that that in the siNC groups (Figure 9G). Importantly, the culture medium of CAFs treated with siNC or siCOL12A1 was collected. The two kinds of conditional medium were added into pancreatic cancer cell PANC-1. The colony formation assay results implied that COL12A1 knockdown reversed the promoting effect of CAF on PANC-1 cells (Figure 9H). The occurrence and development of the solid tumor are accompanied by the connective tissue hyperplasia reaction and the deposition and remodeling of the tumor matrix, which can lead to significant changes in the tumor microenvironment [23,24]. The change in cell polarity and loosening of the adhesion between cells in the tumor environment occurred through alterations in the composition and accumulation mode of the extracellular matrix, resulting in the promotion of tumor growth, invasion, and metastasis [25]. Abnormal extracellular matrix (ECM), as a major component of PDAC stroma, regulated malignant cell behavior, and induced tumor formation and progression [5]. In this study, 123 DEGs were selected in pancreatic cancer from 6 GEO datasets. For enrichment analysis of the overlapping 123 DEGs, the extracellular matrix structural constituent exhibited the most enrichment for molecular function, the extracellular matrix organization was one of the most enriched of the biological processes, and the extracellular space and the extracellular region experienced the highest enrichment for the cellular component. ECM-receptor interaction was one of the most important KEGG pathways for pathway signaling. These results indicated that the change in the ECM played a crucial role in pancreatic cancer genesis and development. Collagen, as the main component of the extracellular matrix, performs some key functions in the development of tumors [26]. Different collagen proteins played different roles in the occurrence and development of pancreatic cancer. Some can promote tumor metastasis, while others can inhibit tumor growth [9,12,27,28,29]. Hence, we hypothesized that certain collagens perform important roles in the occurrence and development of pancreatic cancer. We crossed the DEGs with 32 genes in the collagen family. Six collagen family genes were used for prognostic analysis in pancreatic cancer tissue. Only the COL12A1 gene was found to be notably related to the survival and prognosis of pancreatic cancer, but the others had no significant association, as shown in Figure 1. Therefore, we focused on COL12A1 to explore its mechanism in the occurrence and development of pancreatic cancer. Thirty-one pairs of clinical specimens were used to verify that COL12A1 expression in pancreatic cancer tissue was significantly higher than that in para-cancerous tissue. The result indicated COL12A1 might be a diagnosis and prognosis biomarker in pancreatic cancer. Several published papers have already indicated that COL12A1 might be the key prognosis biomarker for pancreatic cancer. Ding and Chen et al. indicated that MMP14 and COL12A1 constituted the potential combination of prognostic biomarkers in pancreatic cancer based on bioinformatics analysis. Performing bioinformatic analysis, Jing and Chen indicated that COL12A1 was the potential prognosis biomarker in pancreatic cancer [14,15,16,17]. However, the aforementioned results are primarily based on large databases and are thus lacking in vitro and in vivo findings or real-world data. Simultaneously, the source and mechanism of COL12A1 secretion are not evident. Therefore, using bioinformatic analysis and experimental verification, we further explored the source of COL12A1 and investigated the mechanism of its involvement in tumor genesis and development. CAFs are the main contributor to tumor fibrosis, through increasing the synthesis of ECM proteins such as collagens and cross-linking enzymes, which could create a tumorigenic fibrotic environment around the PDAC tumor. Tumor fibrosis was closely associated with pancreatic cancer progression and drug resistance [30,31,32]. In the current study, the single-cell analysis revealed that COL12A1 was mainly distributed in CAF cells but not in tumor cells and other adjacent cells. Furthermore, the results from our cell line analysis indicated that the COL12A1 expression in the extracted para-cancerous fibroblasts was significantly higher than that in pancreatic cancer cells. CIBERSORT, xCELL, TIDE, and EPIC analysis for the dataset indicated that COL12A1 was correlated with CAF infiltration. These results revealed that COL12A1 was mainly derived from CAFs in pancreatic cancer. This finding is consistent with the previously published results related to breast and colon cancers [33,34], which indicated that COL12A1 is mainly expressed in CAF cells. COL12A1 secreted by CAF can change type I collagen tissue, support the pre-invasion microenvironment of metastatic transmission, and promote organ regeneration. Furthermore, we verified that COL12A1 expression could induce CAF cells to express the fibro-activated proteins ACTA2, FAP, and FSP, enhancing the cell invasion and release of inflammatory factors. Simultaneously, COL12A1 could promote tumor cell growth in a paracrine manner. A substantial amount of data has indicated that CAFs act not only as bystanders but are actively involved in the process of cancer initiation, progression, and metastasis. Hence, the CAFs can be considered as the tumor promoter in pancreatic cancer [35,36]. The most commonly exploited CAF biomarkers in PDAC are ACTA2, FAP, vimentin, FSP1, podoplanin (PDPN/gp38), and platelet-derived growth factor receptor alpha and/or beta (PDGFRa/b) [37]. Our results also indicated that the expression of COL12A1 positively correlated with PDGFRB, ACTA2, S100A4, VIM, and FAP expressions at the mRNA level in the TCGA dataset. The CAFs in pancreatic cancer demonstrated heterogeneity for three types: myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs). These three types can be separated by the biomarkers of myCAF, which exhibits the high expression of ACTA2, FAP, and TGF-β, and low expression of IL6, which requires direct interaction with cancer cells in PDAC and might be associated with tumor progression. ICAF, with low ACTA2 expression and high IL6 secretion, can be activated by paracrine factors secreted from tumor cells. However, the location of ICAF was found to be far from the tumor cells and myCAF. Moreover, apCAFs, expressed by MHC class II family genes, exhibit an antioxidant response. The activation and location of the apCAFs need further exploration to be fully understood [35,38,39,40]. Several studies have indicated that the TGF-β pathway can induce fibroblast transformation into myCAF and inhibit iCAF transformation. The findings of our study involving bioinformatic analysis indicated that COL12A1 expression was positively associated with the TGF-B pathway. The knockdown of COL12A1 inhibited ACTA2, FAP, and FSP1 expressions, which might indicate that COL12A1 expression is associated with myCAF transformation and activation. In the CAF subtype analysis, FAP + CAF could induce PDAC progression and indicate worse survival. However, the function of aSMA + CAF was controversial. Athleen. et al. used single-cell RNA sequencing and several genetic mouse models to show that the depletion of FAP + CAF leads to increased survival, but depleting αSMA + CAFs resulted in decreased survival. However, depleting IL6 in a-SMA + CAF could increase gemcitabine sensitivity for the PDAC mice through T cell regulation [41]. Furthermore, Yurina et al., using 215 under-treatment PDAC patient samples, reported that aSMA-dominant and FAP-dominant fibroblast-rich stroma indicated poor prognosis [42]. Sun et al. used in vitro and in vivo experimental results to indicate that CXCR2/CXCL3 enhanced PDAC metastasis by inducing CAF toward myoCAF transformation and upregulated α-SMA expression [43]. Herein, we also found that COL12A1 knockdown can suppress CAF invasion and inhibit CAF biomarker aSMA and FAP expressions and cytokine IL6, chemokine CXCL5, and CXCL10 expression. The bioinformatics analysis also indicated that COL12A1 expression was highly associated with cytokine and chemokine expressions, such as IL6, IL8, CXCL5, and CXCL10 expressions. Furthermore, the colony formation of tumor cells can be suppressed when cultured in a conditional medium, which was collected from siCOL12A1 CAFs but not in the one collected from the group with siNC CAFs. The aforementioned result indicated that CAF expresses COL12A1 and could enhance tumor growth in a paracrine manner through increasing cytokine or chemokine secretion. The above results indicated that COL12A1 might provide new opportunities for TME-targeted therapies in pancreatic cancer. However, the exact mechanism of how COL12A1 effect CAF transformation as well as which subtype CAF was changed and induced tumor growth still not fully elucidated. The key factor that caused the COL12A1-induced tumor growth needs further exploration. Despite many useful inputs, there are several limitations in our research. First, the CAF collected herein were combined with myCAF, iCAF, and apCAF, which did not separate. In further studies, we need to investigate which part of CAFs expressed COL12A1, and thus plays an important role in tumor growth. Second, the prognosis value and pathways were primarily considered by the dataset but not our own clinical samples. Hence, it is possible that in a further study, we need to check OS for our clinical sample for further validation of the results. In this research, we systematically analyzed the mechanism involved in using COL12A1 as a therapy target and prognosis biomarker for pancreatic cancer. Our study demonstrated the potential diagnostic and prognostic value of COL12A1 expression in pancreatic cancer and elucidated the possible molecular mechanism underlying its role in promoting the development of pancreatic cancer in CAFs. These findings indicate that COL12A1 acts as a novel prognosis biomarker and provides new opportunities for TME-targeted therapies in pancreatic cancer. Our study demonstrated the potential diagnostic and prognostic value of COL12A1 expression in pancreatic cancer and elucidated the molecular mechanism underlying its role in CAFs and promoting the development of pancreatic cancer. These findings might provide new opportunities for TME-targeted therapies in pancreatic cancer.

PMC10000543

Zhen Chen,Han Bao,Jingfang Long,Peiqi Zhao,Xiaowei Hu,Hao Wang,Ying Zhang,Jianjing Yang,Qichuan Zhuge,Lei Xia

GBE1 Promotes Glioma Progression by Enhancing Aerobic Glycolysis through Inhibition of FBP1

03-03-2023

glucan branching enzyme 1,Warburg effect,glucose metabolism,fructose-bisphosphatase 1,NF-κB

Simple Summary Due to the poor prognosis of glioma patients and the limitations of glioma treatment, our study aimed to find new targets for glioma on metabolic therapy. Our study reveals a role for glycogen branching enzyme 1 (GBE1) in regulating glioma initiation and progression. We found that the expression of GBE1 correlated with a poor prognosis in glioma patients. Moreover, GBE1 promotes glioma progression by enhancing aerobic glycolysis through the inhibition of fructose–bisphosphatase 1 (FBP1), which reveals GBE1 as a potential target for glioma therapy. Abstract Tumor metabolism characterized by aerobic glycolysis makes the Warburg effect a unique target for tumor therapy. Recent studies have found that glycogen branching enzyme 1 (GBE1) is involved in cancer progression. However, the study of GBE1 in gliomas is limited. We determined by bioinformatics analysis that GBE1 expression is elevated in gliomas and correlates with poor prognoses. In vitro experiments showed that GBE1 knockdown slows glioma cell proliferation, inhibits multiple biological behaviors, and alters glioma cell glycolytic capacity. Furthermore, GBE1 knockdown resulted in the inhibition of the NF-κB pathway as well as elevated expression of fructose-bisphosphatase 1 (FBP1). Further knockdown of elevated FBP1 reversed the inhibitory effect of GBE1 knockdown, restoring glycolytic reserve capacity. Furthermore, GBE1 knockdown suppressed xenograft tumor formation in vivo and conferred a significant survival benefit. Collectively, GBE1 reduces FBP1 expression through the NF-κB pathway, shifting the glucose metabolism pattern of glioma cells to glycolysis and enhancing the Warburg effect to drive glioma progression. These results suggest that GBE1 can be a novel target for glioma in metabolic therapy.

GBE1 Promotes Glioma Progression by Enhancing Aerobic Glycolysis through Inhibition of FBP1 Due to the poor prognosis of glioma patients and the limitations of glioma treatment, our study aimed to find new targets for glioma on metabolic therapy. Our study reveals a role for glycogen branching enzyme 1 (GBE1) in regulating glioma initiation and progression. We found that the expression of GBE1 correlated with a poor prognosis in glioma patients. Moreover, GBE1 promotes glioma progression by enhancing aerobic glycolysis through the inhibition of fructose–bisphosphatase 1 (FBP1), which reveals GBE1 as a potential target for glioma therapy. Tumor metabolism characterized by aerobic glycolysis makes the Warburg effect a unique target for tumor therapy. Recent studies have found that glycogen branching enzyme 1 (GBE1) is involved in cancer progression. However, the study of GBE1 in gliomas is limited. We determined by bioinformatics analysis that GBE1 expression is elevated in gliomas and correlates with poor prognoses. In vitro experiments showed that GBE1 knockdown slows glioma cell proliferation, inhibits multiple biological behaviors, and alters glioma cell glycolytic capacity. Furthermore, GBE1 knockdown resulted in the inhibition of the NF-κB pathway as well as elevated expression of fructose-bisphosphatase 1 (FBP1). Further knockdown of elevated FBP1 reversed the inhibitory effect of GBE1 knockdown, restoring glycolytic reserve capacity. Furthermore, GBE1 knockdown suppressed xenograft tumor formation in vivo and conferred a significant survival benefit. Collectively, GBE1 reduces FBP1 expression through the NF-κB pathway, shifting the glucose metabolism pattern of glioma cells to glycolysis and enhancing the Warburg effect to drive glioma progression. These results suggest that GBE1 can be a novel target for glioma in metabolic therapy. Gliomas are the most common malignant tumors in the central nervous system and are divided into circumscribed gliomas and diffuse gliomas, according to WHO CNS5 in 2021 [1,2]. Glioblastoma (GBM), the most common and fatal diffuse glioma, accounts for 57.3% of gliomas [3]. Treatment of GBM is often unsatisfactory due to the limited extent of surgical resection and the presence of the blood-brain barrier (BBB) and the complex tumor microenvironment, with a median survival of fewer than two years and only 6.9% of patients who survive more than five years after diagnosis [4,5]. Therefore, studying the pathogenesis of glioma and finding the factors driving tumorigenesis and progression are essential for treating glioma. The Warburg effect endows tumor cells with the ability to use aerobic glycolysis to meet their high–metabolite needs, but it also makes tumor metabolism a unique target for targeted therapy. Studies have found that the Warburg effect promotes tumor progression in multiple ways, including by reducing toxic metabolites [6,7], methylating tumor suppressor genes [8,9], and inhibiting immune responses [10,11,12]. Thus, targeting tumor metabolism may inhibit tumor progression from multiple aspects. A recent study found that inhibition of the basic leucine zipper and W2 domain 1 (BZW1) suppresses pancreatic cancer cell proliferation by inhibiting glycolysis under oxygen and glucose deprivation conditions [13]. Ovo Like Zinc Finger 2 (OVOL2), a transcription factor, inhibits the Warburg effect and breast cancer progression by suppressing the expression of glycolytic genes [14]. Moreover, in ovarian cancer, fibrillin-1 (FBN1) knockdown enhances cisplatin sensitivity by inhibiting glycolysis and angiogenesis [15]. Furthermore, the disruption of glycolysis in gliomas suppressed intracranial tumors and prolonged the median survival time of mice [16]. The loss of function of glycogen branching enzymes (GBE1) is the cause of glycogen metabolic disorders such as Glycogen Storage Disease IV (GSD–IV) and Adult Polyglucosan Body Disease (APBD) [17,18]. However, an increasing number of studies have shown its relevance to cancer. On the one hand, GBE1 is highly expressed in acute myeloid leukemia (AML) and maintains abnormal tumor cell proliferation by inhibiting AMPK activity [19]. GBE1 expression is also elevated in lung adenocarcinomas and is associated with worse survival [20]. On the other hand, GBE1 expression is decreased in ovarian cancer, and GBE1 downregulation is correlated with poor clinical outcomes [21]. These studies demonstrate that GBE1 plays different roles in different tumors. However, there has not been enough significant research on GBE1 in gliomas. Herein, we evaluated GBE1’s role in gliomas, confirming that the expression of GBE1 is elevated in gliomas and correlates with a poor prognosis. We then demonstrated through cellular and animal experiments that GBE1 influences FBP1 expression through the NF-κB pathway, which affects the glucose metabolism pattern of glioma cells and promotes the Warburg effect to drive tumor progression. This study provides a potential target for glioma metabolic therapy. Human glioma cell lines U87, ln229, and U251; HEK–293t engineered cells, as well as human umbilical vein endothelial cells (HUVECs), were purchased from the Shanghai Institute of Biosciences and Cell Resources Center (Chinese Academy of Sciences, Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, C11995500BT, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco, 16000044, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, 15070063, Waltham, MA, USA) in an incubator at 37 °C, 5% CO2. Plasmid construction and lentiviral transfection were performed as previously described [22,23]. Oligonucleotides targeting the following mRNA sequences were synthesized by Sangon Biotech (Shanghai, China) (sh-GBE1-1: AAAGGTAGTTATTACTAGTAAA, sh-GBE1-2: TTCGCTACAAGTTCCTAAATAA, and sh-FBP1: TACCAACGTGACAGGTGATCAA) and integrated into a lentiviral vector expressing mCherry fluorescent protein. oe-FBP1 and oe-NC plasmids were provided by Youze Bio (Guangzhou, China). Recombinant plasmids (sh-GBE1, sh-FBP1, and oe-FBP1) and empty plasmids (sh-NC and oe-FBP1) were co-transfected with packaging plasmids (pRSV-Rev, pMDLg pRRE, and VSV-G) into HEK-293t cells to produce lentiviral particles. Glioma cells were infected with the supernatant containing lentiviral particles for 24 h. Infected cells were screened by the BD FACSAria cell sorter (BD, Franklin Lakes, NJ, USA) according to the expression of mCherry. The total RNA of glioma cells was extracted by Trizol (Thermo Scientific, 15596018, Waltham, MA, USA), and then the cDNA libraries were constructed using the RevertAid RT reverse transcription kit (Thermo Scientific, k1691, Waltham, MA, USA). qRT-PCR was performed with the following program for 40 cycles: 95 °C/15 s, 60 °C/15 s, and 72 °C/45 s. The cycle threshold (CT) value of the target RNA was normalized to that of GAPDH. The relative expression was finally calculated by the 2-△△CT method. The qRT-PCR primer sequences were as follows: GBE1-Forward: 5′-GGACTTCCAGCGCAGGTATAA-3′, GBE1-Reverse: 5′-ATCAGCACATCTGTGGACGC-3′, FBP1-Forward: 5′-CCTACTGCCCTCTCTTGCCG-3′, FBP1-Reverse: 5′-CCATGACGAAGCGGGTCAG-3′, GAPDH-Forward: 5′-TGACATCAAGAAGGTGGTGAAGCAG-3′, GAPDH-Reverse: 5′-GTGTCGCTGTTGAAGTCAGAGGAG-3′. The total protein of glioma cells was extracted with RIPA lysis solution (Thermo Scientific, 89900, Waltham, MA, USA) containing a protease phosphatase inhibitor cocktail (Beyotime, p1045, Shanghai, China) and phenylmethanesulfonyl fluoride (PMSF, Beyotime, p1045, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Thermo Scientific, 23227, Waltham, MA, USA). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF, Merck Millipore, IPFL85R, Darmstadt, Germany). They were then blocked with a TBST (TBS, 0.1% Tween) solution containing 5% skimmed milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Finally, they were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. Protein bands were developed using an ECL luminescence reagent (Meilunbio, MA0186, Dalian, China). Details of the antibodies are as follows: Anti-GBE1 (Abcam, ab180596, 1:1000, Cambridge, UK), Anti-FBP1 (Affinity, DF7325, 1:1000, Suyang, China), Anti-HIF1α (CST, 36169, 1:1000, Boston, MA, USA), Anti-MMP9 (Affinity, BF0560, 1:1000, Suyang, China), Anti-VEGFA (Abcam, ab46154, 1:1000, Cambridge, UK), Anti-Bcl-2 (Affinity, AF6139, 1:1000, Suyang, China), Anti-Bax (Affinity, AF0120, 1:1000, Suyang, China), Anti-Caspase-3 (Abcam, ab32351, 1:1000, Cambridge, UK), Anti-Cyclin D1 (Affinity, AF0931, 1:1000, Suyang, China), Anti-Cyclin D1 (Affinity, AF0931, 1:1000, Suyang, China), Anti-p65 (Affinity, AF5006, 1:1000, Suyang, China), Anti-Phospho-p65 (Affinity, AF2006, 1:1000, Suyang, China), Anti-GAPDH (Affinity, AF7021, 1:1000, Suyang, China), Anti-beta-Tubulin (Abcam, ab78078, 1:1000, Cambridge, UK), Goat Anti-Mouse IgG (Biosharp, BL001A, 1:5000, Hefei, China), Goat Anti-Rabbit IgG (Biosharp, BL003A, 1:5000, Hefei, China). Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, MCE, HY-K0301, Monmouth, NJ, USA). Briefly, a CCK-8 reagent was added to the culture medium according to the instructions, then placed in an incubator at 37 °C with 5% CO2 in the dark for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader. U87 cells (1000 per well), ln229 cells (2000 per well), and U251 cells (2000 per well) were seeded in 6-cm dishes and then cultured in an incubator at 37 °C with 5% CO2. A fresh complete medium was replaced every 3 days. After 14 days of culture, cells were fixed with 4% paraformaldehyde (Solarbio, p1110, Beijing, China) and stained with 2.5% crystal violet (Meilunbio, MA0148, Dalian, China). Pictures were finally taken with a digital camera by a researcher who was blinded to the group allocation to calculate the colony formation rate. Colony formation rate = amount of colonies/number of seeded cells. The cell cycle was detected using the DNA content quantification assay (Solarbio, CA1510, Beijing, China). Briefly, cells were fixed with 75% alcohol overnight at 4 °C after digestion with trypsin (Gibco, 25200072, Waltham, MA, USA). RNase A was used the next day to remove RNA. Then PI staining solution was added, and the cells were incubated for 30 min in the dark at 4 °C. The cell cycle was finally detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA). Apoptosis was detected using an Annexin V-FITC apoptosis detection kit (Solarbio, CA1020, Beijing, China). Briefly, cells were digested with an EDTA-free trypsin (Gibco, 15050057, Waltham, MA, USA) and resuspended in a 1× binding buffer. FITC-labeled anti-Annexin V was added for 5 min at room temperature in the dark. Finally, the PI staining solution was added, and cell apoptosis was detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA). Once 90% cell confluence was reached, a wound was created with a 200 μL pipette tip, and floating cells were washed. Fresh serum-free DMEM was added, and three locations were randomly selected and photographed under a microscope (Olympus, Tokyo, Japan) to record the initial wound area. The cells were then incubated at 37 °C with 5% CO2 to continue the culture. Photographs were taken at the same location every 12 h, and the area of scratch reduction was counted as the wound healing area. All the measurements were performed by a researcher who was blinded to group allocation. Transwell chambers (Corning, 3422, New York, NY, USA) were used to further assess cell migration and invasion capabilities. For the migration assay, after 24 h of serum-free starvation, cells were resuspended in serum-free DMEM and seeded at a density of 2 × 105/mL in the upper chamber and in the lower chambers with a complete medium containing 10% FBS. The chambers were then placed in a 37 °C, 5% CO2 incubator for another 24 h. The transwell chambers were coated with Matrigel (Corning, 354234, New York, NY, USA) for the invasion assay. After cells were serum-free starved for 24 h, they were resuspended in serum-free DMEM and seeded at a density of 4 × 105/mL in the upper chamber and the lower chamber with a complete medium containing 10% FBS. The chambers were then placed in a 37 °C, 5% CO2 incubator for 48 h. Cells were fixed with 4% paraformaldehyde after culture, stained with crystal violet after scratching off the upper chamber cells, and finally photographed for counting under a microscope (Olympus, Japan, Tokyo, Japan) by a blinded researcher. Glioma cells (1 × 104 per well) were seeded in 96 well plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. The entire fresh medium was replaced before hypoxia. The plates were then incubated at 37 °C in a hypoxia incubator (5% CO2, 95% N2) for another 24 h, and cell proliferation was detected by CCK-8 reagent every 8 h. U87 (1 × 107 per dish), LN229 (5 × 106 per dish), and U251 (5 × 106 per dish) cells were seeded in 10 cm dishes. After 24 h of normal culture, cells were changed to serum-free DMEM and placed in a 37 °C hypoxia incubator (5% CO2, 95% N2) for another 24 h. The supernatant was collected and purified as conditioned medium (CdM). GFP-labeled HUVEC cells (8 × 104 per well) were seeded in Matrigel-coated 96-well plates after resuspension with CdM. The cells were then placed in an incubator at 37 °C with 5% CO2 for 12 h, and pictures were taken under a fluorescence microscope (Leica, Wetzlar, Germany) every 3 h to record tubule formation. All measurements and observations of tubule formation assays were performed by an investigator blinded to group allocation. For immunofluorescence staining, glioma cells were blocked with 5% bovine serum albumin (BSA, Beyotime, ST023, Shanghai, China) in PBST solution (PBS, 0.4% Triton) for 1 h at room temperature after fixation with 4% paraformaldehyde. The primary antibodies were then added for overnight incubation at 4 °C. The next day, cells were incubated with fluorescence-conjugated secondary antibodies for 1 h at room temperature, and finally, the nuclei were stained with a DAPI staining solution (Solarbio, S2110, Beijing, China). Images were acquired using a fluorescence microscope (Leica, Wetzlar, Germany) by a blinded researcher. For immunohistochemical staining, tumor and adjacent tissues from three glioblastoma patients were collected at the Department of Neurosurgery in the First Affiliated Hospital of Wenzhou Medical University. Tissue sections were dewaxed and hydrated and then soaked in 3% H2O2 for 10 min to remove endogenous catalase. The antigen was then repaired with a citrate-EDTA antigen recovery solution (Beyotime, P0086, Shanghai, China). Tissue sections were then blocked with 5% BSA in PBST for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following day, tissue sections were incubated with HRP-labeled secondary antibodies at 37 °C for 1 h and then developed with the DAB Color Development Kit (Beyotime, P0202, Shanghai, China) for 5 min. Finally, the nuclei were stained with hematoxylin (Beyotime, C0107, Shanghai, China). All measurements and observations were performed by a blinded investigator. The collection of human specimens was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics number: KY2021-R129). The antibody details are as follows: Anti-KI67 (Abcam, ab16667, 1:1000, Cambridge, UK), Anti-GBE1 (Abcam, ab180596, 1:1000, Cambridge, UK), Dylight 488, Donkey anti-rabbit IgG (EarthOx, E032221, 1:500, Millbrae, CA, USA), Goat anti-rabbit IgG (Biosharp, BL003A, 1:5000, Hefei, China). Glioma cells (2 × 104 per well) were seeded in Seahorse XF 96-well culture plates (Agilent, 102601, Santa Clara, CA, USA) and incubated at 37 °C in a 5% CO2 incubator for 24 h. For the glycolytic stress test, cells were changed to detection medium (XF base medium, 2 mM glutamine) the next day, and the change in extracellular acidification rate (ECAR) after the sequential addition of glucose, oligomycin, and 2-Deoxyglucose (2-DG) was detected with the Seahorse XF Pro analyzer (Agilent, Santa Clara, CA, USA). For the mitochondrial stress test, cells were changed to detection medium (XF base medium, 1 mM sodium pyruvate, 2 mM glutamine, and 10 mM glucose) the next day. Then, the change in cellular oxygen consumption rate (OCR) after the sequential addition of oligomycin, FCCP, and rotenone/antimycin A was detected with a Seahorse XF Pro analyzer (Agilent, Santa Clara, CA, USA). A total of 10 BALB-c/nude male mice aged 4 to 6 weeks were purchased from the Shanghai Charles River Experimental Animal Limited Liability Company (Shanghai, China) and housed under specific pathogen-free conditions (SPF) (20–23 °C, 55–60% humidity) at the laboratory animal center, the First Affiliated Hospital of Wenzhou Medical University. Lentivirus (sh-NC or sh-GBE1) infected U87 cells carrying luciferase reporters were prepared. Nude mice were sequentially numbered and randomly divided into two groups (n = 5). Then, 5 × 105 cells were transplanted into the striatum of nude mice according to the mouse brain anatomical atlas (https://atlas.brain-map.org, accessed on 5 May 2022). The optical density values of xenografts were measured every seven days using the IVIS in vivo optical imaging system (PerkinElmer, Waltham, MA, USA). The natural death time of nude mice was recorded for survival analysis. All measurements and observations were performed by a blinded researcher. All animal experiments were approved by the animal ethics committee of Wenzhou Medical University (Ethics number: WYYY-2021–0214). We performed differential expression analysis and Kaplan–Meier survival analysis of GBE1 by gene expression profiling interactive analysis (GEPIA). To further analyze the association of GBE1 expression with glioma grade, IDH mutation, and 1p/19q co-deletion, we obtained RNA-seq and clinical data from 584 glioma patients from the Cancer Genome Atlas (TCGA), including 441 low-grade gliomas (LGG) and 143 glioblastomas. RNA expression profiles were presented as fragments per kilobase of exon model per million mapped fragments (FPKM). Moreover, we obtained the data of 686 glioma patients from the Chinese glioma Genome Atlas (CGGA) and did the same analysis for further demonstration. Finally, based on the TCGA database, ROC curves were generated according to the expression of GBE1 in LGG and GBM. All statistical analyses of this study were performed using GraphPad Prism 9. Student’s t-test was used to determine whether differences between two data groups conformed to the normal distribution, and variance analysis was used to compare data among multiple groups. The homogeneity of variance was tested by the Leneve test. Kaplan–Meier survival curves were compared using the Log-rank (Mantel–Cox) test. Data are presented as mean ± standard deviation (SD), and a two-tailed p-value < 0.05 was considered statistically significant. GBE1 was overexpressed in a variety of tumors. To investigate GBE1 expression in gliomas, we performed a differential expression analysis through the GEPIA database (gepia2.cancer-pku.cn) and found that its expression was significantly higher in both LGG and GBM samples compared to normal samples (Figure 1A). Additionally, the overall survival and disease-free survival of patients in the GBE1 high-expression group were significantly lower than those in the low-expression group (Figure 1B,C). The mRNA expression profiles and clinical information of 584 glioma samples from the TCGA database and 686 glioma samples from the CGGA database revealed a significant positive correlation between GBE1 expression and WHO grade of gliomas (Figure 1D,G). Further, we subdivided the glioma samples according to IDH mutation status and 1p/19q deletion status and found higher GBE1 expression in IDH wild-type gliomas compared to IDH mutant ones (Figure 1E,H). As well as the higher expression of GBE1 in intact 1p/19q samples compared to co-deleted samples (Figure 1F,I). Furthermore, the results of IHC staining of tumor tissues and adjacent normal tissues from three glioma patients confirmed the higher expression of GBE1 in glioma samples (Figure 1J,K). These results suggested that GBE1 expression parallels the malignancy of gliomas. Subsequently, we evaluated the performance of GBE1 in distinguishing glioma grades by ROC curve. Its area under the curve (AUC) was 89.15% compared to 74.21% of KI67 (Figure 1L), indicating that GBE1 can be considered a discriminator of glioma malignancy. Bioinformatics analysis showed a strong association of GBE1 with glioma. To further investigate the role of GBE1 in gliomas, stable GBE1 knockdown and negative control U87, ln229, and U251 cells were constructed by sh-GBE1-1, sh-GBE1-2, and sh-NC lentiviruses, and the knockdown efficiency was verified by qPCR and WB (Figure 2A–C). Since sh-GBE1-1 and sh-GBE1-2 exhibited almost identical knockdown efficiencies, we selected sh-GBE1-1 for subsequent experiments. The cell counting kit-8 assay was then used to detect cell proliferation. The results showed that the proliferation of all three glioma cell lines was inhibited in the GBE1 knockdown group compared with the negative control (Figure 2D). Moreover, cell cycle analysis showed that GBE1 knockdown caused a decrease in cells at the G2/M phase while causing a significant increase in cells at the G0/G1 phase (Figure 2E,F), indicating that GBE1 knockdown arrested the cell cycle at the G0/G1 phase. Furthermore, the results of apoptosis analysis showed that GBE1 knockdown induced apoptosis in all three glioma cell lines (Figure 2G,H). These results indicate that GBE1 knockdown inhibits glioma cell proliferation, and this kind of inhibition results from cell cycle arrest coupled with increased apoptosis. To investigate whether GBE1 knockdown has additional effects on glioma, we assessed the alterations of glioma cells in biological behaviors such as migration, invasion, colony formation, and angiogenesis after GBE1 knockdown. First, a wound-healing assay was performed to assess the migration ability of glioma cells. Since the U87 cells grow in a grid-like pattern and it is difficult to form a dense cell monolayer, U251 cells and LN229 cells were selected for this assay. We found that the healing rate of ln229 and U251 cells was slowed after GBE1 knockdown compared with the negative control (Figure 3A,B). Since the wound healing assay cannot evaluate tumor invasion, a more comprehensive detection of invasion and migration is necessary. Therefore, we performed transwell assays using Matrigel to mimic the extracellular matrix. The results showed that the number of all three glioma cells that successfully crossed the transwell membrane was significantly decreased after GBE1 knockdown compared with the negative control, indicating that both the migration and invasion abilities of glioma cells were inhibited after GBE1 knockdown (Figure 3C,D). Furthermore, the colony formation assay results showed that the colony formation ratios of all three glioma cell lines decreased significantly after GBE1 knockdown compared to the negative control (Figure 3E,F). Moreover, real-time recorded images of the tube formation assay (Figure 3G) showed that conditioned medium (CdM) from GBE1 knockdown glioma cells made HUVECs form fewer tubules compared with the negative control, indicating that GBE1 knockdown impaired the ability of glioma cells to promote angiogenesis (Figure 3H). GBE1 is involved in multiple biological behaviors of glioma cells, including migration, invasion, colony formation, and promotion of angiogenesis, and knockdown of GBE1 will impair these biological behaviors. KI67 is strongly downregulated in resting G0 cells [24], making it a classic indicator of cell proliferation. The immunofluorescence staining of KI67 (Figure 4A) showed that after GBE1 knockdown, the resting cells with low KI67 expression in all three glioma cell lines increased significantly (Figure 4B), which was consistent with the results of cell cycle analysis. Since U251 cells exhibited more conspicuous and stable effects in most of the phenotypic experiments, including the CCK-8 assay, apoptosis detection, wound healing assay, and transwell invasion assay, this suggests that U251 cells were more vulnerable to GBE1 in cell proliferation, apoptosis, migration, invasion, and other classical phenotypes. Hence, we opted for U251 cells to further validate the expression of these phenotype-related proteins. We extracted the total protein of the GBE1 knockdown group and the negative control for western blot analysis. Cyclin D1 is a cell cycle-related protein whose reduced expression will lead to cell cycle arrest in the G1 phase [25,26]. Compared to the negative control, the expression of cyclin D1 was significantly reduced after GBE1 knockdown, which further proved that GBE1 knockdown could transform glioma cells into a resting state and inhibit proliferation (Figure 4C,D). The WB results also showed that GBE1 knockdown caused the downregulation of the invasion-related protein MMP9, the angiogenesis-related protein HIF1α, VEGFa, and the apoptosis inhibitor protein Bcl-2, and increased the expression of Bax and Cleaved-Caspase-3, further confirming the node role of GBE1 in the regulation of the biological behavior of glioma (Figure 4C,D). GBE1 was found to cause promoter methylation of the FBP1 gene in lung adenocarcinoma cells through the NF-κB pathway, which decreased the expression of the FBP1 protein [27]. In glioma, we also observed that knockdown of GBE1 significantly inhibited the phosphorylation level of p65 protein, accompanied by elevated FBP1 expression (Figure 4E,F). To further investigate the relationship between GBE1, the NF-κB pathway, and FBP1, an NF-κB inhibitor was used to treat U251 cells to mimic NF-κB inhibition caused by GBE1 knockdown, and the expression of FBP1 after treatment was examined. QNZ, a neuroprotective inhibitor of the SOC channel, strongly inhibits NF-κB transcriptional activation [28]. The results of WB showed that QNZ (1 μM) decreased the level of phosphorylated p65 protein in U251 cells, while the total amount of p65 remained unchanged. And FBP1 levels were significantly elevated after QNZ treatment compared to vehicle control (Figure 4E,F), which paralleled the effect of GBE1 knockdown, indicating that the conclusion that GBE1 regulates FBP1 expression through the NF-κB pathway is also applicable in gliomas. As a tumor suppressor, FBP1 plays a critical role in the progression of multiple tumors [29,30,31]. Given that previous results showed GBE1 negatively regulated FBP1 expression in U251 cells (Figure 4E,F), to further verify the role of FBP1 in gliomas, we constructed U251 cells with FBP1 knockdown (sh-FBP1) and FBP1 overexpression (oe-FBP1). The knockdown and overexpression efficiencies were detected by Western blot (Figure 5A,B). The results of the CCK-8 assay showed that FBP1 knockdown accelerated U251 cell proliferation, while FBP1 overexpression inhibited the proliferation of U251 cells (Figure 5C). Additionally, wound healing assays showed that, compared to the sh-NC group, FBP1 knockdown significantly accelerated U251 cell migration and nearly closed the wound after 48 h migration. Conversely, compared to the oe-NC group, FBP1 overexpression significantly slowed the wound healing rate (Figure 5D,E). Further, transwell assays also showed that FBP1 knockdown significantly increased the number of cells that crossed the transwell membrane, enhancing U251 cell migration and invasion abilities, while FBP1 overexpression inhibited U251 cell migration and invasion (Figure 5F,G). These results indicate that FBP1 is an unfavorable factor for glioma cell proliferation, migration, and invasion. Increasing FBP1 expression helps to suppress these malignant phenotypes in glioma cells. To investigate whether GBE1 regulates glioma progression through FBP1, we constructed U87 and U251 cell lines in which both GBE1 and FBP1 were knocked down and observed whether the inhibition of glioma by single GBE1 knockdown could be reversed. The knockdown efficiency was verified by qPCR and WB, which showed that in the GBE1 and FBP1 knockdown group of U251 cells, the mRNA and protein content of FBP1 returned to the levels of the negative control (Figure 6A,B). In U87 cells, it was even lower than the negative control (Supplementary Figure S1A,B). The results of the CCK-8 assay showed significantly enhanced cell viability in the group with knockdowns of both GBE1 and FBP1 compared with the single GBE1 knockdown group, even exceeding the negative control (Figure 6C and Supplementary Figure S1C). Meanwhile, the results of cell cycle analysis and apoptosis analysis revealed that the knockdown of FBP1 could reverse the cell cycle arrest and apoptosis caused by GBE1 knockdown, indicating that FBP1 knockdown significantly promoted tumor proliferation, demonstrating the powerful tumor suppressive effect of FBP1 (Figure 6D–G, Supplementary Figure S1D–G). Further studies revealed that glioma cells with knockdowns of both GBE1 and FBP1 showed a significant rebound in migration, invasion, vascularization, and colony formation ability compared with single GBE1 knockdowns (Figure 6H–M, Supplementary Figure S1H–M). Furthermore, the results of WB from U251 cells showed that changes in proteins related to tumor biological behaviors caused by single GBE1 knockdown were also reversed after FBP1 knockdown, which was paralleled to the alterations in tumor phenotypes (Figure 6N,O). These results suggest that the glioma suppressive effect caused by GBE1 knockdown is associated with increased expression of FBP1, and that knockdown of FBP1 will greatly impair this effect. To further investigate why FBP1 inhibited glioma progression, considering the important role of FBP1 in glucose metabolism, we speculated that the glucose metabolic system of glioma cells was affected after GBE1 knockdown, which in turn inhibited glioma progression. Herein, we examined the extracellular acidification rate (ECAR), which reflects the glycolysis level of cells, using the Agilent Seahorse cell metabolic analysis system. The results showed that the basal glycolysis level and the glycolytic reserve capacity of U87 and U251 cells were inhibited after GBE1 knockdown. However, the knockdown of FBP1 reversed the inhibition of glycolysis caused by GBE1 knockdown, indicating that the increased expression of FBP1 is an important reason for the impaired glycolysis of glioma cells caused by GBE1 knockdown (Figure 7A,B). In addition, to evaluate the respiratory capacity of mitochondria, we also detected the cellular oxygen consumption rate (OCR). The results showed that GBE1 knockdown increased the basal respiration level and spare respiration capacity of mitochondria in both glioma cell lines. However, FBP1 knockdown had a limited effect on mitochondrial respiratory function, suggesting that other substances in addition to FBP1 are involved in the elevated mitochondrial respiration caused by GBE1 knockdown (Figure 7C,D). To further verify the alteration of the metabolic pattern of glioma cells caused by GBE1 knockdown, we performed oxygen deprivation assays in U87 and U251 cells. According to the results of the CCK-8 assay, after 24 h of hypoxia, the viability of U87 cells in the control group decreased by 7.74%, whereas that of U87 cells in the GBE1 knockdown group decreased by 29.15%. Additionally, the viability of U251 cells in the control group decreased by 18.25%, compared to a 33.79% decrease in the viability of U251 cells in the GBE1 knockdown group. These results showed that after GBE1 knockdown, the tolerance of glioma cells to an anoxic environment was significantly reduced, which reflected the transformation of cellular metabolic patterns from glycolysis to oxidative phosphorylation after GBE1 knockdown (Figure 7E–H). These results indicate that GBE1 knockdown changes the metabolic mode of glioma cells from glycolysis to mitochondrial oxidative phosphorylation, and increased expression of FBP1 plays an important role in weakening the glycolysis level of glioma cells. To further investigate the effect of GBE1 knockdown on the growth of glioma cells in vivo, U87 cells carrying a luciferase reporter were transplanted into the striatum of nude mice at day four after sh-GBE1 or sh-NC lentivirus infection. The optical density values of xenografts were recorded every seven days by IVIS in vivo optical imaging system measurements (Figure 8A). The results of the bioluminescence imaging showed that the elimination of GBE1 significantly inhibited the growth of U87-derived xenografts (Figure 8B,C) and conferred a significant survival benefit compared to negative controls (Figure 8D). These results demonstrated that GBE1 knockdown effectively inhibited glioma growth in vivo and improved animal survival. Due to the heterogeneity of tumor cells, targeting molecules are not expressed in all tumor cells, making targeting therapies impaired and often unsatisfactory. However, metabolic abnormalities are a common feature of tumor cells [32], making targeting tumor metabolism a different idea for treating glioma. Our study found elevated expression of GBE1 in gliomas and a correlation with a poor prognosis. Furthermore, our findings validated the mechanism by which GBE1 affects glucose metabolism patterns for the first time in glioma (Figure 7I). As one of the causes of glycogen metabolic diseases, recent studies have revealed that GBE1 affects the development and progression of a variety of tumors, such as leukemia, lung adenocarcinoma, and ovarian cancer [19,20,21]. However, the role of GBE1 in glioma is still unclear. Therefore, we analyzed TCGA and CGGA data and found that GBE1 expression was elevated in gliomas and correlated with a poor prognosis. Furthermore, GBE1 was more reliable than KI67 in predicting glioma grade. This was demonstrated when using respective cut-off values for both indexes; GBE1 showed a 20.59% higher specificity than KI67, although both had a sensitivity of 79.72%. In vitro, we found increased apoptosis, arrested cell cycle, and suppressed cell proliferation in glioma cells with GBE1 knockdown. Additionally, GBE1 knockdown also affected glioma cell migration, invasion, colony formation, and angiogenesis abilities. The impairment of these biological behaviors greatly affected the progression of glioma. FBP1, the rate-limiting enzyme of gluconeogenesis, is generally considered a tumor suppressor and shows decreased expression in various tumors, such as renal, prostate, liver, and breast cancer [33,34,35,36]. In lung adenocarcinomas, GBE1 methylates the promoter of FBP1 through the NF-κB pathway and therefore decreases FBP1 expression [27]. Our results showed that the NF-κB pathway was inhibited following GBE1 knockdown in glioma cells, accompanied by elevated FBP1 expression; this paralleled the effects of single NF-κB pathway inhibitors, suggesting that regulation of FBP1 expression by GBE1 via NF-κB is also applicable in glioma. Despite the inhibitory effects of FBP1 on various tumors, it has also been shown that reduced FBP1 expression inhibits ovarian cancer formation and cisplatin resistance in vivo [37]. Furthermore, FBP1 promotes the proliferation, migration, and invasion of esophageal cancer cells by regulating fatty acid metabolism [38]. This suggests that the effects of FBP1 on tumors are multifaceted. Our study confirmed that in glioma cells, FBP1 expression was significantly elevated after GBE1 knockdown. Recently, a study by Son B et al. found that radiation-induced downregulation of FBP1 expression promotes GBM cell migration [39]. Our study found that knockdown of FBP1 promoted the proliferation, migration, and invasion of glioma cells, while overexpression of FBP1 in glioma cells significantly inhibited the malignant phenotype of glioma cells, which paralleled the previous study. In contrast, knockdown of the elevated FBP1 based on GBE1 knockdown restored glioma cell proliferation and impaired tumor biological behavior. The inhibition of glioma caused by GBE1 knockdown was reversed. The above results illustrated that NF-κB pathway inhibition by GBE1 knockdown resulted in elevated FBP1, leading to glioma suppression. The Warburg effect, characterized by aerobic glycolysis, promotes cancer progression [40] and generates many glycolytic intermediates that satisfy multiple anabolic reactions in cells [41]. Based on these theories, many studies targeting aerobic glycolysis have shown good tumor suppressive effects [13,14,15,16], exhibiting the potential of targeting tumor metabolism in tumor therapy. Our study found that FBP1 elevation by GBE1 knockdown suppressed the basal glycolytic rate and the glycolytic reserve capacity of glioma cells; this parallels a previous study on lung adenocarcinoma [27]. Hypoxia is one of the characteristics of the tumor microenvironment. GBE1 knockdown glioma cells in a hypoxic environment exhibit a malfunction in converting oxidative phosphorylation to glycolysis and a decreased tolerance to a hypoxic environment. However, unlike lung adenocarcinoma, GBE1 knockdown increased oxidative phosphorylation in glioma cells and was difficult to reverse with FBP1 knockdown. This phenomenon has also been found in colon cancer and melanoma. Knockdown of glucose-6-phosphate isomerase caused glycolytic inhibition in colon cancer and melanoma cells while elevating the level of oxidative phosphorylation, which investigators consider to be a compensatory response of cells to glycolytic inhibition [42]. However, studies have found a direct inhibition of fructose-1,6-bisphosphate (F16bp), a substrate of FBP1, in rat liver mitochondria [43,44]. Although this explains the elevated oxidative phosphorylation caused by GBE1 knockdown, it cannot explain the inability to reverse this elevation by the knockdown of FBP1. Therefore, further investigation is needed to determine whether the increased level of oxidative phosphorylation caused by GBE1 knockdown is a compensatory response of cells to the inhibition of glycolysis or the influence of other factors. However, it is undeniable that the elevation of oxidative phosphorylation increases the dependency of glioma cells on oxygen and impairs their tolerance to hypoxia, which is beneficial for the treatment of glioma. Furthermore, the intracranial tumor formation assay also validated the inhibitory effect of GBE1 knockdown in a complex tumor microenvironment. Additionally, GBE1 knockdown conferred a significant survival benefit compared to controls. Our study found that GBE1 expression was significantly elevated in gliomas and was correlated with a poor prognosis. GBE1 downregulates FBP1 expression through the NF-κB pathway, causing a shift in the glucose metabolism pattern of glioma cells to glycolysis, enhancing the Warburg effect, and promoting the development of glioma. These findings provide a new potential target for glioma treatment in the metabolic field.

PMC10000544

Xiaoming Li,Sebastian Kempf,Stefan Günther,Jiong Hu,Ingrid Fleming

11,12-EET Regulates PPAR-γ Expression to Modulate TGF-β-Mediated Macrophage Polarization

23-02-2023

soluble epoxide hydrolase,PPAR-γ,macrophage,resolution of inflammation,11,12-epoxyeicosatrienoic acid

Macrophages are highly plastic immune cells that can be reprogrammed to pro-inflammatory or pro-resolving phenotypes by different stimuli and cell microenvironments. This study set out to assess gene expression changes associated with the transforming growth factor (TGF)-β-induced polarization of classically activated macrophages into a pro-resolving phenotype. Genes upregulated by TGF-β included Pparg; which encodes the transcription factor peroxisome proliferator-activated receptor (PPAR)-γ, and several PPAR-γ target genes. TGF-β also increased PPAR-γ protein expression via activation of the Alk5 receptor to increase PPAR-γ activity. Preventing PPAR-γ activation markedly impaired macrophage phagocytosis. TGF-β repolarized macrophages from animals lacking the soluble epoxide hydrolase (sEH); however, it responded differently and expressed lower levels of PPAR-γ-regulated genes. The sEH substrate 11,12-epoxyeicosatrienoic acid (EET), which was previously reported to activate PPAR-γ, was elevated in cells from sEH−/− mice. However, 11,12-EET prevented the TGF-β-induced increase in PPAR-γ levels and activity, at least partly by promoting proteasomal degradation of the transcription factor. This mechanism is likely to underlie the impact of 11,12-EET on macrophage activation and the resolution of inflammation.

11,12-EET Regulates PPAR-γ Expression to Modulate TGF-β-Mediated Macrophage Polarization Macrophages are highly plastic immune cells that can be reprogrammed to pro-inflammatory or pro-resolving phenotypes by different stimuli and cell microenvironments. This study set out to assess gene expression changes associated with the transforming growth factor (TGF)-β-induced polarization of classically activated macrophages into a pro-resolving phenotype. Genes upregulated by TGF-β included Pparg; which encodes the transcription factor peroxisome proliferator-activated receptor (PPAR)-γ, and several PPAR-γ target genes. TGF-β also increased PPAR-γ protein expression via activation of the Alk5 receptor to increase PPAR-γ activity. Preventing PPAR-γ activation markedly impaired macrophage phagocytosis. TGF-β repolarized macrophages from animals lacking the soluble epoxide hydrolase (sEH); however, it responded differently and expressed lower levels of PPAR-γ-regulated genes. The sEH substrate 11,12-epoxyeicosatrienoic acid (EET), which was previously reported to activate PPAR-γ, was elevated in cells from sEH−/− mice. However, 11,12-EET prevented the TGF-β-induced increase in PPAR-γ levels and activity, at least partly by promoting proteasomal degradation of the transcription factor. This mechanism is likely to underlie the impact of 11,12-EET on macrophage activation and the resolution of inflammation. The recruitment of neutrophils and monocytes to inflamed tissue and their differentiation into macrophages is a crucial step in the inflammatory process. However, once the neutrophil respiratory burst subsides, these and other cells, i.e., macrophages, eosinophils and lymphocytes, need to be removed to restore homeostasis [1]. To support the removal of apoptotic cells and tissue debris (efferocytosis), macrophage function is altered and the cells are reprogramed into a pro-resolving phenotype. Polarized macrophages are frequently broadly classified in two main groups, i.e., classically activated (M1) macrophages which are induced by T-helper 1 (Th-1) cytokines, i.e., the combination of bacterial lipopolysaccharide (LPS) and interferon γ (IFN-γ), and alternatively activated (M2) macrophages that have a pro-resolving and pro-angiogenic phenotype, and are induced by Th-2 cytokines [2,3]. The latter group can be further subdivided into more refined phenotypes: M2a, M2b, M2c, and M2d depending on the use of different stimuli such as interleukin (IL)-4 (M2a) or transforming growth factor β (TGF-β) (M2c). However, the phenotypic characterization of macrophages is highly complicated and there are many more distinct genetic fingerprints and metabolic states than are reflected in a basic M0/M1/M2 classification [4,5,6]. Indeed, additional subtypes have been identified such as macrophages stimulated by oxidized phospholipids, oxidized LDL, or hemoglobin [3]. TGF-β is a master immune regulator and checkpoint that has a major impact on immune suppression within the tumor microenvironment [7]. It has also been implicated in poor responsiveness to cancer immunotherapy [8]. In inflamed tissues, macrophage TGF-β synthesis is stimulated by the uptake of apoptotic cells, a step that is essential for the repolarization of pro-inflammatory macrophages into a pro-resolving phenotype (for reviews see [9,10]). Although endothelial TGF-β signaling drives endothelial-to-mesenchymal transition and vascular inflammation [11], there is some controversy about the exact impact of TGF-β on atherogenesis. Rather than promoting vascular inflammation, there is evidence suggesting that TGF-β signaling plays an important role in the protection against excessive plaque inflammation, loss of collagen content, and induction of regulatory immunity (reviewed by [12,13]). The current study set out to determine changes in macrophage gene expression associated with the repolarization of classically activated (M1) macrophages into a pro-resolving phenotype by TGF-β. C57BL/6N mice (6–8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Floxed sEH mice (Ephx2tm1.1Arte) were generated in the C57BL/6N background by TaconicArtemis GmbH (Cologne, Germany) and crossed with Gt(ROSA)26Sortm16(Cre)Arte mice (TaconicArtemis) expressing Cre under the control of the endogenous Gt(ROSA)26Sor promoter to generate mice globally lacking sEH (sEH−/−) as described [14]. Age-, gender- and strain-matched mice were used throughout, where possible littermates were used. In cases where studying littermates was not possible, cells were isolated from age-matched C57Bl/6N mice. Preliminary experiments revealed that responses were comparable in cells from C57Bl/6N and Cre-sEHflox/flox mice and different from those of the sEH−/− (Cre+ sEHflox/flox) mice. For the isolation of bone marrow, mice were sacrificed using 4% isoflurane in air and cervical dislocation. Murine monocytes were isolated from the bone marrow of 8–10-week-old mice and differentiated to naïve (M0) macrophages in RPMI 1640 medium (Invitrogen; Darmstadt, Germany), containing 8% heat inactivated FCS supplemented with M-CSF (15 ng/mL, Peprotech, Hamburg, Germany) and GM-CSF (15 ng/mL, Peprotech, Hamburg, Germany) for 7 days. Cells were kept in a humidified incubator at 37 °C containing 5% CO2. Thereafter M0 macrophages were polarized to classical activated M1 macrophages by treating with LPS (10 ng/mL; Sigma-Aldrich, Munich, Germany) and IFN-γ (1 ng/mL; Peprotech, Hamburg, Germany) for 12 h. Pro-resolving M2c macrophages were repolarized from M1 macrophages by the addition of TGF-β1 (10 ng/mL; Peprotech, Hamburg) for 48 h, as described [6]. Total RNA was extracted and purified from murine macrophages using Tri Reagent (ThermoFisher Scientific, Karlsruhe, Germany) based on the manufacturer’s instructions. Thereafter, RNA was eluted in nuclease-free water, and its concentration was determined (λ260 nm) using a NanoDrop ND-1000 (ThermoFischer Scientific, Karlsruhe, Germany). For the generation of complementary DNA (cDNA), total RNA (500 ng) was reverse transcribed using SuperScript IV (ThermoFischer Scientific, Karlsruhe, Germany) with random hexamer primers (Promega, Madison, WI, USA). Quantitative PCR was performed using SYBR green master mix (Biozym, Hessisch Oldendorf, Germany) and appropriate primers (Table 1) in a MIC-RUN quantitative PCR system (Bio Molecular Systems, Upper Coomera, Australia). Relative RNA levels were determined using a serial dilution of a positive control. The data are shown relative to the mean of the housekeeping gene 18S RNA. Total RNA was isolated from macrophages by using RNeasy Micro kit (Qiagen, Hilden, Germany) based on manufacturer’s instructions. The RNA concentrations were determined by using NanoDrop ND-1000 (ThermoFischer Scientific, Karlsruhe, Germany; λ 260 nm). Total RNA (1 µg) was used as input for SMARTer Stranded Total RNA Sample Prep Kit-HI Mammalian (Takara Bio, Kyoto, Japan). Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q20 in a window of 20 nucleotides and keeping only filtered reads longer than 15 nucleotides [15]. Reads were aligned versus Ensembl mouse genome version mm10 (Ensembl release 101) with STAR 2.7.10a [16]. Aligned reads were filtered to remove: duplicates with Picard 2.25.5 (Picard: A set of tools (in Java) for working with next generation sequencing data in the BAM format), multi-mapping, ribosomal, or mitochondrial reads. Gene counts were established with featureCounts 2.0.2 by aggregating reads overlapping exons on the correct strand excluding those overlapping multiple genes [17]. The raw count matrix was normalized with DESeq2 version 1.30.1 [18]. Contrasts were created with DESeq2 based on the raw count matrix. Genes were classified as significantly differentially expressed at average count >5, multiple testing adjusted p-value < 0.05, and log2FC > 0.585 or <−0.585. The Ensemble annotation was enriched with UniProt data [19]. The PCA, volcano plots and pathway enrichment analysis were generated using http://www.bioinformatics.com.cn/srplot, an online platform for data analysis and visualization. M1 polarized macrophages were treated with either solvent or the PPAR-γ antagonist; GW9662 (10 µmol/L, Merck, Darmstadt, Germany), 2 h prior to repolarization to the M2c phenotype using TGF-β1. Thereafter, cells were incubated in RPMI medium supplement with 0.1% BSA (37 °C, 5% CO2) and containing pHrodo Red Zymosan bioparticles (10 μg/mL, Invitrogen). After 30 min the cells were washed to remove nonphagocytosed material and zymosan uptake was visualized and quantified using an automated live cell imaging system (IncuCyte, Sartorius, Göttingen, Germany). PPAR-γ activity was measured using a luciferase construct (PPRE-X3-Luc, Addgene No. 1015) which contains 3 response elements (AGGACAAAGGTCA) upstream of a luciferase reporter [20]. For transfection, M0 macrophages were incubated in RPMI medium containing 0.1% BSA for 2 h prior to the addition of plasmid (100 ng/mL) and Lipofectamin 3000 Transfection Reagent (ThermoFischer Scientific, Karlsruhe, Germany) according to the manufacturer’s instructions. After 24 h, the cells were polarized to M1 and M2c macrophages and stimulated as described in the results section. Luciferase activity was measured 48 h after cell polarization or stimulation with 11,12-EET (1 µmol/L, Cayman Europe, Tallinn, Estonia) using a commercially available kit (ONE-Glo Luciferase Assay System, Promega, Walldorf, Germany). Cells were lysed in RIPA lysis buffer (50 mmol/L Tris/HCL pH 7.5, 150 mmol/L NaCl, 10 mmol/L NaPPi, 20 mmol/L NaF, 1% sodium deoxycholate, 1% Triton and 0.1% SDS) enriched with protease and phosphatase inhibitors and detergent-soluble proteins were resuspended in SDS-PAGE sample buffer. Samples were separated by SDS-PAGE and subjected to Western blotting as described [21]. Membranes were blocked in 3% BSA, incubated with primary antibodies in the blocking solution and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using Lumi-Light plus Western blotting substrate (Roche, Mannheim, Germany) and captured by an image acquisition system (Fusion FX7; Vilber-Lourmat, Torcy, France). The antibody used to identify PPAR-γ was from Santa Cruz (Texas, USA; Cat. # sc-7196, 1:1000), anti-non muscle myosin was from abcam (Berlin, Germany; Cat. # ab75590, 1:1000), and the anti β-actin antibody was from Linaris (Eching, Germany; Cat. # MAK6019, 1:3000). The secondary antibodies were used were: goat anti-rabbit IgG H and L chain specific peroxidase conjugate, and a goat anti-mouse IgG, H and L chain specific peroxidase conjugate (both 1:20,000; Cat. # 401393 and Cat. # 401253, Merck). Data are expressed as mean ± SEM. Statistical analysis was performed using Student’s t test, or two-way ANOVA with a Tukey’s or Sidak’s post-test. Normalized data were compared using the Kruskal–Wallis rank sum test or Kruskal–Wallis test followed and Dunn’s multiple comparison test (using Prism 9.0.2, GraphPad Software Inc., San Diego, CA, USA) as indicated in the figure legends. Values of p < 0.05 were considered statistically significant. All data associated used this study are present in the paper or the Supplementary Materials. Bone marrow-derived monocytes were isolated from wild-type mice and differentiated to naïve (M0) macrophages in the presence of M-CSF and GM-CSF for 7 days. Thereafter, M0 macrophages were either polarized to classically activated (M1) macrophages by adding lipopolysaccharide (LPS) and interferon (IFN)-γ for 12 h or into pro-resolving M2c by treating M1 macrophages with TGF-β1 for 48 h. RNA-sequencing (RNA-seq) was then performed to identify changes in gene expression associated with macrophage polarization. Principal component analysis (PCA) confirmed that the three groups of macrophages clustered together with clear differences between the polarization types (Figure 1A, Table S1). As expected, the expression of the classical M1 marker genes Nos2, Ptgs2, Il1b, and Nlrp3 were significantly higher in M1 versus M2c polarized macrophages. On the other hand, the typical M2/M2c markers, i.e., Arg1, Vegfa were higher in M2c than in M1 polarized macrophages (Figure 1B). A closer analysis of the genes differentially expressed in M2c versus M1-polarized macrophages revealed additional marked differences, with TGF-β inducing the upregulation of 2952 genes and the downregulation of 2051 genes, including the pro-inflammatory genes Cxcr4, Ptgs2 and Angptl4. One of the genes whose expression was significantly increased in M2c macrophages was Pparg and gene set enrichment analysis identified changes in the expression of several targets of the peroxisome proliferator-activated receptor (PPAR) family of transcription factors (Figure 1C). PPAR-γ-regulated genes induced by TGF-β included Angptl4, Abcd2, Eepd1 and Tmem8. To determine the importance of PPAR-γ on the regulation of selected macrophage genes, we determined the impact of the PPAR-γ antagonist GW9662 on the expression of three selected genes in M2c macrophages, i.e., Cxcr4 (higher in M2c), as well as Ptgs2 and Ptx3 (both higher in M1). While there was no significant effect of PPAR-γ antagonism on Cxcr4 expression, cells treated with GW9662 expressed significantly higher levels of Ptgs2 and Ptx3 than cells treated with solvent (Figure 2A). One characteristic of the latter cells is their ability to phagocytose cell debris. While M2c polarized murine macrophages effectively phagocytosed zymosan, particle uptake was clearly reduced in cells treated with the PPAR-γ antagonist (Figure 2B). These observations imply that PPAR-γ activation is required for the down regulation of some pro-inflammatory genes as well as to support the induction of a pro-resolving phenotype by TGF-β. Consistent with the latter observations, PPAR-γ expression was significantly elevated in M2c versus M1 or M0 macrophages (Figure 3A). Given that M2c polarization was induced by adding TGF-β to M1 polarized macrophages, we determined which TGF-β type I receptor, i.e., activin receptor-like kinase (Alk) 1 or Alk5, mediated the TGF-β-induced increase in PPAR-γ levels. While neither solvent, nor the Alk1 inhibitor; LDN193189 prevented the TGF-β-induced increase in PPAR-γ (Figure 3B), the response was abolished in macrophages pretreated with the Alk5 inhibitor; SD208. Next, we set out to determine whether or not mediators known to regulate PPAR-γ were implicated in the TGF-β-induced changes in PPAR levels and gene expression. Given that arachidonic acid metabolism was one of the pathways altered by TGF-β (see Figure 1C), we focused on the role of the potential role of arachidonic acid epoxides. These fatty acid mediators; such as 11,12-epoxyeicosatrienoic acid (11,12-EET), are reported to activate PPAR-γ [22,23,24,25,26], and their cellular levels are largely determined by the activity of the soluble epoxide hydrolase (sEH). Therefore, a luciferase construct containing three PPAR-γ responsive elements was expressed in macrophages from wild-type mice that were then polarized to the M1 and M2c phenotypes. Consistent with the increase in PPAR-γ protein levels, luciferase activity was clearly increased in the M2c macrophages from wild-type mice (Figure 4A). Deletion of the sEH significantly blunted the latter response, which was reflected in the differential expression of PPAR-γ-regulated genes in M2c macrophages from the two genotypes (Figure 4B, Table S2). Indeed, the well-characterized PPAR-γ-regulated genes Gipr, Vldlr, and Rbp1 were all expressed at significantly lower levels in M2c macrophages from sEH−/− versus wild-type mice. A series of fatty acid epoxides are metabolized by the sEH and it was possible to demonstrate higher 11,12-EET and lower levels of its sEH-generated diol; 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), in M2c polarized macrophages from sEH−/− versus wild-type mice (Figure 4C). Moreover, treating M1 polarized macrophages from wild-type mice with 11,12-EET prior to the repolarization with TGF-β, also decreased PPAR-γ activity (Figure 4D). Comparison of the effects of 11,12-EET versus those of its diol; 11,12-DHET on PPAR-γ protein levels were assessed next. This revealed that the sEH substrate; 11,12-EET, effectively prevented the TGF-β-induced increase in PPAR-γ protein levels in murine macrophages (Figure 5A). 11,12-DHET had no effect. Somewhat unexpectedly, 11,12-EET altered PPAR-γ protein levels without altering Pparg expression (Figure 5B) indicating that 11,12-EET may affect the stability of the PPAR-γ protein. At least in adipocytes, ligand-dependent PPAR-γ activation is associated with its subsequent proteasomal degradation [27]. To determine whether or not 11,12-EET decreased PPAR-γ levels by stimulating its proteasomal degradation, experiments were performed in the absence and presence of the proteasome inhibitor MG132. As before, 11,12-EET, but not 11,12-DHET, decreased PPAR-γ protein levels in M2c polarized macrophages and proteasome inhibition prevented the effect (Figure 5C). The results of this investigation revealed that the TGF-β-dependent repolarization of classically activated (M1) macrophages into a pro-resolving, highly phagocytic phenotype (M2c), relies on the increased expression and activation of PPAR-γ. Deletion of the sEH, to increase cellular levels of fatty acid epoxides, largely prevented TGF-β-induced changes in macrophage gene expression as well as PPAR-γ activation. The effect seen in macrophages from sEH−/− was reproduced in cells from wild-type mice treated with the sEH substrate 11,12-EET and was attributed, at least in part, to the accelerated proteasomal degradation of PPAR-γ. In our study, we set out to determine changes in macrophage gene expression associated with the repolarization of classically activated (M1) macrophages into a pro-resolving phenotype by TGF-β. It is not surprising that repolarization resulted in marked alterations in macrophage gene expression and a decrease in the expression of pro-inflammatory markers. However, the observation that many of the genes increased in TGF-β-treated macrophages were classical PPAR-γ targets, e.g., Abcd2, Eepd1, and Tmem8 was unexpected as TGF-β is a multifunctional cytokine that drives inflammation, fibrosis and cell differentiation, while PPAR-γ activation tends to promote the opposite effects [28]. The impact of TGF-β on gene expression was however consistent with its ability to increase PPAR-γ protein levels as well as transcription factor activity. The changes in gene expression were reflected in functional alterations as zymosan phagocytosis by TGF-β-repolarized macrophages was clearly attenuated in cells treated with a PPAR-γ inhibitor. Our results are consistent with recent reports from other groups that linked the actions of TGF-β with the activation of PPAR-γ signaling (reviewed by [29]). For example, TGF-β signaling and the upregulation of PPAR-γ was reported to be essential for the development and homeostasis of alveolar macrophages [30]. On the other hand, PPAR-γ was reported to interact with Stat3 and Smad3 to interfere with TGF-β signaling and account for the functional antagonism between BMP2 and TGF-β1 pathways in vascular smooth muscle cells [31]. Thus, it seems likely that a complex crosstalk exists between the two pathways. The results of our study also indicate that in macrophages, the TGF-β-induced increase in PPAR-γ expression relies on the activation of Alk5 and as such fits well with a previous report that TGF-β induces M2-like macrophage polarization via Snail-mediated suppression of a pro-inflammatory phenotype, as the induction of Snail is also mediated by Alk5 [20]. PPARs are ligand-inducible transcription factors and are considered important therapeutic targets as they exert anti-atherogenic and anti-inflammatory effects on the vascular wall and immune cells, as well as acting to reduce insulin resistance and dyslipidaemia [32]. However, unlike many receptors that possess a limited number of ligands, there are numerous natural PPAR-γ ligands, in particular mediators derived from polyunsaturated fatty acids [33]. The EETs are among the latter compounds and are generated by the sequential action of cytochrome P450 enzymes and the sEH [34]. These fatty acid mediators are particularly interesting given that their actions have been attributed to PPAR activation [22,23,24,25,26], and the inhibition or deletion of the sEH to increase EET levels has anti-atherosclerotic effects in mouse models [35,36]. In our study, we observed that the activity of PPAR-γ was lower in TGF-β-stimulated macrophages from sEH−/− (EET high) than from wild-type (EET low) mice. While these findings were consistent with the clearly decreased levels of PPAR-γ protein in sEH-deficient macrophages, they seemed to be a direct contradiction of previous reports. The timing of the experiments performed can go a long way to accounting for the observations made as PPAR-γ activity was generally assessed 48 h after TGF-β addition or stimulation with 11,12-EET. Thus, 11,12-EET probably initiates a transient increase in PPAR-γ activity that is terminated by an EET-stimulated pathway that results in PPAR-γ degradation. Given that PPAR-γ levels were not decreased by 11,12-EET in cells treated with MG 132 we propose that 11,12-EET can stimulate the proteasomal degradation of PPAR-γ. Certainly, PPAR-γ levels can be regulated by protein ubiquitination and degradation [27]. Which ubiquitin ligase was activated by 11,12-EET was not studied but there is circumstantial evidence to link 11,12-EET with increased ubiquitination as the cardiomyocyte-specific overexpression of CYP2J2, which generates 11,12-EET and has been reported to promote the ubiquitination of the pattern recognition receptor NLRX1 [37]. Taken together, our results indicate that macrophage levels of the sEH substrate; 11,12-EET, can modulate macrophage polarization by TGF-β, at least partly by promoting the ubiquitination and degradation of PPAR-γ. Given that sEH inhibition prevents the development of atherosclerosis in mice [35,36], and the conversion of inflammatory macrophages to the M2 phenotype drives atherosclerosis regression [38], it may be interesting to determine how much of the phenotype observed can be attributed to changes in PPAR-γ expression.

PMC10000563

Justine N. van der Beek,Ronald R. de Krijger,Rutger A. J. Nievelstein,Axel Bex,Aart J. Klijn,Marry M. van den Heuvel-Eibrink,Annemieke S. Littooij

MRI Characteristics of Pediatric and Young-Adult Renal Cell Carcinoma: A Single-Center Retrospective Study and Literature Review

22-02-2023

renal cell carcinoma,magnetic resonance imaging,pediatrics,radiology,pathology

Simple Summary Whereas renal cell carcinoma (RCC) is the most common renal tumor in adults, pediatric RCC is a rare malignancy. The previous literature focusing on cross-sectional imaging of RCC concerns mainly computed tomography in adults, whereas in children, a different distribution of subtypes is seen, as well as a preference for magnetic resonance imaging (MRI). Therefore, the aim of this study was to identify MRI characteristics of pediatric and young-adult RCC through a case series and literature review focusing on translocation-type RCC (MiT-RCC) and the pediatric and young-adult population. In our review as well as in our case series T2-weighted hypo-intensity seems to be a potential discriminative characteristic. Moreover, an irregular growth pattern and limited diffusion restriction were often described. Nevertheless, we conclude the discrimination of RCC subtypes, and especially the differentiation of RCC from other pediatric renal tumors, remains difficult. Abstract Pediatric renal cell carcinoma (RCC) is a rare malignancy. Magnetic resonance imaging (MRI) is the preferred imaging modality for assessment of these tumors. The previous literature has suggested that cross-sectional-imaging findings differ between RCC and other pediatric renal tumors and between RCC subtypes. However, studies focusing on MRI characteristics are limited. Therefore, this study aims to identify MRI characteristics of pediatric and young-adult RCC, through a single-center case series and literature review. Six identified diagnostic MRI scans were retrospectively assessed, and an extensive literature review was conducted. The included patients had a median age of 12 years (63–193 months). Among other subtypes, 2/6 (33%) were translocation-type RCC (MiT-RCC) and 2/6 (33%) were clear-cell RCC. Median tumor volume was 393 cm3 (29–2191 cm3). Five tumors had a hypo-intense appearance on T2-weighted imaging, whereas 4/6 were iso-intense on T1-weighted imaging. Four/six tumors showed well-defined margins. The median apparent diffusion coefficient (ADC) values ranged from 0.70 to 1.20 × 10−3 mm2/s. In thirteen identified articles focusing on MRI characteristics of MiT-RCC, the majority of the patients also showed T2-weighted hypo-intensity. T1-weighted hyper-intensity, irregular growth pattern and limited diffusion–restriction were also often described. Discrimination of RCC subtypes and differentiation from other pediatric renal tumors based on MRI remains difficult. Nevertheless, T2-weighted hypo-intensity of the tumor seems a potential distinctive characteristic.

MRI Characteristics of Pediatric and Young-Adult Renal Cell Carcinoma: A Single-Center Retrospective Study and Literature Review Whereas renal cell carcinoma (RCC) is the most common renal tumor in adults, pediatric RCC is a rare malignancy. The previous literature focusing on cross-sectional imaging of RCC concerns mainly computed tomography in adults, whereas in children, a different distribution of subtypes is seen, as well as a preference for magnetic resonance imaging (MRI). Therefore, the aim of this study was to identify MRI characteristics of pediatric and young-adult RCC through a case series and literature review focusing on translocation-type RCC (MiT-RCC) and the pediatric and young-adult population. In our review as well as in our case series T2-weighted hypo-intensity seems to be a potential discriminative characteristic. Moreover, an irregular growth pattern and limited diffusion restriction were often described. Nevertheless, we conclude the discrimination of RCC subtypes, and especially the differentiation of RCC from other pediatric renal tumors, remains difficult. Pediatric renal cell carcinoma (RCC) is a rare malignancy. Magnetic resonance imaging (MRI) is the preferred imaging modality for assessment of these tumors. The previous literature has suggested that cross-sectional-imaging findings differ between RCC and other pediatric renal tumors and between RCC subtypes. However, studies focusing on MRI characteristics are limited. Therefore, this study aims to identify MRI characteristics of pediatric and young-adult RCC, through a single-center case series and literature review. Six identified diagnostic MRI scans were retrospectively assessed, and an extensive literature review was conducted. The included patients had a median age of 12 years (63–193 months). Among other subtypes, 2/6 (33%) were translocation-type RCC (MiT-RCC) and 2/6 (33%) were clear-cell RCC. Median tumor volume was 393 cm3 (29–2191 cm3). Five tumors had a hypo-intense appearance on T2-weighted imaging, whereas 4/6 were iso-intense on T1-weighted imaging. Four/six tumors showed well-defined margins. The median apparent diffusion coefficient (ADC) values ranged from 0.70 to 1.20 × 10−3 mm2/s. In thirteen identified articles focusing on MRI characteristics of MiT-RCC, the majority of the patients also showed T2-weighted hypo-intensity. T1-weighted hyper-intensity, irregular growth pattern and limited diffusion–restriction were also often described. Discrimination of RCC subtypes and differentiation from other pediatric renal tumors based on MRI remains difficult. Nevertheless, T2-weighted hypo-intensity of the tumor seems a potential distinctive characteristic. Pediatric renal cell carcinoma (RCC) is a rare renal malignancy [1,2]. Although Wilms tumors (WTs) show the highest prevalence in young children, the incidence of RCC increases in the second decade of life [1,3,4]. Whereas in the Renal Tumor Study Group of the International Society of Pediatric Oncology (SIOP-RTSG), pre-operative chemotherapy is the standard of care for WTs, upfront surgery is recommended for localized RCC [5]. Invasive procedures to determine histology before the start of therapy in young children are discouraged [6,7]. Age and size of the tumor are important factors in the consideration of the diagnosis of pediatric renal tumors as well as in the consideration of performing a biopsy, indicating age >7 years as a criterion to consider tumor biopsy [6]. Thus far, no specific imaging characteristics discriminating RCC from WTs and other non-WTs have been identified [8,9,10]. Magnetic resonance imaging (MRI) is currently the preferred modality for the assessment of pediatric renal tumors within the SIOP-RTSG given its lack of ionizing radiation and excellent soft-tissue contrast. Furthermore, MRI is subject to continuous technical developments, such as the possibility of calculating the apparent diffusion coefficient (ADC) value using diffusion-weighted imaging (DWI) [6,11,12]. MRI could, therefore, play a potential role in the non-invasive discrimination of pediatric renal tumors [13,14,15,16,17]. Contrary to the rarity of RCC in children, this tumor type is the most common renal tumor in adolescents and adults [18,19,20,21]. Nevertheless, childhood RCC shows distinct histological characteristics, possibly related to the different distribution of RCC subtypes. Whereas translocation-type RCC (MiT-RCC), which has been officially recognized since 2004 by the World Health Organization, is the most frequent subtype in children, clear-cell RCC (ccRCC) is the predominant histological subtype in adults [2,5,22,23,24,25]. MiT-RCC is diagnosed based on translocations including transcription factor E3 (TFE3) and EB (TFEB), which are members of the family of microphthalmia transcription factors (MiT) [26,27]. Interestingly, the previous literature has suggested that cross-sectional imaging findings differ between RCC subtypes [13,28,29,30,31,32,33,34,35]. Until today, studies focusing on the MRI characteristics of pediatric RCC are limited in number, although identification of potential specific MRI characteristics of WTs and non-WTs is important for future validation studies [9,25,35]. Therefore, this study aims to retrospectively identify MRI characteristics of pediatric RCC patients at diagnosis through a case series in our center, including a literature review focusing on this topic. Institutional Review Board approval was obtained. For this retrospective study, obtaining further formal consent was waived. All diagnostic MRI scans included were clinically indicated and were performed as the standard of care. Between 2014 and 2019, we identified 6 children with RCC that underwent an MRI scan at diagnosis. The standard of care for localized pediatric RCC is upfront total nephrectomy [22,36]. Only in case of doubt of a WT diagnosis, based on predefined clinical and imaging characteristics, a core needle biopsy was performed. If there was no suspicion of a non-WT, the patients were pre-operatively treated with 4 weeks of vincristine/actinomycin-D (stage I-III) or 6 weeks of vincristine/actinomycin-D/doxorubicin (stage IV/V), according to the SIOP-RTSG protocol. Abdominal MRI for pediatric renal tumors in this study was performed using a 1.5T MRI system (Achieva, Philips Healthcare, Eindhoven, The Netherlands and Ingenia, Philips Healthcare, Eindhoven, The Netherlands). Two patients were scanned in external hospitals at diagnosis before referral to our center (Signa HDxt; GE Healthcare, Boston, USA and Magnetom Avanto; Siemens, Erlangen, Germany). Scan protocols slightly varied but at least consisted of coronal and axial T2-weighted imaging, axial T1-weighted turbo spin-echo and axial DWI with automatically generated ADC maps. Five patients underwent pre- and post-contrast T1-weighted imaging, whereas for one patient, contrast-enhanced MRI was not available (Table 1). Children were awake, sedated or under general anesthesia depending on their ability to cooperate, according to the standard-of-care procedures. Gadobutrol (Gadovist; Bayer B.V., Leverkusen, Germany) was administered intravenously at a dose of 0.1 mL/kg body weight. Hyoscine butylbromide (Buscopan; Sanofi, Paris, France) was administered intravenously at a dose of 0.4 mg/kg body weight to reduce peristaltic artifacts, with a maximum of 10 mg in children ≥6 years and a maximum of 5 mg in children <6 years. All children were screened for contraindications for MRI and those concerning intravenous agents. For the two patients scanned at local hospitals, specifications of gadobutrol and hyoscine butylbromide were not available. The anonymized MRI datasets were transferred to DICOM software Osirix v. 5.5.2 (Pixmero, SARL, Bernex, Switzerland). Two pediatric radiologists (ASL with 13 years of experience and RAJN with 26 years of experience in body MRI, respectively), who were blinded to the histopathological subtype and clinical characteristics but were aware of the pediatric RCC diagnosis, reviewed the diagnostic MRI scans. All diagnostic scans were assessed using a case report form based on previous studies identifying potential specific imaging characteristics of different pediatric renal tumors [9]. The pediatric RCC cases were analyzed focusing on tumor presentation, growth pattern, characteristics of solid components and enhancement pattern, if available. Tumor volume was calculated based on the three dimensions of the tumor times 0.523. Moreover, up to four round-shaped ROIs containing solid areas of the tumor, mainly based on enhancement, were drawn in order to measure the ADC value of the most representative parts of the tumor. To limit inter-observer variability, an instruction form accompanying the case report form was provided. Our national coordinating SIOP-RTSG histopathologist (RRK with 23 years of experience with pediatric renal tumor histopathology) reviewed the available macroscopy and microscopy from the surgically resected tumors and biopsies of all patients following the most recent WHO classification system [27,37]. Following protocol, the dorsal and ventral side and hilar region of the resected specimen were marked with varying color dyes following the instructions of the involved surgeons. The specimens were sliced free-handed in successive 10 to 20 mm transverse macroscopic slices in a cranial to caudal sequence or through longitudinal incision to bivalve the specimen. Due to the small number of patients, inter-observer agreement between the two pediatric radiologists was difficult to assess because Cohen’s kappa is affected by the prevalence of the finding under observation. Only six patients were included in this study, potentially resulting in low values or even an impossible calculation of kappa when focusing on separate characteristics [38,39]. Therefore, the inter-observer agreement was assessed using percentages of observed agreement, including the intra-class correlation coefficient (ICC) for the median ADC values including the regions of interest and for median tumor volumes. ICC values were interpreted as satisfactory >0.75 [40]. A literature review was performed following PRISMA guidelines to reflect on the case series and elaborate on the current knowledge about the MRI appearance of RCC by focusing on the predominant histological subtypes in the pediatric and young-adult population. For this purpose, PubMed, Embase/Medline and Cochrane libraries were searched in November 2021, using the main search terms ‘renal cell carcinoma’ and ‘magnetic resonance imaging’ (Table S1). The study has not been registered. Cross-referencing and a citation check of the included papers were executed using Scopus. Articles were included when they (1) included MRI characteristics of patients with proven RCC; (2) were prospective or retrospective cohort studies, randomized controlled trials or case reports; (3) were written in the English language; and (4) were available in the full-text form. Subsequently, articles focusing on children (<19 years), potentially also including adolescents or young adults (≤35 years) and articles focusing on MiT-RCC were separated to serve as the focus of this literature review. Given the rarity of studies focusing on the MRI characteristics of MiT-RCC, articles focusing on adults were also included for this purpose. With this approach, we guaranteed identification of all relevant articles while subdividing their relevance for our study based on their full-text content. After removal of duplicates, 7012 articles were screened based on title and abstract, leaving 363 articles for full-text screening, resulting in the inclusion of 95 articles. Of these, 13 articles focused on pediatric, adolescent and young-adult RCC, and 13 articles focused on MiT-RCC, with an overlap of 6 articles (Figure 1). In November 2022, the search was updated, with no additional results for articles focusing on children and/or MiT-RCC. The six identified patients in our center had a median age of 12 years (range 63–193 months) (Table 2). Four patients were female, and half of the patients presented with a right-sided tumor. Two/six patients received pre-operative chemotherapy following suspicion of a WT, whereas 4/6 underwent upfront surgery. In one case, RCC was pre-operatively confirmed through tumor biopsy. Three patients had stage 1 disease, whereas the other patients had stage 2 (1/6) and stage 3 (2/6) disease (Table 2). The average post-operative specimen weight was 898.6 g (range 210–2100 g), whereas the maximum post-operative tumor diameter ranged from 2.4 to 12.9 cm (median 6.8 cm). The post-operative weight of the specimen was missing for one patient, of which the largest tumor diameter was 9.5 cm (Table 2). Five patients were tested for MiT-RCC, resulting in 2/5 MiT-RCC cases (Table 2, Figure 2 and Figure 3). In 4/5 cases, FISH was used, whereas in the two most recent cases, also RNA sequencing was performed, resulting in a rearrangement of TFE3 and SFPQ in the sixth patient. Two patients were diagnosed with ccRCC, and in one patient, the subtype could not be specified. The first patient, who was not tested for MiT-RCC, showed an FH mutation in the context of a hereditary leiomyomatosis and RCC cancer syndrome (Table 2) [41]. For the 5-year-old patient diagnosed with ccRCC, the FISH for MiT-RCC was not conclusive, and RNA sequencing for further analysis of TFEB was not available. The median observed agreement between the two observers was 83% (range 33.3%–100%). The few imaging characteristics with low observed agreement were discussed between the two radiologists, and mismatching concepts were resolved (Table S2). Furthermore, the inter-reader agreement for median tumor volume was excellent, with an ICC of 0.991 (95% 0.941–0.999). Therefore, the imaging characteristics found by the first reader (ASL) were reported (Table 2). Tumor volume ranged from 29 to 2191 cm3, with varying locations. The shape of the tumors was predominantly lobulated (4/6), and margins were well-defined in a majority of the patients (4/6). Capsule rupture was seen in only 2/6 cases, which was defined as an interruption of the hypo-intense capsule of the tumor. None of the cases presented with a tumor thrombus. Concerning hemorrhage and necrosis, these components were present in 3/6 and 1/6 cases, respectively. Cysts were present in 2/6 cases, whereas fatty tissue and subcapsular fluid were not observed (Table 2). The tumors presented mainly homogeneously (4/6), with a predominant hypo-intense appearance on T2-weighted imaging and iso-intense appearance on T1-weighted imaging. Almost all cases showed a homogeneous enhancement pattern, varying from mild to strong enhancement (Table 2). There was no obvious consistency concerning MRI characteristics within patients based on histological subtype (Table 2, Figure 2, Figure 3). Inter-reader agreement was excellent for median ADC values with an ICC of 0.942 (95% CI 0.639–0.992) (Table S3). Therefore, only the median surfaces of ROIs and median ADC values measured by the first reader (ASL) were reported (Table 2). The median ADC values ranged from 0.70 to 1.20 × 10−3 mm2/s. The MiT-RCC cases and the case diagnosed as ccRCC but with inconclusive TFE results showed the lowest ADC values, ranging from 0.70 to 0.98 × 10−3 mm2/s (Table 2, Figure 2 and Figure 3). We identified thirteen studies focusing on MRI findings of pediatric RCC, with a total of 25 patients (Figure 1, Table 3) [19,24,42,43,44,45,46,47,48,49,50,51,52]. Ages ranged from 4 to 33 years, with four studies also including young adults ≤35 years [19,24,48,51]. Six studies focused on MiT-RCC, whereas other histological subtypes represented ccRCC, papillary type RCC (pRCC), chromophobe RCC (chrRCC), renal medullary carcinoma (RMC) and other rare RCC types. The location of all reported pediatric RCC tumors in the identified articles varied from central to peripheral (Table 3). On T1-weighted imaging and T2-weighted imaging, tumors appeared predominantly heterogeneously, whereas no clear predominant intensity was seen for one of these sequences. Accordingly, enhancement pattern on contrast-enhanced MRI was reported mostly as heterogeneous. Cysts, when specified, were found in only three cases, whereas the presence of necrosis and/or hemorrhage was often not specified [24,43,52]. Regional lymph node involvement and/or metastases to lymph nodes were reported in five studies (Table 3) [19,24,42,43,51]. In a study of seven patients, Wang et al. reported positive regional lymph node status in four patients and positive cervical lymph node status in one patient [19]. Blitman et al. reported two patients with vascular tumor involvement of the renal vein and three patients with encasement of the vascular pedicle out of a total of six patients, all with infiltrative tumors with ill-defined margins (Table 3) [51]. Only one study specified findings of DWI, reporting the iso-intense appearance of the tumor on the b500 DWI sequence compared to the renal parenchyma [50]. Concerning MRI characteristics of RCC subtypes other than MiT-RCC in children, Zou et al. reported a case of a 17-year-old male with von Hippel–Lindau disease with bilateral renal cysts and ccRCC (Table 3) [46]. This patient showed T2-weighted hyper-intensity and T1-weighted hypo-intensity, whereas enhancement was limited on contrast-enhanced imaging. Koetter et al. described a 16-year-old female at 31 weeks’ gestation presenting with a large, heterogeneous cystic–solid mass, which was histologically diagnosed as pRCC [43]. Another reported pRCC that presented as a complex cyst containing bloody elements, whereas a pediatric chrRCC showed a well-defined T1-weighted hypo-intense and T2-weighted hyper-intense tumor with necrosis (Table 3) [45,52]. Finally, RMC has been described as a very rare and malignant renal tumor, especially in children and young adults, and is often seen in RCC patients with sickle-cell traits [28,51,53]. Noreña-Rengifo et al. described a 12-year-old male with an intermediate enhancing mass on T1-weighted imaging with evident retroperitoneal lymphadenopathies, similar to the reported regional adenopathy identified on MRI in a retrospective study by Blitman et al. (Table 3) [42,51]. Thirteen studies focusing on MRI characteristics of MiT-RCC were identified, including the six identified studies focusing on pediatric patients with MiT-RCC (Figure 1, Table 3 and Table 4) [13,19,24,44,47,48,50,54,55,56,57,58,59]. There was a total of 46 patients, who were aged 4–76 years old, with MiT-RCC included in the identified articles. Whereas the tumor location was again highly variable among patients, overall, there was a majority showing hyper-intensity on T1-weighted imaging and hypo-intensity on T2-weighted imaging, with a heterogeneous enhancement pattern. Wang et al. reported 8/9 patients with necrosis, and 7/9 patients with hemorrhage, whereas in other studies, these characteristics were often not specified [19]. The tumor composition and growth pattern of MiT-RCC was very heterogeneous, although a substantial part of the cases seems to present with an infiltrative and/or irregular growth pattern. Fifteen patients presented with lymph node involvement; however, four studies lacked information concerning this characteristic. Reported metastatic sites were liver and/or lungs in a total of three patients [55,57]. DWI characteristics were reported in 5 studies for a total of 23 patients [13,50,54,55,57]. Overall, diffusion restriction seemed limited in these cases, with, for instance, Tohi et al. reporting no restriction and Chen et al. reporting a relatively high signal on the ADC map [54,57]. Razek et al. showed a mean ADC value of 1.50 ± 0.97 for four patients [13]. In our case series, the 14-year-old female patient in particular showed a typical presentation of MiT-RCC based on these findings in the previous literature. The tumor showed an ill-defined tumor with capsule invasion and an infiltrative growth pattern, appearing hypo-intense on T2-weighted imaging with a relatively high median ADC value (Table 2 and Table 4, Figure 2). The presentation of the 16-year-old male patient with MiT-RCC seemed less typical (Table 2 and Table 4, Figure 3). The RCC subtypes most frequently occurring in children and adolescents besides MiT-RCC are ccRCC, pRCC and chrRCC (Table 3) [3,18]. Knowledge of MRI characteristics of these subtypes is based mainly on adult studies. A retrospective study of Wang et al. focused on the MRI characteristics of 57 adult RCC patients, in which ccRCC and pRCC showed hemorrhage in 20–25% of the cases compared to no evidence of hemorrhage for chrRCC [60]. Moreover, a very high percentage of cystic necrosis was seen in ccRCC and pRCC, resulting in a significant difference of this characteristic compared to chrRCC, for which no cases were seen. Compared to ccRCC, other RCC subtypes often show a less aggressive growth pattern on MRI, which is illustrated by a higher numbers of cases with well-defined margins, less peripheral invasion and less extension of the tumor [60,61]. Oliva et al. described the MRI-features of 21 pRCCs and 28 ccRCCs, concluding that pRCC typically presents with T2 hypo-intensity, whereas ccRCC typically shows T2 hyper-intensity [62]. This finding, as well as the occurrence of increased enhancement in ccRCC compared to pRCC and chrRCC, has often been reported in the previous literature [35,63,64,65]. Furthermore, ccRCC seems to show significantly higher ADC values than pRCC and chrRCC [64,66,67]. There seems to be a lack of specific imaging characteristics for discrimination of pediatric RCC and its subtypes based on MRI characteristics alone [6,9,10]. Nevertheless, imaging plays an increasingly important role in the diagnosis and follow-up of pediatric renal tumors and in the discrimination of different renal tumor types [28,68,69]. The heterogeneous diagnostic appearance of our patients was in line with findings in the identified literature and with previous studies stating that RCC is often indistinguishable from WTs based on MRI characteristics alone [70,71,72,73]. Part of the included patients showed cysts, necrosis and hemorrhage; however, none of these characteristics were explicitly found in all patients [74]. Calcifications have often been reported as common findings in pediatric RCC; however, MRI does not allow for a trustworthy assessment of calcifications and was, therefore, not included as an imaging characteristic in our case report form [28,69,75]. Despite the recommendation of the SIOP-RTSG to use MRI for cross-sectional imaging of renal tumors, various countries still perform abdominal CT scans in these patients. One of the largest studies focusing on CT characteristics of pediatric RCC to date also reported a widely variable radiological appearance, often with the presence of calcifications [49]. Nevertheless, calcifications can also be seen in WTs, making discrimination based on this imaging characteristic difficult given the rarity of pediatric RCC and other non-WTs [76,77]. Finally, the findings in our case series were in concordance with the frequently reported localized presentation and small size of pediatric RCC [6,75]. Whereas MiT-RCC is the most frequent histological subtype in children, we reported only two out of six patients with a proven TFE translocation. The MRI characteristics of these two patients were quite different from one another. MiT-RCC, similar to ccRCC, is often described as a relatively aggressive tumor in terms of growth pattern and tumor extension as well as prognosis [28,35,60,78,79,80,81]. Nevertheless, only one MiT-RCC case showed an infiltrative growth pattern with capsule rupture, whereas the second MiT-RCC case and both ccRCC cases had well-defined margins with the presence of a pseudocapsule, without any signs of aggressive growth. In general, capsule rupture remains difficult to assess. Concerning the discrimination between histological RCC subtypes, the predominantly reported T2-weighted hypo-intensity in MiT-RCC is also often described for pRCC and chrRCC, whereas ccRCC classically demonstrates high intrinsic T2-weighted signal intensity [31,33,35,82,83]. Nonetheless, knowledge of specific MRI characteristics of MiT-RCC remains limited, given the rarity of MiT-RCC in adult patients and its relatively recent recognition as an official subtype by the WHO [27]. Whereas in adult RCC, the main focus is often the discrimination of histological subtypes, in pediatric RCC, discrimination from the much more frequently occurring WTs in the early diagnostic stages is of great importance [6,7,9]. WTs have a very heterogeneous presentation at diagnosis and are, most often, large intra-renal tumors with a pseudocapsule [74,84,85]. Whereas an irregular growth pattern and absence of a capsule are often described as common for RCC in the previous literature, we observed a majority of well-defined margins and the presence of a pseudocapsule in our case series. Nonetheless, an enhancing capsule has also been reported as a characteristic of MiT-RCC [25,28,57]. MRI characteristics reported to be typical for RCC will still not be discriminative given the heterogeneous appearance of WTs. Nevertheless, WTs often appear hyper-intense on T2-weighted imaging, which is opposite to the T2-weighted hypo-intensity in a majority of our cases with RCC, as substantiated by the findings in the previous literature [28,69]. Finally, RCC is often reported to be smaller than WTs [7,10,57]. Following SIOP-RTSG protocols, based on the suspicion of a non-WT, a biopsy is recommended for children ≥10 years of age and for children between 7 and 10 years old with a tumor volume <200 mL [10]. In our case series, tumor volume was relatively low, except for the expected large FH-RCC case (case nr. 1). In the previous literature, tumor volume ranged widely; however, often only the largest diameter was reported [7,57,74,77,86]. Overall, there seems to remain a lack of pathognomonic MRI characteristics for the discrimination of pediatric RCC from other renal malignancies in children, as well as for the differentiation of histological subtypes [6,9,10]. Nevertheless, DWI has shown an increasing potential reliability for the non-invasive discrimination of renal lesions [15,16,87,88]. Whereas only one included pediatric study focused on the diffusion restriction of pediatric RCCs, our literature review confirmed results from previous overviews stating adult clear-cell RCC has shown significantly higher ADC values compared to non-clear-cell RCC [17,32,50,87,89]. In contrast, our case series showed the three lowest median ADC values in the ccRCC and MiT-RCC cases, whereas also relatively high ADC values were reported. In WTs, relatively low ADC values can be observed, varying among histological WT subtypes [12,16]. In children, discrimination of common histological RCC subtypes, as well as discrimination from WTs based on DWI, therefore, remains difficult. Nonetheless, the female patient with MiT-RCC in our case series appeared to have a typical presentation in the light of previous reports, showing potential discriminative MRI characteristics for TFE-positive tumors. Future studies may focus on validating adult findings in the pediatric population and explore the relationship between ADC values and common pediatric RCC subtypes combined with other typical MRI characteristics. Over the past decades, differences between adult and pediatric RCC have increasingly been appreciated. Concerning imaging studies, the direct comparison of the pediatric and adult population has become even more complicated by the preference of CT in the adult population, whereas MRI has developed as the preferred imaging modality within the SIOP-RTSG [6,8]. Nevertheless, MRI also plays an increasingly important role in the adult population, mainly due to its ability to perform quantitative measurements [32,90]. Therefore, when searching the literature databases for MRI characteristics of pediatric RCC and MiT-RCC, the literature about the adolescent and adult population cannot be ignored. Not only because knowledge of MR imaging of these cases is scarce, but also because they are often embedded in studies focusing on adolescents and/or adults as well. Concerning cut-off values for age classification, we focused on the predefined range of 18–35 years for the ‘adolescents and young adults’ often used in Europe. However, this classification varies around the world [91,92]. Our study has a few limitations, mainly based on its retrospective nature and small study population. The limited number of patients did not allow any statistical analysis or strong conclusions. Furthermore, scan parameters were inconsistent due to not as yet centralized care. Nevertheless, these cases served mainly as an illustration accompanying the literature review in this developing field of research. In this way, this descriptive study contributes to the increasing knowledge of pediatric RCC and its diagnostic presentation on MRI. Concerning the reported imaging characteristics by two independent observers, there was excellent inter-observer agreement [39,40]. The small number of patients in this study does not allow for strong conclusions concerning validity of the use of the CRF in other populations. For a few years, MRI has been the preferred imaging modality for imaging pediatric renal tumors within the SIOP-RTSG protocol. This case series represents one of the largest retrospective reports so far, including an extensive review focusing on MRI characteristics of RCC in the pediatric and young-adult population. The reported cases showed a varying presentation of different pediatric RCC subtypes on MRI, in line with the published literature. Nevertheless, based on this study, T2-weighted hypo-intensity of the tumor has been shown to be a potential distinctive characteristic for the discrimination of RCC from other renal tumors that are prevalent at this age, especially WTs. Future studies should focus on larger study populations through international collaboration, also exploring innovative techniques such as DWI as a non-invasive biomarker.

PMC10000565

Hwang-Ju Jeon,Kyeongnam Kim,Chaeeun Kim,Sung-Eun Lee

Antimelanogenic Effects of Curcumin and Its Dimethoxy Derivatives: Mechanistic Investigation Using B16F10 Melanoma Cells and Zebrafish (Danio rerio) Embryos

22-02-2023

curcumin derivative,melanogenesis,MC1R signaling pathway,zebrafish,melanin production,whitening agent

Regulation of melanin production via the MC1R signaling pathway is a protective mechanism of the skin of living organisms against exposure to ultraviolet rays. The discovery of human skin-whitening agents has been one of the most intense pursuits of the cosmetic industry. The MC1R signaling pathway is activated by its agonist, alpha-melanocyte stimulating hormone (α-MSH), and mainly regulates melanogenesis. Here, we evaluated the antimelanogenic activities of curcumin (CUR) and its two derivatives, dimethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), in B16F10 mouse melanoma cells and zebrafish embryos. CUR and BDMC reduced the α-MSH-induced melanin production in B16F10 cells and also downregulated the expression of the melanin-production-related genes Tyr, Mitf, Trp-1, and Trp-2. Moreover, the biological activity of these two compounds against melanogenesis was confirmed in in vivo experiments using zebrafish embryos. However, the highest concentration of CUR (5 µM) resulted in slight malformations in zebrafish embryos, as indicated by acute toxicity tests. In contrast, DMC did not show any biological activity in vitro or in vivo. Conclusively, BDMC is a strong candidate as a skin-whitening agent.

Antimelanogenic Effects of Curcumin and Its Dimethoxy Derivatives: Mechanistic Investigation Using B16F10 Melanoma Cells and Zebrafish (Danio rerio) Embryos Regulation of melanin production via the MC1R signaling pathway is a protective mechanism of the skin of living organisms against exposure to ultraviolet rays. The discovery of human skin-whitening agents has been one of the most intense pursuits of the cosmetic industry. The MC1R signaling pathway is activated by its agonist, alpha-melanocyte stimulating hormone (α-MSH), and mainly regulates melanogenesis. Here, we evaluated the antimelanogenic activities of curcumin (CUR) and its two derivatives, dimethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), in B16F10 mouse melanoma cells and zebrafish embryos. CUR and BDMC reduced the α-MSH-induced melanin production in B16F10 cells and also downregulated the expression of the melanin-production-related genes Tyr, Mitf, Trp-1, and Trp-2. Moreover, the biological activity of these two compounds against melanogenesis was confirmed in in vivo experiments using zebrafish embryos. However, the highest concentration of CUR (5 µM) resulted in slight malformations in zebrafish embryos, as indicated by acute toxicity tests. In contrast, DMC did not show any biological activity in vitro or in vivo. Conclusively, BDMC is a strong candidate as a skin-whitening agent. Natural products have been widely used in various fields, including biological, pharmaceutical, and nutraceutical areas, with the intention of obtaining and exploiting their beneficial effects, such as anticancer, anti-inflammatory, antifungal, antibacterial, and antimelanogenesis functions. These natural products are found in various organisms in Nature, including microorganisms, animals, and plants [1]. Plants are the main source of natural products, and as such the usage of plant extracts in the pharmaceutical and cosmetic industries has been increasing, partly owing to the negative perception associated with animal-origin extracts [1]. Numerous research groups are exploring the beneficial effects of new bioactive plant extracts or isolated plant-derived chemical compounds. Their studies attempt to demonstrate the biological mechanisms of valuable candidates, and suggest their potential usage. Investigating the underlying biological mechanisms of the active compounds of natural products is crucial for confirming the lack of harmful side effects in humans. In order to investigate the molecular mechanisms and potential toxicity of natural products, both in vitro and in vivo methods are employed, including cell cultures and animal experiments [1,2]. However, recently introduced ethics regarding the use of animals in research have enforced the pursuit of alternative animal models, such as small fish [2]. The zebrafish is an animal that adheres well to the requirements of alternative experimental animal models. Their genome is very similar to that of humans, they have a high reproduction rate, and their early development is achieved within 96 h after fertilization. Moreover, because of the clear chorion of the embryo, the entire developmental process can be easily observed [3,4,5]. As a result of these advantages, zebrafish have been used in various fields of biological research, including those of natural products. Recently, the global cosmetic market has seen large growth [6]. In addition, whitening products are one of the most popular products in the cosmetics industry. Based on this, the exploration of new candidates for whitening agents is spotlighted in this field. Whitening agents play an important role in the cosmetic industry. Many studies have attempted to discover new agents, such as arbutin and kojic acid, with improved skin-whitening effects [6]. The main focus of such studies is to suppress the activation of the melanocyte-specific melanocortin-1 receptor-tyrosinase (MC1R-TYR) signaling pathway in melanocytes. The MC1R signaling pathway is the primary pathway regulating melanin production [7,8]. MC1R is activated by the alpha-melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), and ACTH-secreting phaeochromocytoma (ASP) ligands [7]. Once activated, the α-MSH-induced cAMP pathway stimulates the activation of TYR [7,9]. The binding of α-MSH to MC1R on the cell membrane activates adenylate cyclase and leads to an increase in the levels of intracellular cAMP [7,9]. Subsequently, cAMP-dependent protein kinase A (PKA) phosphorylates cAMP response element-binding protein (CREB), leading in turn to the phosphorylation of MITF, which acts as a DNA-binding transcription factor of melanin-production-related genes, including TYR: a rate-limiting enzyme of melanogenesis. In particular, MITF regulates the expression of TYRP-1 and TYRP-2 proteins by binding to M-box in the tyrosinase distal element of these genes [7,10]. Curcumin (CUR), dimethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are active polyphenolic compounds of turmeric (Curcuma longa), a member of the ginger family, called curcuminoids. Although turmeric originates from India, it has been grown in many other places, including China and Southeast Asia [11,12]. Owing to its special taste and flavor, turmeric has been used over the past few centuries as a coloring agent, insect repellent, and antimicrobial agent. Because of the increased interest in natural products in the past several decades, turmeric has also been used as a nutraceutical, dietary supplement, and functional food [13,14]. Curcuminoids have been well known to exhibit a broad spectrum of bioactivities, including antioxidant, anti-inflammatory, antimicrobial, antifungal, and anticancer effects [14,15,16]. These beneficial effects have been established both in vitro and in vivo by many studies using cell lines and mouse models, respectively [11,13]. In this study, we evaluated the antimelanogenic effects of curcuminoids in vitro and in vivo using a mouse melanoma cell line (B16F10) and zebrafish embryo model, respectively, to explore new whitening agent candidates for use in cosmetics. Concomitantly, we performed acute toxicity tests to ensure the safety of using these compounds in vertebrates. CUR, DMC, BDMC, α-melanocyte stimulating hormone (α-MSH), and kojic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used in this study were of molecular biology grade. The B16F10 mouse melanocarcinoma cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Culturing cells were performed as described before [17]. Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GE Healthcare, Chicago, IL, USA) containing 10% fetal bovine serum (Corning, Corning, NY, USA) and 1 % penicillin/streptomycin (GE Healthcare, Chicago, IL, USA). Cells were cultured in a CO2 incubator (BINDER, Tuttlingen, Germany) under conditions of humidified atmosphere with 5% CO2 at 37 °C. Cells used in experiments were kept at as low a passage number as possible. The proliferation of B16F10 cells was evaluated using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions [17]. Specifically, 1 × 103 cells were seeded on a 96-well plate and cultured for 24 h to recover. After recovery, the medium was changed with fresh medium containing CUR (5, 10, 20, 40, and 80 μM), DMC (5, 10, 20, 40, and 80 μM), or BDMC (5, 10, 20, 40, and 80 μM). Treated cells were incubated for an additional 48 h and then 20 μL of MTS solution per 100 μL media was added to each well. After an additional incubation for 4 h, the plate was placed at 25 °C for 30 min, followed by determining absorbance at 490 nm using the Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Data were normalized to the absorbance value of the control and represented as percentage ratio. Total melanin contents were determined according to the method previously reported [18]. Briefly, B16F10 cells were pretreated with 200 nM α-MSH to induce melanogenesis, and then incubated for 72 h with phenol-red-free DMEM supplemented with 10% FBS and antibiotics. Kojic acid (200 μM), CUR (5 and 10 μM), DMC (5 and 10 μM), or BDMC (5 and 10 μM) was added to the media. After incubation, media were collected for measuring extracellular melanin contents, whereas 200 μL RIPA lysis buffer was added to harvested cells for determination of intracellular melanin contents. To isolate intracellular melanin, lysed cells were centrifuged at 4 °C and 13,000× g for 15 min and the pellet was resolved in 10% DMSO solution containing 1 N NaOH at 95 °C for 2 h. Melanin contents were determined by measuring absorbance at 470 nm using a spectrophotometer. For normalization of samples, protein concentration was determined by the BCA method as previously described [18]. Isolation of total RNA and determination of the level of mRNA was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to a previously described method [19]. Briefly, total RNA was extracted from samples using the Trizol reagent (Ambion, Austin, TX, USA), and the absorbance at 260 nm, 260/280 nm, and 260/230 nm was measured for quantity and quality check of extracted RNA using the Multiskan GO microplate reader. Subsequently, 5 μg cDNA was synthesized from extracted total RNA using the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The obtained cDNA was used as the template for the determination of the levels of Mitf, Tyr, Trp-1, and Trp-2 mRNAs. Accordingly, qRT-PCR was performed using the Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. The primers used in qRT-PCR are listed in Table S1. All data were normalized to the level of Gapdh mRNA. The activity of intracellular tyrosinase was determined according to a previously reported method [18]. Briefly, α-MSH-pretreated B16F10 cells were incubated with various concentrations of test compounds (200 nM kojic acid; 5 and 10 μM CUR, DCM, or BDCM) for 72 h. Cells were then harvested in a lysis buffer containing proteinase inhibitors and centrifuged at 13,000× g and 4 °C for 15 min to collect the supernatant. For measuring the oxidation rate of L-DOPA, 20 μL of collected sample and 80 μL of 40 mM L-DOPA were mixed and placed in each well of a 96-well plate. Samples were incubated at 37 °C for 2 h and their absorbance was measured at 475 nm using a microplate reader. Absorbance values were normalized to the protein concentration of each sample. AB strain zebrafish were generously provided by Prof. Tae-Lin Huh from the Korean Zebrafish Resource Bank, Kyungpook National University (Daegu, Republic of Korea) and kept in the laboratory in an automatic flow-through system under conditions of 26 ± 1 °C and 8 h:16 h day and night ratio for more than eight generations. Fish were cared for according to the modified ZFIN general fish care method previously reported [19]. The night before the experiment, 10 pairs of fish were mated in order to obtain embryos. Early in the morning, embryos were collected, and healthy embryos (>80% fertilization ratio) were transferred to E3 media. Fifteen healthy embryos were placed in each well of a 6-well plate, followed by the addition at the shield stage of E3 media containing kojic acid (8 mM) and test compounds (CUR, DMC, or BDMC; 1.25, 2.5, and 5 μM each). After treatment, the developmental stage, abnormal development, and phenotype were checked every 24 h until 72 hpf. The phenotype of embryos was photographed at 72 hpf using an Olympus BX53 microscope equipped with a DP80 CCD camera (Olympus, Waltham, MA, USA). After documentation, embryos were homogenized with CETi lysis buffer (Translab, Daejeon, Republic of Korea), and melanin concentration was determined by measuring absorbance at 475 nm using a microplate reader. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). One-way ANOVA with Tukey’s post hoc test was performed for multiple comparisons. All data are presented as the mean ± standard deviation (SD). A p < 0.05 was considered statistically significant. To set the dose range of CUR, DCM, and BDCM, we performed a Tetrazolium Assay (MTS assay). The inhibition ratio was calculated and represented as a percent compared with the control (Figure 1). We found that the inhibition ratio of the proliferation of B16F10 cells treated with 10 µM of CUR, DCM, and BDCM was 12.31 (±7.90)%, 18.03 (±16.70)%, and 15.14 (±6.74)%, respectively. We also observed that at concentrations of curcuminoids above 20 μM, the percent inhibition ratios of treated cells were above 20% (28.13, 23.26, and 22.38% for cells treated with 20 μM CUR, DCM, and BDCM, respectively). Considering this result, we set the highest dose of curcuminoids to 10 μM when we evaluated their antimelanogenic effect in B16F10 mouse melanoma cells. We detected that both the extracellular and intracellular melanin contents were elevated in α-MSH-induced B16F10 cells, exhibiting a 1.88- to 2.75-fold and 2.23- to 3.29-fold increase for extracellular and intracellular melanin content, respectively, compared with those in the control (Figure 2). Overall, treatment with α-MSH induced a 2.17- to 3.04-fold increase in total melanin contents in B16F10 cells (Figure 2). We also observed that the stimulation of melanin production in B16F10 cells was suppressed by 1.39- and 1.98-fold after the addition of 5 μM CUR or BDMC, respectively, in comparison to that in the control (Figure 2). We further noticed that at its highest concentration (10 μM), CUR reduced both the extracellular and intracellular melanin contents by 1.59- and 1.31-fold in comparison to those in the control (Figure 2). Likewise, the amount of extracellular and intracellular melanin was 1.69- and 1.28-fold higher after the administration of 10 μM BDMC than that in the control (Figure 2). In contrast, we observed that the addition of 5 and 10 μM DMC increased the production of total melanin in a dose-dependent manner, resulting in 2.94- and 3.43-fold higher total melanin contents, respectively, than those in the control. Moreover, we found that both 10 μM CUR and DMC inhibited the α-MSH-induced increase in tyrosinase activity, keeping it to 1.46- and 1.27-fold higher than that in the control (Figure 2). We subsequently evaluated the antimelanogenic properties of CUR, DMC, and BDMC at the molecular level. We detected that the α-MSH-induced increases in the mRNA levels of Mitf, Tyr, Trp-1, and Trp-2 in B16F10 cells were dramatically reduced following treatment with CUR, DMC, or BDMC (Figure 3). We specifically found that the α-MSH-induced increase in the level of Mitf mRNA was reduced 0.10-, 0.19-, and 0.08-fold following administration of 10 µM CUR, DMC, or BDMC, respectively, compared with that in the control (Figure 3). Likewise, we observed that the expression of Tyr and Trp-1 was reduced in B16F10 cells following administration of CUR, DMC, or BDMC (5 and 10 µM) in a dose-dependent manner (Figure 3). In contrast, the expression of Trp-2 was unaltered or slightly reduced following treatment with a low or high dose of curcuminoids, respectively (Figure 3). In particular, the expression of Trp-2 was reduced by 0.28-, 0.53-, and 0.43-fold following 10 µM CUR, DMC, or BDMC administration, respectively, in comparison to that in the control (Figure 3). Before evaluating the antimelanogenic effect of curcuminoids in early-stage zebrafish embryos, we performed acute toxicity tests to set the range of treatment dose. We accordingly observed various malformations in zebrafish embryos treated with 5 μM CUR. More specifically, we found that the spine was curved, and the tail tip was also curved to one side in embryos at 72 hpf (Figure 4). The survival rate was also slightly decreased at 72 hpf (Figure 4). However, these effects disappeared at concentrations of curcuminoids below 2.5 μM. Interestingly, we observed neither abnormal development nor death in zebrafish treated with DMC or BDMC (Figure 4). We observed the pigmentation of zebrafish as dark dots in the overhead view of zebrafish embryos at 72 h post fertilization (hpf) (Figure 5a). These dots are located between the top of the embryo head and its trunk. Kojic acid, a known antimelanogenic compound, reduces the synthesis of melanin in developing zebrafish embryos, and was thus used here at a concentration of 8 mM as positive control (Figure 5a,b). We determined that kojic acid inhibited the production of melanin by 41% compared with that in the control. We also found that CUR and BDMC inhibited the formation of dark dots on the surface of zebrafish skin in a dose-dependent manner (Figure 5a). More specifically, we detected that administration of 5 µM CUR (highest tested concentration) inhibited the production of melanin in zebrafish by 50% (Figure 5b). Finally, we noticed that although BDMC showed the highest bioactivity in in vitro experiments, it was not the most effective compound in in vivo experiments. In particular, we observed that the relative amount of melanin in zebrafish embryos exposed to 1.25, 2.5, and 5 µM BDMC was 0.92-, 0.84-, and 0.53-fold lower, respectively, than that in the control (Figure 5b). In this study, we evaluated the potential antimelanogenic effects of CUR, DMC, and BDMC. Among the tested curcuminoids, CUR and BDMC tended to weaken the α-MSH-induced production of melanin. These two compounds reduced not only the intracellular, but also the extracellular melanin contents. Moreover, CUR suppressed the α-MSH-induced increase in tyrosinase activity in B16F10 cells [7]. Notably, the inhibitory effect of CUR was stronger than that of another well-known whitening agent, kojic acid, which has already been reported in many previous studies [20,21,22]. Although the pharmacokinetics study of curcuminoids reported poor bioavailability of these chemicals [23], skin-whitening reagents were not designed for oral administration, but topical cosmetics. In order to overcome this limitation, researchers tried to use curcumin in encapsulated form, and this advanced formulation was very promising [24]. In a previous study, 10 μM CUR was reported to suppress tyrosinase activity by 47.85% [25]. This study’s reported suppression ratio was slightly lower than that in our study; however, their experimental standard deviation was greater. Despite small differences in the inhibition ratio, other studies have also reported the inhibitory effect of CUR against tyrosinase activity and melanin production in B16F10 cells [20,21] and human melanocytes [22,26]. Based on these studies, CUR has strong antimelanogenic activity and can thus be considered a strong candidate whitening agent. In contrast to its antityrosinase activity [27], the antimelanogenic effect of BDMC has not been reported previously. Nonetheless, our in vitro results suggested that BDMC has great potential as a skin-whitening agent. Our quantitative analysis of the mRNA levels of melanin-production-related genes in B16F10 cells revealed that the α-MSH-induced upregulation in the expression of these genes was ameliorated following administration of these three curcumin derivatives. CUR-induced reduction in the level of key proteins in melanogenesis, including MITF, tyrosinase, TRP-1, and TRP-2 was previously reported [21], but changes in the levels of mRNA were not reported. As reported in a previous study, CUR downregulated the expression of members of the MITF-tyrosinase signaling pathway at the protein level [21]. Likewise, our study suggested that the mRNA levels of melanogenesis-related genes were also reduced following treatment with CUR. Combining these findings and those of previous studies, we concluded that CUR inhibits melanogenesis by suppressing the expression of melanogenesis-related genes regulated by the MC1R signaling pathway, including MITF, TYR, TRP-1, and TRP-2 [7,28,29]. Administration of DMC downregulated the expression of melanogenesis-related genes; however, the degree of inhibition was lower than that of CUR and BDMC. In addition, DMC did not lead to a reduction in the total melanin contents in B16F10 cells. To explain these results, further studies are needed. Similar to CUR, BDMC also ameliorated the α-MSH-induced activation of the melanogenesis signaling pathway. To date, BDMC was only known to inhibit tyrosinase [27], which is obviously an enzyme that plays a very important role in the synthesis of melanin; however, melanogenesis is much more tightly regulated by the melanogenesis signaling pathway [7,29]. Hence, we approached it here from a diverse perspective and found that BDMC reduced not only the production of melanin in B16F10 melanoma cells, but also the mRNA level of melanin-production-regulating genes including Mitf, Tyr, Trp-1, and Trp-2. This finding suggested that administration of CUR or BDMC ameliorated the MC1R signaling pathway-induced upregulated expression of melanin-production-related genes [7]. In vivo evaluation of the biological activity of a natural compound is one of the most important steps in exploring the potential medicinal use of natural products, because sometimes the biological activity of a compound evaluated in an in vitro system might be different from that exhibited in an in vivo system [18,30]. In addition, the rapid exploration of new candidates is important in the screening phase to reduce time and cost. Therefore, many research groups have been trying to develop faster and more effective in vivo evaluation systems [3,19]. Many studies, including our own, have shown the effectiveness of the zebrafish embryo model for screening compounds with inhibitory activity against melanogenesis [3,17,31]. The main biosynthetic pathways in zebrafish are very similar to those in humans [3]. In addition, the targets of well-known inhibitors of melanogenesis were shown to be similar, and their efficacy has already been confirmed in both the zebrafish embryo model and humans [32,33]. In this study, we screened three curcumin derivatives for their antimelanogenic properties using zebrafish embryos. None of the tested curcuminoids showed acute toxicity at concentrations below 5 μM in 96 hpf zebrafish. However, at a concentration of 5 μM, both abnormal development of zebrafish embryos and lethality were confirmed. Notably, 5 μM CUR was shown to result in the tail bending of zebrafish embryos. A previous study also reported the malformation of zebrafish embryos in the presence of CUR [34]. In that study, the toxic effects of CUR were reported to occur in a dose-dependent manner [34], with a minimum effective concentration of 5 μM, which is consistent with our findings [34]. Considering the malformations observed in zebrafish embryos, we concluded that CUR is cytotoxic in zebrafish embryos during early development. Both CUR and BDMC exhibited outstanding inhibition properties in the in vivo test. Importantly, the inhibitory effect of both curcuminoids was stronger than that of kojic acid, a compound used as positive control in our study and one of the most popular whitening agents in the cosmetic industry [6]. In addition, the concentration of kojic acid was 8 mM, which was much higher than that of CUR (5 µM) or BDMC (5 µM). Another compound frequently used as positive control in antimelanogenic studies, arbutin, has also been used at a concentration range of 10 to 20 mM [3,35,36], with both compounds, arbutin and kojic acid, being reported to have similar activity and working concentrations [3]. Compared with these positive control compounds, the working concentration of our two candidate agents was much lower (5 µM for curcuminoids vs. 10 mM for the two positive controls) in the in vivo investigation of melanin production. Hence, these findings suggested that our curcuminoid candidate compounds have great potential to be used as whitening agents in cosmetics. In conclusion, as all these results demonstrate, CUR and BDMC have outstanding inhibition effects on melanin production. Considering the toxic effect of CUR on zebrafish embryos during early-stage development, BDMC may be used as a whitening agent for melanogenesis by inhibiting melanin-production-related proteins TYR, TRP-1, and TRP-2 in both in vitro and in vivo.

PMC10000578

Laura Boyero,José Francisco Noguera-Uclés,Alejandro Castillo-Peña,Ana Salinas,Amparo Sánchez-Gastaldo,Miriam Alonso,Johana Cristina Benedetti,Reyes Bernabé-Caro,Luis Paz-Ares,Sonia Molina-Pinelo

Aberrant Methylation of the Imprinted C19MC and MIR371-3 Clusters in Patients with Non-Small Cell Lung Cancer

25-02-2023

DNA methylation,C19MC and MIR371-3 clusters,miRNA–target mRNA expression,prognosis,non-small cell lung cancer (NSCLC)

Simple Summary Aberrations in DNA methylation profiles may alter the expression of key miRNAs in non-small cell lung cancer. In this study, we focus on the analysis of the imprinted C19MC and MIR371-3 miRNA clusters due to their oncogenic role. We identified the DNA methylation status and discovered its deregulated target genes in this disease. Additionally, we found five downstream target genes that were correlated with worse overall survival in non-small cell lung cancer. We conclude that C19MC and MIR371-3 are key players in lung cancer because their polycistronic epigenetic regulation leads to differential tumor expression, affecting downstream targets with prognostic value. Abstract Epigenetic mechanisms have emerged as an important contributor to tumor development through the modulation of gene expression. Our objective was to identify the methylation profile of the imprinted C19MC and MIR371-3 clusters in patients with non-small cell lung cancer (NSCLC) and to find their potential target genes, as well as to study their prognostic role. DNA methylation status was analyzed in a NSCLC patient cohort (n = 47) and compared with a control cohort including COPD patients and non-COPD subjects (n = 23) using the Illumina Infinium Human Methylation 450 BeadChip. Hypomethylation of miRNAs located on chromosome 19q13.42 was found to be specific for tumor tissue. We then identified the target mRNA–miRNA regulatory network for the components of the C19MC and MIR371-3 clusters using the miRTargetLink 2.0 Human tool. The correlations of miRNA-target mRNA expression from primary lung tumors were analyzed using the CancerMIRNome tool. From those negative correlations identified, we found that a lower expression of 5 of the target genes (FOXF2, KLF13, MICA, TCEAL1 and TGFBR2) was significantly associated with poor overall survival. Taken together, this study demonstrates that the imprinted C19MC and MIR371-3 miRNA clusters undergo polycistronic epigenetic regulation leading to deregulation of important and common target genes with potential prognostic value in lung cancer.

Aberrant Methylation of the Imprinted C19MC and MIR371-3 Clusters in Patients with Non-Small Cell Lung Cancer Aberrations in DNA methylation profiles may alter the expression of key miRNAs in non-small cell lung cancer. In this study, we focus on the analysis of the imprinted C19MC and MIR371-3 miRNA clusters due to their oncogenic role. We identified the DNA methylation status and discovered its deregulated target genes in this disease. Additionally, we found five downstream target genes that were correlated with worse overall survival in non-small cell lung cancer. We conclude that C19MC and MIR371-3 are key players in lung cancer because their polycistronic epigenetic regulation leads to differential tumor expression, affecting downstream targets with prognostic value. Epigenetic mechanisms have emerged as an important contributor to tumor development through the modulation of gene expression. Our objective was to identify the methylation profile of the imprinted C19MC and MIR371-3 clusters in patients with non-small cell lung cancer (NSCLC) and to find their potential target genes, as well as to study their prognostic role. DNA methylation status was analyzed in a NSCLC patient cohort (n = 47) and compared with a control cohort including COPD patients and non-COPD subjects (n = 23) using the Illumina Infinium Human Methylation 450 BeadChip. Hypomethylation of miRNAs located on chromosome 19q13.42 was found to be specific for tumor tissue. We then identified the target mRNA–miRNA regulatory network for the components of the C19MC and MIR371-3 clusters using the miRTargetLink 2.0 Human tool. The correlations of miRNA-target mRNA expression from primary lung tumors were analyzed using the CancerMIRNome tool. From those negative correlations identified, we found that a lower expression of 5 of the target genes (FOXF2, KLF13, MICA, TCEAL1 and TGFBR2) was significantly associated with poor overall survival. Taken together, this study demonstrates that the imprinted C19MC and MIR371-3 miRNA clusters undergo polycistronic epigenetic regulation leading to deregulation of important and common target genes with potential prognostic value in lung cancer. Lung cancer remains the most common cause of mortality worldwide, and tobacco exposure increases its risk of developing [1]. Furthermore, the incidence of lung cancer is significantly higher in patients diagnosed with chronic obstructive lung disease (COPD), reflecting the impact of smoking habits in both pathologies [2,3]. Approximately 85% of all lung cancers are non-small cell lung cancer (NSCLC). Histologically, NSCLC is classified as adenocarcinoma (ADC), squamous cell carcinoma and large cell carcinoma (SCC) [4]. Molecular analyses have led to advances in our understanding of NSCLC genetics and even in the identification of biomarkers that can predict its occurrence [5]. This includes the role of microRNAs (miRNAs) in the disease, involved in the complexity of gene expression regulation. Therefore, a single miRNA can exert its regulatory function on several target mRNAs, and a particular target can be regulated by multiple miRNAs [6]. There are numerous miRNAs involved in cancer-relevant processes, and many of them are clustered on the genome and act in coordinated regulatory networks. Furthermore, some evidence has even been provided suggesting that several miRNAs are able to identify patients with an increased risk of developing lung cancer, as well as COPD [7,8]. For a more extensive review of oncomiRs in lung cancer, see [9,10]. In this context, epigenetics appears to play an important role in the regulation of miRNA expression levels [11]. Aberrations in methylation profiles can promote silencing of tumor suppressor microRNAs or overexpression of oncogenic miRNAs (oncomiRs) [12]. For example, a significant upregulation of the miR-17-92 cluster has been reported in lung cancer [13]. In addition, some oncomiRs can be located in imprinted genomic regions. Such is the case of the imprinted delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) cluster, which includes two large miRNA clusters between other coding and non-coding transcripts, and has been reported to contribute to tumorigenesis in the lung, leukemia, breast, and hepatoblastoma, among others [14,15,16,17,18]. In addition, alterations in other clusters located in imprinted regions are also attracting interest due to their downstream targets and their involvement in oncogenic and drug resistance mechanisms, such as the chromosome 19 microRNA (C19MC) and MIR371-3 clusters [19,20,21]. The C19MC and MIR371-3 clusters are located on chromosome 19q13.42. The first cluster includes forty-six miRNA genes and the second one contains four miRNAs (miR-371, miR-372, miR-373 and miR-373*). Both clusters are only expressed from their paternal allele, so they are functionally haploid and, furthermore, they are expressed mainly in embryonic tissue, particularly in the placenta [22,23]. However, aberrations that involve some miRNAs from both these clusters have been linked to tumoral processes, such as immunomodulation, angiogenesis, invasion, and cell reprograming [21,24,25,26,27,28,29]. In fact, some miRNAs through exosomes have been proposed as specific cell-to-cell communication mediators [21,27,30]. Regarding lung cancer, the regulation of imprinted C19MC and MIR371-3 has not been extensively and systematically reviewed in patients with NSCLC. For this reason, we have analyzed the methylation profile of the C19MC and MIR371-3 clusters in lung tumors compared to non-tumoral lung tissue in NSCLC patients. We have also assessed the methylation patterns of both clusters in COPD patients to study their association in a population at high risk of developing lung cancer. In addition, we were able to experimentally identify and validate deregulated targets, as well as their prognostic role in the disease. The present study was carried out on 70 subjects from the Virgen del Rocio University Hospital (Seville, Spain). Samples were divided into 2 cohorts according to the underlying pathology. The first cohort consisted of 47 NSCLC patients who had undergone surgical resection at an early clinical stage. During surgical resection, adjacent normal and tumor tissue samples were collected from all patients and immediately frozen at −80 °C until further use. The clinical characteristics of patients with NSCLC (n = 47) are summarized in Supplementary Table S1. The second cohort was used as control without lung cancer (n = 23). This control cohort consisted of COPD patients and non-COPD subjects (Supplementary Table S1) who had undergone bullectomy or bronchoscopic biopsy with a negative diagnosis of lung cancer. Both cohorts were used for the analysis of the methylation profile. The protocol of the study and the use of human samples were approved by the Ethics Committee of our hospital (1381-N-21). Written informed consent was obtained from all patients included in the study. Genomic DNA was extracted from 15 mg adjacent normal and tumor tissue samples using the QIAamp DNA mini kit (QIAGEN, Hilden, Germany). DNA was quantified using the QuantiFluor dsDNA system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. DNA methylation status at the CpG sites within the C19MC and MIR371-3 clusters was identified using the Illumina Infinium Human Methylation 450 BeadChip (Illumina Inc., San Diego, CA, USA). 500 ng of DNA were treated with sodium bisulfate using the EZ DNA Methylation™ Kit and cleaned with the ZR-96 DNA Clean-up Kit™ (Zymo Research, Irvine, CA, USA). Subsequently, the following steps were performed: amplification, hybridization and imaging. Intensity data was analyzed with Illumina’s GenomeStudio, from which, β-scores (i.e., the proportion of total fluorescence signal from the methylation-specific probe or color channel) were obtained. Infinium HD-based assays included sample-dependent and sample-independent controls for the highest quality data. The methylome data was processed using the R/Bioconductor package RnBeads [31]. After a quality check, intensity normalization was performed by SWAN method [32] and converted to β values. The probes were tested for differential methylation with the limma linear model followed by empirical Bayes methods for the comparisons of interest [33]. Statistical significance was established using the Benjamini–Hochberg false discovery rate (FDR) with a value lower than 0.05. The DNA methylation status and CpG chromosomal location were displayed using the Circos software [34]. Furthermore, the methylation data was visualized by the Wash U Epigenome Browser [35]. Strong validated miRNA–mRNA interactions were identified for the C19MC and MIR371-3 miRNA clusters with the miRTargetLink 2.0 Human tool. miRTargetLink collects information from various databases, including the sources miRBase (v.22.1) and miRTarBase (v.8) [36,37]. Gene expression analysis on tumor and normal lung tissue were analyzed from the Cancer Genome Atlas datasets (TCGA) using GEPIA 2.0 (http://gepia2.cancer-pku.cn/#index, accessed on 6 September 2021). GEPIA is an interactive web server that compiles data from TCGA and GTEx projects, using a standard processing pipeline. This tool allows for a customizable analysis of the collected data [38]. The molecular function and proposed biological process of the experimentally verified mRNAs were determined using the PANTHER program (http://pantherdb.org, accessed on 29 September 2021). The PANTHER classification system contains a comprehensive, annotated “library” of phylogenetic trees of gene families designed to classify proteins (and their genes) to facilitate high-throughput analysis [39]. Besides, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to know the molecular interaction network (https://www.genome.jp/kegg/pathway.html, accessed on 3 October 2021) [40]. Transcriptome profiling data from TCGA datasets were downloaded using the CancerMIRNome tool [41]. MicroRNA and mRNA expression data from primary tumors were retrieved from the LUAD (Lung Adenocarcinoma) and LUSC (Lung Squamous Carcinoma) datasets included in TCGA. Only samples labeled as “tumor” and expression level (Log2 Counts per Million (CPM)) > −3.322 were used for the analyses. To test the association between paired miRNA–mRNA profiles, the Pearson correlation coefficients and p-values were computed. p-values lower than 0.05 were considered statistically significant. To analyze the prognosis associated with the target genes of the C19MC and MIR371-3 miRNA clusters, the Kaplan–Meier survival plots to overall survival time were obtained using the Kaplan–Meier (KM) plotter website [42], where unprocessed. CEL files from the Gene Expression Omnibus (GEO), the European Genome-phenome Archive (EGA) and the Cancer Genome Atlas (TCGA) repositories were normalized in the R environment. The datasets included in the Kaplan–Meier plotter website are GSE3141, GSE4573, GSE8894, GSE14814, GSE19188, GSE29013, GSE31210, GSE37745, EGA and TCGA (n = 1715 patients). The best-performing threshold from computed lower and upper quartiles was used as cut-off point for the definition of high and low expression of the analyzed genes. Overall survival (OS) was determined from the date of diagnosis to the date of death. p-values lower than 0.05 were considered statistically significant. To evaluate the potential role of the C19MC and MIR371-3 miRNA clusters in lung cancer, we analyzed the methylation status of both of them in human lung tissues from a NSCLC patient cohort (n = 47) and a control cohort (n = 23) of the Virgen del Rocio University Hospital (Seville, Spain). The methylation profile of these clustered miRNAs, which are located on chromosome 19q13.42, were evaluated in human tumor samples compared to paired non-tumoral tissue by using the Illumina Infinium Human Methylation 450 BeadChip. The methylation levels in lung cancer versus paired non-tumoral tissues are represented in Figure 1A and Supplementary Table S2. Patients with lung cancer at our hospital showed DNA hypomethylation at 50 miRNAs included in the C19MC and MIR371-3 clusters after standardisation with non-tumoral control samples. Statistically significant differences (adjusted p-value < 0.05) were detected in the large C19MC cluster (46 miRNAs) and the closely distal MIR371–3 cluster (miR-371a, miR-371b, miR-372 and miR-373). Among all miRNAs, miR-520b, miR-520c, miR-520f, miR-526a1, miR-1283-1 and miR-1283-2 showed greater changes in the DNA hypomethylation pattern in NSCLC patients (Figure 1B,C and Table 1). We next analyzed the DNA methylation pattern of both clusters in patients at high risk of developing lung cancer, such as patients with COPD. In these patients, we found that the C19MC cluster methylation profile showed no statistical differences compared to the control group (Figure 1B and Table S2). We even found that miRNA-520e, miR-524 and miR-516b2 were hypermethylated in COPD patients versus the non-tumoral control group (Figure 1B). However, these differences did not reach statistical significance. In the case of the MIR371-3 cluster, changes in the DNA methylation levels were negligible between both patients groups (Figure 1C and Table S2). To analyze genomic features associated with different mechanisms of the transcriptional regulation of the C19MC and MIR371-3 miRNA clusters, we used the Wash U Epigenome Browser [33] to display the epigenomic mapping of both clusters (19q13.42) on the reference human genome (hg19; chr19: 54,030,000–54,430,000 genomic coordinates) (Figure 2A). Thus, it is shown which transcriptional mechanisms (direct regulators and structural determinants) act at each locus throughout both clusters (Figure 2B). We observed that enhancers are distributed outside the regions where the C19MC and MIR371-3 miRNA clusters are located (yellow histograms). In these same regions, we found low activity of polycomb protein-mediated epigenetic regulator (grey histograms). In addition, we identified two heterochromatin-rich domains located in the C19MC cluster region (purple histograms). One of these highly condensed regions is observed at 5′ of the C19MC miRNA cluster. Interestingly, the 5′ and 3′ flanking regions of both clusters showed transcription initiation activity, marked by small active transcriptional start sites (TSS) shown by red histograms. No other region of the C19MC and MIR371-3 miRNA clusters showed active TSS. Behind these active TSS, we found regions with strong transcription activity (green histograms) (Figure 2B). We identified a CpG island in the 5′ region at the beginning of the C19MC miRNA cluster (~54,150,000 bp; CpG count: 86; and citosine base count plus guanine base count: 762) and another in the MIR371-3 miRNA cluster (~54,270,000 bp; CpG count: 23; and citosine count plus guanine count: 157) (green lines) (Figure 2C). However, the CpG sites were distributed throughout both clusters (white lines) (Figure 2D). The CG-content was similar from 54,030,000 to 54,430,000 bp on chromosome 19 (Figure 2E). Due to lung cancer-specific hypomethylation, in order to evaluate the potential role of the C19MC and MIR371-3 miRNA clusters as biomarkers in different histological subtypes of lung cancer, we analyzed the methylation pattern in SCC versus adenocarcinoma, pre-normalizing each patient with the methylation status of the matched non-tumoral tissue. The DNA-methylation levels of the C19MC and MIR371-3 clusters were consistently hypomethylated in both histological subtypes in comparison with non-tumoral tissue (Table S2). Furthermore, these differences were statistically significant for both clusters (C19MC, p < 0.001; MIR371-2, p = 0.030) (Figure 3). We have graphically represented a network with experimentally validated miRNA–mRNA interactions for each component of the C19MC and MIR371-3 clusters in order to study their functional relevance (Figure 4). According to the data included in miRTargetLink software [36] and considering only strong evidence targets, a total of 115 genes were found targeted by at least one of the aforementioned miRNAs. In the network of the C19MC miRNA cluster, the nodes with several connections were those corresponding to miRNA-512-5p, miR-518a-5p, miR-519b-3p, miR-519a-3p, miR-519d-3p, miR-520a-3p, miR-520c-3p, miR-520g-3p, miR-520h, miR-524-5p and miR-525-3p (Figure 4A). Among the MIR371-3 cluster compounds, miR-371a-3p, miR-372-3p and miR-373-3p showed a high number of connections. The common target genes possessed a high interaction grade with different miRNAs simultaneously (Figure 4B and Table 2). Of these targets, we identified six genes (CD44, CDKN1A, MTOR, SIRT1, TGFBR2 and VEGFA) that were targeted by miRNAs from both clusters. We explored the transcriptional levels of the previously identified validated target genes in cancer and normal lung tissues in the TCGA/GTEx data available in GEPIA2 [38]. Of the 115 target genes validated by miRTargetLink, 31 were significantly underexpressed in at least one of the histological subtypes of NSCLC compared to non-tumor tissue (Table 3 and Figure 5). Seventeen of them showed significant differences in both histological subtypes of lung cancer (adenocarcinoma and SCC). It is worth highlighting those genes that we found targeted by several miRNAs from both clusters, such as the tumor suppressor genes (TSG) CDKN1A (regulated by miR-372-3p, miR-512-5p, miR-515-3p, miR-519a-3p, miR-519b-3p, miR-519e-3p and miR-520a-3p) and TGFBR2 (targeted by miRNA-372-3p, miRN-373-3p and miR-520a-3p). As displayed in Figure 6, we classified the validated targets by gene ontology (GO) molecular function and biological processes using the PANTHER software [39]. The main GO molecular functions were binding (GO:0005488) (39.0%), catalytic activity (GO:0003824) (26.8%) and transcription regulator activity (GO:0001071) (14.6%). The primary binding types were protein binding (GO:0005515) and organic cyclic compound binding (GO:0097159). In the case of catalytic activity, we primarily found hydrolase (GO:0016787), transferase (GO:0016740) and catalytic (GO:0140096) activities. Validated targets displayed three main biological processes: response to stimulus (GO:0050896) (15.0%), biological regulation (GO:0065007) (17.7%) and cellular process (GO:0009987) (19.5%). In the latter, we found represented cell communication (GO:0030234), signal transduction (GO:0007165) and cellular metabolic process (GO:0044237). Finally, we found that 31% of validated targets with aberrant expression in lung cancer were classified in cancer by the KEGG pathways (adjusted p = 7.0 × 10−3). Since we had compelling data on the functional relationship between miRNA–target for the C19MC and MIR371-3 miRNA clusters, as well as differential expression of the validated target genes in lung cancer, we tested whether miRNA–target correlations extended to primary tumors. We studied these correlations using external expression data obtained from the TCGA datasets through the CancerMIRNome tool [41]. We found 11 significant negative correlations for lung adenocarcinoma (Figure 7A), all of them for miRNAs included only in the C19MC cluster. On the other hand, 8 were found for SCC (Figure 7B), this time including miRNAs from both clusters. It should be noted that CDKN1A was the target gene with the highest number of significant negative correlations (p < 0.05) in both adenocarcinoma (miRNA-512-5p, miRNA-512-3p, miRNA-520a-3p and miRNA-520h) and SCC (miRNA-515-3p, miRNA-519b-3p, miRNA-520a-3p and mir-372-3p). Furthermore, DAPK2 was also negatively correlated with miRNA-520g-3p and miRNA-520h in both histological subtypes of lung cancer (p < 0.01). Other significant correlations for adenocarcinoma were PTK2B-miRNA-517c-3p (p < 0.001), FOXF2-miRNA-519a-3p (p = 0.001), TGFBR2-miRNA-520a-3p (p = 0.009), MICA-miRNA-520c-3p (p = 0.007) and TECEAL1-miRNA-520g-3p (p = 0.037); while for SCC, they were JAG1-miRNA-524-5p (p < 0.001) and KLF13-miRNA-372-3p (p = 0.003). To evaluate whether the genes targeted by the C19MC and MIR371-3 clusters with significant negative correlations were associated with clinical outcomes in patients with lung cancer, we analyzed their gene expression levels according to OS data using the KM Plotter website [42]. We found that five target genes were also significantly associated with worsening OS (Figure 8). These genes were FOXF2 (HR = 0.66, 95% CI = 0.58–0.75, p < 0.001), KLF13 (HR = 0.61, 95% CI = 0.52–0.72, p < 0.001), MICA (HR = 0.75, 95% CI = 0.66–0.86, p < 0.001), TCEAL1 (HR = 0.72, 95% CI = 0.63–0.82, p < 0.001) and TGFBR2 (HR = 0.66, 95% CI = 0.58–0.75, p < 0.001). The expression levels of the rest of the genes did not show significant differences regarding the OS in patients with lung cancer. In the present study, we identified the methylation pattern of the imprinted C19MC and MIR371-3 clusters in patients with NSCLC. Specifically, we identified that all compounds of both clusters are hypomethylated in tumor lung tissue compared to paired normal lung tissue. Importantly, this imprinted cluster-specific methylation signature is restricted to lung cancer, as it is absent in patients with COPD, who have an increased risk of developing lung cancer. Furthermore, these epigenetic changes observed in patients with NSCLC are negatively associated with the expression of relevant target genes in the disease; even five of them were significantly associated with the prognosis of this disease. This methylation profile is consistent with the epigenetic features found at the 19q13.42 locus, in which, the C19MC and MIR371-3 miRNA clusters are located. Epigenetic modifications are characterized by not altering the nucleotide sequence, but by regulating genetic structure and expression reversibly. Early events in tumorigenesis and its progression are related to DNA methylation, histone modifications, nucleosome remodeling and miRNA, which are key epigenetic players [43]. In the particular case of C19MC and MIR371-3 miRNA clusters, the mechanisms involved in regulation were the presence of enhancers, CpG islands, active TSS, heterochromatin-rich domains, as well as low activity of polycomb complexes. In addition, these differences were evident for both histological subtypes of NSCLC. Thus, we found that although all subtypes are hypomethylated, this is more accentuated in SCC than in lung adenocarcinoma. At the same locus, the Protein Tyrosine Phosphatase Receptor type H (PTPRH) has also been confirmed to be hypomethylated and this is correlated with increased gene expression and leads to a poor prognosis in NSCLC [44,45]. Therefore, the epigenetic features of this region have a significant effect on the disease. Members of the C19MC and MIR371-3 clusters are expressed almost exclusively in human embryonic stem cells (hESC) and rapidly down-regulated during the differentiation process [46,47,48]. However, they are over-expressed in cancer [20,48], suggesting a tumorigenic role and a possible maintenance function of tumor-associated progenitor cells for these clusters when reactivated [49]. Higher expression of members of the C19MC and MIR371-3 clusters has also been reported in thyroid adenomas [50] and parathyroid carcinomas [48], germ cell tumors [29,51,52], retinoblastoma [53], breast cancer [54], gastric adenocarcinoma [55] and esophageal cancer [56,57], among others. And this increase affects tumor growth, differentiation, progression and aggressiveness and, ultimately, patient survival. This cluster overexpression phenomenon also occurs in other sets of miRNAs in NSCLC, such as the miR-23a/27a/24-2 cluster, which has predictive value in early stages and stimulates postoperative progression by inducing tumor suppressor gene silencing [58]. Activation of all miRNAs from the DLK1-DIO3 locus has also been described for human lung adenocarcinoma samples, which is associated with cell stemness and its targets are involved in embryogenesis [59]. Moreover, it is hypomethylated in current and former smokers with NSCLC, suggesting a relevant role in the pathogenesis of lung cancer [15]. The 14q32 miRNA cluster is another example of up-regulation due to DNA hypomethylation in metastatic lung adenocarcinoma patients. Overexpression of this cluster induces cell migration and invasion and has prognostic value [60]. Furthermore, overexpression of the miR-17-92 cluster, which is a highly conserved oncogene cluster, has been frequently reported in lung cancer, especially in the small cell lung cancer subtype, promoting cell growth [61,62]. Therefore, the gene regulation mechanism mediated by miRNAs is very interesting due to its ability to associate with mRNAs of multiple targets. On the other hand, we identified a total of 115 strongly validated targets for the C19MC or MIR371-3 clusters, and 31 of them presented a significant lower transcriptional level in NSCLC tissue compared to normal. In other words, we identified only those miRNA–mRNA interactions that had been experimentally validated previously. Of these, only six genes were targeted by members of both clusters: CD44, CDKN1A, MTOR, SIRT1, TGFBR2 and VEGFA. In addition, these miRNA–target interactions were verified with TCGA data for primary NSCLC tumors, corroborating significant miRNA–target negative correlations in both clusters and histological subtypes of the disease. Interestingly, redundancy is observed in the miRNAs belonging to C19MC and MIR371-3, since many of them share targets, as it can be inferred from our results. An explanation for this is the high degree of homology that has been described for some C19MC miRNAs through a seed region (5′-AAGUGC-3’), which can be found in several members of the cluster at different positions [20,46]. This suggests that, in addition to a polycistronic regulation, the targets of these miRNAs are common or share functions. Bioinformatic predictions for this seed region relate these miRNAs to cellular proliferation and apoptosis [20,63]. Something similar occurs with the MIR371-3 cluster, which has identical seed sequences that are similar to its murine miR290-295 homolog [64]. In this study, we even found that this redundancy occurs between the two clusters C19MC and MIR371-3. The CDKN1A and DAPK2 genes are notable for being negatively correlated with and targeted by multiple miRNAs from these clusters in the two most common subtypes of NSCLC (lung adenocarcinoma and SCC). CDKN1A, also known as p21, plays a critical role in the cellular response to DNA damage, and its overexpression results in p53-mediated cell cycle arrest [65]. It has been reported that CDKN1A/p21 can be blocked in NSCLC by oncomiRs, such as miR-212/132 [66] or miR-93. The latter can act directly or indirectly, thereby inhibiting liver kinase B1 (LKB1) [67]. This promotes proliferation and metastases through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in NSCLC. Other authors have also confirmed the relationship between CDKN1A/p21 and members of the MIR371-3 cluster in hESCs [68], specifically miR-372. On the other hand, DAPK2 is a serine/threonine kinase that promotes cell apoptosis and autophagy [69] by activating the oncogenic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway [70], which sensitizes resistant cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated death [71]. DAPK2 expression has been reported to be significantly associated with the poor prognosis in NSCLC [70,72]. As in this study, DAPK2 is also downregulated by miR-520h in breast cancer [73] and miR-520g in epithelial ovarian cancer [74] and it contributes to chemoresistance. Finally, we found that five of the genes targeted by members of the C19MC and MIR371-3 clusters correlated with worse OS in lung cancer. These genes were FOXF2, KLF13, MICA, TCEAL1 and TGFBR2. They are known transcription regulators and oncogenes, some involved in immune evasion and resistance to immune checkpoint inhibitors [75], which places them in the focus of current cancer research. For example, according to our results, decreased FOXF2 expression has been reported as an independent predictor of poor prognosis for patients with early-stage NSCLC [76]. FOXF2 deregulation ought to an aberrant DNA methylation status has also recently been identified for gastric cancer [77]. In the case of MICA, Okita et al. reported that PD-L1low/MICA/Bhigh is associated with a better clinical outcome in patients with stage I-IIIA NSCLC. On the other hand, both oncogenic and tumor suppressor roles have been attributed to the transforming growth factor beta (TGF-β) pathway, depending on both the type of tumor and its stage [78]. In this matter, TGFBR2 [79] has recently been proposed as a tumor suppressor with prognostic value in early-stage NSCLC; however, there is no prior evidence of an association between KLF13 and lung cancer prognosis, so further research is required. Our study has the limitation that the mechanism underlying the alterated methylation status of the C19MC- and MIR371-3-imprinted clusters was not evaluated. Cancer-related gene hypomethylation is common in solid tumors, which could contribute to increased expression of oncogenes. In this case, miRNAs may act as oncomiRs by inhibiting the expression of tumor suppressor genes. DNA methylation is a dynamic process regulated by the action of DNA demethylases and DNA methyltransferases (DNMT) [80]. Alterations in the expression or activity of these enzymes can lead to changes in DNA methylation patterns that can trigger alterations in key genes involved in cancer development. For example, Zhang et al. have reported that DNMT1, DNMT3A and DNMT3B show frequency alterations in approximately 3% to 5% of lung cancer patients from the cBioPortal datasets [81]. However, further research is needed to validate the mechanisms in detail, e.g., in organoid in vitro and/or in preclinical in vivo models, to fully understand the mechanisms upstream involved in the aberrant methylation of both clusters and to assess the potential therapeutic applications of targeting them. In addition, we identify relevant miRNA–mRNA interactions in NSCLC patients. In other words, the regulation of the expression of these genes can be partly explained by the activity of these miRNAs; nevertheless, it should be noted that additional mechanisms may also be involved in the regulation of these genes, such as deletion, amplification, mutation, fusion and multiple alterations, and even other miRNAs not included in these clusters, as their regulation can be mediated by the action of many miRNAs. Despite these limitations, this study provides important insights into the consequences of these epigenetic alterations in NSCLC patients and highlights potential targets for future research and therapy. On the other hand, another limitation would be the sample size of patients with COPD included in the study. We have analyzed the DNA methylation pattern of the C19MC and MIR371-3 clusters in a group of COPD patients because they have a three- to six-fold increased risk of developing lung cancer, even if they quit smoking [2]. In our study, we found no statistical differences between the control group without lung cancer and COPD. However, sample size is an important consideration because it may affect the accuracy and reliability of the results. A larger size should be considered to conclude the effect of methylation status of both clusters in this disease. In conclusion, this study demonstrates that the imprinted C19MC and MIR371-3 clusters undergo polycistronic epigenetic regulation that leads to differential tumor expression in NSCLC patients. These differences, in turn, deregulate the expression of important and common target genes, many of them with a clear oncogenic and regulatory role in this disease, highlighting five genes (FOXF2, KLF13, MICA, TCEAL1, TGFBR2) that have also a potential prognostic value in NSCLC patients. All these characteristics make the different components of both clusters an interesting target in oncology that needs to be further investigated; it would even be interesting to evaluate the use of methylation agents as an alternative approach to lung cancer therapy.

PMC10000581

Lara Tinacci,Deyan Stratev,Mariyana Strateva,Georgi Zhelyazkov,Ralica Kyuchukova,Andrea Armani

An Authentication Survey on Retail Seafood Products Sold on the Bulgarian Market Underlines the Need for Upgrading the Traceability System

02-03-2023

seafood labeling,authenticity,mislabeling,DNA barcoding,PCR–RFLP analysis seafood,marking

Economically motivated or accidental species substitutions lead to economic and potential health damage to consumers with a loss of confidence in the fishery supply chain. In the present study, a three–year survey on 199 retail seafood products sold on the Bulgarian market was addressed to assess: (1) product authenticity by molecular identification; (2) trade name compliance to the list of official trade names accepted in the territory; (3) adherence of the list in force to the market supply. DNA barcoding on mitochondrial and nuclear genes was applied for the identification of whitefish (WF), crustaceans (C) and mollusks (cephalopods—MC; gastropods—MG; bivalves—MB) except for Mytilus sp. products for which the analysis was conducted with a previously validated RFLP PCR protocol. Identification at the species level was obtained for 94.5% of the products. Failures in species allocation were reconducted due to low resolution and reliability or the absence of reference sequences. The study highlighted an overall mislabeling rate of 11%. WF showed the highest mislabeling rate (14%), followed by MB (12.5%), MC (10%) and C (7.9%). This evidence emphasized the use of DNA–based methods as tools for seafood authentication. The presence of non–compliant trade names and the ineffectiveness of the list to describe the market species varieties attested to the need to improve seafood labeling and traceability at the national level.

An Authentication Survey on Retail Seafood Products Sold on the Bulgarian Market Underlines the Need for Upgrading the Traceability System Economically motivated or accidental species substitutions lead to economic and potential health damage to consumers with a loss of confidence in the fishery supply chain. In the present study, a three–year survey on 199 retail seafood products sold on the Bulgarian market was addressed to assess: (1) product authenticity by molecular identification; (2) trade name compliance to the list of official trade names accepted in the territory; (3) adherence of the list in force to the market supply. DNA barcoding on mitochondrial and nuclear genes was applied for the identification of whitefish (WF), crustaceans (C) and mollusks (cephalopods—MC; gastropods—MG; bivalves—MB) except for Mytilus sp. products for which the analysis was conducted with a previously validated RFLP PCR protocol. Identification at the species level was obtained for 94.5% of the products. Failures in species allocation were reconducted due to low resolution and reliability or the absence of reference sequences. The study highlighted an overall mislabeling rate of 11%. WF showed the highest mislabeling rate (14%), followed by MB (12.5%), MC (10%) and C (7.9%). This evidence emphasized the use of DNA–based methods as tools for seafood authentication. The presence of non–compliant trade names and the ineffectiveness of the list to describe the market species varieties attested to the need to improve seafood labeling and traceability at the national level. Global fish consumption has grown steadily in the last five decades, driven by a combination of population growth, rising incomes and changes in food habits, as well as strong expansion in fish production [1]. Aquatic foods are increasingly recognized for their key role in food security and global nutrition. In addition, they represent an invaluable source of income and employment with a direct impact on the livelihoods of a substantial percentage of the population, mainly in Asia and Africa. According to FAO, the amount of seafood destined for human consumption in 2020 was estimated to be 20.2 kg per capita, doubling the average of 9.9 kg per capita noted in 1960. While a plateau has been reached for the volumes of aquatic products obtained from fishing activities, this trend is set to grow further, mainly due to the expansion and modernization of aquaculture systems. The increase in per capita consumption is also favorably influenced by the speeding–up of the supply chain and the improvement of transport logistics, which have led to the exponential expansion of the global product offer [1]. The European Union (EU) holds a considerable position in this scenario, with consumption estimates of around 25 kg per capita per year [2]. However, a significant variation between Member States exists. Seafood prices, purchasing ability of the consumer and net income, and culturally–driven dietary preference are the factors most frequently affecting the variability of consumption at EU and international levels [3]. In Bulgaria, apparent domestic seafood consumption appears to be limited, ranging from 5.3 kg to 7.5 kg per capita per year, and an average value of 6 kg per capita, according to EUMOFA and the Bulgarian Ministry of Agriculture for the years 2019–2020 [2,4]. In addition, according to data published by the EU Commission on consumer habits regarding fishery and aquaculture products for the year 2021, the consumption rate generally does not exceed a monthly frequency [5]. Evidence of this limited consumption is also attested by a consumer survey conducted in 2018 by Stancheva [6], from which emerged that seafood products have an average consumption rate not exceeding 1–2 times per month by about the 46% of the respondents, while only 26% declared weekly consumption. The low buying appetite towards seafood is generally due to (1) the consumer’s lack of perception of the benefits of a regular consumption and (2) the medium/high price of seafood [7]. These aspects could therefore constitute major limiting factors to the expansion of seafood market demand in Bulgaria. However, despite the unchanged consumption of fresh water and marine seafood from both inland waters and the coast of the Black Sea, an increase in fresh, chilled, frozen and variously processed marine seafood imports (fish, crustaceans and mollusks) of EU and extra–EU origin on the Bulgarian market has been recently reported [8]. This evidence was also previously highlighted in a survey conducted in 2019, in which the presence of imported seafood, especially at the large retail level, was described as mostly pre–packaged fresh and frozen [9]. It is well known that globalization and the complexity of the supply chain, together with the distribution system, can potentially expose the seafood market to an increased opportunity for the perpetration of deceptive behaviors [10,11]. Fraudulent incidents are frequently described, leading to misdescriptions, mislabeling and economically motivated species substitutions, resulting in consumer economic damage and loss of confidence in the sector chain. These incidents could potentially include health risks whenever substitution events include the illicit presence of toxic species or the omission of allergens [11,12,13]. To ensure safety, transparency, and fair trading, which are founding principles of EU food law [14,15], specific provisions have been issued for the labeling and presentation of the sale of fishery products by EU Regulation No.1379/2013 [16]. Pursuant to this regulation, the Member States are delegated to publish and update a list reporting the official seafood trade names corresponding to the species’ scientific names accepted in the national territories for product sales. According to the current EU legislation, the responsibility for the correctness of the information of the product to the consumer lies directly with the food business operator who prepares the product for sale; the organization of official controls on labeling is entrusted to a central competent authority identified by each member state [15,16,17]. Specifically, in Bulgaria, the responsibility for official controls in this area is delegated to the Bulgaria Food Safety Agency (BFSA; http://www.bfsa.bg, accessed on 30 October 2022). Nonetheless, fraudulent incidents in the seafood supply chain are still well documented both at the international and EU level [11,12,13], although data as regards labeling compliance and mislabeling seafood rates in Central–Eastern and Eastern European countries are rarely reported [16]. In this respect, the regulation also promotes control measures aimed at verifying the identity of products through the use of available technology, including DNA testing, to deter fraudulent substitution practices. Several DNA–testing techniques have been successfully applied in market surveys to verify the labeling compliance of commercial seafood collected at retail. Particularly, sequencing–based methods like FINS and DNA barcoding based on the analysis of mitochondrial (COI, cytb; 16S rRNA) targets, are recognized as valuable tools for seafood species identification. Nuclear targets (rhodopsin, Phosphoenolpyruvate Carboxykinase (PEPCK), and Polyphenolic Adhesive Protein (PAP)) have been additionally selected in the presence of introgression or hybridization phenomena [18,19,20]. Additionally and alternatively, PCR–restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), or single–stranded conformational polymorphism (SSCP) on both mitochondrial and nuclear targets have been applied as targeted methods to design genus or species–specific assays [19,20]. In this regard, the validity of an RFLP protocol on the PAP gene for species–specific discrimination of bivalve mollusks—mussels—belonging to the genus Mytilus sp., has been confirmed [21]. The exposure of the Bulgarian market to fraudulent phenomena was first documented in a pilot study conducted in 2017 [22]. In that survey, a DNA–barcoding approach applied to assess the product’s authenticity highlighted an overall substitution rate of 17.7%. The species substitutions were equally distributed between unprocessed and processed products (smoked and marinated products). Particularly, five plausibly deceptive incidents for economic gain were finally pointed out, two of which consisted in the replacement of Gadus chalcogramma (Alaska pollock) with Pangasianodon hypophthalmus (striped catfish) and the remaining three cases in the replacement of Illex argentinus (Argentine squid) with Dosidicus gigas (Humboldt squid). The remaining mislabeling incidents, consisting of species substitution between species of similar commercial value, were linked the improper training of the operators in species morphological identification [22]. Furthermore, the study highlighted the urgency of the revision of the Bulgarian official list of seafood trade names. This evidence was reaffirmed in a further study on the validity and accuracy of the new official Bulgarian list of seafood trade and the list evolution and adherence to the Bulgarian market’s trend [23]. Considering the evidence acquired in the pilot study on products belonging to two taxonomic classes (fish and gastropod mollusks) purchased at retail in a single city (Stara Zagora) [22], a three–year survey was conducted for the assessment of the authenticity of seafood products belonging to five distinct taxonomic classes, purchased in retail settings in four different cities across the country. The products’ molecular identification to verify the compliance of the labeled species designations was conducted by means of DNA barcoding and PCR–RFLP based on mitochondrial and nuclear markers. Furthermore, the labeled designations were checked against the Bulgarian official list of seafood trade names to verify the use of authorized commercial designations. Finally, the comparison of the labeled designation with the accepted designation in force was performed to verify the adherence of the official list to the products basket available at retail. In the present study, the sampling plan for selecting the seafood categories was structured by considering the characteristics of the Bulgarian market in terms of production, imports and exports [8] and the most frequent seafood fraud incidents reported in a previous study [22] and at the EU level. Within the fish taxonomical class, the sampling was targeted on white fish (WF) since Tinacci et al. [22] observed that deliberate substitutions mainly involved Alaskan pollock (Gadus chalcogrammus) products. This choice was also performed in response to clear evidence of deliberate substitution phenomena involving valuable WF, particularly those belonging to the Gadidae and Merluccidae families, both at the international and EU levels [12,24,25,26,27]. In addition, in a nationwide survey conducted on the Bulgarian fish retail market, Gadidae and Merluccidae were reported to be among the products (frozen and filleted) most frequently found on sale, especially in large retail settings [9]. The survey also revealed the association of a few generic commercial designations for a considerable number of species belonging to the two families characterized by heterogeneous commercial values. This aspect was considered by the authors as favoring consumers’ confusion on fish value and market exposure to deceitful incidents for economic gain. [9]. Within cephalopods class, the number of products was mainly selected in relation to the results of the pilot study and the sampling was primarily targeted at squid products, for which three substitutions were shown in the limited sampling of products under analysis (N = 11) [22]. Moreover, the sampling was also extended to crustaceans, mollusks bivalves, and gastropods classes by virtue of the increased interest in the Bulgarian market, showing crustaceans to be second in terms of nationwide production and import, followed by mollusk categories [8]. For gastropod mollusks (MG), the sampling was deliberately reduced as, according to bibliographic data, their consumption is mostly limited to the Rapana venosa species [28]. The products were purchased from large distribution retailers in four different towns (Stara Zagora, Shumen, Varna and Dobrich) from March 2019 to July 2021, and the sampling resulted in the collection of 199 seafood products, including whitefish (WF; N = 100), cephalopod mollusks (MC; N = 40), crustaceans (C; N = 38), mollusks bivalves (MB; N = 16), and mollusks gastropods (MG; N = 5). Therefore, the different proportion of products collected for the five product categories (WF = 100, 50.3%; MC = 40, 20.1%; C = 38, 19.1%; MB = 16, 8.0%; MG; N = 5, 2.5%) more or less reflects the weight of the various categories on the national market. The products were transferred to the Department of Food Quality and Safety (Faculty of Veterinary Medicine, Trakia University). Each product was classified according to Regulation (EC) No. 852/2004 [29] as unprocessed (fresh, frozen, beheaded, cleaned and filleted) or processed (marinated and precooked), then provided with a progressive numerical code, which was recorded in a single file together with labeling information (Table S1). Finally, 1–5 g of muscle tissue were collected, dehydrated by means of 95% ethanol and sent to FishLab (Department of Veterinary Science, University of Pisa) for molecular identification. Total DNA extraction from each dehydrated tissue sample was performed according to the salting–out procedure proposed by Armani et al. [30], starting from 50 to 100 mg of tissue. Final DNA concentrations and quality were checked with a Nanodrop ND–1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) according to the manufacturer guidelines and absorbance ratios A260/A280 > 2.0 and A260/A230 >1.8 were set as minimum values of nucleic acid purity. The selection of the molecular target followed the decisional procedure (decision tree) proposed by Tinacci et al. [31]. A fragment of 655–658 bp of the Cytochrome C oxidase subunit I (COI) gene was chosen as an elective target for species identification for all the product categories. Additionally, two mitochondrial gene coding for the enzyme cytochrome b and 16S ribosomal RNA (cytb and 16SrRNA) and one nuclear gene coding for the enzyme Phosphoenolpyruvate Carboxykinase (PEPCK) were applied in the study to enhance the discriminatory molecular ability when the elective target alone failed to achieve species identification according to the seafood product’s taxonomical class under analysis. Products labeled as Mytilus sp. (Table S1) were only tested by the PCR–RFLP technique for the analysis of a non–repetitive region of the nuclear Polyphenolic Adhesive Foot Protein (PAP) gene as described in Giusti et al. [21]. Details on elective and additional targets, primer pairs and amplification protocol applied in the study are summarized in Table 1. After amplification, 5 µL of each PCR product was checked on a 1.8% agarose gel previously stained with GelRed™ Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The presence of the expected amplicon and the final concentration was verified by comparison with the standard molecular marker SharpMass™50–DNA (Euroclone Spa, Pero; Milano, Italy). A concentration equal to or greater than 5 μg/μL of PCR product was set as a threshold value for subsequent sequencing reaction according to the sequencing laboratory operative procedures and sent to Eurofins Genomics laboratories (Eurofins Genomics, Ebersberg, Germany). The obtained sequences were aligned and edited with Clustal W in BioEdit version 7.0.9 [38], and the final sequences were queried against the reference sequences available in GenBank (http://www.ncbi.nlm.nih.gov, accessed on 30 July 2022 ) and, in case of COI, the BOLD (http://www.boldsystems.org/, accessed on 30 July 2022) databases. The species was finally allocated by setting target–specific identity score cut–offs as specified below. For the COI and cytb targets, a top match identity score >98% was applied for species allocation [31,39]; for the 16SrRNA and PEPCK targets, an identity score of 100% was defined as the cut–off threshold for species identification [31,40,41]. Furthermore, for species allocation with the additional targets, the Neighbor–joining method [42] and Kimura 2–parameter model [43] with 1000 bootstrap re–samplings were applied to infer distance–based dendrograms in MEGA–11 [44]. The information reported on the labels was verified against the mandatory requirements of Regulation (EU) No. 1379/2013 [16]. Specifically, for each product included in the scope of Article 35, commercial designation of the species and its scientific name, an indication of production method, catch or farming area for aquaculture products, fishing method and thawing before the sale were considered. The scientific names were verified by consulting the official databases listed in Article 37 of the Regulation (FAO Sealife base; FAO Fishbase, World Register of Marine Species (WorMS) and ASFIS databases). Thus, a direct comparison between the molecular results and the labeled scientific names was performed. The products were declared non–compliant if the molecularly identified species did not match the scientific names labeled on the product. Furthermore, the adherence of the labeled designations against the list of accepted scientific names in force during the sampling period, included in Ordinance no. 4 of 13.01.2006 [45], was verified. The labeled designations were finally compared with the new list, published with Ordinance No. 13 of 30.11.2021 [46], published after the completion of the sampling phase. The comparison with both lists was made to assess the evolution of the list of official designations in terms of adherence to the Bulgarian market supply. The definition of the sampling strategy should be based on a preliminary study of the different product categories available on the market to better define the representative sample size for each taxonomical class [12]. Thus, the sample products collected and analyzed in this study reflect the weight of the different seafood taxonomical classes and products as assessed in the preliminary sampling strategy (see Section 2.1). The study highlighted the availability at large retail levels of a fairly limited variety of species (Figure 1) in agreement with several findings from an extensive survey conducted in 2019 on the Bulgarian seafood market, both at the local and large retail levels, to assess the diversity of available retail fish products [9]. When limiting the comparison to retail data, the survey had already revealed increased attention towards marine imported products of Atlantic and Pacific origins. This finding showed a clear evolution of the consumption trend compared to a study conducted by Stancheva [6], from which the consumers’ preferences appeared to be mainly oriented towards local freshwater products or locally caught marine fish products in accordance with national culinary traditions. Prominent species were immediately identifiable for each taxonomical class (WF, MC, MG, C). In detail, in WF, G. chalcogrammus accounted for 46% of products collected in the study, followed by Argentine hake (Merluccius hubbsi) (23%). The C and MC categories were led respectively by Penaeus vannameii and Dosidicus gigas representing 58% and 60% of the products, respectively. A greater equilibrium of species is observed within the MB in which the species M. chilensis is predominant but in balance with M. galloprovincialis, while the third species Perna canaliculus, was only occasionally available for sampling. With regard to MG, consumer interest, as previously introduced, is focused exclusively on R. venosa, the only species sampled for the taxonomical class and, according to Keskin et al. [47], one of the most exploited species along Black Sea coasts. Overall, 76.4% (N = 152) of the samples were made of unprocessed products (75.4% frozen and 1% chilled) and the remaining 23.6 % (N = 47) of distinct types of processed products (14% precooked frozen, 5.0% marinated and 4.5% canned) with different frequencies among the categories included in the study (Figure 2A). A different presentation of the products at purchase for the different taxonomic classes was observed (Figure 2B). In particular, the greatest variability was observed in WF where, however, the prevalence of variously cut products (59% in fillets and 9% in slices) compared to whole products can be observed. In 100% of the MC products, a standard level of processing was observed with the absence of whole products at retail. A similar assessment applies to MB, where 100% of the products presented for sale in whole form without shells. In MG, a distribution between whole products without shells and sliced products was observed where the aquatic organism was no longer immediately recognizable as it had been reduced to a pulp. For class C finally, a clear predominance of peeled products (68.6%, 26/38) over whole specimens (31.6%, 12/38) was observed. Thus, according to the results, the retailers imported from EU and non–EU producers seem to almost exclusively target frozen unprocessed and precooked products and less frequently canned products, except for MB. This evidence agrees with the data collected in Todorov [8], confirming the Bulgarian large retail fishery market trades are mainly oriented towards imports of prepared (frozen cleaned, filleted or sliced) and processed (canned, marinated) products of EU and non–EU origin. This is in agreement with the description also given by Vindigni and colleagues [48] on the type of products most imported at the EU level based on data provided by the EU Commission in 2020. It is also interesting to note the degree of preparation found not only in processed products (ready–to–eat or breaded precooked) but also in unprocessed products. This observation is consistent with that reported by Stancheva [6] regarding consumer demand for easy–to–use, fresh, frozen or canned products. Total DNA sample was successfully extracted from all 199 products included in the study. All the 199 DNA samples were successfully amplified producing 208 PCR products (COI N = 186; cytb N = 11; 16S rRNA N = 8; PEPCK N = 3) for further sequencing and 13 PCR products (belonging to products labeled as Mytilus sp.) to be analyzed by RFLP (Table S2). All the 208 PCR products intended for post–sequencing analysis returned readable sequences. The length of the final sequences and the results of the post–sequencing analysis are shown in Table S2. Overall, a final species allocation was reached in 188 out of 199 products included in the study (94.5%) using: (1) a COI barcoding approach in 159 products (F = 81; MC = 39; MB = 3, C = 35); (2) or by the analysis of an additional target in 16 products (F = 11; MG = 5); (3) by the application of a PCR–RFLP protocol specifically for Mytilus sp. species allowed the final species allocation in 13 MB products as detailed in Figure 3. Specifically, 10 MB were finally assigned to the species M. chilensis, and the remaining 3 MB products (MB–3, MB–8, and MB–16) were assigned to the species M. galloprovincialis. In particular, 11 WF products finally identified as M. productus (N = 7) and M. gayi (N = 4) by the analysis of COI and cytb targets (Section 3.2.1) and five MG products were finally assigned at the species level by the analysis of COI and 16S rRNA targets (Section 3.2.2). In the remaining 11 products, the analysis exclusively led to a genus–level allocation with the assignment of 8 F products to Alepocephalus sp. and 3 C products to Metapenaeus sp. (N = 2) and Heterocarpus sp. (N = 1). Nevertheless, all the data obtained were sufficient for the subsequent verification of labeling compliance. Major limitations to the successful species identification could be due to (1) low target barcode resolution, (2) the low reliability of reference sequences or (3) the absence of reference sequences for the comparative analysis, as already highlighted by Fernandes et al. [18]. Low target barcode resolution is typically described in recently diverged and/or closely related species that are geographically isolated or are in the presence of species complexes and hybrids [49,50]. In the present study, a low resolution of COI barcodes was highlighted during the analysis of products finally identified as belonging to species from Merluccius sp. and products finally not assigned at species level belonging to Metapenaeus sp. and Heterocarpus sp. Limitations in species allocation have been described in the analysis of hake products (Merluccius sp.) for which the evaluation of different mitochondrial targets such as cytb or Control Region barcodes have been proposed in numerous studies [51]. In the present study, the use of cytb, in additional to COI, was conclusive for the product allocation to two species, M. productus and M. gayi. In fact, the similarity analysis on the COI target computed with BLASTn and K2P analysis computed with BOLD IDs had shown overlapping identity values within the threshold set for species–specific identification. The distance–based dendrogram produced on cytb target by including reference sequences belonging to 11 Merluccius sp. species and 11 sequences obtained in this study from products shows distinct and discrete species clustering and the allocation of the products sequences within the cluster M. products (F15, F16, F17, F46, F63, F66, F95) and M. gayi (F61, F79, F81, F82) clusters (Figure S1). Thus, in accordance with the multitarget approach proposed by Tinacci et al. [31], the introduction of an additional target offered a decisive improvement of the species disambiguation and the final allocation of the products at the species level. With respect to Metapenaeus sp., although the DNA barcoding technique offers an efficient tool for species identification to ensure traceability and identity verification of crustacean products [52,53] in the present study, objective limitations were highlighted to the final species allocation within the genus even despite the use of the additional target 16S rRNA or PEPCK. Similar considerations can be applied to the failure of species identification for a product (C10) identified as belonging to the genus Heterocarpus sp. Limits in molecular identification of penaeid shrimps were also highlighted by Rajkumar [54]. In this respect, several authors concurred in identifying the gene targets selected in the present study as valid for the identification of Penaeid shrimp species, nonetheless emphasizing the importance of selecting the bioinformatics method to be applied for an accurate definition of the relationships and clustering of the various species within genera clades [18,52,55]. Reference sequences’ unreliability and absence have been extensively debated and generally attributed to incorrect species–sequence associations or the submission of sequences belonging to not taxonomically validated specimens [56,57,58]. The presence of reference sequences of uncertain and debatable reliability was highlighted in the study during the post–sequencing analysis of COI target barcodes obtained from an MC product (MC32) and from five MG products (MG1, MG2, MG3, MG4 and MG5). In the first case, MC32 was finally assigned to the species Notodarus sloanii despite an initial overlapping top identity match of the MC32 COI sequence with the reference sequences of Nototodarus sloanii (100–99.84%), and 4 reference sequences (DQ373957–60) deposited by Carlini et al. [59] for Illex argentinus (100–99.84%). The four sequences were excluded by assuming the hypothesis of a potential error occurring during the morphological identification of the specimens. The assumption was strengthened by the results of a further cross–BLAST analysis of the sequences deposited by Carlini et al. [59] against the GenBank repository of all I. argentinus COI reference sequences from which the identity values lower than 85% were highlighted for each of the 4 investigated sequences. A similar assumption was pursued for the final allocation at the species level of the five MG products included in the study. In this respect, the post–sequencing analysis of the COI barcodes obtained from five MG products highlighted overlapping similarity scores within the ID score threshold (98%) with R. venosa, R. bezoar and three reference sequences deposited for Turbo cornutus (HM180929, HM180930 and HM180931) deposited by Kim at al. [60]. The three sequences were excluded following a cross–BLAST analysis against the GenBank repository of all Turbo cornutus COI sequences from which identity values lower than 78% were highlighted for each of them. Thus, the final allocation of the products to the species Rapana venosa was achieved through distance analysis conducted on the target 16S rRNA (Table S3; Figure S2), which had been previously successfully applied in a comprehensive phylogeny study on Rapaninae, Muricidae taxonomical group in association with COI, 18S rRNA, 12S rRNA [61]. In the case of Alepocephalus sp., given the low resolution of the elective target for species–specific identification revealed during BLAST analysis, the analysis was not conclusive due to the lack of deposited reference sequences for the additional target gene provided by the operative protocol (cytb). This evidence underlined a major limitation of the DNA barcoding technique and the need to continuously update reference databases. The need for continuous updating of the comprehensive DNA barcode reference libraries has been extensively emphasized in a study conducted by Weigand and colleagues [62] on the Barcode of Life Data Systems (BOLD) and NCBI GenBank databases. From this study a clear lack of homogeneity in the deposit of sequence records among taxonomic groups emerged among geographic regions. The authors also highlighted that the presence of a large proportion of species (up to 50%) in several taxonomic groups is only represented by private data with obvious implications on the actual possibility of their use. The authors, therefore, emphasize the need for a coordinated and systematic action of database improvement to close the information gap and to maximize phylogenetic representativeness, thereby yielding to the collection of reference barcodes of representative species from missing orders, families and genera. It is first appropriate to emphasize that according to Regulation (EU) No. 1379/2013 [16], the labeling obligations stated in Article 35 do not apply to processed products which, except for marinated and smoked products, do not fall within the scope of the provision. For other products, the application of the regulatory requirements is exclusively subject to the FBO’s will, although strongly advocated by the EU Parliament to promote informed consumer choice at the time of purchase [63]. Voluntary extensions of the regulation’s requirements for the labeling of out–of–scope products have been documented in numerous studies of species identification in variously processed seafood [64,65,66,67]. This considered the labels’ analysis confirmed for all the products within the scope of Regulation (EU) No. 1379/2013 [16] (N = 160), the proper application of Article 35, indicating commercial designation and species scientific name, an indication of production method, catching area, and thawing process. The only exception was represented by the fishing method, which was not declared in 6.8% of the products (11/160) (Table S1). This confirmed the substantial labeling compliance of products sold at large retail, as already highlighted by Tinacci et al. [9]. Interestingly, the analysis of the data also revealed the voluntary application of the Regulation requirements in the remaining products (N = 39), consisting of breaded precooked (N = 26) and canned (N = 13) products. In all these products the commercial designation and scientific name, origin and catching area was reported while the fishing method was only reported in 38.46% (15/39) of the products. Notwithstanding, since any information included on the label, whether mandatory or voluntarily introduced, is subject to transparency and authenticity obligations according to Regulation (EU) No. 1169/2011 (article 7) [15], all products under study were included in the calculation of the mislabeling rate overall and for the individual product categories (F, MC, MB, MG and C) discussed below. In the comparison of the molecular results with the labeled scientific names, the presence of 22 substitutions out of 199 products, corresponding to an overall mislabeling rate of 11%, was found All substitution incidents are presented in Table 2. The overall mislabeling rate appears to be lower than in the pilot study [22], which stood at around 17%. The highest percentage was found in WF (14%), followed by MB (12.5%) and MC (10%). The overall percentage and percentages per taxonomical class fall within the substitution range (4–14%) identified by Luque and Donlan [13] in a meta–analysis study conducted on scientific data and publications produced up to 2017. In particular, the WF mislabeling rate appears next to the percentage (12.9%) highlighted by Minoudi et al. [68] in a market survey conducted on the Greek market and mostly directed toward whitefish species. In this regard, we have to highlight that in 2015, the frequency and impact of fraud perpetrated on this category prompted the EU Commission to organize a coordinated control program across all member states to assess the extent of mislabeling in the fishery sector with a specific focus on the whitefish market [69]. Although not internationally harmonized, the definition of species substitution is broadly described as the intentional deception of a food product for economic gain or to conceal other illegal actions such as illegal, unreported and unregulated fishing achieved through the misrepresentation of food products or alteration of the associated documentation [11,70,71]. Conversely, in addition to fraudulent incidents, the existence of involuntary substitutions due to the lack of adequate training in morphological species identification of fishermen and operators at the first sale level should also be emphasized [72]. Both incidents result in damage to both consumers and food business operators and underline the fishery sector’s vulnerability and the need to acquire in–depth knowledge of seafood chain traceability systems and individual business practices [11,25]. Within WF substitution, incidents between Alaska pollock (G. chalcogrammus) and species belonging to Merluccius sp. of medium commercial value were frequently observed, followed by substitutions of Atlantic cod (G. morhua) with Saithe (Pollachius virens) and substitutions of hake species (Merluccius productus and Merluccius hubbsi) of medium commercial value replaced with Gadidae species of lower commercial value (Micromesistius australis). The substitutions observed in the study are all widely described and attributable to fraudulent incidents for economic gain [12,25,73,74]. Similar evidence within the Gadidae and Merluccidae families was found by Minoudi et al. [68], wherein they identified misidentification during fishing and plausible fraudulent actions perpetrated at the product distribution level as possible causes of substitution. The study of the natural geographical distribution of substitute and replaced species could provide useful elements to potentially determine the origin of the fraud, as most potentially perpetrated at the first sale or at an intermediate level during the products processing [75]. In the present study, the analysis of the labeled origins revealed a mostly non–Mediterranean supply mainly oriented to products of Atlantic or Pacific origin. Within WF, the fishing areas most frequently highlighted at purchase were Northeast/Northwest Pacific (FAO 61/67), corresponding to the distribution area of G. chalcogrammus. Less frequent but nonetheless relevant were Northeast/Northwest Atlantic (FAO 21/27) and Southeast Atlantic (FAO 41), corresponding to the distribution areas of Atlantic cod (G. morhua), Saithe (P. virens), Baird’s smooth–head (Alepocephalus bairdii) and a few hake species (M. hubbsi, Macruronus sp.) all appreciably represented in large–scale distribution. Product of Mediterranean origin and, in particular, Black Sea origin (FAO 37.4) was exclusively found in products labeled as Merlangius merlangus (whiting), a fish species belonging to the Gadidae family of local interest and of medium commercial value. Thus, from the observation of the results collected in Table 3, fraudulent substitution phenomena within WF possibly occurred both at fishing/first sale and during processing or packaging. In detail, fraudulent substitutions at the first–sale level are plausibly conceivable for replaced and substitute species that share the geographic area, as in the substitution of G. chalcogrammus with M. productus or G. morhua with P. virens. Conversely, fraudulent substitution phenomena occurring at a more advanced stage of the chain (processing, packaging and distribution) are speculated in cases where a geographically distant substitute species were highlighted, such as in the substitution of G. chalcogrammus with M. hubbsi and M. productus with M. hubbsi or M. australis. In terms of environmental sustainability, it is pertinent to emphasize that the perpetration of substitution between geographically distant products could also conceal the attempt to reallocate products belonging to illegal fishing [25]. Within MC, Humboldt squid (D. gigas) was verified as the dominant species on the market, bringing the Southwest and Southeast Pacific (FAO 81/87) into a prominent position among exporting areas. The species was also the most frequently substituted species in the pilot study [22] and in the studies concerning mislabeling of cephalopod products on the Chinese and EU retail market [76,77]. In fact, D. gigas has been thoroughly described as one of the elective substitute species, especially due to its high availability and low commercial value, which render the species an appealing candidate in the perpetration of deceptive frauds for economic gain [77,78]. In MB products, the analysis of products origin highlighted a market orientation towards imported products belonging to North Atlantic (FAO 27) or South Pacific (FAO 81/87), specifically represented by the Mediterranean mussel (M. galloprovincialis), Chilean mussel (M. chilensis) and New Zealand green–lipped mussel (Perna canaliculus). The only two substitutions encountered consisted of the replacement of M. galloprovincialis with M. chilensis. Similar substitutions are described in products imported from Chile and have been attributed to unintentional accidents related to the coexistence of the two species in fishing and aquaculture areas along Chilean coasts [21]. However, this observation does not apply to one of the two mislabeled products in the study (MB13), a canned marinated product, for which the clear North Atlantic (FAO 27) origin of the product was declared on the label and a fraudulent action is clearly hypothesized and collocable at the product processing stage. Lastly, in terms of C–class from the data collected at purchase, the market supply appeared directed to aquaculture products (P. vannamei) of Asian origin (Vietnam, Bangladesh, India) except for the sporadic presence of P. borealis of the Northwestern Atlantic origin and Metapenaeus sp./Penaeus sp. products from the Indian Ocean. Thus, with respect to the substitutions highlighted, given the considerations outlined above concerning the difficulties encountered in the morphological species identification [52] and given the high degree of overlap of the geographical distribution areas of the substituted and substituted species (Sealifebase.org), it is plausible to affirm the occurrence of involuntary substitution phenomena for these products. Nevertheless, this aspect further underlines the need to promote and improve the operator’s awareness and skills for species recognition, also implementing molecular identification systems that can be used at processing plants [79]. Improved traceability tools represent, in fact, an essential support for FBOs who, in any case, hold the responsibility of verifying the identity of their products for consumer protection in accordance with EU legislation [31]. In this regard, rapid identification methods have been promoted and developed in recent years for the most traded species, potentially applicable to self–monitoring by various operators, especially at the distribution level [80,81,82]. Table 3 presents the results of the comparison between the scientific names found in association with the products and the official list of accepted trade names in force during the sampling period [45], and the updated list [46]. The comparison of the labeled scientific names and those listed in the ministerial ordinance, including the accepted designations, clearly highlighted the ineffectiveness of the list in force at the time of sampling in describing the basket of species present on the market. This aspect is extensively investigated and described by Tinacci et al. [23] in a study aimed at assessing the validity and accuracy of the new official Bulgarian list of seafood trade names in compliance with EU requirements. The authors, in this regard, stated that there was a clear contradiction between the official records listed in the ordinance and the market needs. The authors particularly emphasized the ineffectiveness of the list in describing imported products that are widely available on the Bulgarian market and especially at the large retail level, which is even more evident in the newly promulgated list, in which some previously included scientific names of relevance, such as G. morhua or Pollachius virens, have disappeared. Therefore, an urgent need for further revision and expansion of the list of official denominations is advisable and pivotal to meet the obligation of periodic updating imposed by Regulation (EU) No. 1379/2013 (Article 37) [16] and to provide FBOs with an effective tool to guarantee consumer rights on informed choice. In the revision of the list, as pointed out by Tinacci et al. [22], Tinacci et al. [23] and in the present study, all taxonomical classes should be considered since, albeit to a lesser extent, besides fish, both mollusks and crustaceans are variously represented on the national market. An example of exponential evolution and expansion of the list of official denominations in relation to market needs is presented by Tinacci et al. [83] for the Italian context. In a comparative retrospective analysis of the lists promulgated for the marketing of seafood products on the national territory from 2002 to 2017, the authors highlighted the market inputs at the origin of the evolution of the list, which were driven both by the demand of the average Italian consumer and by the ethnic groups and migrant populations permanently present on the national territory. Finally, regarding taxonomical validity, three invalid names, referring to an obsolete classification were highlighted (Table 3). It is, admittedly, true that updating scientific designations is an extremely challenging issue, given the continuous advancement in fish and seafood phylogeny research [83]. In this regard, therefore, in conjunction with the revision of the list in which, as highlighted, these names are not yet included, the promotion of a specific FBO training would be appropriate to monitor and encourage the replacement of obsolete names and update the labeling of new product batches. The overall mislabeling rate of 11% and the analysis of the substitution incidents that emerged in the study highlighted the need to promote the implementation of DNA–based monitoring systems oriented towards supplier selection to be applied amongst FBOs at various levels of the production chain (processing and retail). This could be reduce involuntary substitutions and prevent deceptive practices to protect both seafood supply chain and consumers’ rights. In this light, an integrated approach for species–specific polymorphisms analysis, through the association of different DNA analytical methods, may represent a useful and effective strategy for univocal seafood identification. In addition, this study confirms how the Bulgarian fish market, with reference to large–scale retail sales, still appears to be targeting a limited, albeit expanding, number of species. This aspect is stressed by the apparent inadequacy of the list of official names currently in force in the territory to describe the variety of products on sale. Therefore, as pointed out in a previously published study by the authors [23], a further update and expansion of the carnet of official commercial designations authorized in the territory are required. The data obtained from this survey could constitute inputs for implementing a monitoring plan promoted by governmental agencies in agreement with seafood stakeholders (wholesalers and sellers) for seafood authentication, contributing to the transparency of the seafood market at the national level.